$0 8 - Issue 1.pdf · reports the e ect of soaking, autoclaving, roast-ing or germination on the nutritional quality and functional properties of cashew our. 2 Materials and Methods

International Journalof Food Studies

ISSN 2182-1054

Volume 8 | April 2019

International Journal of Food Studies

The International Journal of Food Studies (IJFS) a journal of the ISEKI_Food Association is an international peer-

reviewed open-access journal featuring scientific articles on the world of Food in Education Research and Industry

This journal is a forum created specifically to improve the dissemination of Food Science and Technology

knowledge between Education Research and Industry stakeholders Manuscripts focusing on Food related

Education topics are particularly welcome The IJFS also accepts original research works dealing with food processing

design storage and distribution including effects on productrsquos safety and quality and food chain sustainability The

journal is also open to other food-related topics such as food security and food policy

Editor-in-Chief

PROFESSOR CRISTINA L M SILVA

Catholic University of Portugal - College of Biotechnology

Rua Arquiteto Lobatildeo Vital 172 4200-374 Porto Portugal

Vice Editors-in-Chief

Professor Margarida Cortez Vieira

High Institute of Engineering of University of Algarve

Estrada da Penha 139 8005-139 Faro Portugal

Professor Paulo Joseacute do Amaral Sobral

University of Satildeo Paulo - Faculty of Animal Science and

Food Engineering Av Duque de Caxias Norte 225

Campus Fernando Costa ndash USP CEP 13635-900

Pirassununga Satildeo Paulo Brazil

Associate Editors

Professor Liliana Tudoreanu

University of Agronomic Sciences and Veterinary

Medicine Bucharest Romania

University of Algarve Portugal

University of Satildeo Paulo - Faculty of Animal

Science and Food Engineering Brazil

Professor Petras Rimantas Venskutonis

Kaunas University of Technology Lithuania

Professor Rui Costa

College of Agriculture - Polytechnic Institute of

Coimbra Portugal

Dr Rui Cruz

Professor Tanaboon Sajjaanantakul

Kasetsart University Thailand

Professor Victoria Jideani

Cape Peninsula University of Technology South

Africa

Advisory Board

Afam Jideani

University of Venda South Africa

Antoacutenio Vicente

Universidade do Minho Portugal

Brian McKenna

University College Dublin Ireland

Elisabeth Dumoulin

Paris Institute of Technology for

Life France

Ferruh Erdogdu

Ankara University Turkey

Gerhard Schleining

BOKU Austria

Gustavo V Barbosa-Canovas

Washington State University

United States of America

Gustavo Gutieacuterrez-Loacutepez

National School of Biological

Sciences Mexico

Joseacute Antoacutenio Teixeira

Kristberg Kristbergsson

University of Iceland Iceland

Mustapha El Idrissi

Mohamed Premier University

Morocco

Pablo Ribotta

School of Exact Physics and

Natural Sciences Argentina

Paul Singh

University of California - Davis

United States

Paola Pittia

University of Teramo Italy

Peter Ho

University of Leeds United

Kingdom

Pilar Buera

School of Exact and Natural

Sciences Argentina

Sam Saguy

Hebrew University of Jerusalem

Israel

Teresa Brandatildeo

Catholic University of Portugal

Portugal

V Prakash

Central Food Technological

Research Institute India

Venkatesh Meda

University of Saskatchewan

Canada

INTERNATIONAL JOURNAL

OF FOOD STUDIES

Volume 8 nordm 1 (2019)

(Published 18 April 2019)

CONTENTS

1 The Impact of Processing Methods on Chemical Composition Mineral Bioavailability and Functional Properties of

Nigerian-Grown Cashew Flour

MAKINDE FOLASADE MARIA amp JOEL IFEOLUWA HANNAH

14 Use of Response Surface Methodology (RSM) for Composite Blends of Low Grade Broken Rice Fractions and Full-fat

Soybean Flour by a Twin-screw Extrusion Cooking Process

NAHEMIAH DANBABA IRO NKAMA amp MAMUDU HALIDU BADAU

30 Nutritional and Antioxidant Potential of Rice Flour Enriched with Kerstingrsquos Groundnut (Kerstingiella geocarpa) and

Lemon Pomace

OLUGBENGA O AWOLU amp MAGOH A OSIGWE

41 Effect of Chestnut and Acorn Flour on Wheat Wheat-Barley Flour Properties and Bread Quality

MARIE HRUŠKOVAacute IVAN ŠVEC amp IVANA KADLČIacuteKOVAacute

58 Stability of Vitamin C in Broccoli at Different Storage Conditions

NASSER AL-HABSI SITHARA SURESH AMANI AL-YHMEDI MARWA AL-SHORYANI MOSTAFA I WALY amp

MOHAMMAD SHAFIUR RAHMAN

68 Physico-Chemical Composition and Antimicrobial Protein Content of Early Lactation Donkey Milk

MARIA ASPRI KALLIS SOUROULLAS CHRISTINA IOANNOU amp PHOTIS PAPADEMAS

76 Study of the Self-Stabilization Ability of Tzatziki (a Traditional Greek Ready-to-Eat Deli Salad)

STAVROS LALAS VASSILIS ATHANASIADIS IOANNA KARAGEORGOU ELENI BOZINOU amp VASSILIS G

DOURTOGLOU

87 Moisture Sorption Isotherm and Thermal Characteristics of Freeze-Dried Tuna

MOHAMMAD SHAFIUR RAHMAN MOHAMMED KHALFAN AL-KHUSAIBI KUTAILA ABBAS AL-FARSI

ISMAIL MOHAMED AL-BULUSHI AISHA ABUSHELAIBI amp NASSER AL-HABSI

97 Antioxidant Indices and Amino Acid Composition of Phenolic Containing Lima Beans (Phaseolus lunatus) After

Simulated Human Gastrointestinal Digestion

SULE O SALAWU OLUWASEUN M FOLORUNSO AKINTUNDE A AKINDAHUNSI amp ALINE A BOLIGON

111 Moisture Sorption Isotherm and Thermodynamic Properties of Jamun (Syzygium cumini L) Powder made from Jamun Pulp

and Seed

INDIRA DEY PAUL amp MADHUSWETA DAS

International Journal of Food Studies IJFS April 2019 Volume 8 pages 1ndash13

The Impact of Processing Methods on Chemical CompositionMineral Bioavailability and Functional Properties of

Makinde Folasade Mariaa and Joel Ifeoluwa Hannaha

a Department of Food Science and Technology Bowen University Iwo Osun State NigeriaCorresponding authorsademakinyahoocomTel +2348072673097

Received 23 October 2017 Published online 18 April 2019

Abstract

In this study the effect of processing methods on the nutritional quality and functional properties ofcashew (Anacardium occidentale Linn) kernels were investigated The kernels were soaked autoclavedroasted or germinated at varying time duration raw kernel served as control The samples were anal-ysed for chemical mineral bioavailability and functional properties Data was subjected to analysisof variance and means were separated by the Duncan multiple range test The result of chemicalcomposition analyses revealed that raw cashew kernels contained 355plusmn008 moisture 213plusmn005protein 450plusmn015 fat 253plusmn002 fibre 159plusmn002 ash 261plusmn001 carbohydrate 52175 Kcalgenergy 221009plusmn002mgkg calcium 171254plusmn003mgkg magnesium 6004plusmn001 mgkg iron and3674plusmn002mgkg zinc Tannin phytate and oxalate concentrations in the raw cashew kernel were1014plusmn003 mgkg 9930plusmn002mgkg and 1103plusmn003mgkg respectively Increased fat ash and fibrelevels were noted for treated samples compared to raw kernels Mineral concentrations were increasedsignificantly by various treatments compared to raw kernel however germination resulted in the high-est increase of mineral content A reduction trend was observed in phytate oxalate and tannin con-centrations in the treated samples with respect to increased processing time Consequently varioustreatments influenced the bioavailability of mineral elements Treated samples exhibited significantdifferences in loose and packed bulk densities water and oil absorption capacities when compared toraw kernels Germination shows potential to generate not only much needed nutrients in cashew forhuman development but also improved bioavailability of nutrients and functionality compared to otherprocessing methods This approach can used in Community Nutrition and Emergency Feeding Pro-grammes in developing countries where the consequence of anti-nutritional factors may worsen theincidence of malnutrition and disease

Keywords Cashew Nutrient Mineral bioavailability Functionality Processing effects

1 Introduction

The nutritional inadequacies in developing coun-tries that arise from high cost of animal pro-teins have necessitated the use of other proteinfood sources such as those from plant originsTree nuts such as almonds cashews hazel nuts

macadamias pecans pine nuts pistachios andwalnuts are readily available and offer great po-tential as a ready to eat food for human con-sumptionThe cashew tree (Anacardium occidentale Linn)is a native of Brazil and widely cultivated acrossthe coastal regions of the tropics (Gibbon amp

Copyright copy2019 ISEKI-Food Association (IFA) 107455ijfs812019a1

2 Maria and Hannah

Pain 1985) The major producing countries ofthe cashew nut are Tanzania India Mozam-bique Srilanka Kenya Madagascar ThailandMalaysia Indonesia Nigeria Senegal Malawiand Angola Cashew was introduced into Nige-ria in 16th century however the cultivation hasspread to western eastern and northern states(Hammed Anikwe amp Adedeji 2008) It is im-portant to note that cashew nut is underutilizedin Nigeria as only about 10 is utilized locally(FAO 2008)The cashew fruit is made up of the apple in whichthe kernel is embedded The kernel has high nu-trient value and consists of 212 protein 220carbohydrate and 470 fat it offers 575 kcalof energy per 100g and various minerals (Sathe1994) As with other tropical nuts the cashewcomes with associated issues such as the pres-ence of secondary plant metabolites that are alsoknown as anti-nutritional factors (ANFs) Theseinclude tannin phytate and trypsin inhibitorwhich bind with minerals such as Fe Zn andother similar metals and protein respectivelyThese metabolites subsequently interfere withnutrient utilization when fed to human (Ngatchicet al 2013)This activity remains a major draw-back to the direct use of cashew as food ingredi-ent Several processing techniques have been re-ported by various investigators that may enhancethe nutritional quality of food materials and alsoreduce or destroy the anti-nutrient present inthem (Esenwah amp Ikenebomeh 2008 S lupski2011) The knowledge of how processing condi-tions modify bioavailability of nutrients trappedin the food matrix is indeed a critical factor to beconsidered in the development of food for variousage groups in addition to cost of raw ingredientsprocessing cost and sensory acceptability of suchfoods (Abebe Stoecker Hinds amp Gates 2006)Similarly the functional performance of plant-based protein is also important in determiningits usefulness in food systems This study addsto the current knowledge of cashew nutrition andreports the effect of soaking autoclaving roast-ing or germination on the nutritional quality andfunctional properties of cashew flour

2 Materials and Methods

21 Materials

Cashew nuts were purchased from a local marketin Kogi State Nigeria All chemicals used were ofanalytical grade and were purchased from SigmaChemical Company St Louis MO USA

22 Preparation of cashew flour

Cashew nuts were sun dried to facilitate shellingThe dried nuts were shelled manually with theuse of hammer to obtain the kidney shaped ker-nels The kernels were divided into five por-tions one potion was kept as a whole kernel (con-trol) second portion was soaked in distilled wa-ter third portion was autoclaved fourth portionwas roasted and the last portion was germinated

Soaking

Cashew kernels (50g) were soaked in tap waterin a ratio 110 (wv) at room temperature (25plusmn2oC) for 6 12 and 18h respectively The soakedkernels were washed twice with water followedby rinsing with distilled water and then driedin an oven at 60 oC to a constant weight ac-cording to Kaur and C Kapoor (1990) Thedried samples were milled using Philips labora-tory blender (HR2811 model) to produce flourand sieved using a 60 mm mesh The flour ob-tained was packed in a glass container and storedin a refrigerator maintained at 8 oC prior to use

Autoclaving

Cashew kernels (50g) were autoclaved for 5 10and 15 min respectively at 121 oC according toKaur and C Kapoor (1990) The autoclavedkernels were dried at 60 oC to a constant weightThe dried samples were milled into flour usingPhilips laboratory blender (HR2811 model) andsieved using a 60 mm mesh The flour obtainedwas packed in a glass container and stored in arefrigerator maintained at 8 oC prior to use

IJFS April 2019 Volume 8 pages 1ndash13

Impact of processing on nutritional and functional properties of cashew flour 3

Germination

Cashew kernels (500g) were sorted manually forviable kernels The selected kernels were plantedin a basket containing wood sawdust as themediumsubstrate for growth The planted ker-nels were watered daily The kernels were allowedto germinate at 35plusmn2 oC for 7 days producingradical protrusion of about 05cm The germi-nated kernels were washed with distilled waterdrained and dried in a hot air oven at 60 oCfor 24 h according to Ijarotimi Oluwalana andOgunedojutimi (2012)The dried samples weremilled into flour using Philips laboratory blender(HR2811 model) and sieved using a 60 mmmesh The flour obtained was packed in a glasscontainer and stored in a refrigerator maintainedat 8 oC prior to use

23 Chemical analyses

Determination of proximatecomposition

Standard methods according to the AOAC(2005) were used to determine moisture (AOAC-92510) fat (AOAC-200305) by soxhlet extrac-tion and ash (AOAC-92303) by combustionProtein (AOAC-96052) content (Nx625) wasdetermined by micro Kjeldahl method Carbohy-drate content was determined by difference Theenergy value was estimated (kcalg) by multiply-ing the percentage crude protein crude lipid andcarbohydrate by the recommended factor (244837 and 357 respectively) as described by Ek-nayake Jansz and Nair (1999) All analyseswere carried out in triplicate

Determination of mineralconcentration

The method according to the AOAC (2005) wasused for the determination of mineral contentTwo grams of the pulverized sample was placedin a crucible and ignited in a muffle furnace at550 oC for 12 h The resulting ash was dis-solved in 10 mL of 10 HNO3 and heated slowlyfor 20 min After heating it was filtered andthe filtrate used for the determination of min-eral content The mineral constituents (Ca Mg

Fe and Zn) were analyzed separately using anatomic absorption spectrophotometer (HitachiZ6100 Tokyo Japan)

Determination of anti- nutritionalcompounds

Determination of phytate content

The phytate content of the flours was deter-mined using method described by Oladele Os-undahunsi Yemisi and Adebowale (2009)Two(2g) grams of each finely ground flour samplewas soaked in 20mL of 02N HCl and filteredAfter filtration 05mL of the filtrate was mixedwith 1mL ferric ammonium sulphate solution ina test tube boiled for 30min in a water bathcooled in ice for15min and centrifuged at 3000rpm for 15min One millilitre of the supernatantwas mixed with 15mL of 22-pyridine solutionand the absorbance measured with a spectropho-tometer at a wavelength of 519nm The concen-tration of phytic acid was obtained by extrapola-tion from a standard curve using standard phyticacid solution

Determination of oxalate content

The titration method described by Oladele et al(2009) was used to determine the oxalate con-tent One gram of the sample was weighed in a100mL conical flask 75mL 3N H2SO4 was addedand stirred intermittently with a magnetic stir-rer for 1h The contents were then filtered usingWhatman No1 filter paper From the filtrate a25mLaliquot was taken and titrated while main-tained at a temperature of 80-90 oC against 01NKMnO4 solution until a faint pink colour per-sisted for at least 30 sec

Determination of tannin content

Tannin content was determined by the methoddescribed by Mugabo Afoakwa Annor andRwubatse (2017) One gram of sample wasdispersed in 10mL distilled water and agitatedThe dispersion was left to stand for 30 min atroom temperature (20 plusmn 2 oC) then centrifuged25mL of the supernatant (extract) was pipet-ted into a 50mL volumetric flask Similarly 25

4 Maria and Hannah

mL of standard tannic acid solution was pipet-ted into a separate 50mL flask One millilitre ofFolin-Denis reagent was measured into each flaskfollowed by 25mL of saturated Na2CO3solutionThe mixture was made up to mark in a 50mL vol-umetric flask and incubated for 90 min at roomtemperature The absorbance was measured at250 nm with a spectrophotometer (Jenway model6000)

Determination of functionalproperties

The loose and packed densities were determinedusing the method described by Ikpeme Os-uchukwu and Oshiele (2010) Water and oil ab-sorption properties of the cashew flour were de-termined following methods of Adebayo Ogun-sina and Gbadamosi (2013) with slight modi-fications A flour sample (1g) was mixed with10mL distilled water for water absorption and10mL of oil for oil absorption in a Philips labo-ratory blender (HR2811 model) at high speed for30 sec Samples were allowed to stand for 30minat room temperature then centrifuged (UniscopeEngland) at 2000rpm for 30 min The volume ofsupernatant in a graduated cylinder was notedDensity of water was taken to be 1gmL and thatof oil was determined to be 0993gmL Analysiswas performed in triplicate

Mineral bioavailability determination

The bioavailability of minerals (calcium andmagnesium iron and zinc) was calculated as re-ported by Woldegiorgis Abate Haki and Ziegler(2015) The molar ratio between anti-nutrientand mineral was obtained after dividing the moleof anti-nutrient with the mole of mineral Themole of phytic acid was calculated as the mea-sured value of phytic acid divided by the molec-ular weight of phytic acid (6608) whereas themole of mineral (Ca or Mg Fe or Zn) was cal-culated as the measured value of the mineral di-vided by the individual mineral molecular weight(Fe 558 Zn 654 Ca 400 Mg 243)

24 Statistical analysis

Determinations were carried out in triplicatesand the error reported as standard deviationof the mean Analysis of Variance (ANOVA)was performed and the least significant differ-ences were calculated with the SPSS software forwindows (release 1700 SPSS Inc Chicago ILUSA) Significance was accepted at p le 005 lev-els

3 Results and Discussions

The compositions of raw and treated cashewflours are presented in Table 1 The moisturecontent the flour was in the range of 268plusmn010to 478plusmn008 Soaking of cashew kernel for18 h (sample S3) resulted in highest value(478plusmn008) while lowest moisture content(268) was exhibited in the cashew kernel flourroasted for 15min (sample R3)The lowest valuerecorded in sample R3 may be attributed toa greater moisture loss by evaporation duringroasting This result is in agreement with thefindings of Vickers Peck Labuza and Huang(2014) who reported that roasting processesdecreased moisture content of almond kernelsThe moisture content of roasted cashew flourwas however lower than 590 reported byOgungbenle and Afolayan (2015) The proteincontent of the control sample (213plusmn005) andgerminated sample (2221plusmn054) were signif-icantly higher compared to soaked autoclavedand roasted flours respectively Roasting re-sulted in a decrease in protein content over timecompared to raw cashew flour This is by virtueof the Maillard reaction which is an interactionbetween the carbonyl group of a reducing sugarand the free amino group from an amino acidor protein Similarly soaking (6-18h) and auto-claving (5-15 min) of cashew kernel resulted inlower protein values compared to raw kernel dueleaching of soluble proteins into processing wa-ter However the observed significant increasein protein value of germinated cashew flourcould be attributed to the liberation of boundproteins during germination Earlier studies alsoreported that during germination carbohydratesare mobilized to synthesize amino acids for the

growing seedling (Ocheme amp Chinma 2008)There was significance difference (ple005) inthe fat content of the flour samples The highestvalue (530plusmn008) was observed in sampleS3 while the lowest value (450plusmn015) wasexhibited in the control (sample C) Fat contentof cashew kernel flour in this study increasedwith all processing techniques applied Thehigher value noted in soaked flours compared toraw flour is perhaps due to the fact that soakinginitiated the cleavage of the protein-lipid orcarbohydrate-lipid linkages thereby facilitatingthe easy extraction of the oil by the extractingsolvent (Madigan Martinko Parker et al1997) Similarly the higher lipid content inroasted sample compared to the control couldbe explained by the fact that oil bodies and theendoplasmic network were largely destroyed andthe volume of extracellular pores enlarged duringroasting (Grundy et al 2015) The fat contentpresently reported for roasted cashew flour washigher than that reported by Ogungbenle andAfolayan (2015) However it is important tonote that lipid oxidation a critical factor inlimiting shelf life may be controlled to a largeextent by roasting of nuts Non-enzymaticreactions facilitate the release of antioxidantsthough the formation of acrylamide has beenobserved only in almonds and only at excessiveroasting temperatures (Perren amp Escher 2013)Furthermore in terms of its physical behaviourduring mastication roasted nuts were foundto be more brittle and crunchy and producedmore loose particles post chewing than raw nuts(Vickers et al 2014)The ash content of the flour samples ranged from159plusmn015 to 266plusmn005 with the lowest valueobserved in the control sample The significantincrease in ash content of roasted samplescompared to raw sample may be attributedto concentration of the organic compoundsin the former during roasting Soaking andautoclaving of cashew kernels with time alsoimproved the ash content The increase in ashcontent observed in soaked kernels could beattributed to leaching of some constituentsnotably anti-nutrients into the processing waterwhich in turn improved mineral availabilitySimilarly higher ash content is expected espe-cially at above ambient temperature employed

during autoclaving through the swollen andruptured cell walls which permeate water andsoluble constituents Germination process alsoimproves the ash content of cashew flour Thefibre content of the flour samples ranged from253plusmn002 to 576plusmn012 with the lowestvalue observed in the control sample Thecarbohydrate content of flour samples rangedfrom 158plusmn016 to 261plusmn001 Carbohydratehas the highest composition by percentagein raw kernel flour (control) with a value of261plusmn001The lower range recorded for soakedand autoclaved samples respectively may bedue to leaching of soluble carbohydrate (iesugars) into the processing water Similarlythe decrease in carbohydrate content of theroasted cashew kernel with time could be dueto Maillard reaction which occurred betweenamino acids amines aldehydes and carbonylgroup of reducing sugars at high temperatureto produce the roasted cashew flavour Thedecreased carbohydrate content in germinatedcashew flour (sample G) could be explained bythe fact that hydrolysis of carbohydrate to sugaroccurs during germination The energy contentof treated cashew samples was significantlydifferent to that of the control sample Energyvalue of the germinated flour was significantly(ple005) lower than that of other flour samplesGenerally long-term consumption of nuts isassociated with a decreased risk of weightgain and obesity (Bes-Rastrollo et al 2009)Flores-Mateo Rojas-Rueda Basora Ros andSalas-Salvado (2013) in recent studies observedno association between nut consumption andweight gain as demonstrated in a meta-analysisof clinical trials Additionally it is quite im-portant to note that there was spontaneousreduction of energy intake subsequent to nutconsumption relative to a no-load or alternateload condition That is the majority probably65-75 of the energy reportedly provided bynuts is offset by lower energy intake at latereating events Surprisingly no single componentof nuts appears responsible for this effect Thefatty acid profile is often mentioned but it doesnot appear to be responsible for spontaneousreduction of energy intake because dietarycompensation to walnuts almonds and peanutis comparable and they vary markedly in pro-

6 Maria and Hannah

Table 1 Proximate composition of cashew flours

Sample Moisture Protein Fat Fiber Ash Carbohydrate Energy() () () () () () (Kcalg)

A1 380eplusmn010 1969efplusmn003 4516bplusmn002 385fplusmn010 244eplusmn004 2205dplusmn011 51550b

A2 394fplusmn010 1963eplusmn010 4751eplusmn010 376dplusmn010 251fplusmn010 2263eplusmn010 52634d

A3 399fgplusmn006 1942dplusmn003 4825gplusmn071 301bplusmn003 256gplusmn006 2277fdplusmn003 52698f

S1 461hplusmn002 2111iplusmn004 5106iplusmn011 382eplusmn005 167bplusmn004 1676cplusmn099 54218i

S2 469iplusmn010 2057gplusmn010 5281jplusmn010 377dplusmn010 176cplusmn010 1640bplusmn010 55076j

S3 478jplusmn008 2093hplusmn087 5300kplusmn008 363cplusmn005 187dplusmn004 1597aplusmn016 55105k

R1 304cplusmn004 1913cplusmn002 4529cplusmn004 576iplusmn012 247efplusmn003 2443iplusmn014 53097g

R2 279bplusmn010 1795bplusmn010 4766fplusmn010 552hplusmn010 254fgplusmn010 2354hplusmn010 52675e

R3 304cplusmn004 1773aplusmn005 4887hplusmn009 518gplusmn002 266hplusmn005 2291gplusmn018 53398h

G 279bplusmn010 2221jplusmn054 4568dplusmn016 380eplusmn002 272iplusmn003 2202dplusmn002 51200a

C 268aplusmn008 2125jplusmn005 4503aplusmn015 253aplusmn002 159aplusmn002 2614jplusmn001 52175c

Data are mean values of triplicate determination plusmn standard deviation Means within a row with different superscript are significantly different (ple005)

A1-cashew kernels autoclaved for 5min A2-cashew kernels autoclaved for 10min A3-cashew kernels autoclaved for 15min S1-cashew kernels soaked for 6 h

S2-cashew kernels soaked for 12 h S3-cashew kernels soaked for 18 h R1-cashew kernels roasted for 5min R2-cashew kernels roasted for 10min R3-cashew

kernels roasted for 15min G-cashew kernels germinated for 7days C-raw cashew kernels

portions of monounsaturated fatty acids andPUFA (USDA 2002)The mineral concentrations of raw and treatedcashew flours are presented in Table 2 Inparallel with the observed increase in ashcontent in treated kernels there was increase inmineral levels relative to the raw cashew flourThe highest concentration of mineral elementswas noted in germinated samples followedby roasting autoclaving and soaking Theenhancement of mineral concentrations duringgermination may be due to decomposition ofanti-nutrient (notably phytate) thus releasingthe bound nutrients This result agrees with thereport of Ihemeje Ukauwa and Ekwe (2015)who reported higher mineral concentrations ingerminated walnut compared to raw walnutthough there was depletion as the germinationtime increased The only factor that couldaccount for higher mineral concentrations inroasted samples is the observed decrease inlevel of anti-nutrients Similarly soaked andautoclaved samples had higher mineral concen-trations than the raw sampleThe anti-nutritional factors in the raw andtreated flour samples are presented in Table 3The phytate oxalate and tannin contents of rawand treated cashew kernel flours were found tobe in a range of 5027plusmn003 to 9933plusmn003mgkg

462plusmn003 to 1103plusmn002mgkg and 447plusmn004to 1014plusmn003mgkg respectively Generallyflour samples from soaked germinated auto-claved and roasted cashew kernels producedsignificantly lower phytate oxalate and tanninconcentrations that were typical of products thathad received some degree of treatment duringpreparation However the anti-nutritionalfactors were better deactivated by germinationroasting and autoclaving compared to soakingof the kernels Furthermore when the impact ofdifferent heat treatment (roasting and autoclav-ing) was compared to germination germinationwas found to be more efficient in terms ofdeactivation of anti-nutrients as shown in Table3 Water uptake of dry dehiscent nuts causesa rise in general metabolic activity and theformation of a seedling from the embryo duringgermination Such growth requires phosphorusthat is mobilized from the main storage form iephytic acid and the process is accompanied withincrease in phytase activity The lower phytateand oxalate concentrations observed in roastedand autoclaved samples respectively may beattributed to the fact that they are heat labileSimilarly observed decrease in anti-nutrients insoaked samples could be attributed to losses dueto leaching during soaking and rinsing of thekernels

Table 2 Mineral concentrations of cashew flours (mgkg)

Sample Calcium Magnesium Iron Zinc

A1 331200fplusmn003 190467eplusmn003 6983eplusmn002 6497cplusmn004A2 344511gplusmn003 196323gplusmn003 7097hplusmn003 6854dplusmn003A3 355302iplusmn004 199753iplusmn003 7205iplusmn003 7257iplusmn003S1 223124bplusmn005 186942bplusmn003 6739cplusmn003 6309bplusmn002S2 225743cplusmn003 188431cplusmn003 6812dplusmn003 6879eplusmn003S3 228967dplusmn003 189233dplusmn003 6998fplusmn015 7060gplusmn003R1 324033eplusmn005 192942fplusmn003 6532bplusmn004 6976fplusmn002R2 346544hplusmn003 198748hplusmn003 7023gplusmn003 7233hplusmn003R3 358527jplusmn005 206033jplusmn004 7512jplusmn005 7795kplusmn005G 366442kplusmn003 211805kplusmn002 7922kplusmn003 7596jplusmn003C 221009aplusmn002 171254aplusmn003 6004aplusmn001 3674aplusmn002

Data are mean values of triplicate determination plusmn standard deviation

Means within a row with different superscript are significantly different (ple005)

Table 3 Anti-nutrients content of cashew flours (mgkg)

Sample Phytate Oxalate Tannin

A1 6941iplusmn002 712hplusmn002 648fplusmn006A2 5712eplusmn002 693gplusmn002 623eplusmn002A3 5394bplusmn004 660fplusmn004 609dplusmn002S1 6586hplusmn005 924jplusmn003 714iplusmn002S2 5926fplusmn002 754iplusmn002 694fplusmn002S3 5592dplusmn003 616cplusmn005 673gplusmn003R1 7043jplusmn002 620dplusmn004 648fplusmn003R2 6254gplusmn002 513bplusmn002 603cplusmn002R3 5027aplusmn002 462aplusmn003 573bplusmn005G 5561cplusmn003 638eplusmn003 447aplusmn004C 9933kplusmn003 1103kplusmn002 1014jplusmn003

8 Maria and Hannah

The presence of anti-nutrients in human di-ets affects nutrient absorption in infants andadults Phytate chelates metal ions such ascalcium magnesium zinc copper and iron toform insoluble complexes that are not readilyabsorbed from the gastrointestinal tract Itseffect on mineral elements especially in thecase of iron is intense as even small amounts ofphytic acid inhibit iron absorption in biologicalsystems (Hurrell et al 1992) Iron occurs in twoforms in foods as haem and as non-haem ironHaem iron which is present as haemoglobin andmyoglobin is absorbed directly as the intact ironporphyrin complex Haem iron is well-absorbed(15-35) and little influenced by physiologicalor dietary factors The absorption of non-haemiron varies widely and is influenced by dietarycomponents and iron status of the individual(Monsen et al 1978) In essence differentmethods to reduce phytic acid during foodpreparation will have a positive effect on ironabsorption However it is important to notethat ascorbic acid has been shown to increaseiron absorption and to at least partly overcomethe inhibiting effect of phytic acid (HallbergBrune amp Rossander 1989) The mechanism ofthis effect is thought to be due to the preventionof the formation of insoluble iron compoundsnot available for absorption and due to thereduction of ferric to ferrous iron (Hallberget al 1989) Tannin has been implicated inthe formation of complexes with protein andminerals thereby limiting their availability Thetoxicity effects of the tannin may not be signifi-cant since the total acceptable tannic acid dailyintake for a man is 560 mg (Gemede amp Fekadu2014) Similarly anti-nutritional activity ofoxalates lies in their ability to form complexeswith metals like calcium zinc magnesium andiron making them unavailable for absorptionHowever it is imperative to note that the riskof calcium deficiency due to the consumptionof oxalate-rich plants has been reported to beminimal as humans are able to efficiently usevery low amounts of calcium in food (ReddyBalakrishnan amp Salunkhe 1978) Moreso theconcentrations of oxalate reported in this studyare unlikely to pose toxicity problems to mansince it is below the toxic levels of 2-5g (Oke1969) Generally the amount of anti-nutrients

in nuts is highly variable the levels found ina specific food probably depends on growingconditions harvesting techniques processingmethods testing methods and even the age ofthe food being tested (Bello Salami-Jaji SaniAbdulhamid amp Fakai 2013)The mineral bioavailability of raw and treatedflours is presented in Table 4 Phytic acid has sixstrongly dissociated protons (pKn 11-29) andsix weaker dissociated protons (pAa 46-120) asearlier reported by Cosgrove and Irving (1980)Phytate chelates metal ions to form insolublecomplexes Phytate works in a broad pH-region(20-120) as reported by De Carli RossoSchnitzler and Carneiro (2006) Therefore atpH values that normally occur in foods andunder physiological conditions (pH 1 to 10)phytic acid being negatively charged has thepotential to bind cations or positively chargeddivalent and trivalent mineral ions such as Zn2+Fe2+3+ Ca2+ Mg2+ Mn2+and Cu2+ (Fred-lund Isaksson Rossander-Hulthen Almgren ampSandberg 2006) The pH is also an importantfactor influencing the solubility of phytate itbeing more soluble at lower than at higherpH values (Torre Rodriguez amp Saura-Calixto1991) The molar ratio of phytate to ironcalcium magnesium and zinc were reducedwhen treatments (soaking autoclaving roastingand germination) were applied The molarratio of phytate to zinc and calcium were farbelow the recommended maximum value 15 and024 in all the flour samples indicating betterbioavailability for absorption Similarly rawand treated cashew flours had phytate to ironratio within the recommended value of less than1 (Walingo 2009) As with iron zinc and cal-cium it is assumed that magnesium-phytic acidcomplexes are formed in the intestine which areinsoluble at a pH gt 6 (Champagne 1988) andthus are not absorbable However the stabilityof the magnesium-phytic acid complex is weakerthan phytic acid complexes with iron copperand zinc (Vohra Gray amp Kratzer 1965) Anysignificant effect due to the reduction in thelevel of anti nutrients in these samples shouldbe expected in the bioavailability of mineralelements Therefore the reduction achievedfor the anti-nutrient in treated samples whencompared to raw sample could be directly

Table 4 Mineral bioavailability of cashew flours

SamplePhytate Fe Phytate Ca Phytate Mg Phytate Znmolar ratio molar ratio molar ratio molar ratio

A1 008abplusmn000 nd nd 012dplusmn000A2 007aplusmn000 nd nd 008abplusmn000A3 007aplusmn000 nd nd 007aplusmn000S1 008abplusmn000 nd nd 010cplusmn000S2 007aplusmn000 nd nd 008abplusmn000S3 006aplusmn000 nd nd 008abplusmn000R1 009bplusmn000 nd nd 010cplusmn000R2 008abplusmn000 nd nd 009bplusmn000R3 006aplusmn000 nd nd 006aplusmn000G 006aplusmnplusmn000 nd nd 007aplusmn000C 014cplusmnplusmn000 nd nd 027eplusmn000

nd - not detected

Table 5 Functional properties of cashew flours

SampleBulk density (gmL) WAC OACLoose Packed (mL100g) (mL100g)

A1 045bplusmn001 063dplusmn001 9000bplusmn015 16600eplusmn017A2 045bplusmn001 060cplusmn001 9400cplusmn115 18200gplusmn115A3 045bplusmn002 058bplusmn001 10000dplusmn045 20000hplusmn028S1 045bplusmn001 055aplusmn003 9600cdplusmn017 17300fplusmn014S2 044bplusmn001 055aplusmn001 10200dplusmn115 20700iplusmn115S3 044bplusmn003 055aplusmn002 11000eplusmn014 22500jplusmn021R1 049cplusmn001 057bplusmn001 9000bplusmn014 13700bplusmn015R2 049cplusmn001 060cplusmn001 10700eplusmn115 14600cplusmn115R3 053dplusmn002 066eplusmn002 11000fplusmn007 15000dplusmn014G 049cplusmn001 061cplusmn002 9500cplusmn035 16500eplusmn021C 040aplusmn001 065eplusmn001 8700aplusmn011 13000aplusmn012

WAC-Water absorption capacity OAC- Oil absorption capacity

10 Maria and Hannah

related to this improved availability In essencebioavailability of nutrients in processed foodproducts could be enhanced when they containminimal amount of residual anti nutritionalfactors This is of great concern in CommunityNutrition and Emergency Feeding Programmesin developing countries where the consequenceof anti-nutritional factors may worsen incidenceof malnutrition and disease among infants andother vulnerable groupsThe functional properties of raw and treatedflours are as shown in Table 5 Determinationof functional properties of food ingredient isessential for the development of different foodproducts especially for children Processingmethods had significant effect (ple005) on thefunctional parameters under consideration Theloosed bulk density value ranged from 044plusmn001to 053plusmn002gmL Roasting significantly(Ple005) increased the loosed bulk density ofcashew flour Sample R3 had the maximumvalue while the control sample had the leastThe increase in loose density of the flour sampleas a result of soaking and germination could beattributed to enzymatic activities The packedbulk densities of the flour samples ranged from055plusmn001 to 066plusmn002gmL The reduction inpacked density observed in germinated samplemight have been as a result of decrease in weightof the flour owing to the breakdown of complexdenser compounds inherent in cashew kernelinto simpler ones (Gernah Ariahu amp Ingbian2011) Similarly Ihemeje et al (2015) in earlierstudies observed a reduction in bulk densityof germinated walnut compared to raw sampleThe decrease in packed density of the floursample as a result of soaking could also beattributed to enzymatic activities The highvolume per gram of flour material is importantin relation to its packaging It is desirable tohave high bulk density in that it offers greaterpackaging advantage as greater quantity maybe packed within a constant volume (AdepejuGbadamosi Adeniran amp Omobuwajo 2011)Moreso higher bulk density is important factorin convalescent child feedingWater absorption capacity (WAC) offlour samples ranged from 870plusmn011 to1100plusmn014mL100g Water absorption capacityof food product is an index of the maximum

amount of water the product absorbs andretains and it is important to soften and increasedigestibility (Ijarotimi et al 2012) Differencesin WAC of raw and treated flour samples couldbe as a result of differences in the content ofpolar amino acid residues of proteins or chargedside chains which have an affinity for watermolecules (Jitngarmkusol Hongsuwankul ampTananuwong 2008) The oil absorption capac-ities (OAC) of cashew flours were in the rangeof 1300plusmn012 to 2250plusmn021mL100g The oilabsorption capacity values were generally higherthan those of the water absorption capacity Ev-idence on oil absorption of the samples indicatethat there was significant (p le 05) differencein the values obtained in raw and treatedsamples Autoclaving and roasting significantlyincreased the oil absorption compared to thecontrol The increased oil absorption capacity ofheat-processed samples may be due to the de-naturation and dissociation of their constituentproteins that occur on heating which unmasksthe non-polar residues from the interior of theprotein molecule (Odoemelam 2005) Simi-larly germination improved the oil absorptioncapacity of cashew flour compared to the rawflour The increase in oil absorption capacityof germinated sample may be attributed to theincreased activity of lipolytic enzymes whichproduce more free fatty acids during sprouting(Ihemeje et al 2015)

4 Conclusion

The present study has demonstrated that pro-cessing of cashew kernel by soaking roastinggermination or autoclaving had significant ad-justments in the nutrient density and functionalproperties Various processing methods sig-nificantly decreased the levels of phytate ox-alate and tannin with corresponding improve-ment in mineral bioavailability Germinationshows potential to generate not only muchneeded nutrients in cashew for human but alsoimproved bioavailability of nutrients and func-tionality compared to other processing methods

References

Abebe Y Stoecker B Hinds M amp GatesG (2006) Nutritive value and sensory ac-ceptability of corn- and kocho-based foodssupplemented with legumes for infant feed-ing in southern ethiopia African Journalof Food Agriculture Nutrition and Devel-opment 6

Adebayo W A Ogunsina B S amp GbadamosiS O (2013) Some physico-chemical andfunctional properties of kariya (hildegardiabaterii) kernel flours Ife Journal of Sci-ence 15 (3) 477ndash488

Adepeju A B Gbadamosi S Adeniran A ampOmobuwajo T O (2011) Functional andpasting characteristics of breadfruit (arto-carpus altilis) flours African Journal ofFood Science 5 529ndash535

AOAC (2005) Association of Official Analyti-cal Chemists Official Methods of Analysisof the Association of Analytical ChemistsInternational Gathersburg MD USA Of-ficial methods 200508

Bello F Salami-Jaji J Sani I AbdulhamidA amp Fakai I M (2013) Evaluationof some antinutritional factors in oil-freewhite sesamum indicum l seed cake In-ternational Journal of Food Nutrition andSafety 4 (1) 27ndash33

Bes-Rastrollo M Wedick N M Martinez-Gonzalez M A Li T Y Sampson Lamp Hu F B (2009) Prospective study ofnut consumption long-term weight changeand obesity risk in women American Jour-nal of Clinical Nutrition 89 (6) 1913ndash1919doi103945ajcn200827276

Champagne E T (1988) Effects of PH onmineral phytate protein mineral-phytateand mineral fiber interactions-possible con-sequences of atrophic gastritis on mineralbioavailability from high-fiber foods Jour-nal of the American College of Nutrition7 (6) 499ndash508

Cosgrove D J amp Irving G C J (1980) Inosi-tol phosphates Their chemistry biochem-istry and physiology Studies in organicchemistry Elsevier Scientific Pub Co Re-trieved from https books google pt booksid=C0k3AQAAIAAJ

De Carli L Rosso N D Schnitzler E ampCarneiro P I B (2006) Estudo da esta-bilidade do complexo aiexclcido fatico e o aonni(ii) Food Science and Technology 26 19ndash26 doi101590S0101-20612006000100004

Eknayake S Jansz E R amp Nair B M (1999)Proximate composition mineral and aminoacid content of mature canavalia gladiataseeds Food Chemistry 66 (1) 115ndash119

Esenwah C N amp Ikenebomeh M J (2008)Processing effects on the nutritional andanti-nutritional contents of african locustbean (parkia biglobosa benth) seed Pak-istan Journal of Nutrition 7 (2) 214ndash217

FAO (2008) Agriculture food and nutrition forafrica Rome 385-387

Flores-Mateo G Rojas-Rueda D Basora JRos E amp Salas-Salvado J (2013) Nutintake and adiposity Meta-analysis of clin-ical trials American Journal of ClinicalNutrition 97 (6) 1346ndash1355 doi103945ajcn111031484

Fredlund K Isaksson M Rossander-HulthenL Almgren A amp Sandberg A S(2006) Absorption of zinc and retentionof calcium Dose-dependent inhibition byphytate Journal of Trace Elements inMedicine and Biology 20 (1) 49ndash57 doi101016jjtemb200601003

Gemede H F amp Fekadu H (2014) Nutritionalcomposition antinutritional factors and ef-fect of boiling on nutritional composition ofanchote (coccinia abyssinica) tubers Jour-nal of Scientific and Innovative Research3 (2) 177ndash188

Gernah D I Ariahu C C amp Ingbian E K(2011) Effects of malting and lactic fer-mentation on some chemical and functionalproperties of maize (zea mays) AmericanJournal of food technology 6 (5) 404ndash412

Gibbon D P amp Pain A (1985) Crops of thedrier regions of the tropics Intermediatetropical agriculture series Longman Re-trieved from https books google pt booksid=K5C 4gAQAAMAAJ

Grundy M M L Grassby T Mandalari GWaldron K W Butterworth P J BerryS E E amp Ellis P R (2015) Effect of mas-tication on lipid bioaccessibility of almondsin a randomized human study and its im-

12 Maria and Hannah

plications for digestion kinetics metabo-lizable energy and postprandial lipemiaAmerican Journal of Clinical Nutrition101 (1) 25ndash33 doi10 3945 ajcn 114 088328

Hallberg L Brune M amp Rossander L (1989)Elevated dosages of vitamins Benefits andhazards In H S P W Brubacher (Ed)(pp 103ndash108) International journal for vi-tamin and nutrition research SupplementHans Huber Publishers Retrieved fromhttps books google pt books id =GRdrAAAAMAAJ

Hammed L Anikwe J amp Adedeji R (2008)Cashew nuts and production developmentin nigeria American-Eurasian Journal ofScientific Research 3 (1)

Hurrell R F Juillerat M A Reddy MLynch S Dassenko S A amp Cook J D(1992) Soy protein phytate and iron ab-sorption in humans The American journalof clinical nutrition 56 573ndash8

Ihemeje A Ukauwa O amp Ekwe C (2015) Ef-fects of cooking and germination on physio-chemical properties and sensory attributesof african walnut (tetracarpidium conopho-rum) International Journal of Pharmacol-ogy Phytochemistry and Ethnomedicine 193ndash102

Ijarotimi O S Oluwalana I B amp Ogunedo-jutimi M O (2012) Nutrient composi-tion functional sensory and microbial sta-tus of popcorn-based complementary foodsenriched with cashew nut flour AfricanJournal of Food Agriculture Nutrition andDevelopment 12 (5) 684ndash5374

Ikpeme C A Osuchukwu N amp OshieleL (2010) Functional and sensory prop-erties of wheat (aestium triticium) andtaro flour (colocasia esculenta) compositebread African Journal of Food Science4 (5) 248ndash253

Jitngarmkusol S Hongsuwankul J amp Tananu-wong K (2008) Chemical compositionsfunctional properties and microstructureof defatted macadamia flours Food Chem-istry 110 23ndash30

Kaur D amp C Kapoor A (1990) Starch andprotein digestibility of rice bean (vigna um-bellata) Effects of domestic processing and

cooking methods Food Chemistry 38 263ndash272

Madigan M T Martinko J M Parker J etal (1997) Brock biology of microorganismsPrentice hall Upper Saddle River N J

Monsen E R Hallberg L Layrisse M Heg-sted D M Cook J D Mertz W ampFinch C A (1978) Estimation of avail-able dietary iron The American journal ofclinical nutrition 31 134ndash41

Mugabo E Afoakwa E O Annor G ampRwubatse B (2017) Effect of pretreat-ments and processing conditions on anti-nutritional factors in climbing bean floursInternational Journal of Food Studies6 (1) 34ndash43

Ngatchic J T M Sokeng S D NjintangN Y Maoundombaye T Oben J ampMbofung C M F (2013) Evaluationof some selected blood parameters andhistopathology of liver and kidney of ratsfed protein-substituted mucuna flour andderived protein rich product Food andChemical Toxicology 57 46ndash53 doi10 1016jfct201302045

Ocheme O amp Chinma C (2008) Effects ofsoaking and germination on some physico-chemical properties of millet flour for por-ridge production Journal of Food Technol-ogy 6 (5) 185ndash188

Odoemelam S A (2005) Proximate composi-tion and selected physicochemical proper-ties of the seeds of african oil bean (Penta-clethra marcrophylla) Pakistan Journal ofNutrition 4 382ndash383

Ogungbenle H N amp Afolayan M F (2015)Physical and chemical characterization ofroasted cashew nut (anacardium occiden-tale) flour and oil International Journalof Food Science and Nutrition Engineering5 (1) 1ndash7

Oke O L (1969) The role of hydrocyanic acid innutrition World Review of Nutrition andDietetics 11 170ndash98

Oladele K A Osundahunsi F O YemisiO A amp Adebowale A Y (2009) Influ-ence of processing techniques on the nutri-ent and antinutrient of tiger nut (cyperusesculentus l) World Journal of Dairy andFood Sciences 4 88ndash93

Perren R amp Escher F E (2013) Improvingthe safety and quality of nuts In L JHarris (Ed) (Chap Impact of roasting onnut quality pp 173ndash197) Woodhead Pub-lishing Series in Food Science Technologyand Nutrition Elsevier Science Retrievedfrom httpsbooksgoogleptbooksid=wlpEAgAAQBAJ

Reddy N R Balakrishnan C V amp SalunkheD K (1978) Phytate phosphorus and min-eral changes during germination and cook-ing of black gram (phaseolus mungo) seedsJournal of Food Science 43 (2) 540ndash543

Sathe S (1994) Solubilization and elec-trophoretic characterization of cashew nut(anacardium occidentale) proteins FoodChemistry 51 319ndash324

S lupski J (2011) Evaluation of the effect ofpretreatment and preservation on macro-and microelements retention in flageolet(Phaseolus vulgaris l) bean seeds ActaScientiarum Polonorum Technologia Ali-mentaria 10 (4) 475ndash485

Torre M Rodriguez A R amp Saura-Calixto F(1991) Effects of dietary fiber and phyticacid on mineral availability Critical re-views in food science and nutrition 30 1ndash22

USDA (2002) United states department of agri-culture agricultural research service na-tional nutrient database for standard Re-trieved from httpwwwnal usdagovfnicfoodcomp

Vickers Z Peck A Labuza T amp Huang G(2014) Impact of almond form and mois-ture content on texture attributes and ac-ceptability Journal of Food Science 791399ndash1406

Vohra P Gray G A amp Kratzer F H (1965)Phytic acid-metal complexes Proceedingsof the Society for Experimental Biology andMedicine 120 (2) 447ndash449

Walingo M K (2009) Indigenous food process-ing methods that improve zinc absorptionand bioavailability of plant diets consumedby the kenyan population African Journalof Food Agriculture Nutrition and Devel-opment 9 523ndash535

Woldegiorgis A Z Abate D Haki G D ampZiegler G R (2015) Major minor and

toxic minerals and anti-nutrients compo-sition in edible mushrooms collected fromethiopia Food Processing Technology 6134ndash142

Use of Response Surface Methodology (RSM) for CompositeBlends of Low Grade Broken Rice Fractions and Full-fat

Nahemiah Danbabaa c Iro Nkamab and Mamudu Halidu Badauc

a Food Technology amp Value addition Research Program National Cereals Research Institute (NCRI) BadeggiPMB 8 Bida Niger State Nigeria

b Department of Food Science and Technology University of Nigeria Nsukka Enugu State Nigeriac Department of Food Science and Technology University of Maiduguri Nigeria

Corresponding authordnahemiahgmailcomTel +2348039736494

Received 3 November 2017 Published online 18 April 2019

Abstract

In this study seventeen (17) composite blends of broken rice fractions and full-fat soybean for-mulated using response surface methodology and central composite design within a range of barreltemperatures (100-140oC) initial feed moisture content (15-25) and soybean composition (8-24)were extruded with a twin-screw extruder and the expansion and color indices were optimized Theresults indicated a significant (plt005) effect of extrusion conditions on the responses Fitted predictivemodels had coefficients of 889 957 973 954 and 952 respectively for expansion indexbulk density lightness redness and yellowness The p-value and lack-of-fit tests of the models couldwell explain the observed variability and therefore could be used to establish production setting for thetwin-screw extruder The optimum extrusion conditions were found to be 130 oC (barrel temperature)20 (feed moisture level) and 23 feed soybean composition and optimum responses in terms of bulkdensity expansion index lightness redness and yellowness chroma indices were 021 g cmminus3 1289171 313 and 245 respectively This indicates that optimum conditions can be established in twin-screw extrusion cooking of broken rice fractions and full-fat soybean composite blends that can resultin product of low bulk and maximum expansion with a satisfactory light yellow product color that canbe used to produce products that valorize broken rice and reduce qualitative postharvest loss

Keywords Broken rice Soybean Extrusion Response surface methodology Optimization

1 Introduction

Rice (Oryza sativa L) is an important source offood for a large number of the population in sub-Saharan Africa (SSA) its production and utiliza-tion is an important economic program for mostgovernments in the region Though productiondoes not match consumption demand and thereare huge import bills at the detriment of othercritical sectors significant improvement has been

made over the last few years in increasing yieldper ha in most countries But little improve-ment has been recorded in the postharvest valuechain of the production system (Manful Quayeamp Gayin 2004 Ndindeng et al 2015) Recentlyresearch investment for the reduction of posthar-vest losses has resulted in increased interest inthe utilization of rice flour from low grade brokenrice fractions for the production of value addedproducts (Chinma Anuonye Simon Ohiare amp

Broken rice and full-fat soybean twin extrusion cooking 15

Nomenclature

QPL Qualitative postharvest loss

SSA sub-Saharan Africa

RSM Response surface methodology

CCRD Central composite rotatable design

EC Extrusion cooking

FARO Federal agricultural research oryza

NCRI National Cereals Research Institute

EI Expansion index

BD Bulk density

L Lightness

a Redness

b Yellowness

D Deviations

RD Relative deviation

BRT Barrel temperature

FMC Feed moisture content

FSC Feed soybean composition

DOE Designed of experiment

Danbaba 2015 Danbaba Nkama amp Badau2016 Danbaba et al 2017) mainly due to itshigh expansion during extrusion ease of diges-tion high calcium content low glycemic indexlow sodium attractive white colour low glutenlow allergenicity and low cost as it is primarilyproduced from low grade broken rice fractions(Danbaba et al 2016 Kadan Bryant amp Pep-perman 2003) However rice is relatively low inprotein (6-8 g100 g db) and its amino acids lim-ited by lysine (Chaiyakul Jangchud JangchudWuttijumnong amp Winger 2009) Interestinglylysine content of rice is higher (35-4016 g Ni-trogen) than that of other common cereals suchas corn sorghum and millet and therefore caneasily be improved nutritionally by the additionof high lysine food materials to attain 55 g16 gN considered adequate for humansThe utilization of soybean to complement ce-real protein has been increasing rapidly in mostparts of Africa Soybean is a rich source of ly-sine hence rice-soybean foods can provide bal-anced dietary protein Uses of locally-grownhigh protein crops and shelf stable and afford-able recipes have been proposed for addressingprotein-energy malnutrition (PEM) in develop-ing countries (Asefa amp Melaku 2017) combin-ing this with processing technologies that resultin shelf stable convenient and consumer accept-

able products has also been advocated (Iwe VanZuilichem Ngoddy amp Ariahu 2001) (Iwe 2003)Extrusion cooking technology and blends of cere-als and legumes have been used in several partsof Africa to produce products that are nutri-ent dense consumer acceptable with improvedphysical and functional qualities (Danbaba et al2017 Filli 2011 2016 Gogoi Oswalt amp Choud-hury 1996) During extrusion enzymes are par-tially or totally inactivated anti-nutritional fac-tors destroyed and microbial load significantlyreduced thereby improving widespread uses oflegumes (Abd El-Hady Mostafa El-Samahy ampEl-Saies 1998) In other parts of the world thetechnology has been employed to produce foodssuch as breakfast cereals snack foods pastaproducts extruded bread modified starchesbeverage powders meat and cheese analoguestextured vegetable proteins and meat products(Abd El-Hady et al 1998 Moore Sanei Heckeamp Bouvier 1990) The process has high versa-tility efficiency low cost of operation and highyields with relatively no waste generationBut to maximize the use of EC in new food prod-uct development it is important to be able toalter process conditions to produce consumer ac-ceptable products improved physical and func-tional properties minimized cost of productionby adopting low-cost raw materials maintained

16 Nahemiah et al

high quality standards and minimized losses dur-ing production (Chaiyakul et al 2009) This canonly be achieved by systematically selecting ap-propriate production conditions using designedexperiments (DOE) (Abd El-Hady et al 1998Danbaba et al 2016 Moore et al 1990) Us-ing DOE Eggum Juliano Ibabao and Perez(1986) and Asare Sefa-Dedeh Sakyi-Dawsonand Afoakwa (2004) studied the behaviour oflow amylose rice flour blended with cowpea dur-ing extrusion cooking Effects of protein contentand extrusion parameters on sensory and physi-cal properties of extruded high protein glutinousrice-based snacks were reported by Chaiyakul etal (2009) and Asare et al (2004) while the ef-fect of the addition of cowpea and groundnuton the quality of rice-based extrudates has alsobeen reported (Asare et al 2004) In relationto rice-soybean extrusion Coutinho BatistaCaliari and Soares Junior (2013) studied the ef-fect of moisture and extrusion temperature onbroken rice blended with by-products of soy-bean processing Marengo et al (2016) and Gargand Singh (2010) used partially defatted soy-bean blended with rice to produce snacks whileNoguchi Kugimiya Haque and Saio (1982) re-ported on the physical and chemical character-istics of extruded rice flour and soybean proteinisolate Omwamba and Mahungu (2014) devel-oped extruded ready-to-eat snacks from blendsof rice sorghum and soybean In the currentstudy effects of moisture extrusion temperatureand amount of full-fat soybean flour on the phys-ical and other functional characteristics of twinscrew extruded snacks were evaluated Also lit-tle or no information is available on the extru-sion of full-fat soybean flour blended with bro-ken parboiled rice fractions on physical indices ofextruded snacks Response surface methodology(RSM) and central composite design are math-ematical techniques used for optimizing processconditions where multiple independent variablesinfluence the response value In food process-ing the main objective of using this DOE is tolocate more precise combinations of the processvariables that will have the highest probability ofproducing food products of desired quality andalso to describe interrelations between the inde-pendent variables and product quality (Colonna1989 Nwabueze 2007) for sustainable quality as-

suranceLittle work regarding the effects of extrusiontemperature initial feed moisture content andratio of broken parboiled rice fractions to full-fat soybean blends on the physical and chemi-cal properties of rice-extruded foods has been re-ported Therefore the objective of the currentstudy is to produce extruded foods from blendsof low grade broken parboiled rice fractions andfull-fat roasted soybean flour optimize the pro-cess parameters of twin-screw extruder param-eters and to develop and validate mathemati-cal models for the relationship between the re-sponse variables and the process variables usingdesigned experiments We believe that this willprovide new sources of cheap and available rawmaterials reduce qualitative rice postharvest lossand provide nutritious convenient foods for mit-igating protein-energy-malnutrition in SSA

Broken fractions of parboiled improved highamylose (2897) rice variety (Federal Agricul-tural Research Oryza - FARO 52) and full-fat soybean (TGX-1448-2E) flour were obtainedfrom the National Cereals Research Institute(NCRI) Badeggi Niger State Nigeria The ma-terials were manually sorted washed and sundried (32plusmn2 oC) for 6 h to 1011plusmn008 and941plusmn004 (db) moisture content respectivelybefore storing under refrigeration until requiredfor analysis Seventeen (17) rice-soybean formu-lations containing between 14 to 28 kg100 kgsoybean were mixed and moisture content ad-justed (Danbaba et al 2016) to between 15 and25 g100 g (db) (Table 2) by adding a given quan-tity of water to each sample and mixed throughin a laboratory mixer for 5 min before standingfor 3hr prior to extrusion at specified tempera-tures (Table 2) Preliminary experiments wereconducted by extruding 8 samples with moistureand soybean contents ranging between 110 and30 and 4 and 30 respectively and extrudedat a temperature of 70 to 140 oC Extruded sam-ples that were not burnt and had expanded ap-preciably were selected and their processing con-ditions were used for the main experiments Ex-trusion cooking was carried out in a twin-screw

extruder (Model - SLG 65 Jinan Saibaino Tech-nology Development Co Ltd China) poweredwith a 16 HP electric motor screw speed of 300rpm and length to diameter ratio of barrel (201)

21 Experimental design andExtrusion Cooking

Response surface methodology (RSM) in a five-level (-α -1 0 1 +α) central composite rotat-able design (CCRD) of three variables (X1 =barrel temperature X2 = feed moisture contentX3 = feed soybean composition) was used (Ta-ble 1) where plusmnα is equal to 1682 and defines thedistance of the axial runs from the design cen-tre (Montgomery 2001) The experimental runsconsisted of 17 trials (8 cube points 6 star pointsand 3 centre points) The expansion (EI) bulkdensity (BD) and colour characteristics (L ab) were set as the response variables (y1 y2y3 y4 and y5) respectively Experimental datawere fit to a second order polynomial responsesurface model equation 1

Y = b0+

nsumi=1

biix2i +

nminus1sumi=1

nsumj=i+1

bijxixj+ei

(1)where Y is any response variable n is the num-ber of independent variables xi and xj are codedvariables ba is the intercept bi bii and bij arethe linear quadratic and interaction effects re-spectively and ei is the error term The good-ness of fit of the models were measured by con-sidering the coefficient of determination (R2 andAdjusted-R2) the adequacy of the quadraticmodels was confirmed by the analysis of variance(ANOVA) using Fishers test value (F-value) andlack-of-fit test For statistical analysis the nat-ural variables values (Xi) were transformed tocoded forms according to equation 2

xi =xi minus x0δx

Where Xi = coded (dimensionless) value of theindependent variables X0= the value of Xi at thecentre point and δi= the step change in Xi andthe results are presented in Table 2 Optimiza-tion of process variables was based on the maxi-mum values of all the responses (EI BD and L

a b) Responses were individually assignedto different importance levels varying between 1and 5 and optimized based on higher desirabilityfor expansion index L b and lower desirabil-ity for bulk density and a The deviations (D)and relative deviations (RD) between the experi-mental values and those predicted by the modelsfor the responses at the optimal condition werecalculated by equations 3 and 4 respectively

Deviation (D) = Y ndashΥ (3)

Relative deviation (RD) =D

Υtimes 100 (4)

where Y is the experimental data and Υ is theresponse predicted by the model

22 Moisture contentdetermination

The moisture content of feed materials and ex-truded samples was determined using the dryoven method with accuracy of about four digits(00000 g g)

23 Bulk density (BD) andExpansion Index (EI)

Extruded samples were analysed for EI by usingthe mean diameter of 10 readings and ER calcu-lated as the ratio of diameter of the extrudatesand diameter of extruder die (3mm) (Alvarez-Martinez Kondury amp Harper 1988) After mea-suring the weight per unit length of the extrudedsamples the BD was calculated using the rela-tionship

πd2L(5)

where BD = Bulk density (g cmminus3) d = Diam-eter of the extrudate (cm) L = Length per gramof the extrudate (cm gminus1)

24 Color characteristicsdetermination

Colors of the extruded foods were measured witha Monilta Color Reader (CR-10 Minolta CoLtd Tokyo Japan) using the L a b sys-tem where the L value is a measure of product

18 Nahemiah et al

Table 1 Codes ranges and levels of independent variables in RSM design

Levels of Barrel Temperature Feed Moisture content Feed Compositionvariables (oC A) (g 100gminus1 sample B) (g 100gminus1 sample C)

-α (-168) 8636 116 26Low (-1) 1000 150 80Medium (0) 1200 200 160High (1) 1400 250 240+α (+168) 1536 284 295

Level of each variable was established based on preliminary extrusion trials The distance of the axial points

from the center point was plusmn 168 and calculated from Equation α = (2n)14 where n is the number of variables

lightness a chromatic color attribute of absoluteblack (0) to absolute white (100) a value meansmixed red-to-green color of samples ranging fromnegative on the red side to positive on the greenside (a+ = 0 - 60 for red and a- = 0-(-60) forgreen) and the b value means mixed blue-to-yellow colour of samples ranging from negativeto positive value (b+ = 0 - 60) for yellow and b-= 0-(-60) for blue

Homogeneous variance is a necessary pre-requisite for linear regression model development(Filli 2011) Therefore to reduce the variabil-ity within the response variables the naturalvariable values (Xi) were transformed to codedforms according to equation 2 For the standard-ized data one-way analysis of variance (ANOVA)was carried out to determine significant differ-ences (ple005) among the treatment combina-tions using response surface analysis of Minitab16 software Fishers Least Significance Differ-ence (LSD) was used to check equality of vari-ances Results are presented as mean of threereplications

3 Results and Discussion

31 Experimental results ofextrusion exercise

Based on the seventeen (17) composite blends ofbroken rice fractions and full-fat soybean floursformulations were grouped into 5 based on the

extrusion temperature 860 oC (1) 100 oC (4)120 oC (7) 140 oC (4) and 154 oC (1) (Table2) These extrusion temperatures were selectedbased on preliminary exercise (results not pre-sented) Digital images showing physical charac-teristics of snacks as they emerged from the die atsteady state at each experimental condition arepresented in Fig 1 and internal macro structurein Fig 2 There were great variations in physicalappearance in terms of size colour and puff lev-els and surface smoothness therefore indicatingvariability due to differences in extrusion condi-tions They were mostly in the form of cylindri-cal puffed strands with different sizes and surfacetextures Samples containing lower moisture con-tent (8) and extruded at temperatures above100 oC had slightly expanded size and roughersurface than those extruded at higher moisturecontents As the moisture level increased to24 strands became thinner and smoother sur-faced These observations are similar to those re-ported by Nwabueze (2007) that thin-smooth tothin-fine-smooth extrudates were obtained whenAfrican bread fruits blended with soybean andyellow maize were extruded at a higher moisturelevel These indicate the need to systematicallydefine optimum moisture level for the extrusionof rice-full-fat soybean blends for optimum prod-uct qualityThe extrudates also had varying honeycomb cel-lular structure of different numbers and sizeswith samples 2 5 8 10 11 12 13 and 14 show-ing more and larger spaces (Fig 2) Struc-turally starch-based extruded foods have beenreported to have a honeycomb cellular structure

Figure 1 Digital images of expanded rice-full fat soy blends extruded at different conditions

Figure 2 Cross section showing void spaces in rice-full fat soy blends extruded at different conditions

20 Nahemiah et al

(Faubion amp Hoseney 1982 Lai Guetzlaff amp Ho-seney 1989) and the size and number of the cellsformed have been reported to be dependent onthe feed moisture content and the compositionof protein and lipids in feed material as demon-strated in this study Similar reports were putforward by Faubion and Hoseney (1982) Lai etal (1989) Lai and Kokini (1991)In the reports of Agbisit Alavi Cheng Heraldand Trater (2007) Yagci and Gogus (2008) ithad been demonstrated that variation of feedmoisture content and temperature during extru-sion cooking affected the degree of expansionthrough transformation of the extrusion matrixleading to differences in void space formation ofpuffed products From the observed cross sec-tion of the extrudates it could be inferred thatsamples 2 5 8 10 11 12 13 and 14 would havelow breaking strength high crispiness and bettersensory acceptabilityVarying physical appearance of the extrudates interms of color (Fig 1) is an indication of vary-ing response of the different formulations to dif-ferent extrusion conditions and therefore clearlydefines the need to locate appropriate processingconditions to have products of acceptable colorThough change in total reducing sugar variationduring the process is not reported variation insurface color of the different extruded samplesmay be attributed to browning reactions and pig-ment degradation that take place during the pro-cess (Altan McCarthy amp Maskan 2008)

32 Effects of individual variablesand interaction on responses

Bulk density

Table 3 shows the mean effects of the barrel tem-perature initial feed moisture content and feedsoybean composition on the BD of extruded ricefull fat soybean The experimental values for BDrange between 0035 and 0358 g cmminus3 with amean value of 0098 g cmminus3 The deviation whichmeasures the difference between the observedvalue and the predicted value ranged between0021 and 0329 g cmminus3 and the relative deviationwas 600 and 11528 indicating wide variationbetween different treatments in terms of BD It

may be observed from the results that the BDincreased as the feed moisture content decreasedfrom 25 to 15 g100 g sample in runs 3 and 5respectively while relative deviation which indi-cates percentage deviation of the observed valuefrom the predicted value was highest (11528)at run 6 and lowest (600) at run 3 Our obser-vations coincide with those reported by Kulka-rni and Joshi (1992) and Garg and Singh (2010)that there is a strong correlation between ini-tial feed moisture content and BD of extrudatesAs the value of the initial moisture content in-creased the value of BD decreased and viceversa Chang and El-Dash (2003) and Leonel deFreitas and Mischan (2009) also reported sim-ilar findings for extruded cassava starch Theyreported that moisture content of 24-26 as ob-served in this study followed by barrel temper-ature of 120-200 oC resulted in a significant ef-fect on expansion and therefore bulk density Atlow moisture content there is an increased shearforce in the extruder barrel which increases resi-dence time and gelatinization of starch and sub-sequently increases bulk density as observed inthese results BD is an important quality param-eter in the production of extruded products as itindicates expansion at the die in all directions Inthis study barrel temperature and feed soybeancontent did not show significant impact on theBD (Table 3) even though several authors (CaseHamann amp Schwartz 1992 Hernandez-NavaBello-Perez San Martin-Martinez Hernandez-Sanchez amp Mora-Escobedo 2011) reported thatBD value decreases when the extrusion temper-ature is increased due to aggravated starch gela-tinization The lack of agreement between thisstudy and earlier reports may be due to high tem-perature and possible contribution of soybean oilcontent to the flow of the matrix in this studyMinimal addition of soybean flour was also ob-served to affect BD At 8 g100 g sample of soy-bean flour in a blend processed at high moisturecontent the highest BD was recorded (Table 3)These observations may be attributed to the be-haviour of protein and oil in the formulationssince friction and shear forces during extrusionmay cause extensive interlacing between proteinmolecules and lead to their texturization and be-cause high protein content promotes denser andmore rigid extrudates (Hernandez-Nava et al

Table 2 Outline of experimental design with design points coded and uncoded values of independentvariables

Runs PointsCoded variables Natural variablesA B C BRT FMC FSC

Group 1 (100oC extrusion temperature)1 Factorial point -1 -1 -1 100 15 83 Factorial point -1 1 -1 100 25 85 Factorial point -1 -1 1 100 15 247 Factorial point -1 1 1 100 25 24Group 2 (140oC extrusion temperature)2 Factorial point 1 -1 -1 140 15 84 Factorial point 1 1 -1 140 25 86 Factorial point 1 -1 1 140 15 248 Factorial point 1 1 1 140 25 24Group 3 (120oC extrusion temperature)11 Star point 0 -168 0 120 116 1612 Star point 0 168 0 120 284 1613 Star point 0 0 -168 120 20 2614 Star point 0 0 168 120 20 29515 Centre point 0 0 0 120 20 1616 Centre point 0 0 0 120 20 1617 Centre point 0 0 0 120 20 16Group 4 (86oC extrusion temperature)9 Star point -168 0 0 864 20 16Group 5 (154oC extrusion temperature)10 Star point 168 0 0 1536 20 16

BRT = barrel temperature oC FMC = feed moisture content () FSC = feed soybean composition

A = Barrel temperature B = Feed moisture content C = Feed composition Duplicate runs were

carried out at all design points and averages recorded The experimental runs were randomized

2011 Jorge et al 2008)

Expansion index

Generally high expansion index is a desirableproperty in puffed extruded foods as this typi-cally indicates a lighter crispier product Ex-pansion index has been shown to be dependenton extrusion conditions as well as blend for-mulations (Maskus amp S 2015) significant fac-tor affecting expansion ratio (Ding AinsworthTucker amp Marson 2005) In this study the EIincreased from 1257 at 100 oC barrel tempera-ture to 13380 when the temperature increasedto 120 oC with concurrent reduction in initialfeed moisture content (Table 3) HagenimanaDing and Fang (2006) reported reduction in EI

as the moisture level increased from 16 to 22in a twin-screw extrusion of rice flour In ourwork similar observations were seen increased EIwas observed as a moisture content reduced from25 to 20 indicating a narrow range of moistureis required for optimum product expansion of riceflour (between 16 and 22) Relative deviationranging between 9961 and 10031 (Table 3) isalso an indication of a narrow range in percentagevariability between observed and predicted val-ues High moisture level reduces shear strengthand energy input to the material resulting in de-creased moisture evaporation at the die exit andtherefore negatively affects expansion In ourstudy the use of soybean flour increased the pro-tein level and therefore reduced the starch levelin the blend which may have impeded full starch

22 Nahemiah et al

Table 3 Mean experimental predicted and relative deviation results for bulk density and expansionindex of extruded broken-rice full-fat soybean formulations

RunsBulk density (g cmminus3) Expansion index ()

Y D RD Y D RD

1 0089 0092 10337 1278 1282 100312 0058 0041 6897 1311 1307 99693 0035 0021 6000 1257 1258 100084 0156 0162 10385 1309 1308 99925 0358 0329 9190 1307 1308 100086 0072 0083 11528 1299 1298 99927 0075 0081 10800 1317 1316 99928 0039 0028 7179 1327 1329 100159 0142 0146 10282 1288 1283 996110 0059 0059 10000 1315 1315 1000011 0147 0151 10272 1289 1289 1000012 0048 0045 9375 1295 1295 1000013 0058 0062 10690 1298 1298 1000014 0154 0148 9610 1338 1338 1000015 0059 0058 9831 1335 1335 1000016 0058 0057 9828 1337 1338 1000717 0059 0056 9492 1336 1338 10015Mean 0098 0095 9511 1308 1308 10000SD 0076 0073 1433 222 224 016LSD 0012 - - 212 - -

Y = experimental results D = deviation which represent fitted value RD = Relative

deviation SD = Standard deviation Equation on which responses are predicted =

βo + β1X1 + β2X2+ β3X3+ β11X21 + β22X2

2 + β33X23 + β12 X1X2 + β13 X1X3

+ β23 X2X3 Mean in the same column having differences greater than LSD are

significantly different (ple005) according to Fishers Least Significance Test

gelatinization with resultant reduction of expan-sion index from 1338 to 1257 representingabout a 605 reduction Yu Ramaswamy andBoye (2013) and Devi et al (2013) in separatestudies reported a reduction in expansion ratiowith the addition of protein This result indi-cates that the EI of extruded products dependson starch gelatinization as an increasing starch-protein ratio leads to formation of a continuousstarch matrix that affects its expansion as it exitsthe die

Color characteristics of extrudedfoods

In extruded starch-based puffed products coloris one of the important quality indicators

Change in color may indicate extent of browningreactions such as Maillard reaction carameliza-tion and degree of cooking and possibly pigmentdegradation that occurs during heat treatments(Altan et al 2008 Danbaba et al 2016) As-sessment of the color components of the extrudedsamples showed that the value of the L compo-nent varied from 031 to 1031 with a mean valueof 580 as compared to 8734 for the raw ma-terials before extrusion indicating a decrease inluminosityL value was highest at temperatures between100 oC and 120 oC feed moisture levels of 15 to20 and soybean composition of 8 to 16 (Table3) At moisture content less than 20 and extru-sion temperature greater than 100 oC there was

darkening of extruded starch mainly due to in-creased residence time and viscosity ie lesser Lvalue Our results were in agreement with thoseof Gutkoski and El-Dash (1999) who reportedthat with initial moisture content of 17 to 24and barrel temperature of 90 oC to 150 oC lumi-nosity decreased linearly with barrel temperaturein an extruded oat product The deviation rangeof 006 to 1032 and relative deviation of 1935to 12446 is an indication of variation amongtreatments in terms of this parameterThe chroma a (redness) values ranged from 073to 521 and the deviation and relative devia-tion were between 092 5913 and 813 14091which indicates slight variability in this color pa-rameter during extrusion cooking considering theobserved value can vary from -60 to +60 andlow RD indicates slight variability in percent-age The highest value occurred at a temperaturerange of 120 to 140 oC feed moisture level of 15to 20 and feed soybean content of 8 to 16(runs 2 and 17) (Table 2 and 4) indicating theneed to optimize the process variables to locatethe most suitable point that will give acceptablequality in terms of this parameter Responses ofthe b color index which represents transitionfrom blue to yellow on the chroma chart variedaccording to the treatment (from 1313 to 2880)indicating that all samples had a yellow colorThe increase of barrel temperature from 120 to154 oC (runs 13 and 10) (Table 2 and 4) increasedb color intensity at 20 moisture content and26 soybean content of the feed materials Highmoisture level resulted in brighter products sinceincrease in moisture decreases the residence timeproviding less non-enzymatic darkening of ex-truded products (Badrie amp Mellowes 1991)

33 Fitting and validating RSMmodels for optimizingproduction processes

RSM models to optimize the relationship be-tween extrusion cooking variables and responsevariables in terms of expansion and color indicesare outlined in the regression model equation4-8 The coefficients with only one factor (X1X2 and X3) represent the effect of that singlefactor while those with two factors (X1X2

X1X3 X2X3) are the interaction terms andX2

1 X22and X2

3 are the square terms Positiveor negative coefficients indicate synergisticor antagonistic contributions to the observedresponses The results of ANOVA performedon the RSM models to assess the significanceof the linear quadratic and interactive effectsof the independent variables on the dependentvariables are presented in Tables 5From equation 6 it is clear that X1 and X2

negatively affected bulk density at both linearand quadratic levels while only X1X3 and X2X3

reduced this response at the interaction levelAll three factors positively impacted expansionat linear levels and negatively affected expan-sion index at square levels (equation 7) Thisdemonstrated the inverse relationship betweenbulk density and expansion index of extrudateunder linear and square processing conditionsEquation 8 indicated that increasing barreltemperature (X2) resulted in increased lightnessat linear and interaction levels X1 and X3linearly affected redness positively and all thefactors positively affected this factor at squarelevels (equation 9) Temperature only seemedto negatively affect the yellow color index atboth linear and square levels but interactionsbetween X1X3 and X2X3 negatively impactedyellow color formation (equation 10)

Bulk density(g cmminus3) = 0110 minus 0027X1minus0027X2 + 0029X3 minus 0002X2

1 minus 0007X22+

0001X23 + 0053X1X2 minus 0053X1X3minus

0043X2X3

(R2 = 0957 R2adj = 0946)

Expansion index = 1324 + 0012X1 + 0010X2+

0014X3 minus 0008X21 minus 0004X2

2 minus 0004X23+

0004X1X2 minus 0007X1X3 + 0012X2X3

(R2 = 0889 R2adj = 0860)

Lightness (L) = 16773 minus 2021X1 + 0132X2minus0447X3 minus 1135X2

1 + 0173X22 minus 0330X2

0625X1X2 + 0649X1X3 minus 1282X2X3

(R2 = 0973 R2adj = 0966)

24 Nahemiah et al

Table 4 Mean experimental predicted and relative deviation results for color characteristics of extrudedbroken-rice full-fat soybean formulations

RunsLightness (L) Redness (a) Yellowness (b)

Y D RD Y D RD Y D RD

1 1031 1032 10010 09 097 10778 1601 1591 99382 367 355 9673 521 513 9846 2326 2341 100643 713 692 9705 157 154 9809 210 2097 99864 223 242 10852 23 187 8130 221 2198 99465 551 545 9891 075 092 12267 237 23497 991436 139 173 12446 364 341 9368 2649 2667 100687 673 698 10371 32 302 9438 1717 1717 100008 54 552 10222 201 168 8358 1313 1338 101909 69 697 10101 263 249 9468 255 2549 999610 031 006 1935 435 486 11172 288 286 993111 716 712 9944 261 253 9693 237 2348 990712 76 746 9816 11 155 14091 1655 1656 1000613 642 656 10218 102 117 11471 1478 1492 1009514 538 506 9405 073 095 13014 1481 1447 977015 841 844 10036 136 13 9559 2312 2316 1001716 1031 1032 10010 09 097 10778 1601 1591 993817 367 355 9673 521 513 9846 2326 2341 10064Mean 580 579 9665 232 232 10417 2055 3297 15239SD 284 - - 153 - - 475 - -LSD 104 - - 021 - - 202 - -

Y = experimental results D = deviation which represent fitted value RD = Relative deviation SD = Standard deviation

Equation on which responses are predicted = βo + β1X1 + β2X2+ β3X3+β11X21 + β22X2

2 + β33X23 + β12 X1X2 +

β13 X1X3 +β23 X2X3 Mean in the same column having differences greater than LSD are significantly different (ple005)

according to Fishers Least Significance Test

Redness (a) = 2521 + 0431X1 minus 0162X2+

0051X3 minus 1135X21 + 0368X2

2 + 0154X23+

0001X1X2 minus 0493X1X3 minus 0364X2X3

(R2 = 0954 R2adj = 0942)

Yellowness (b) = 22060 + 00936X1 minus 0170X2+

1770X3 minus 0720X21 + 0602X2

2ndash1652X23+

(R2 = 0952 R2adj = 0939)

It is clear from equations 4-8 and probabilityvalues (Table 5) that the bulk density expan-sion indices and color parameters were all signif-icantly (p lt 005) affected by varying barrel tem-perature initial feed moisture content and feed

soybean composition as predicted by the mod-els The R2 and adjusted R2 values were R2=0957 R2

adj = 0946 for bulk density R2= 0889

R2adj = 0860 (expansion index) R2 = 0886

R2adj = 0870 (L) R2 = 0954 R2

adj = 0942

(redness) and R2 = 0952 R2adj = 0939 (yel-

lowness) (equation 4-8 respectively) The R2

and adjusted R2 defines the proportion of vari-ability in the observed responses which are ac-counted for by the fitted models (Danbaba etal 2016 Filli 2011 Singh Sekhon amp Singh2007) and the closer the values are to unity thebetter the empirical model fits the experimen-tal data (Lee amp Wang 1997) thus the resultsof this study suggest that the models appropri-ately defined the process behaviour by explain-ing 946 860 870 942 and 939 of

Table 5 Analysis of variance (ANOVA) and lack-of-fit p-values R2 and adjusted R2 and estimates ofthe coefficients of the second-order response surface

Expansion index Bulk density Lightness (L) Redness (a) Yellowness (b)Coefficient p-value Coefficient p-value Coefficient p-value Coefficient p-value Coefficient p-value

b0 1324 0000 0110 0003 16773 0000 2521 0022 2206 0017Linearb1 0012 0001 -0027 0001 -2021 0002 0431 0000 0094 0002b2 0010 0002 -0027 0002 0132 0000 -0162 0000 -2093 0000b3 0014 0000 0029 0000 -0447 0000 0051 0001 -0170 0010Quadraticb12 -0008 0051 -0002 0001 -1135 0003 0368 0002 1770 0000

b22 -0004 0000 -0007 0004 0173 0012 0154 0003 -0720 0000

b32 -0004 0001 0001 0008 -0330 0017 0001 0021 -0602 0016

Interactionb1b2 0004 0002 0053 0027 0625 0001 -0493 0002 -1652 0000b1b3 -0007 0001 -0053 0001 0649 0002 -0364 0000 -1265 0001b2b3 0012 0001 -0043 0000 1282 0000 0031 0000 -1945 0003R2 0889 - 0957 - 0973 - 0954 - 0952 -R2

ajusted 0860 - 0946 - 0966 - 0942 - 0939 -

Lack-of-fit 0710 - 0071 - 0319 - 0082 - 0982 -

Model on which the coefficients were calculated y = bo+ b1X1 + b2X2 +b3X3 + b21 X2

1 + b22 X2

2 + b23 X2

3 +b12 X1 X2 + b13 X1 X3 + b23 X2 X3 + ε

the variations observed in bulk density expan-sion index lightness redness and yellowness re-spectively Additionally the p-values of lack-of-fit test for the models were not significant (plt 005) which indicates that the models canbe used for the prediction of the observed re-sponses within the experimental domain stud-ied Yagci and Gogus (2008) were of the opin-ion that with significant probability values (p lt0001) and non-significant lack-of-fit tests fittedsecond-order models could be adequately used aspredictor models regardless of low coefficients ofdetermination It is clear and appropriate there-fore to conclude that the developed models ade-quately approximate the response variables con-sidered in this study and can be used satisfac-torily for the prediction of any value of the re-sponses within the defined experimental range

Optimization process variables

In this study simultaneous numerical optimiza-tion of the independent and dependent variableswas conducted using a Minitab response opti-mizer To meet the intended target requirementplaced on each response variable and indepen-dent factor in this experiment a goal was set foreach independent and dependent variable An-

uar Adnan Saat Aziz and Taha (2013) GuptaVerma Jain and Jain (2014) and Danbaba etal (2016) reported that for locating optimumconditions using numerical methods there wasa need to set goals which may either be nonemaximum minimum target or range for bothresponse and independent variables that couldbe combined into one desirable function In thiswork the barrel temperature feed moisture con-tent and feed soybean composition were all setwithin range (100 to 140 oC 15 to 25 8 to24) as defined by the preliminary study (Ta-ble 1) while the goal for the response variablesof bulk density expansion index and lightnesschroma levels were set at maximum and a andb set at minimum (Table 6) Anuar et al (2013)reported that when goal was set at 5 the re-sponse target objective was to meet the objectiveof getting response at a maximum levelResults of the numerical optimization thereforeindicated that the optimum extrusion variableswere 130 oC (barrel temperature) 20 (feedmoisture level) and 23 (feed soybean compo-sition) (Table 6) while optimum responses interms of bulk density expansion index lightnessredness and yellowness indices were 021g cmminus31289 171 313 and 245 respectively Thisindicates that the optimum conditions can be es-tablished in twin-screw extrusion cooking of low-

26 Nahemiah et al

Table 6 Constraints and goals applied to locate optimum conditions for processing parameters andresponses for rice-full-fat soybean based extruded foods

Variables Goal Lower limit Upper limit Importance Optimum

Extrusion variablesBarrel Temperature (oC) In range 100 140 3 130Moisture content ( wb) In range 8 24 3 20Blend composition ( ) In range 15 25 3 23Response variablesBulk density (gcmminus3) Maximize 0035 036 5 021Expansion index () Maximize 1257 1338 5 1289Lightness (L) Maximize 103 183 3 171Redness (a) Minimize 075 435 3 313Yellowness (b) Minimize 131 288 3 245

grade fractions of parboiled rice and full-fat soy-bean composite blends which can result in prod-uct of low bulk and maximum expansion withsatisfactory light yellow color

4 Conclusions

Based on the RSM models proposed optimumextrusion cooking conditions for broken rice frac-tions and full-fat soybean were established at130 oC 20 g water per 100g sample and 23 gsoybean per 100 g rice with overall desirabilityconditions of 0996 while the response variableswere all desirable The optimum formulation ex-pands favorably at the die (1289) produceslight weight product with desirable light yellowcolor similar to commercial extruded breakfastor complimentary cereals Based on this resultmore alternative raw materials are at the dis-posal of food industries in Nigeria and indeedSub-Saharan African countries that are recom-mended to produce expanded instant foods whichcould be blended with legumes to improve nutri-tion and reduce malnutrition and even produceproducts for food aid among migrant communi-ties

Acknowledgements

Financial support received from Africa RiceCentre (AfricaRice) and Global Affairs Canada(GAC) under the Project number A034968 on

ldquoEnhancing food security in Africa through theimprovement of rice postharvest handling mar-keting and the development of new rice- basedproductsrdquo is highly appreciated

References

Abd El-Hady E A Mostafa G A El-SamahyS K amp El-Saies I A (1998) Produc-tion of high fiber corn extrudates Jour-nal of Agricultural Science Mansoura Univ23 (3) 1231ndash1245

Agbisit R Alavi S Cheng E Herald T ampTrater A (2007) Relationships betweenmicrostructure and mechanical propertiesof cellular cornstarch extrudates Journalof Texture Studies 38 (2) 199ndash219 doi101111j1745-4603200700094x

Altan A McCarthy K L amp MaskanM (2008) Twin-screw extrusion of bar-leyndashgrape pomace blends Extrudate char-acteristics and determination of optimumprocessing conditions Journal of Food En-gineering 89 (1) 24ndash32 doi10 1016 j jfoodeng200803025

Alvarez-Martinez L Kondury K P amp HarperJ M (1988) A general model for expan-sion of extruded products Journal of FoodScience 53 (2) 609ndash615 doi10 1111 j 1365- 26211988tb07768x eprint https onlinelibrary wiley com doi pdf 10 1111j1365-26211988tb07768x

Anuar N Adnan A F M Saat N AzizN amp Taha R M (2013) Optimiza-tion of extraction parameters by using re-sponse surface methodology purificationand identification of anthocyanin pigmentsin melastoma malabathricum fruit Scien-tific World Journal doi10 1155 2013 810547

Asare E K Sefa-Dedeh S Sakyi-DawsonE amp Afoakwa E O (2004) Applica-tion of response surface methodology forstudying the product characteristics of ex-truded rice-cowpea-groundnut blends In-ternational Journal of Food Sciences andNutrition 55 (5) 431ndash439 doi10 1080 09637480400003238

Asefa B amp Melaku E T (2017) Evaluation ofimpact of some extrusion process variableson chemical functional and sensory proper-ties of complimentary food from blends offinger millet soybean and carrot AfricanJournal of Food Science 11 (9) 302ndash309doi105897AJFS20171608

Badrie N amp Mellowes W A (1991) Effect ofextrusion variables on cassava extrudatesJournal of Food Science 56 1334ndash1337doi101111j1365-26211991tb04766x

Case S E Hamann D D amp SchwartzS J (1992) Effect of starch gelatiniza-tion on physical-properties of extrudedwheat-based and corn-based products Ce-real Chemistry 69 (4) 401ndash404

Chaiyakul S Jangchud K Jangchud A Wut-tijumnong P amp Winger R (2009) Ef-fect of extrusion conditions on physical andchemical properties of high protein gluti-nous rice-based snack LWT - Food Scienceand Technology 42 (3) 781ndash787 doi10 1016jlwt200809011

Chang Y K amp El-Dash A A (2003) Ef-fects of acid concentration and extrusionvariables on some physical characteristicsand energy requirements of cassava starchBrazilian Journal of Chemical Engineer-ing 20 (2) 129ndash137 doi10 1590 S0104 -66322003000200006

Chinma C E Anuonye J C Simon O COhiare R O amp Danbaba N (2015) Ef-fect of germination on the physicochemicaland antioxidant characteristics of rice flour

from three rice varieties from nigeria FoodChemistry 185 454ndash458 doi10 1016 j foodchem201504010

Colonna P (1989) Extrusion cooking of starchand starchy products Extrusion Cooking247ndash319

Coutinho L S Batista J E R Caliari M ampSoares Junior M S (2013) Optimizationof extrusion variables for the production ofsnacks from by-products of rice and soy-bean Food Science and Technology 33 (4)705ndash712

Danbaba N Nkama I amp Badau M (2016)Application of response surface methodol-ogy (rsm) for the production and optimiza-tion of extruded instant porridge from bro-ken rice fractions blended with cowpea In-ternational Journal of Nutrition and FoodSciences 5 105ndash116 doi1011648jijnfs2016050213

Danbaba N Nkama I Bada M H GbenyiD I Idakwo P Y Ndindeng S A ampMoreira J (2017) Multiple parameter op-timization of hydration characteristics andproximate compositions of rice-soybean ex-truded foods Open Access Library Journal4 (02) 1

Devi N L Shobha S Tang X Shaur S ADogan H amp Alavi S (2013) Develop-ment of protein-rich sorghum-based ex-panded snacks using extrusion technologyInternational Journal of Food Properties16 (2) 263ndash276 doi10 1080 10942912 2011551865

Ding Q-B Ainsworth P Tucker G amp Mar-son H (2005) The effect of extrusion con-ditions on the physicochemical propertiesand sensory characteristics of rice-basedexpanded snacks Journal of Food Engi-neering 66 (3) 283ndash289 doi10 1016 j jfoodeng200403019

Eggum B O Juliano B O Ibabao M G Bamp Perez C M (1986) Effect of extrusioncooking on nutritional value of rice flourFood Chemistry 19 (3) 235ndash240 doihttpsdoiorg1010160308-8146(86)90073-7

Faubion J M amp Hoseney R C (1982) Hightemperature short time extrusion cookingof wheat and flour IIEffect of moisture con-tent and flour type on extrudate proper-

28 Nahemiah et al

ties Cereal Chemistry 59 (6) 329ndash333 Re-trieved from httpaacnetorg

Filli K (2011) Application of response sur-face methodology for the study of composi-tion of extruded millet-cowpea mixtures forthe manufacture of fura A Nigerian foodAfrican Journal of Food Science 5 (16)884ndash896

Filli K (2016) Physicochemical properties ofsorghum malt and bambara groundnutbased extrudates Journal of Food Scienceand Technology Nepal 9 55ndash65 doi10 3126jfstnv91012075

Garg S K amp Singh D S (2010) Optimizationof extrusion conditions for defatted soy-rice blend extrudates Journal of Food Sci-ence and Technology-mysore 47 (6) 606ndash612 doi101007s13197-010-0117-y

Gogoi B K Oswalt A J amp Choudhury G S(1996) Reverse screw element(s) and feedcomposition effects during twin-screw ex-trusion of rice flour and fish muscle blendsJournal of Food Science 61 (3) 5990ndash595doi101111j1365-26211996tb13165x

Gupta K Verma M Jain P amp Jain M(2014) Process optimization for produc-ing cowpea added instant kheer mix us-ing response surface methodology Journalof Nutrition Health and Food Engineering1 (5) 00030 doi1015406jnhfe20140100030

Gutkoski L C amp El-Dash A A (1999) Ef-fect of extrusion process variables on phys-ical and chemical properties of extrudedoat products Plant Foods for Human Nu-trition 54 (4) 315ndash325 doi10 1023 A 1008101209353

Hagenimana A Ding X amp Fang T (2006)Evaluation of rice flour modified by ex-trusion cooking Journal of Cereal Science43 (1) 38ndash46 doi101016jjcs200509003

Hernandez-Nava R G Bello-Perez L A SanMartin-Martinez E Hernandez-SanchezH amp Mora-Escobedo R (2011) Ef-fect of extrusion cooking on the func-tional properties and starch componentsof lentilbanana blends Response surfaceanalysis Revista Mexicana De IngenieriaQuimica 10 (3) 409ndash419

Iwe M O (2003) The science and technology ofsoybean Rejoint Communication ServicesLtd Uwani Enugu Nigeria

Iwe M O Van Zuilichem D J Ngoddy P Oamp Ariahu C C (2001) Residence timedistribution in a single-screw extruder pro-cessing soy-sweet potato mixtures LWT -Food Science and Technology 34 (7) 478ndash483 doi101006fstl20010785

Jorge R-R Alma M-A Silvina D RolandoG David B-A amp Luis C-G (2008) Ex-trusion of a hard-to-cook bean (phaseolusvulgaris l) and quality protein maize (zeamays l) flour blend LWT - Food Scienceand Technology 41 (10) 1799ndash1807 doi101016jlwt200801005

Kadan R S Bryant R J amp Pepperman A B(2003) Functional properties of extrudedrice flours Journal of Food Science 68 (5)1669ndash1672 doi101111j1365-26212003tb12311x

Kulkarni S D amp Joshi K C (1992) Potatostarch soy blends Possible effects of starchproperties on few aspects of end productsIndian Food Packer 46 38ndash38

Lai C S Guetzlaff J amp Hoseney R C (1989)Role of sodium-bicarbonate and trappedair in extrusion Cereal Chemistry 66 (2)69ndash73

Lai L S amp Kokini J L (1991) Physicochem-ical changes and rheological properties ofstarch during extrusion (a review) Biotech-nology Progress 7 (3) 251ndash266 doi10 1021bp00009a009

Lee C L amp Wang W L (1997) Biologi-cal Statistics (Beijing Peoplersquos Republic ofChina Ed) Science Press

Leonel M de Freitas T S amp MischanM M (2009) Physical characteristics ofextruded cassava starch Scientia Agri-cola 66 (4) 486ndash493 doi101590S0103-90162009000400009

Manful J T Quaye W amp Gayin J (2004)Feasibility study on parboiled rice qual-ity improvement programme in some se-lected communities around tamale in thenorthern region of ghana Report forthe Food Security and Rice ProducersOrganization Project (FSRPOP) CSIR-FRIREMJT2004020

Marengo M Akoto H Zanoletti M CarpenA Buratti S Benedetti S SaaliaF et al (2016) Soybean-enriched snacksbased on African rice Foods 5 (2) 38

Maskus H amp S A (2015) Extrusion process-ing and evaluation of an expanded puffedpea snack product Journal of Nutritionand Food Sciences 5 378 doi10 4172 2155-96001000378

Montgomery D C (2001) Design and analysisof experiments John Wiley and Sons IncNew York

Moore D Sanei A Hecke E amp BouvierJ M (1990) Effect of ingredients on phys-icalstructural properties of extrudatesJournal of Food Science 55 (5) 1383ndash1387Retrieved from 101111j1365-26211990tb03942x

Ndindeng S A Manful J Futakuchi K Mor-eira J Graham Acquaah S Oussou P Candia A (2015) The first interna-tional congress on postharvest loss preven-tion developing measurement approachesand intervention strategies for smallhold-ers In Rome Italy October 4-7 2015(pp 31ndash33)

Noguchi A Kugimiya W Haque Z amp SaioK (1982) Physical and chemical charac-teristics of extruded rice flour and rice flourfortified with soybean protein isolate Jour-nal of Food Science 47 (1) 240ndash245 doi101111j1365-26211982tb11069x

Nwabueze T U (2007) Nitrogen solubil-ity index and amino acid profile of ex-truded african breadfruit (t africana)blends Nigerian Food Journal 25 (1) 23ndash35 doi104314nifojv25i133651

Omwamba M amp Mahungu S M (2014) Devel-opment of a protein-rich ready-to-eat ex-truded snack from a composite blend ofrice sorghum and soybean flour Food andNutrition Sciences 5 (14) 1309 doi10 4236fns2014514142

Singh B Sekhon K S amp Singh N (2007) Ef-fects of moisture temperature and level ofpea grits on extrusion behaviour and prod-uct characteristics of rice Food Chemistry100 (1) 198ndash202 doi101016jfoodchem200509042

Yagci S amp Gogus F (2008) Response sur-face methodology for evaluation of physicaland functional properties of extruded snackfoods developed from food-by-productsJournal of Food Engineering 86 (1) 122ndash132 doi101016jjfoodeng200709018

Yu L Ramaswamy H S amp Boye J (2013)Protein rich extruded products preparedfrom soy protein isolate-corn flour blendsLWT - Food Science and Technology50 (1) 279ndash289 doi10 1016 j lwt 2012 05012

Nutritional and Antioxidant Potential of Rice Flour Enrichedwith Kerstingrsquos Groundnut (Kerstingiella geocarpa) and

Lemon Pomace

Olugbenga O Awolua and Magoh A Osigwea

a Department of Food Science and Technology Federal University of Technology PMB 704 Akure OndoState Nigeria

Corresponding authorooawolufutaedung

Received 6 December 2017 Published online 18 April 2019

Abstract

This study was designed to enhance the nutritional quality antioxidant properties and productutilization potentials of locally produced lsquoIgbemorsquo rice flour by adding Kerstingrsquos groundnut and lemonpomace Kerstingrsquos groundnut is an underutilized legume while lemon pomace is a byproduct of lemonutilization both meant to enhance the protein quality antioxidant potential and fibre contents of thecomposite flour The dependent variables were minerals composition amino acid profile antioxidantsand antinutrients properties in-vitro protein digestibility and in-vitro carbohydrate digestibility Theresult showed that blends with higher lemon pomace of 1000 g had the best calcium iron potassiumand magnesium contents and antioxidant contents while blends with highest Kerstingrsquos groundnut(2000 g) had the best zinc content The anti-nutrients in the blends were generally low and safe forconsumption

Keywords Antioxidants Composite flour Kerstingrsquos groundnut Response surface methodology Riceflour

1 Introduction

The increase in production and consumersrsquo ac-ceptability of rice flour in the production of non-gluten baked products is promoting further re-search into rice flour utilization In additionto its non-gluten characteristics rice flour hasbeen found to possess good nutritional qualitiesRice as a cereal serves as a basic food sourcefor over half the world population whilst it pro-vides about 80 of the food intake as ready-to-eat convenience and inexpensive gluten-freesnacks (Awolu Oluwaferanmi Fafowora amp Os-eyemi 2015) Nutritionally cereals are impor-tant sources of carbohydrates dietary fibre andvitamins (Katina et al 2005) but they are defi-

cient in lysine Cereal-based flours are thereforesupplemented with legumes as credible source oflysine (Awolu et al 2015 Awolu Omoba Ola-woye amp Dairo 2017) In addition legumes serveas sources of protein and minerals needed forhealth growth and developmentKerstingrsquos groundnut (Kerstigiella geocarpaHarms) is an underutilized legume it is richin essential minerals protein and amino acidsThe crop is indigenous to Africa and a viablealternative to high protein content foods (Bay-orbor Dzomeku Avornyo amp Opoku-Agyeman2010) Kerstingrsquos groundnut has not been fullyexploited and its nutritional importance has notbeen fully evaluatedLemon (Citrus limon) comes after orange and

Antioxidants Potential of Enriched Rice Flour 31

mandarin in the order of citrus importance It isrich in vitamin C minerals dietary fibre essen-tial oils organic acids carotenoids and flavonoids(Gonzalez-Molina Dominguez-Perles Morenoamp Garcia-Viguera 2010)This study produced composite flour comprisingrice Kerstingrsquos groundnut and lemon pomaceThe addition of Kerstingrsquos groundnut was meantto enhance its protein content and mineral com-positions whilst lemon pomace enhanced its fi-bre contents Kerstingrsquos groundnut and lemonpomace has been found to be rich in antioxidantshence it was expected that their addition wouldenhance the antioxidant capacities of the com-posite flour

21 Materials

Igbemo rice was sourced from Igbemo-Ekiti Ek-iti State Kerstingrsquos groundnut (Kerstingiellageocarpa Harms) was sourced from Oyingbomarket Lagos Lemon fruits were sourced atOja-Oba Akure Ondo State All reagents wereof analytical grade

22 Preparation of Flours

Preparation of rice flour

About 5 g of Igbemorsquo rice grains were manuallycleaned dry-milled using hammer mill sievedthrough 210 microm particle size sieves and thenstored in a sealed plastic container at room tem-perature for further processing (Awolu et al2015)

Preparation of Kerstingrsquos groundnutflour

Kerstingrsquos groundnut seeds (20 kg) were par-boiled for 35 min manually dehulled oven driedat 65 oC until constant weight was obtained andhammer-milled into fine particles The flour wassubsequently kept inside a sealed plastic con-tainer prior to usage (Awolu et al 2015)

Preparation of lemon pomace flour

Lemon pomace was produced according to themethod described by Kolodziejczyk MarkowskiKosmala Krol and Plocharski (2007) Freshlemon fruits were washed with warm water toremove tough dirt and dust from the fruits Thewashed fruit was pulverized with a sharp knifeand blended into slurry using a Kenwood blender(BL-237) The juice was extracted from theslurry using a muslin cloth whilst the wet lemonpomace was oven dried (Gen-lab hot air ovenmodel DHG-91011SA) at 60 oC for 18 h Thedried pomace was blended into fine flour usingthe blender

23 Experimental design for thedevelopment of flour blends

Optmization of the proximate properties of thecomposite flour was carried out using the opti-mal mixture model design of response surfacemethodology (Design expert 8031 trial ver-sion) The independent variables were rice flour(7030 ndash 8500) Kerstingrsquos groundnut flour(1000 ndash 2000) and lemon pomace (500 ndash1000) while the dependent variables were theproximate composition

24 Proximate compositiondetermination of thecomposite flour

The moisture content crude protein fat ashcontent and crude fibre of the composite flourblends were determined according to the stan-dard methods of AOAC (2005)

25 Minerals analysis

Mineral analysis was determined according tothe AOAC (2005) method The sample wasashed and about 15 mL of 6 N HCl was addedto it and transferred to a 100 mL volumetricflask Distilled water was used to make up to the100 mL mark Atomic Absorption Spectroscopy(AAS) was used for analysis of all the mineralsexcept potassium and sodium which were ana-lyzed using a flame emission spectrophotometer

32 Awolu and Osigwe

(Model A-6200 Shimadzu Corporation and Ky-oto Japan) Standards for sodium and potas-sium were prepared from their chloride salts

26 Amino acid profiledetermination

The sample was hydrolyzed using hydrochloricacid (6 N) for 24 h at 110 oC in a vial un-der vacuum and N2 atmosphere evaporated anddissolved in sodium citrate buffer (pH 22) andthe hydrolysates were analyzed by using a HPLCcombined with a Pickering PCX5200 derivatizer(Pickering Laboratories Inc USA) and ion ex-change column (30 times 250 mm 8 microm) Theamino acids were identified spectrophotometri-cally by measuring at 570 nm (Benitez 1989)

27 Evaluation of AntioxidantsProperties

Determination of ABTS scavengingability

Aqueous 2 2rsquo-azino-bis (3-ethylbenthiazoline-6-sulphonic acid) (ABTS) scavenging ability so-lution (78 M) with K2S2O8 (245 mM finalconc) was left in the dark for 16 h and theabsorbance adjusted at 734 nm to 0700 withethanol About 02 mL of the appropriate dilu-tion of the extract was added to 20 mL of ABTSsolution and the absorbance was read at 732 nmafter 15 min The Trolox (6-hydroxy-2 5 7 8-tetramethylchromane-2-carboxylic acid) equiva-lent antioxidant capacity was subsequently cal-culated (Re et al 1999)

Determination of DPPH free radicalscavenging ability

The stock reagent solution (1 times 10minus3 M was pre-pared by dissolving 22 mg of DPPH in 50 mLof methanol and stored at 20 oC The workingsolution (6 times 10minus5 M) was prepared by mixing 6mL of stock solution with 100 mL of methanol toobtain an absorbance value of 08 plusmn 002 at 515nm Exactly 01 mL each of extract solutionsof different concentrations were vortexed for 30 swith 39 mL of DPPH solution and left to react

for 30 min the absorbance at 515 nm was thenrecorded A control with no added extract wasalso analyzed DPPH Scavenging activity wascalculated using Eq (1) (Lee Mulugu York ampOrsquoShea 2007)

DPPH =Abcontrol minusAbsample

Abcontroltimes 100 (1)

Where Ab = Absorbance

Total flavonoids

About 05 mL aliquot of 20 g Lminus1 AlCl3 ethano-lic solution was added to 05 mL of extract solu-tion The absorbance at 420 nm was measuredafter 1 h at room temperature The presence offlavonoids was indicated by a yellow colourationExtract samples were evaluated at a final concen-tration of 01 mg mLminus1 (Eq 2) and expressed asquercetin equivalent (QE) based on the calibra-tion curve (Ordonez Gomez Vattuone amp Lsla2006)

C = 000255 timesAb (R2 = 09812) (2)

Where Ab is the absorbance and C is the con-centration (mg QE gminus1 DW)

28 Determination ofAntinutrients

About 1 g of sample was weighed into 100 mLconical flask 75 mL 3 M H2SO4was added andstirred for 1 h with a magnetic stirrer Ex-actly 25 mL of the filtrate (Whatman filter paperNo1) was taken and titrated hot against 005 MKMnO4 solution until a faint pink colour whichpersisted for at least 30 sec was formed The ox-alate content was calculated by taking 1 mL of005 M KMnO4 as equivalent to 22 mg oxalateusing Eq (3) (Day amp Underwood 1986)

Oxalate(mg100g) =Titrevaluetimes 22 timesDF

Where 22 mg = mass equivalent value of 1mLof 005 M KMnO4 solutionDF = Dilution factor (total volume of sampledivided by volume of portion used for titration)W = Sample weight in g

Determination of phytic acid content

About 2 g of sample was weighed into 250 mLconical flask 100 mL of 2 concentrated HClwas thereafter added allowed to soak for 3 hand filtered The filtrate (50 mL) was pipettedinto 250 mL beaker with 107 mL ammoniumthiocyanate solution added as an indicatorand titrated with standard iron III chlorideFeCl3 solution (containing 000195 g ironmL)until a brownish yellow colour appeared andpersisted for five minutes The phytic acid con-tent was calculated using Eq (4) (Russell 1980)

PHY =000195 times VFeCl3 timesDF

WSample(4)

PHY = Phyticacid (gkg)VFeCl3 = volume of FeCl3 consumedWSample = sample weightDF = Total volume of extraction solventaddedvolume of aliquot taken for the titration

About 02 g of sample was placed in a test tube10 mL of 1 HClmethanol was added the testtube was capped continuously shaken for 20 minand then centrifuged at 2500 rpm for 5 min Ex-actly 1 mL of the supernatant was pipetted intofresh tubes the absorbance was set at zero and1 mL blank solution was mixed with 5 mL 4 HClmethanol and 5 mL vanillin reagent in a testtube The sample and blank test tubes were incu-bated for 20 min at 30 oC Absorbance was readat 500 nm and concentration of condensed tan-nins was determined from standard curve Tan-nin concentration was expressed in as follows(Trease amp Evans 1978)

Tannic content =(C times 10)

200times 100 (5)

WhereC= Concentration corresponding to the opticaldensity10 = Volume of extract (mL)200 = sample weight (mg)

Determination of saponin content

About 20 mL of 20 aqueous ethanol was addedto 10 g of the ground sample and agitated witha magnetic stirrer for 12 h at 55 oC The solu-tion was filtered through Whatman No1 filterpaper and the residue re-extracted with 200 mL20 aqueous ethanol The extract was reducedto 40 mL under vacuum and 20 mL diethyl etheradded in a separating funnel and shaken vigor-ously The aqueous layer was recovered whilethe ether layer was discarded The pH of theaqueous solution was adjusted to 45 by addingNaOH and the solution shaken with 60 ml n-butanol The combined butanol extracts werewashed twice with 10 mL of 5 aqueous NaCland evaporated to dryness in a fume cupboardto give a crude saponin (Hudson amp El-Difrawi1979)

Determination of trypsin inhibitor

Tris-buffer (005 M pH 82) containing 002M CaCl2 605 g tris- (hydroxymethyl)aminomethane and 294 g CaCl2middot2H2O were dis-solved in 500 mL of distilled water the pH wasadjusted to 82 and the volume made up to 1 Lwith distilled water About 20 mL of trypsin so-lution was added to 10 g of the extracted samplein a test tube and then placed in a water bathat 37 oC Exactly 5 mL hydrated Benzoyl-DL-arginene-p-nutoanilide (BAPA) solution was dis-solved in dimethyl sulfoxide previously warmedto 37 oC The reaction was terminated 10 minlater by adding 1 mL of 30 acetic acid Af-ter thorough mixing the contents of each tubewere filtered through Whatman No1 paper andthe absorbance was measured against the blank(Kakade 1974)

29 In-vitro protein (IVPD)determination

Composite flour samples (200 mg) were weighedinto an Erlenmeyer flask and mixed with 35mL of porcine pepsin (15 g of pepsin in 01 MKH2PO4 pH 20) Samples were digested for 2h at 37 oC digestion was stopped by additionof 2 mL of 2 M NaOH and the samples werecentrifuged (4900 xg at 40 oC) for 20 min after

34 Awolu and Osigwe

which the supernatant was discarded Theresidues were washed and centrifuged twicewith 20 mL of buffer (01 M KH2PO4 pH70) Undigested nitrogen was determined usingKjeldahl method Digestibility was calculatedusing Eq (6) (Aboubacar Axtell Huang ampHamaker 2001)

IV PD =(Nsample minusNUndigested)

Nsampletimes 100 (6)

210 In-vitro carbohydratedigestibility determination(IVCD)

Exactly 4 mL of phosphate buffer (pH 66) 1mL of sodium chloride and 1 mL of α amylaseenzyme was added to 5 mL of the sample at roomtemperature and mixed thoroughly Aliquots(02 mL) of the mixture were taken at zero and10 h (complete hydrolysis was predetermined)after addition of the enzyme and dispensed into10 mL Lugolrsquos iodine solution (1100 dilution)The absorbance was measured at 620 nm andthe invitro carbohydrate digestibility wascalculated using Eq (7) (Shekib Eliraqui ampAbobakr 1988)

IV CD =Abstime=0 minusAbstime=1h

Abstime=0times 100 (7)

where Abs is the Absorbance

31 Proximate composition andfunctional properties of thecomposite flour

The result of the proximate composition is pre-sented in Table 1 The moisture content of thecomposite flour (620 to 681 g100g) was withinthe acceptable range (le 10) for flours to en-sure shelf stability In addition the variation inmoisture content of the composite flour had a

low R-squared and the adjusted R-squared val-ues of 03496 and 00244 respectively which wasan indication that it would not support mois-ture hence the composite flour would have agood shelf lifeThe ash content of the composite flour rangedbetween 098 and 181 g100g The ash contentincreased significantly (p le 005) as the levelsof lemon pomace incorporation increased TheR-squared and adjusted R-squared values were07546 and 07168 respectively The crude pro-tein of the flour samples ranged between 751 and1299 g100g The protein content increased sig-nificantly (p le 005) as the level of Kerstingrsquosgroundnut incorporation increased A similar re-sults have been obtained for composite flour con-sisting of rice cassava and Kerstingrsquos groundnutflours (Awolu et al 2015) The ANOVA indi-cated that the model and model term (linear mix-ture AB AB (A-B)) were significant (p le 005)while the R-squared and the adjusted R-squaredvalues were 09975 and 09937 respectively Thehigh adjusted R-squared value for protein con-tent showed that the protein had high positiveeffect on the composite flourThe fat content of the flours ranged from 400- 444 g100 g In comparison wheat flour hadfat content of 133 g100g The fat content de-creased with increased incorporation of rice andlemon pomace flours The composite flour maybe better enhancer of flavour and fat-soluble vi-tamins than wheat flour In addition the fatvalues of the composite flour should not pos-sess any negative effect in terms of rancidityThe crude fibre values ranged from 252 to 378g100gThe result indicated that the addition ofKerstingrsquos groundnut flour and lemon pomace in-creased the crude fibre content of the compositeflour The ANOVA indicated that the model andmodel terms (linear mixture AB AB(A-B) forcrude fibre were significant (p le 005) while theR-squared and adjusted R-squared were 09975and 09937 respectivelyThe carbohydrates content of the flours rangedbetween 6203 - 7298 g100g The model andmodel terms (linear mixture component ABAC A2BC ABC2) were significant (p le 005)while the R-squared and adjusted R-squaredwere 09945 and 09882 respectively

Table 1 Proximate Composition of Composite Flour

RunVariables (g) Proximate composition (g100g)

A (g) B (g) C (g) Moisture Ash Protein Fibre Fat CHO

1 7984 1016 1000 640 181 751 371 400 66572 7500 2000 500 620 125 1299 289 428 67393 8350 1150 500 650 105 856 280 400 72094 7030 2000 970 630 176 1257 334 430 62035 7698 1575 725 646 170 912 321 427 67986 7984 1016 1000 647 130 843 362 400 66187 8228 1000 771 630 148 841 370 400 68408 7500 2000 500 681 130 128 290 444 66759 8500 1000 500 640 098 841 268 401 725210 7316 1907 776 662 168 1272 318 436 636711 8228 1000 771 680 160 846 372 411 676012 8500 1000 500 641 106 799 266 400 728813 8031 1468 500 643 132 955 252 420 709814 7744 1255 1000 641 180 900 378 410 649115 7356 1643 999 630 174 1162 309 419 631016 7030 2000 970 630 176 1263 330 424 6207

A - Rice Flour B - Kerstingrsquos groundnut C- Lemon pomace CHO - Carbohydrate

32 Minerals composition ofoptimized composite flour

The result of the mineral composition of thecomposite flour is presented in Table 2 Ironcontents in the composite flour ranged between282 and 347 mg1 00g Iron deficiency is themost common nutrient disorder worldwide asit accounts for 50 of the cases of anaemia(World Health Organization 2001) Iron con-tents reported in rice Kerstingrsquos groundnutand lemon pomace flours were 082 mg100g1000 mg100g and 14765 mg100g respectively(Adeyeye amp Faleye 2007 Atukorale 2002Janati Beheshti Feizy amp Fahim 2012) Theiron content in composite flour increased signifi-cantly (p le 005) as the level of lemon pomaceincreasedZinc values ranged from 237 to 267 mg100gZinc content in the composite flour increasedsignificantly (p le 005) as the levels of Ker-stingrsquos groundnut increased The values ofzinc obtained in this study was higher than058 ndash 066 mg100g reported for wheat basedcomposite flour enriched with lsquoorarudirsquo (Vahigna

sp) (Onoja et al 2014)Magnesium values ranged between 454 and 464mg100g The magnesium content increasedsignificantly (p le 005) as the lemon pomaceincreased Magnesium is a cofactor in about300 enzyme systems which play regulatoryroles in several biochemical reactions in thebody such as protein synthesis muscle andnerve function blood glucose control and bloodpressure regulation (Laurant amp Touyz 2000)It also promotes strong bones strong and keepsheart rhythm steady (Twum et al 2015)Potassium ranged between 12177 -16633mg100g Potassium content increased sig-nificantly (p le 005) with increasing lemonpomace flour content Potassium plays a vitalrole in maintaining osmotic balance and pH ofthe body fluids regulating muscle and nerveirritability controlling glucose absorption andenhancing normal retention of protein duringgrowth (National Research Council 1980)Calcium values ranged from 5565 to 6510mg100g Lemon pomace had been reported tocontain about 845250 mg100g calcium content(Janati et al 2012) The calcium content of

36 Awolu and Osigwe

the composite flour increased significantly (ple 005) as lemon pomace increased Calciumfunctions primarily in the development of strongbones

33 Antioxidant properties of thecomposite flour

The results of the antioxidant property of com-posite flour are presented in Table 3 The DPPHfree radical scavenging ability ranged from 3957to 4510 DPPH of the composite flour in-creased significantly (p le 005) as lemon pomaceincreasedThe ABTS scavenging ability of the compositeflour ranged between 2381 and 2540 mMolgThe ABTS also increased significantly (p le 005)as lemon pomace increased The increase in bothDPPH scavenging activities and ABTS as a re-sult of the addition of lemon pomace is a justifica-tion for its addition which is to improve antioxi-dant capacities In addition lemon pomace flourhad positive effect on the flavonoids content ofthe composite flour an increase in level of lemonpomace in the composite flour brought about acorresponding significant (p le 005) increase inthe flavonoid contents Flavonoids are majorpolyphenolic components of foods and displayanti-inflammatory anti-allergic and anti-canceractivities (Crozier Clifford amp Ashihara 2008)

Antinutritional properties ofcomposite flour

The results of antinutritional properties of thecomposite flour are presented in Table 4 Phytatecontent of the composite flour ranged between229 and 250 mg100g Run 2 with the highestKerstingrsquos groundnut content (20 g100g) hadthe overall highest antinutrients contents How-ever the antinutrients contents were within rec-ommended level that is safe for human consump-tion the recommended toxicity level of phytatesfor humans is 2 ndash 5 gday (Hassan Umar ampUmar 2004) while the phytate content in thecomposite flour was far less than this valueThere was no significant (p gt 005) difference be-tween the tannin contents of the composite flourof the samples The tannin content obtained in

this study was also very safe for consumption(Ikpeme Ekpeyoung amp Igile 2012) This lowresult indicated that the composite flour wouldhave good protein digestibility as high proteincontents interferes with protein digestibilityThe trypsin inhibitor activity of the compositeflour ranged between 026 and 034 mg100gThe higher the Kerstingrsquos groundnut content thehigher the trypsin inhibition activity As with al-ready mentioned antinutrients the trypsin inhi-bition levels were very minimal and safe for hu-man consumption A trypsin inhibitor activitycontent ranging from 401 to 4601 mg100g hasbeen reported for Acha-Soybean composite flour(Ikpeme et al 2012)Oxalates ranged from 073 to 087 mgg Thelevel of oxalate obtained is also low and safe forhuman consumption The toxicity of oxalates is2 ndash 5 gday (Hassan et al 2004) The saponincontent obtained in this study was also low andsafe for human consumption

In-vitro carbohydrate digestibilityand in-vitro protein digestibility ofthe composite flour

Research has shown that nutrient compositionof foods is not enough to determine nutrientbio-availability (Julian et al 2007) hence theneed for in-vitro (starch and protein) digestibil-ity analyses The result of in-vitro carbohydratedigestibility is presented in Table 5 The resultsshowed that the sample with highest rice con-tent (run 7) had the overall best carbohydratedigestibility Rice content dictates the extent ofthe carbohydrate digestibility the higher the ricecontent the higher the carbohydrate digestibil-ity and vice versa In addition digestion timerather than digestion temperature enhanced car-bohydrate digestibility Carbohydrate digestibil-ity was higher in samples with the same digestiontemperature but higher digestion time hence di-gestion at 60 min was higher than digestion at30 minThe results of in-vitro protein digestibility arepresented in Table 6 Unlike carbohydrate di-gestibility where the sample with the highestrice (with highest carbohydrate content) had thehighest digestibility protein digestibility had the

Table 2 Minerals Composition of the Composite Flour

SampleCalcium Potassium Zinc Iron Magnesium

(mg100g) (mg100g) (mg100g) (mg100g) (mg100g)

Runs 2 5565 plusmn 121c 12177 plusmn 059c 267 plusmn 005a 282 plusmn 011b 454 plusmn 012c

Runs 7 6111 plusmn 118b 13900 plusmn 058b 255 plusmn 010a 333 plusmn 014a 458 plusmn 018b

Runs 14 6510 plusmn 096a 16633 plusmn 058a 237 plusmn 005b 347 plusmn 008a 464 plusmn 012a

values are mean plusmn standard deviation of triplicate samples

values on the same column with the same superscript are not significantly different at p le 005

Run 2 = 75 g100g rice 20 g100g Kerstingrsquos groundnut 5 g100g lemon pomace flours

Run 7 = 8228g100g rice 10 g100g Kerstingrsquos groundnut 771 g100g lemon pomace flours

Table 3 Antioxidant Properties of the Composite Flour

Sample DPPH () ABTS (mMolg) Flavonoids (mg QE gminus1)

Runs 2 3957 plusmn 135c 2381 plusmn 008c 121 plusmn 001c

Runs 7 4273 plusmn 305ab 2440 plusmn 040b 128 plusmn 001a

Runs 14 4510 plusmn 189a 2540 plusmn 024a 126 plusmn 001b

values are mean plusmn standard deviation of triplicate samplesvalues on the same column with the same superscript are not significantly different at p le 005Run 2 = 75 g100g rice 20 g100g Kerstingrsquos groundnut 5 g100g lemon pomace floursRun 7 = 8228g100g rice 10 g100g Kerstingrsquos groundnut 771 g100g lemon pomace floursRun 14 = 7744 g100g rice 1255 g100g Kerstingrsquos groundnut 10 g100g lemon pomace flours

Table 4 Antinutritional Properties of the Composite Flour

SampleOxalate Saponin Tannin Phytates Trypsin

(mg100g) () (mg100g) (mg100g) ()

Runs 2 087 plusmn 001a 331 plusmn 020a 003 plusmn 000a 2505 plusmn 002a 034 plusmn 012a

Runs 7 073 plusmn 002c 282 plusmn 004b 003 plusmn 000a 2403 plusmn 002b 026 plusmn 002c

Runs 14 081plusmn 004b 244 plusmn 001c 003 plusmn 000a 2289 plusmn 001c 027 plusmn 004b

values are mean plusmn standard deviation of triplicate samples values on the same column with the same superscript are not significantlydifferent at p le 005Run 2 = 75 g100g rice 20 g100g Kerstingrsquos groundnut 5 g100g lemon pomace floursRun 7 = 8228g100g rice 10 g100g Kerstingrsquos groundnut 771 g100g lemon pomace floursRun 14 = 7744 g100g rice 1255 g100g Kerstingrsquos groundnut 10 g100g lemon pomace flours

Table 5 In-vitro Carbohydrate Digestibility of Composite Flour

SampleDigested Starch

50 oC for 60 min 50oC for 30 min 40oC for 60 min 40oC for 30 min

Run 2 2867 plusmn 080b 1048 plusmn 005c 2547 plusmn 012c 1005 plusmn 009c

Run 7 3000 plusmn 045a 2562 plusmn 003a 2844 plusmn 042a 2336 plusmn 042a

Run 14 2854 plusmn 002b 1578 plusmn 007b 2788 plusmn 011b 1245 plusmn 008b

38 Awolu and Osigwe

Table 6 In-vitro Protein Digestibility Determination (IVPD) of the Composite Flour

Sample Digestibility 10min Digestibility 15min

Runs 2 6926 plusmn 001c 6727 plusmn 019c

Runs 7 7505 plusmn 023b 7378 plusmn 048b

Runs 14 7759 plusmn 004a 7831 plusmn 002a

Table 7 Amino Acid Profile of the Composite Flour

Essential Concentration Non-essential Concentrationamino acid g100g protein amino acid g100g protein

Leucine 680 Glycine 389Lysine 453 Alanin 409

Isoleucine 392 Serine 313Phenylalanine 408 Cystine 121

Valine 397 Aspartic acid 701Methionine 240 Glutamic acid 954Histidine 255 Proline 284Threonine 319 Hydroxyproline 576

Tryptophan 186 Citrulline 378Arginine 255

highest digestibility when Kerstingrsquos groundnut(main protein component) was only 1255 ofthe composite flour The effect of higher antinu-trients (though at safe levels) in run 2 must haveaccounted for the lowest protein digestibility Infact run 2 with the highest protein content hadthe lowest protein digestibility As with carbohy-drate digestibility the higher the digestion timethe higher the digestibility

Amino acid profile of the compositeflour

The amino acid profile of the composite flour ispresented in Table 7 Leucine was the highest(680 mg100g) essential amino acid followed bylysine (453 g100g) Lysine is a major limitingamino acid in cereals and the increase in lysine inthe composite flour could be as a result of Kerst-ingrsquos groundnut incorporation Lysine promotesprotein synthesis and thus it is important for

growth and maintenance of the body (Awolu etal 2017) Glutamic acid and aspartic acid werethe most abundant amino acids in the compositeflour with values of 954 g100g and 701 g100grespectively Glycine together with other essen-tial amino acids such as alanine arginine andphenylalanine forms polypeptides that promotegrowth and tissue healing (Davies amp Reid 1979)

4 Conclusions

The utilization of rice flour in the production ofnutritionally rich baked products would be en-hanced by the addition of Kerstingrsquos groundnutand lemon pomace While Kerstingrsquos groundnutenhanced the protein content and minerals com-position lemon pomace enhanced its fibre con-tent and antioxidant potentials The addition ofKerstingrsquos groundnut at level of 20 g100g of thecomposite flour was considered nutritionally safe

In essence composite flour consisting rice Kerst-ingrsquos groundnut and lemon pomace at the blendratios carried out in this study would be bene-ficial for consumption in terms of its nutritionalcomposition and antioxidant capacities withoutnegative antinutritional factors

References

Aboubacar A Axtell J D Huang C P ampHamaker B R (2001) A rapid protein di-gestibility assay for identifying highly di-gestible sorghum lines Cereal Chemistry78 (2) 160ndash165 doi10 1094 CCHEM 2001782160

Adeyeye E amp Faleye F (2007) Chemical com-position and the food properties of (ker-stingiella geocarpa) harms seeds

AOAC (2005) Official methods of analysis 15thedn (Gaithersburg S edn) AOAC PressWashington DC USA pp 78- 90

Atukorale D P (2002) Nutritional value of riceDaily News March 1

Awolu O O Oluwaferanmi P M FafoworaO I amp Oseyemi G F (2015) Optimiza-tion of the extrusion process for the pro-duction of ready-to-eat snack from ricecassava and kerstingrsquos groundnut compos-ite flours LWT-Food Science and Technol-ogy 64 (1) 18ndash24 doi101016jlwt201505025

Awolu O O Omoba O S Olawoye O ampDairo M (2017) Optimization of pro-duction and quality evaluation of maize-based snack supplemented with soybeanand tiger-nut (cyperus esculenta) flourFood Science amp Nutrition 5 (1) 3ndash13doi101002fsn3359

Bayorbor T Dzomeku I Avornyo V ampOpoku-Agyeman M (2010) Morphologi-cal variation in kerstingrsquos groundnut (ker-stigiella geocarpa harms) landraces fromnorthern ghana Agriculture and BiologyJournal of North America 1 (3) 290ndash295

Benitez L V (1989) Amino-acid and fatty-acidprofiles in aquaculture nutrition studies InS S Desilva (Ed) Fish nutrition researchin asia (Vol 4 pp 23ndash35) Asian Fish-eries Society Special Publication 3rd Asian

Fish Nutrition Network Meeting BangkokThailand Jun 06-10 1988 INT DEV RESCTR Manila Asian Fisheries Soc

Crozier A Clifford M N amp Ashihara H(2008) Plant secondary metabolites Oc-currence structure and role in the humandiet John Wiley amp Sons

Davies N T amp Reid H (1979) An evaluationof the phytate zinc copper iron and man-ganese contents of and zn availability fromsoya-based textured-vegetable-proteinmeat-substitutes or meat-extendersBritish Journal of Nutrition 41 (3) 579ndash589 doi101079BJN19790073

Day R A amp Underwood A L (1986) Quanti-tative analysis 5th edition laboratory man-ual Includes bibliographical references andindex Englewood Cliffs NJ Prentice-Hall

Gonzalez-Molina E Dominguez-Perles RMoreno D A amp Garcia-Viguera C(2010) Natural bioactive compounds of cit-rus limon for food and health Journal ofPharmaceutical and Biomedical Analysis51 (2 SI) 327ndash345 doi10 1016 j jpba 200907027

Hassan L G Umar K J amp Umar Z (2004)Antinutritive factors in tribulus terrestris(linn) leaves and predicted calcium andzinc bioavailability Journal Tropical Bio-science 7 33ndash36

Hudson B J F amp El-Difrawi E A (1979)The sapogenins of the seeds of four lupinspecies Journal of Plant Foods 3 (3) 181ndash186 doi1010800142968X197911904227

Ikpeme C Ekpeyoung I amp Igile G O (2012)Nutritional and sensory characteristics ofan infant food based on soybean seeds(glycine max) and tigernut tubers (cype-rus esculenta) British Journal of AppliedScience and Technology 2 (4) 356ndash366

Janati S S F Beheshti H R Feizy J ampFahim N K (2012) Chemical compositionof lemon (Citrus limon) and peels its con-siderations as animal food GIDA-Journalof Food 37 (5) 267ndash271

Julian A G I O B Inyang C M A AC U O amp V I M (2007) Protein dis-persibility index and trypsin inhibitor ac-tivity of extruded blends of achaaoybean

40 Awolu and Osigwe

A response surface analysis AmericanJournal of Food Technology 2 (6) 502ndash511

Kakade M L (1974) Biochemical basis forthe differences in plant protein utiliza-tion Journal of Agricultural and FoodChemistry 22 (4) 550ndash555 doi10 1021 jf60194a014

Katina K Arendt E Liukkonen K H Au-tio K Flander L amp Poutanen K (2005)Potential of sourdough for healthier cerealproducts Trends in Food Science amp Tech-nology 16 (1-3) 104ndash112 2nd InternationalSymposium on Sourdough Brussels BEL-GIUM OCT 2003 doi101016jtifs200403008

Kolodziejczyk K Markowski J Kosmala MKrol B amp Plocharski W (2007) Applepomace as a potential source of nutraceu-tical products Polish Journal of Food andNutrition Sciences 57 (4) 291ndash295

Laurant P amp Touyz R M (2000) Physiolog-ical and pathophysiological role of magne-sium in the cardiovascular system Impli-cations in hypertension Journal of Hyper-tension 18 (9) 1177ndash1191 doi10 1097 00004872-200018090-00003

Lee Y-S Mulugu S York J D amp OrsquoSheaE K (2007) Regulation of a cyclin-cdk-cdk inhibitor complex by inositol py-rophosphates Science 316 (5821) 109ndash112 doi101126science1139080

National Research Council (1980) Drinking wa-ter and health Volume 3 WashingtonDC National Academy Press

Onoja U Akubor P Ivoke N I AtamaC Onyishi G Felicia E EjereV (2014) Nutritional composition func-tional properties and sensory evaluation ofbreads based on blends of orarudi (vignasp) and wheat flour Scientific Researchand Essays 9 (24) 1019ndash1026 doi105897SRE20146104

Ordonez A A L Gomez J D VattuoneM A amp Lsla M I (2006) Antioxidantactivities of sechium edule (jacq) swartzextracts Food Chemistry 97 (3) 452ndash458

Re R Pellegrini N Proteggente A Pan-nala A Yang M amp Rice-Evans C(1999) Antioxidant activity applying animproved abts radical cation decolorization

assay Free Radical Biology and Medicine26 (9-10) 1231ndash1237 doi10 1016S0891-5849(98)00315-3

Russell J A (1980) A circumplex model of af-fect Journal of Personality and Social Psy-chology 39 (6) 1161ndash1178 doi10 1037 h0077714

Shekib L A Eliraqui S M amp AbobakrT M (1988) Studies on amylase in-hibitors in some egyptian legume seedsPlant Foods for Human Nutrition 38 (4)325ndash332 doi101007BF01091730

Trease G E amp Evans W C (1978) A textbook of pharmacognosy 11edBailliere-Tindall London

Twum L A Kottoh I D Asare I KTorgby-Tetteh W Buckman E S ampAdu-Gyamfi A (2015) Physicochemicaland elemental analyses of banana compos-ite flour for infants British Journal of Ap-plied Science amp Technology 6 (3) 276ndash284

World Health Organization (2001) Iron de-ficiency anaemia Assessment preventionand control A guide for programme man-agers Geneva WHO

Effect of Chestnut and Acorn Flour on Wheat Wheat-BarleyFlour Properties and Bread Quality

Marie Hruskovaa Ivan Sveca and Ivana Kadlcıkovaa

a Department of Carbohydrates and Cereals University of Chemistry and Technology PragueCorresponding author

IvanSvecvschtczTel +420 220 443 206

Abstract

Additions of barley flour alone or with combination of chestnut and acorn flour (30 30+530+10) were aimed at increasing the dietary fibre content in wheat bread In this regard enhance-ment by acorn flour elevated the dietary fibre by a greater extent (up to 780) compared to barley orchestnut flours Increasing the proportion of non-traditional raw materials also influenced flour pastingproperties during the amylograph test as well as the farinograph and extensigraph properties of non-fermented dough In contrast to the wheat flour analysis of Falling Number and Zeleny values showeda decrease in technological potential of flour composites of approximately 30 Water absorption in-creased about 2 percentage points mainly with enhancement by chestnut flour All the non-traditionalraw materials slowed dough development whilst dough softening degree differed according to actualcomposition Dough viscous and elastic properties worsened as shown by a decrease of in energy ab-sorbed depending on the type and the addition of the non-traditional products Changes in flourcomposition were reflected in amylograph viscosity maximum which became lower with increasingamounnts of chestnt and acorn flour A significant worsening of the bread specific volume as well asof bread shape (vaulting) corresponded with a partial dilution of the gluten matrix Compared to thewheat bread 10 chestnut flour caused bread size to diminish to less than one half of the wheat loafStatistically the principal features were water absorption dough softening degree and extensigraphenergy together with specific bread volume In terms of wheat flour and bread quality the influence ofbarley flour overcame the effects of adding chestnut or acorn flours

Keywords Wheat-barley composite flour Chestnut flour Acorn flour Bread Principal componentanalysis

1 Introduction

For hundreds of years fine wheat flour repre-sented a ldquowhite goldrdquo accessible for the richerminority of society Owing to this plebeian nu-trition was based on rye or barley together withlegumes and pseudocereals In this regard ediblefruits from trees were rarely used and served pri-marily as a feed for domestic animals Neverthe-less in times of crop failure caused eg by flood-ing or local wars humans gratefully ate plant

products such as chestnut or acorn to stay aliveLater the industrial revolution supressed socialdiscrepancies and wheat flour gradually becamethe basic raw material for nearly all social classesAlternative plant raw materials have been forgot-ten and only with the rising incidence of lifestylediseases have we returned to their intentionalconsumptionChestnuts fruits of tree Castanea sativa Millare traditionally treated by hydrothermal pro-

42 Hruskova et al

cess to transform them into flour Compared towheat chestnut flour contains much more simplesugars and dietary fibre (ca 2 vs 16 and 2vs 26 respectively) but approximately a halfprotein content (6 against 10-15 Giovannelli(2009) and CREA (2015a 2015b) respectively)In limited amounts chestnut flour alone or incombination with rice flour may improve volumeof biscuits (Hegazy Kamil Hussein amp Bareh2014) or gluten-free bread (Demirkesen MertSumnu amp Sahin 2010)Acorns are fruits from several species of oaktree especially holm oak Quercus rotundifoliaor Q ilex To produce acorn flour fruits tradi-tionally undergo drying or roasting peeling andmilling A further step may be flour debitter-ing ie tannins extraction The nutrition char-acteristics of this naturally gluten-free flour arementioned by Silva et al (2016) half the proteincontent (4-5) ten-times higher fat content (10-14) and around seven-times higher dietary fibrelevel (13-17) compared to wheat flour Past-ing properties of acorn starch differ from thatfrom chestnut - on viscograph curves no peakand trough points were identified during slurryheating and holding at a higher constant tem-perature The viscosity in 40th min of the testwas about approximately one-third lower just foracorn flour (900 vs 1300 Brabender units Cor-reia and Beirao-da-Costa (2010)) In Sardiniaacorn bread was part of local folklore and itspreparation was a ceremony with religious con-notations (Pinna 2013)Korus Witczak Ziobro and Juszczak (2015)found that only the lowest (20) replacementof corn and potato starches in gluten-free breadrecipe led to a rise in the final product vol-ume When 50 wheat flour was replaced byacorn meal composite cookies differed from con-trol in texture appearance and flavour slightlythey were still considered as acceptable (Sabrin2009)Barley (Avena sativa L) is the one of the ba-sic cereals ie plants bred for human nutritionfor the longest time in ancient Egypt emmerwheat and barley served for bread and beer pro-duction (Samuel 1997) In barley flour pro-tein content is usually around 10 forming alower portion than in wheat flour and but thereverse is true for the polysaccharides (close to

80) A substantial part of the biopolymersin the latter form β-glucans and arabinoxylans- Belcredi Ehrenbergerova Belakova and Vac-ulova (2009) analysed 12 different samples anddetermined level of both components to be be-tween 278-608 and 366-546 respectivelyThese polysaccharides may help in preventionof lifestyle diseases - the European Commissionapproved health claims for barley and oat β-glucans (EFSA 2011) Within the Czech Re-public barley flour found its fixed place amongbakery raw materials and together with egchia and nopal flour it extended the range ofbread types produced As reported Gill Vasan-than Ooraikul and Rossnagal (2002) quality ofwheat-barley bread depends also on barley type(regular waxy) For 15 substitution of wheatflour by regular barley one bread loaf volumewas larger and crumb softer than for counter-part containing waxy barley flour Similarly themolar mass of barley β-glucans predeterminesthe resulting bread properties In terms of in-creasing the specific bread volume and reducingthe crumb firmness addition of β-glucans char-acterised by molar masses of 203 x 105 and 100 x105 resulted in preference of the loaves made withthe higher mass β-glucans (Skendi BiliaderisPapageorgiou amp Izydorczyk 2010) These au-thors mentioned that 06 of such β-glucan typeimproved rheological properties of wheat doughas shown by prolongation of both farinographdough development and stability during knead-ing With respect to dough machinability β-glucans supported dough extensigraph elasticityelasticity-to-extensibility ratio increased almosttwice (052 for weak bakery wheat control lsquoDionrsquoand 095 for that composite dough) The rise indough elasticity alleviates the expansion of poresin dough during fermentation and thereby it in-creases bread crumb softness (compressibility)The aim of this work was to evaluate basic ana-lytics viscous and elastic behaviour of wheat orwheat-barley flour composites containing two dif-ferent amounts of chestnut or acorn flour Bak-ing potential of the flour composites preparedwas evaluated directly in a laboratory bakingtrial To distinguish the influence of the non-traditional plant material types and dosage lev-els results were statistically analysed by princi-pal components

Dough amp Bread Quality from Wheat-AcornChestnut Flours 43

21 Wheat and barley flour

Two samples of white wheat flour (WF1 andWF2) used as a bases for bi- and tri-compositeblends were produced by the Czech commer-cial mill Delta Prague in years 2015 and 2016They were characterised by protein contents of112 and 132 Zeleny values of 50 and 39 mLand Falling Number values of 341 and 432 s re-spectively Barley flour (BF) was supplied bythe Czech commercial mill Kresın (productionyear 2015) and was described by protein con-tent Zeleny value and Falling number as 92323 mL and 119 s respectively Total dietary fibre(TDF) ratios were similar (340 318 and 425for WF1 WF2 and BF respectively Table 1) Inthe cereal premixes WF1BF and WF2BF barleyflour replaced 30 of WF1 or WF2 (mixing ratio3070 ww respectively)

22 Non-traditional flour samples

Further non-traditional plant materials namelychestnut (C) and acorn (A) flour were bought inlocal specialised food shops The former was pro-duced by Sonnentor KrauterhandelsgesellschaftmbH (Austria) and the latter was from Bioob-chod Czech Republic (agriculture productionEU mentioned on a label) Declared protein con-tents were 49 and 41 respectively Ratio ofTDF was determined analytically as 1352 and4449 respectively Replacement levels of bothwheat flour and wheat-barley premix were cho-sen as 5 or 10 wt (coding eg WF1+5CWF2+10A WF1BF+5A)

23 Analytical tests

Analytical testing of wheat controls and pre-pared flour composites consisted of the techno-logical parameters Falling number (ISO 3039)and Zeleny sedimentation value (ISO 5529) de-termined in two replications each In correspon-dence to EU legislation (Regulation 11692011)TDF was calculated from data for pure flour sam-ples (wheat barley chestnut and acorn flourAOAC method 99143) Due to method repeata-

bility determined earlier (actual standard devia-tion 022 percent points) it was possible to carryout the measurement with a single repetition

24 Rheological behaviour

Viscous behaviour was determined according toICC method No 1261 using the Brander Amy-lograph (Germany) (80 g of solids 450 mL ofdistilled water) Rheological characteristics ofwheat and composite non-fermented dough wererecorded using the Farinograph and the Exten-sigraph (Brabender Germany ISO 5530-12013and ISO 5530-22013 respectively) Due topreparation of leavened bread whose proofingtakes 50 min only the Extensigraph data after60 min of dough resting was considered

25 Bread baking trial

Bread preparation and assessment of its charac-teristics were carried out as described in previouswork (Svec amp Hruskova 2004) Leavened doughwas prepared using the Farinograph with the ba-sic formula 3000 g wheat flour 120 g yeast 51g salt 45 g sugar 30 g margarine and sufficientdistilled water needed for preparation of doughto a consistency of 600 plusmn 20 Brabender units(consistency based on experience of the ResearchInstitute of Milling-Baking Industry Czech Re-public) Recipe water additions were determinedto be in the range 535-625 on basis of flourweight ie from 1605 mL to 1875 mL In theevaluation of flour composites wheat flour wasreplaced by wheat-chestnutacorn blends wheat-barley premix or wheat-barley-chestnutacornmixture Commercial French-type yeast rdquoFalardquoand the Czech margarine rdquoPerlardquo (fat content40) were used (producers Lesaffre Czech Re-public and Unilever Czech Republic respec-tively) Dough fermentation and leavening took50 and 45 min at constant temperature (30 oCRH 95) 70 g dough pieces were moulded man-ually then placed on a baking plate Baking for14 min was performed in a laboratory oven (Bak-ery Research Institute Poland) preheated to 240oC steamed was created immediately after fullbaking plate insertion by injection 50 mL of dis-tilled water After two-hour cooling under lab-

44 Hruskova et al

Table 1 Barley chestnut and acorn flour influence on polysaccharide pasting properties

Flour flour composite

Analytics Amylograph testTFD Falling Temperature Viscosity

number of maximum maximum() (s) (oC) (BU)

WF1 340 341 de 850 cd 460 cWF2 318 420 f 884 de 940 dBF 425 107 a nd ndWF1BF 372 304 c 790 b 355 cWF2BF 350 168 ab 690 a 215 abWF1+5C 391 323 cd 858 cd 415 cWF1+10C 441 317 cd 880 de 420 cWF2+5A 523 408 f 960 f gt1000 dWF2+10A 729 388 ef 910 ef gt1000 dWF1BF+5C 421 286 c 820 bc 345 bcWF1BF+10C 470 276 c 820 bc 360 cWF2BF+5A 554 178 ab 700 a 190 aWF2BF+10A 758 185 b 720 a 185 a

Repeatability 022 25 020 420Number of groups

nd 6 12 (a - l) 6 12 (a - l) 4 12 (a - l)(of total)Distinguishing rate nd 50 50 33

TDF - total dietary fibre content feature excluded from ANOVA (calculated ie dependent values for flour composites tested) nd - notdeterminedWF - Wheat flour WF1BF WF2BF - wheat-barley flour premix 7030 wt respectively BF A C - barley acorn and chestnut flourrespectivelya-f - means in columns with the same letter were not statistically different (p gt 005)Number of groups - number of homogenous groups statistically differing in their averages identified by different letters (a-f)Total number of groups - number of tested samples (N = 12 corresponding letters a-l)Distinguishing rate () = 100 Number of homogenous groupsTotal number of groups (=12)

oratory conditions the specific volume and theshape (height-to-diameter ratio) was determinedin triplicate To measure the volume of buns thetraditional volume displacement method withrapeseeds was used For crumb firmness (orsoftness) evaluation gravitational penetrometerPNR-10 (Petrotest Instruments Germany) wasemployed This apparatus was equipped witha stainless steel hemisphere (25 mm diameter)locked in a metal screw holder (total weight 150g) Crumb samples were cut out of the centreof the bun halves (height and diameter of crumbcylinders 35 mm and 30 mm) and the penetra-tion depth was determined after a compressiontime of 5 s (five replications for each sample)In eight-point sensory analysis three skilled as-sessors were employed thus results of the test

have informative character only On a hedonicscale summary sensory profile could reach val-ues from 8 to 24 points as the best and the un-acceptable bread respectively (the higher scorethe higher extent of negative changes) Qualityattributes evaluated were bun shape (vaulting)crust colour and shine crust thickness and crisp-ness crumb elasticity and porosity aroma andtaste crumb chewiness and stickiness to palateFor single parameters bread quality was classi-fied into three categories 1 - optimal typical2 - acceptable (soft) change non-typical flat 3- unacceptable strange (negative unfavourablechange) Within the evaluation values 15 or 25points were also allowed This part of researchfollowed the tenets of the Declaration of Helsinkipromulgated in 1964 and was approved by the

Experimentation Committee of the UCT Praguewhich issued informed consent

The scatter of the data was first described byANOVA (Tukey HSD test p lt 005) using Sta-tistica 130 software (StatSoft Inc USA) Sec-ondly correlation analysis was calculated be-tween flour features (analytical parameters rheo-logical tests) and bread quality attributes Multi-variate analysis by principal components (PCA)was conducted for 14 representative parame-ters selected on basis of the correlation ma-trix as well as the PCA results of the com-plete dataset For ANOVA and PCA the fac-tors considered were composite flour base (wheatwheat-barley wheat-barley-chestnutacorn) andenhancement level of non-traditional plant mate-rial (0 510 chestnutacorn BF30 BF30+510chestnutacorn)

31 Effects of non-traditionalplant materials on propertiesof wheat polysaccharides

Analytical tests

According to the TDF contents in the non-traditional materials tested the amount of di-etary fibre increased in composite flours upto twice that in wheat flour alone mainly byaddition of acorn flour (from 318 for WF2to 729 and 758 samples WF2+10A andWF2BF+10A Table 1) When both WF stan-dards were compared there was a clear differenceshown in the Falling number test it reflecteddiverse weather conditions during two consecu-tive harvest years For the barley flour samplethe value reached about half that level signify-ing higher amylase activity andor a higher rateof damaged starch Mechanically physically orenzymatically damaged starch is more accessibleto amylases which contributes to lowering of thevalue of the Falling number Between WF1BFand WF2BF premixes a significant drop was ob-served in the latter case (Falling numbers 304 and

168 s) compared to values of 420 and 341 s forWF1 and WF2 respectively (Table 1)

Pasting behaviour

For WF controls and the 10 flour compositesdifferences in amylograph viscosity maxima cor-responded with level of Falling number (r = 092p lt 0001 data not shown) Besides amylo-graph maximum viscosity partial differentiationof the flour composites was obtained at the tem-perature corresponding to this maximum Ta-ble 1 shows that there was a lesser influence ofchestnut flour and strong one of acorn and bar-ley flour A negative influence of chestnut flourconfirmed results by Hegazy et al (2014) usingthe RVA test - the viscosity for wheat-chestnutflour 9010 (ww) was clearly lower than forwheat control (2036 vs 2651 mPas decrease ca23) Conversely as expected higher TDF con-tent due to additions of acorn flour caused an in-crease of the maximal viscosity that exceeded theamylograph technical limit of 1000 Brabenderunits (BU) In combination with BF high amy-lase activity in the alternative cereal flour sup-pressed the thickening effect of the acorn flourFor wheat-barley-chestnut composites it seemedthat chestnut flour partially buffered the effect ofBF Contrary to our results direct comparisonof pasting of the chestnut and the acorn starchesconducted Yoo Lee Kim and Shin (2012) by us-ing of the RVA proof showed lower peak viscosityfor the acorn starch than chestnut starch (4874mPas against 5640 mPas respectively) Foracorn starch these authors determined that thiswas caused by the smaller size of starch granulesslightly higher total amylose content as well as aweaker resistance to swelling (features swellingpower and solubility at 80 oC)

32 Effect of non-traditional plantmaterials on properties ofwheat proteins

Analytical tests

Baking quality of proteins was satisfactory forboth wheat controls - the demanded minimum inpraxis is 35 mL Additions of all non-traditional

46 Hruskova et al

materials induced predictable decrease of sedi-ment volumes during the Zeleny test - the great-est drop was observed for 5 or 10 of chestnutflour The combination of barley and especiallyacorn flour reduced the technological quality ofcomposite flour by approximately one half (Table2) Reason for this difference may have lain inthe different granulation of the alternative ma-terials - chestnut flour appearance was obviouslysmoother (like a wheat flour) compared to theroughly milled acorn one On the contrary BFwas produced in industrial mill and its granula-tion was comparable to wheat flour

Viscous and elastic behaviour

Two principal properties of wheat proteins areelasticity and extensibility expressed as theirratio For bakery products the extensigraphelasticity should be 2 or 25-times higher thanthe extensibility optima of the parameters liein ranges 450 - 600 BU and 140 - 170 mmrespectively In addition extensigraph energytaken as area under the curve gives a complexoverview of flour technological quality (Prıhodaamp Hruskova 2017) its empirical minimum is 100cm2 (Kovarıkova amp Netolicka 2011) With re-spect to the present work both wheat flour con-trols WF1 and WF2 fulfilled the demand for goodtechnological quality (the ratios 203 and 281the energies 139 and 115 cm2 respectively Ta-ble 2) In our results baking quality of WF1could be considered as better than WF2Both chestnut and acorn flour added alone(WF1+C WF2+A) noticeably increased the en-ergy level The shape of the curves ie doughmachinability were affected by enhancementlevel - the elasticity-to-extensibility ratios roseby about twice and three-times respectively (Ta-ble 2) It could be supposed that non-glutenchestnut and acorn proteins (albumins) first sup-ported the dough extensibility and secondly theelasticity too In combination with barley flourthe influence of these alternative plant materi-als was obvious - the BF proteins released in thenon-fermented dough caused it to be more elas-tic and with lower tensile strength Changes inthe shape of the recorded curves were reflectedin extensigraph energy - flour composites basedon the stronger WF1 were negatively influenced

by a larger extent For the wheat-barley-acorncounterparts acorn fibre supported dough elas-ticity ie it showed a certain potential to cor-rect such quality loss On the other hand asthe trend registered in the plot attests there wasa stepwise decline in dough handling properties- the higher the wheat flour replacement ratethe greater the increase in extensigraph ratio (upto 145 and 111 for samples WF1B+10C andWF2B+10A respectively) Gonzaga BatistaGuine and Correia (2015) tested wheat-acornblends mixed in ratios of 9010 and 8515 (ww)comparing flour from two Spanish wheat varietiesCerealis and Ceres Acorn flour was prepared ona laboratory scale with grain size of less than 10mm As we found these authors confirmed theclear-cut rise in the extensigraph ratio accordingto acorn flour ratio and dough resting time

33 Effect of non-traditional plantmaterials on wheat doughduring kneading

For both wheat dough controls behaviour dur-ing kneading was closer than indicated the Ze-leny test - the WF2 sample had a little shorterdough development time but about 50 longerdough stability Water absorption values of 677and 651 as well as dough softening degrees of50 and 40 BU could be considered as compa-rable and satisfactory for enhancement by non-traditional plant materials With increasing por-tion of chestnut flour water absorption of thecomposites tended to decrease while dough de-velopment was somewhat slowed (time was pro-longed) and degree of dough softening slightlyincreased (Table 3) Hegazy et al (2014) ar-rived at different results owing to differences intechnological quality of WF1WF2 and Egyp-tian wheat flour - 10 replacement by chest-nut flour improved dough rheological properties(eg water absorption had been increased from565 to 60 and stability was shortened from90 min to 75 min) The authors characterisedtheir base material by protein content about 2percent points lower (980) and with approxi-mately half dough development time (15 min) -the Egyptian sample had weaker baking qualitythan both our controls

Table 2 Barley chestnut and acorn flour influence on technological quality of proteins and extensigraphparameters of wheat flour

Analytics Extensigraph testZeleny value Ratio Energy

(mL) (1) (cm2)

WF1 50 g 203 a 1395 cdWF2 38 ef 281 b 1153 bcBF 22 a nd ndWF1BF 31 cd 650 e 1137 bcWF2BF 27 abc 478 d 648 aWF1+5C 40 f 396 c 1481 cdWF1+10C 39 f 528 d 1462 cdWF2+5A 34 de 388 c 1359 cdWF2+10A 31 cd 642 e 1562 dWF1BF+5C 29 bc 1190 g 872 abWF1BF+10C 26 ab 1449 h 864 abWF2BF+5A 25 ab 689 e 898 abWF2BF+10A 23 a 1109 f 770 ab

Repeatability 1 013 8Number of groups

7 12 (a - l) 8 12 (a - l) 4 12 (a - l)(of total)Distinguishing rate 58 67 33

- extensigraph parameters determined after 60 min of dough restingWF - Wheat flour WF1BF WF2BF - wheat-barley flour premix 7030 wt respectively BF A C - barley acorn and chestnut flourrespectivelya-h - means in columns the same letter were not statistically different (p gt 005)Number of groups - number of homogenous groups statistically differing in their averages identified by different letters (a-h)Total number of groups - number of tested samples (N = 12 corresponding letters a-l)Distinguishing rate () = 100 Number of homogenous groupsTotal number of groups (=12)nd - not determined

The farinograph behaviour of the wheat-acornflour demonstrated a similar course - wateramounts rose by about 2 percent points onlydough development time was increased four-times and dough stability fell from 110 min to900 min (Table 3) Farinograph test results forwheat-acorn composites differs according wheatflour base (Gonzaga et al 2015) - in the caseof variety Cerealis 10 acorn flour lowers wa-ter absorption (600 vs 557) and but in-creases it in the case of the Ceres variety (575vs 593) They also showed that in termsof dough development time there is a shorten-ing from 40 to 20 min in the former and nosignificant change in the latter Comparing ourown amylograph and farinograph results acornpolysaccharides are likely to have a better ab-

sorption capacity at lower temperatures thanthey are when heated This premise confirmedresults by Yoo et al (2012) that during the coldphase (beginning) of the RVA test acorn starchdemonstrates a higher viscosity than chestnutstarch (5594 mPas against 4483 mPas respec-tively) Yoo et al (2012) proposed that the rea-sons for this behaviour are to be found in thedifferent lengths of amylose chains and in con-tent of so-called intermediate materials (slightlybranched amylopectin with molar mass betweenamylopectin and amylose) As far as is knownboth starch components influence the propertiesof water suspension in opposite ways - the amy-lopectin supports starch swelling while amyloselowers the viscosity

48 Hruskova et al

Table 3 Barley chestnut and acorn flour influence on farinograph behaviour of wheat flour

Flour flour compositeWater absorption Dough development Dough stability MTI

() (min) (min) (BU)

WF1 677 cd 375 ab 750 b 50 abWF2 651 a 275 a 1100 d 40 aWF1BF 700 e 1000 f 1700 e 30 aWF2BF 680 d 450 b 350 a 120 eWF2+5A 661 ab 850 e 450 a 50 abWF2+10A 675 cd 1050 f 900 c 70 bcWF1BF+5C 682 d 400 b 400 a 100 deWF1BF+10C 675 cd 400 b 400 a 100 deWF1+5C 666 bc 800 de 900 c 50 abWF1+10C 655 ab 650 c 750 b 70 bcWF2BF+5A 683 d 700 cd 800 bc 90 cdWF2BF+10A 685 d 825 e 1100 d 50 ab

Repeatability 02 4 020 020

Number of groups5 12 (a - l) 6 12 (a - l) 5 12 (a - l) 5 12 (a - l)(of total)

Distinguishing rate 42 50 42 42

MTI - mixing tolerance index (dough softening degree) BU - Brabender unitWF - Wheat flour WF1BF WF2BF - wheat-barley flour premix 7030 wt respectivelyBF A C - barley acorn and chestnut flour respectivelya-f - means in columns with the same letter are not statistically different (p gt 005)Number of groups - number of homogenous groups statistically differing in their averages identified by different letters (a-f)Total number of groups - number of tested samples (N = 12 corresponding letters a-l)Distinguishing rate () = 100 Number of homogenous groupsTotal number of groups (=12)

34 Effect of non-traditional plantmaterials on baking testresults

During leavened dough preparation recipe wa-ter additions were evaluated in the ranges 580- 625 and 535 - 595 within the chestnutand acorn sample subgroups respectively (Ta-ble 4) For flour composites based on WF1 theinitial water amount of 625 decreased as thechestnut content increased (similarly for sampleswith the WF1BF base) reflecting an increasingportion of sucrose in dough (Moreira ChenloTorres amp Prieto 2010) Conversely KucerovaSottnikova and Nedomova (2013) found increas-ing recipe water amount during wheat breadpreparation that had been enhanced by 1 and3 of bamboo fibre Bamboo fibre consists ap-proximately from 97 cellulose characterised byhigher swelling capacity Within the subgroupwith bases of WF2 and WF2BF a decreasingtrend in water amount was observed This was

the reverse pattern compared to the farinographwater absorption In spite of a significant riseof TDF content (Table 1) bread formula com-ponents such as salt sugar and fat may have re-stricted the incorporation of water molecules intothe coating of protein chains and so lowered to-tal amounts of water that had to be addedThe quality of the bread samples was differenti-ated according to absence or presence of BF indough recipe (Figure 1 triplicates of WF1 sam-ples numbered 1-3 WF1BF ones 7-9 WF2 sam-ples 4-6 WF2BF ones 10-12) Although chest-nut and acorn flour were added in small por-tions both materials decreased specific breadvolume (Demirkesen et al 2010) as well as mod-ifying bread shape Chestnut flour contains fi-bre characterised by rigid structures (Demirke-sen et al 2010) which limits the expansion ofpores in the dough mass during fermentation(Collar Santos amp Rosell 2007) DallrsquoAsta etal (2013) even tested 50 replacement of softwheat flour by chestnut flour such product had

Figure 1 Barley acorn and chestnut flour influence on wheat bread properties Samples coding 1 ndashWF1 2 ndash WF1+5C 3 ndash WF1+10C 4 ndash WF1BF 5 ndash WF1BF+5C 6 ndash WF1BF+10C (numbers 7-12similarly within WF2-acorn subset) Variable coding hd ndash height-to-diameter ratio of bread bun Datavariance analysis bread shape lsquoa-brsquo (in red) specific bread volume lsquoa-frsquo (in blue) crumb penetrationlsquoa-frsquo (in green) ndash values signed by the same letter are not statistically different (p lt 005)

a clearly lower specific volume (13plusmn02 cm3gvs 28plusmn04 cm3g for wheat control) homoge-nous porosity with correspondingly higher ratioof pores smaller than 0049 mm2 We also foundthat there was a strong relationship between spe-cific bread volume and crumb penetration (r =093 p lt 001 Table 5) with somewhat hardercrumb for bread with acorn flour (Table 4)

35 Sensory analysis of bread

With the sensory profiling a value of 8 pointsmeant the lowest score ie optimal bread qual-ity in terms of all 8 quality attributes Replac-ing 30 of WF gave a somewhat moister butsticker mouthfeel perhaps due to the presence ofβ-glucans (+05 point) and non-typical flavour

(+05 point) - the bread score reached 9 pointsFor composite bread variants the most affectedparameters were taste and aroma chewiness andstickiness For the most enriched bread thescores rose 145 points (Figure 2) where the max-imum 24 points represented unacceptable breadso this bread was of worse sensory quality byabout 41Taste of chestnut flour itself is fruity and sweetdue to the higher content of simple sugars(DallrsquoAsta et al 2013) Within WF+chestnutsamples free sugars contributed to darkening ofthe surface of the final products (Man PuaceanMuste Muresan amp Francu 2012) further wors-ening was noticed with the development of anelastic non-crispy crust and denser sticky crumbThe more compact crumb perhaps limited water

50 Hruskova et al

Table 4 Baking test results as affected by barley chestnut and acorn flour additions

Flour flour compositeRecipe water Crumb penetration Bread sensory scoreaddition () (mm) (points)

WF1 625 i 208 f 80 aWF2 610 h 168 e 85 aWF1BF 605 gh 103 d 90 abWF2BF 620 i 66 ab 100 bWF1+5C 600 fg 48 ab 95 abWF1+10C 580 c 51 ab 100 bWF2+5A 590 de 143 e 80 aWF2+10A 595 ef 159 e 80 aWF1BF+5C 585 cd 96 cd 105 bWF1BF+10C 584 c 72 bc 90 abWF2BF+5A 560 b 52 ab 105 bWF2BF+10A 535 a 40 a 145 c

Repeatability 01 09 05

Number of groups9 12 (a - l) 6 12 (a - l) 3 12 (a - l)(of total)

Distinguishing rate 75 50 25

WF - Wheat flour WF1BF WF2BF - wheat-barley flour premix 7030 wt respectivelyBF A C - barley acorn and chestnut flour respectivelya-i - means in columns with the same letter are not statistically different (p gt 005)Number of groups - number of homogenous groups statistically differing in their averages identified by different letters (a-i)Total number of groups - number of tested samples (N = 12 corresponding letters a-l)Distinguishing rate () = 100 Number of homogenous groupsTotal number of groups (=12)

evaporation and together with the higher activ-ity of the amylases (lower Falling number) ledto a higher occurrence of sticky dextrins than inwheat control A partial improvement of overallperception brought about by the addition of thechestnut flour when added at over 10 was dueto the increase in sweet taste In combinationwith BF these characteristics were maintainedthough mouthfeel stickiness vanished but typi-cal barley flavour was detected anew Man et al(2012) summarised that 10 of that alternativeplant raw material led to the best quality prod-uct in terms of taste odour and colour Theseauthors found significant and positive changes inbread crumb elasticity although bread porositywas influenced negativelyIn terms of sensory acceptability bread at-tributes got worse for acorn flour additions over10 - taste and aroma became woody with a par-tial acid aftertaste For WFBF-acorn samples

woody flavour was identified for the sample with10 acorn flour in the recipe During mastica-tion acorn flour seemed to be milled roughly somouthfeel sensation was also ldquosandyrdquo with somestickiness One positive effect of acorn flour wasthe semi-darkening effect on the bread mainlyfor recipes containing 10 or more

36 Correlation analysis

The reduced correlation matrix confirmed thatbread quality was predetermined by polysaccha-rides and proteins properties (Table 5) In termsof the analytical and rheological tests the resultsof the farinograph proof were less influenced bydough recipe modification - only water absorp-tion influenced specific bread volume negatively(r = -062 p lt 005 data not shown)For starch and its constituents correlation anal-ysis verified a similar relationship between the

develo

taddit

(degre

record

52 Hruskova et al

Figure 2 Barley acorn and chestnut flour influence on sensory profile of wheat bread

Figure 3 Principal components (PC) biplot of barley chestnut and acorn flour influence on wheat flourdough and bread properties and quality Samples coding WF1 WF2 ndash wheat flour (controls) WF1BFWF2BF ndash wheat-barley flour premixes 7030 wt WF1+5C WF2+10A ndash flour composites containing5 or 10 of chestnut and acorn respectively Variables coding TDF ndash total dietary fibre contentFN ndash Falling number Zeleny ndash Zeleny sedimentation value WAF ndash farinograph water absorption MTIndash mixing tolerance index (dough softening degree) ERA 60rsquo EEN 60rsquo ndash extensigraph ratio and energyafter 60 min of dough resting AMA Tmax ndash amylograph maximum and proper temperature RWA ndashrecipe water addition SBV ndash specific bread volume BRS ndash bread shape (height-to-diameter ratio) PENndash crumb penetration SEN ndash sensory analysis

54 Hruskova et al

screening Falling number method the detailedamylograph pasting test and specific bread vol-ume and crumb penetration (Pearsonrsquos r be-tween 063 and 078) In case of dietary fibrepolysaccharides it seems they played more a im-portant role at lower temperatures during doughmixing (in water absorption) and in bread sen-sory evaluation (r = -073 and 076 respectivelyp lt 005)Estimation of baking quality of protein (Zelenyvalue) in flour composites played a significantrole in the prediction of bread quality with ex-ception of bread shape it was correlated withfour further bread attributes (mostly with cal-culated p lt 001) Likewise for extensigraphelasticity-to-extensibility ratio and energy it wasdemonstrated that their principal effect was onbun size development (eg correlations to spe-cific bread volume r = 082 -083 and 081 re-spectively p lt 001 Table 5)Finally specific bread volume could also be es-timated on the basis of recipe water addition aswell as backwards on the basis of crumb penetra-tion (r = 052 r = 093 p lt 001 respectively)It supported the premise that bread volume isbuilt on 3D crumb structure (2D porosity) andits physico-mechanical propertiesIn summary the tighter the relationships be-tween the recorded features the higher the ex-tent of their explanation by multivariate statis-tical exploration

37 Principal component analysis

In a biplot of loadings and scores the first twoprincipal components (PC) explained 74 ofdata ie 55 was accounted for PC1 and 19for PC2 (Figure 3) The third PC explained 20of variance of the features TDF extensigraph ra-tio recipe water addition and bread shape Inaddition PC4 explained both farinograph fea-tures ie for the water absorption and themixing tolerance index (38 of trait variabilitydata not shown) Fourteen representative vari-ables were split into four main groups demon-strating relationships to bread quality Specificbread volume and crumb penetration were posi-tively influenced by protein quality (Zeleny valueextensigraph energy recipe water addition) and

negatively on polysaccharide pasting behaviour(Falling number and both amylograph features)It could be presumed that the increasing rate ofTDF had an impact on dough rheological prop-erties (water absorption mixing tolerance indexand extensigraph ratio) and bread characteristicsincluding sensory profileAs mentioned above the absence or presence ofBF in dough or bread recipe was crucial to dis-tinguish between the flour composites Along thePC1 axis barley flour lowered baking potential ofbi-composite mixtures The PC2 could be associ-ated with addition level of non-traditional plantmaterials In this regard contradictory effectswere identified for 10 of chestnut and 5 ofacorn flour - the former had a worsening effectbut the latter an improving effect (ie specificbread volumes 253 and 347 mL100 g respec-tively)In summary wheat flour composites with 10of chestnut flour and wheat-barley ones with 5of chestnut or acorn flour could be recommendedfor bakery use

4 Conclusion

Enhancement of wheat flour by barley chest-nut and acorn flours contributed to a dietary fi-bre increase of twice the control level In flourcomposites these non-traditional materials alsointroduced non-gluten proteins Such composi-tion changes influenced the technological qualityof both polysaccharides and proteins - values ofFalling number and Zeleny test were lowered sig-nificantly The effects of chestnut and flour weredifferent reflecting a wide granulation of the ma-terials Pasting properties of flour compositeswere recorded during the amylograph test andsamples could be differentiated according to thetemperature of the viscosity maximum With ex-ception of wheat-acorn samples containing 5 or10 of the acorn flour viscosity maxima were de-termined to be about 10-25 lower in compari-son to wheat controls In this regard effect ofbarley flour was stronger than that of chestnutor acorn flourRecipe modification also led to changes in doughphysico-mechanical properties - non-gluten ma-terials had more influenceon elasticity than ex-

tensibility of non-fermented dough Extensi-graph elasticity-to-extensibility ratio rose to ap-proximately double for wheat-chestnut wheat-acorn and wheat-barley mixtures For tri-composite blends this increase reached a stillhigher level Modified polysaccharide and pro-tein properties also significantly influenced rheo-logical behaviour during dough kneading Waterabsorption was lessened by additions of chest-nut flour corresponding to the higher portion ofsucrose in the flour Conversely acorn and bar-ley fibre gradually increased the amount of wa-ter necessary to reach the required dough con-sistency by up to 3 percent points Dilution ofthe gluten matrix in the dough led to prolon-gation of dough development time and to par-tial loss of dough resistance to overmixing Suchdough modification caused lower specific breadvolumes and variation in the shape of the bunsin general the higher the enhancement level thelower the bread volume Lower bread sizes alsomeant denser crumb as shown by firmness mea-surement with the penetrometer Sensory pro-files of bread variants differed primarily on basisof the alternative material added The wheat-barley and wheat-acorn samples were consideredto be less acceptable due to characteristic bar-ley aroma and taste and bitter taste plus acidaftertaste of acorn flour Wheat-chestnut coun-terparts had a light sweet taste but a partialstickiness was identified during mastication To-tal sensory score of wheat-barley-acorn breadwas clearly worse than the wheat-barley-chestnutone but in both cases scores could be cate-gorised as ldquoacceptable with some reservationrdquoAs a compromise between fibre content and finalproperties of a bakery product wheat flour com-posites with 10 of chestnut flour and wheat-barley ones with 5 of chestnut or acorn flourcould be recommended for practical usage

Acknowledgements

The study was conducted under the grant NoQI111 B053 of the National Agency of Agricul-tural Research (NAZV) Czech Republic

References

Belcredi N B Ehrenbergerova J BelakovaS amp Vaculova K (2009) Barley grainas a source of health-beneficial substancesCzech Journal of Food Sciences 27 S242ndashS244 6th Chemical Reactions in Food Con-ferene 2009 Prague CZECH REPUBLICMAY 13-15 2009

Collar C Santos E amp Rosell C M (2007) As-sessment of the rheological profile of fibre-enriched bread doughs by response surfacemethodology Journal of Food Engineering78 (3) 820ndash826 doi10 1016 j jfoodeng 200511026

Correia P R amp Beirao-da-Costa M L(2010) Chestnut and acorn starch proper-ties affected by isolation methods Starch-starke 62 (8) 421ndash428 doi10 1002star 201000003

CREA (2015a) Il Centro di Ricerca Alimenti eNutrizione Tabelle di composizione deglialimenti - 3 - Farina di frumento tipo00 Retrieved from http nut entecra it646tabelle di composizione20 deglialimentihtmLidalimento=0002205Campquant=100

CREA (2015b) Il Centro di Ricerca Alimentie Nutrizione Tabelle di composizione deglialimenti - 11 - Farina di castagne Retrievedfrom httpnut entecra it646tabelledi composizione degli 20alimentihtmL20idalimento=5040105Campquant=100

DallrsquoAsta C Cirlini M Morini E RinaldiM Ganino T amp Chiavaro E (2013) Ef-fect of chestnut flour supplementation onphysico-chemical properties and volatiles inbread making Lwt-food Science and Tech-nology 53 (1) 233ndash239 doi101016jlwt201302025

Demirkesen I Mert B Sumnu G amp SahinS (2010) Utilization of chestnut flour ingluten-free bread formulations Journal ofFood Engineering 101 (3) 329ndash336 doi101016jjfoodeng201007017

EFSA (2011) Panel on dietetic products nu-trition and allergies - scientific opinionon the substantiation of health claims re-lated to beta-glucans from oats and bar-ley and maintenance of normal blood ldl-

56 Hruskova et al

cholesterol concentrations (id 1236 1299)increase in satiety leading to a reductionin energy intake (id 851 852) reduction ofpost-prandial glycaemic responses (id 821824) and ldquodigestive functionrdquo (id 850) pur-suant to article 13(1) of regulation (ec) no19242006 EFSA Journal 9 (6) doi10 2903jefsa20112207

Gill S Vasanthan T Ooraikul B amp Rossna-gal B (2002) Wheat bread quality as in-fluenced by the substitution of waxy andregular barley flours in their native andcooked forms Journal of Cereal Science36 (2) 239ndash251 doi101006jcrs20020459

Giovannelli C (2009) La castagna Dal boscoalla tavola tradizione ed innovazione thechestnut From wood to tabel traditionand innovation (Masterrsquos thesis Univer-sita degli studi di Padova Italy) Retrievedfrom httptesi cabunipdit218291TESI CASATAGNApdf

Gonzaga M Batista M Guine R amp Cor-reia P (2015) Development and char-acterization of wheat breads with acornflour In ICEUBI2015-international confer-ence of engineering Engineering for soci-ety

Hegazy N A Kamil M M Hussein A M Samp Bareh G F (2014) Chemical andtechnological properties of improved bis-cuit by chestnut flour International Jour-nal of Food Nutritional Science 3 (6) 7ndash15 Retrieved from http www ijfans com Volume 203 20Issue 206 2 20IJFANS20A0343-14pdf

Korus J Witczak M Ziobro R amp JuszczakL (2015) The influence of acorn flour onrheological properties of gluten-free doughand physical characteristics of the breadEuropean Food Research and Technology240 (6) 1135ndash1143 doi10 1007 s00217 -015-2417-y

Kovarıkova D amp Netolicka V (2011)Vzdelavacı material pro predmet Techno-logicka prıprava (Quality of cereals forprocessing in mill Educational materialfor subject Technological training) (CStrednı prumyslova skola potravinarskaPardubice Pardubice Ed) Retrieved from

httpswwwspspasczesf-technologicka-priprava

Kucerova J Sottnikova V amp Nedomova S(2013) Influence of dietary fibre additionon the rheological and sensory properties ofdough and bakery products Czech Journalof Food Sciences 31 (4) 340ndash346

Man S Puacean A Muste S Muresan Camp Francu A-V (2012) Chestnut flour ad-dition influence on bread quality Journalof Agroalimentary Processes and Technolo-gies 18 (2) 150ndash154

Moreira R Chenlo F Torres M D amp PrietoD M (2010) Influence of the particle sizeon the rheological behaviour of chestnutflour doughs Journal of Food Engineering100 (2) 270ndash277 doi101016j jfoodeng201004009

Pinna C (2013) Acorn bread A traditionalfood of the past in sardinia (italy) Jour-nal of Cultural Heritage 14 (3 Supple-ment) S71ndashS74 Science and Technologyfor the Safeguard of Cultural Heritage inthe Mediterranean Basin doi10 1016 j culher201211012

Prıhoda J amp Hruskova M (2017) Hodnocenıkvality Aplikace doporucenych prıstrojumetod a interpretace vysledku pro praxi(Quality evaluation Application of rec-ommended apparatuses methods and re-sults interpretation for praxis) In P Sprumyslovych mLynu (Ed) (pp 104ndash108)

Sabrin M (2009) Characterization of acornmeal (Masterrsquos thesis Johnson amp WalesUniversity USA) Retrieved from httpsgetdlibsugaedupdfssabrin michael d200908 mspdf

Samuel D (1997) Cereal foods and nutrition inancient egypt Nutrition 13 (6) 579ndash580doi101016S0899-9007(97)00027-0

Silva S Costa E M Borges A Car-valho A P Monteiro M J amp PintadoM M E (2016) Nutritional characteriza-tion of acorn flour (a traditional componentof the mediterranean gastronomical folk-lore) Journal of Food Measurement andCharacterization 10 (3) 584ndash588 doi10 1007s11694-016-9340-1

Skendi A Biliaderis C G Papageorgiou Mamp Izydorczyk M S (2010) Effects of two

barley beta-glucan isolates on wheat flourdough and bread properties Food Chem-istry 119 (3) 1159ndash1167 doi10 1016 j foodchem200908030

Svec I amp Hruskova M (2004) Image dataof crumb structure of bread from flour ofczech spring wheat cultivars Czech Jour-nal of Food Sciences 22 (4) 133ndash142

Yoo S-H Lee C-S Kim B-S amp ShinM (2012) The properties and molecularstructures of gusiljatbam starch comparedto those of acorn and chestnut starchesStarch 64 (5) 339ndash347 doi101002star201100104

Stability of Vitamin C in Broccoli at Different StorageConditions

Nasser Al-Habsia Sithara Suresha Amani Al-Yhmedia Marwa Al-ShoryaniaMostafa I Walya and Mohammad Shafiur Rahmana

a Department of Food Science and Nutrition College of Agricultural and Marine Sciences Sultan QaboosUniversity P O Box 34-123 Al-Khod Oman

Corresponding authorsitharasuresh88gmailcomTel +968-2414-1273

Received 5 May 2018 Published online 18 April 2019

Abstract

In this study the retention of vitamin C in fresh broccoli stored at different temperatures (ie chillerroom cooking and roasting or baking 5-120oC) was investigated The thermal stability of vitamin Cin broccoli was analysed at 5 20 45 60 70 80 110 and 120oC The vitamin C content was measuredby the indophenol titration method Vitamin C was affected negatively at all stored temperatures Thedegradation of vitamin C was modelled by first-order reaction kinetics and the reaction rate constantswere observed as 903Ouml10minus8 and 565Ouml10minus3 sminus1 when stored at 5oC and 120oC respectively Theactivation energy was estimated as 742 kJmol within the temperature range used in this study Thelowest decay of vitamin C was observed during the chilling condition The data on retention of vitaminC in broccoli could be used to determine their stability when stored as raw and when heated atdifferent temperatures

Keywords Ascorbic acid First-order reaction Cooking Chilling Activation energy

1 Introduction

Vitamins serve as an essential component inmetabolism and could be used to protect thehuman body against diseases such as cancercataracts and cardiovascular diseases It isshown from epidemiological studies that a highintake of vegetables and fruits is correlated witha low risk of diseases due to their antioxidantshealth functional components and vitamins suchas ascorbic acid (vitamin C) carotenoids and to-copherols (Bergquist Gertsson amp Olsson 2006Wootton-Beard amp Ryan 2011) Broccoli hasgained considerable attention due to its health-promoting ability which has been attributedto bioactive phytochemicals such as nitrogen-sulfur compounds (glucosinolates and isothio-

cyanates) phenolic compounds (chlorogenic andsinapic acid derivates and flavonoids) and vi-tamins (Dominguez-Perles Carmen Martinez-Ballesta Carvajal Garcia-Viguera amp Moreno2010 Suresh Al-Habsi Guizani amp Rahman2017)Among the vitamins vitamin C is one of themost important for maintaining human health(Hamad 2009 Kumar Ajay Kumar Raghu ampManjappa 2013) Fruits and vegetables con-tain a high amount of ascorbic acid VitaminC in the natural forms of L-ascorbic acid (L-AA) and L-dehydroascorbic acid (L-DA) is foundin foods (Ismail 2013) It is a highly unsta-ble molecule and could decrease during domes-tic and industrial processing through enzymaticand non-enzymatic reactions (Munyaka Makule

Stability of Vitamin C in Broccoli 59

Oey Van Loey amp Hendrickx 2010) There-fore processing treatments like peeling bruis-ing and cutting fruits or vegetables into piecesand air exposure of the disrupted cells causeoxidation of L-AA in the presence of ascorbicacid oxidase (AAO) and this decreases the re-tention of ascorbic acid (Munyaka et al 2010)The degradation can be triggered by many fac-tors such as moisture temperature light pHmetal ions and oxygen (Munyaka et al 2010Spinola Mendes Camara amp Castilho 2012)Enzymatic degradation involves oxidation of L-AA to dehydroascorbic acid (L-DA) and loss ofits antiscorbutic activity (ie ability to pre-vent scurvy) The antiscorbutic activity can belost rapidly and irreversibly by hydrolysis of L-DA to 23-diketogulonoic acid (23-DKG) (Jainamp Mulay 2014) The oxidation is catalyzed bythe enzymes AAO and ascorbic acid peroxidase(AAP) (Venkatesh amp Park 2014)The stability of vitamin C depends on manyfactors such as the types of fruits and vegeta-bles growing conditions level of maturity stor-age and processing conditions (Masamba amp Mn-dalira 2013) For instance about 8-25 of ascor-bic acid is lost when apples are peeled Thebest retention of vitamin C is possible with freshor minimal processing compared with thermaland drying processing methods (Oyetade 2012Spinola Mendes Camara amp Castilho 2013)The ripening of fruits causes gradual loss of vi-tamin however this can be slowed down by re-frigeration (Oyetade 2012) Phillips Council-Troche McGinty Rasor and Teresa Tarrago-Trani (2016) studied the stability of vitamin Cin fruit and vegetable homogenates stored at dif-ferent temperatures (10 -20 and -55oC) and ob-served that the degradation depended on thestorage temperatures as well as the types offruits and vegetables Maximum losses were ob-served as 23 and 94 after 1 and 7 days ofstorage respectively The vitamin C stabilityin three types of juices (pineapple guava andbaobab) was influenced by storage temperaturestorage time and types of preservatives used Atroom temperature vitamin C in pineapple juice(347 mg100 ml) decreased to 899 after 2months of storageNath Bagchi Misra and Deka (2011) studiedthe weight loss ascorbic acid chlorophyll β-

carotene and total antioxidant activity decayin fresh broccoli stored at 15oC and 4oC Theascorbic acid (ie 130 mg100 g FW) decay ofthe stored samples at 15oC and 4oC (open at-mosphere) were 929 and 292 respectivelyafter 6 days Favell (1998) studied the stabil-ity of vitamin C in peas (three varieties) greenbeans broccoli and spinach at ambient chilland frozen conditions They observed that thedecrease of ascorbic acid in peas after post-harvest varied from 26 to 31 mg100 g respec-tively depending on the variety The samplesstored at 4oC (chilled) showed little change inthe first 3 days but reduced steadily at 2-3per day On the other hand the loss was muchfaster for the peas stored at ambient tempera-ture (20oC) and the loss was about 10 per dayover the first 7 days The ascorbic acid contentof freshly harvested broccoli was reported withinthe 770-930 mg100 g sample A steady losswas observed for broccoli stored at ambient stor-age with only 44 ascorbic acid retained after 7days of storage and 28 after 14 days Howeverat chilled temperature the retention was muchbetter with no loss after 7 days of storage and80 retention even after 21 days Most of thestudies determined percent losses during a prede-termined storage time There are relatively fewreports available in the literature on the degrada-tion kinetics of vitamin C in fruits and vegetables(Ariahu Abashi amp Chinma 2011) Yet the reac-tion order rate constant and activation energyare essential for predicting food quality loss dur-ing storage as well as thermal processing (NishaSinghal amp Pandit 2005)There are negligible studies in the literature onthe loss of vitamin C in broccoli during storageand processing over a wide temperature rangeThe objective of this study was to determine thestability of vitamin C at different temperatures(ie 5-120oC) The experiments were performedin isothermal conditions and the selected temper-atures could be used to simulate the conditions ofchilling and room temperature storage cookingand roasting or baking conditions The reactionrate was modelled by first-order reaction kinet-ics

60 Al-Habsi et al

21 Sample Collection andPreparation

Fresh broccoli (Brassica oleracea variety Cal-abrese) grown in Oman were purchased from alocal supermarket The vegetables were washedin tap water to remove the dirt adhering to themand was spread on tissue paper to absorb the ex-cess surface water Only florets were cut from thebunch and eight batches of 200 g samples wereplaced into different aluminium cells and storedat temperatures of 5 20 45 60 70 80 110 and120oC Previous reports showed that dependingon the type of vegetables and fruits and effects oftemperature (low and high) more or less heat-ing time would be needed to measure the vita-min C content (Hal Bosschaart Twisk Verkerkamp Dekker 2012 El-Ishaq amp Obirinakem 2015Polinati Kremer Faller amp Fialho 2010) Con-sidering this aspect different time frames wereused for different storage temperatures Theheating time and temperature for broccoli sam-ples are shown in Table 1 Samples were takenfrom each cell at different time intervals and theirvitamin C content was measured

22 Measurement of AscorbicAcid in Broccoli

Vitamin C was measured according to theAOAC (1990) using a titration method with 26-dichloro-indophenol reagent Samples of 100 gbroccoli were weighed at different time intervalsfrom each storage temperature and blended with100 ml metaphosphoric acid (3) The solutionwas then vacuum filtered and transferred to a 100ml volumetric flask Ten ml of the diluted samplesolution was then titrated against the standard-ized dye The dye was standardized by a knownconcentration of ascorbic acid solution (01 mgAAml) The end-point was indicated by the ap-pearance of a light pink colour The result wasexpressed as mg100 g fresh broccoli sample andreplicated three times

23 Reaction Kinetics

The loss or degradation of vitamin C is com-monly modelled by the first-order reaction as fol-lows

)= minusk1t (1)

where C is the concentration of vitamin C in asample at time t (mgg sample) C0 is the initialconcentration of vitamin C in the sample (mggsample) k1 is the first-order rate constant (sminus1)and t is the storage time (s)In most of the isothermal experiments an initiallag period was observed and the above equationwas modified with an intercept rather forcing theintercept to zero as Equation 1 (Rahman et al2015) The experimental data was fitted with alinear equation with an intercept as follows

)= minusk1t+ a (2)

where a is the intercept The rate constant wasdetermined from the slope of a linear plot Ln(CCo) versus t Activation energy of first-orderkinetics was estimated using the Arrhenius equa-tion as

Ln(k1) = minus(Ea

)+ b (3)

where R is the universal gas constant (8314Jmol K) Ea is the activation energy (Jmol) Tis the temperature (K) and b is the pre-exponentfactor The activation energy was estimated fromthe slope of the linear plot of Ln k1 versus 1T

24 Statistical Analysis

Each experiment was replicated three timesand the regression analysis of Equations 2 and3 were performed using Microsoft Excel (MS-Excel 2016) The regression coefficient was con-sidered as the goodness of the regression equa-tion

Table 1 Temperature and heating scheme for broccoli

Broccoli

Temperature 5 20 45 60 70 80 110 120(degC) (Days) (h) (h) (min) (min) (min) (min) (min)

Time 0 0 0 0 0 0 0 01 5 525 10 5 4 3 23 195 725 27 15 9 6 521 255 875 40 25 14 9 835 425 1000 55 40 22 13 1149 1125 70 50 32 19 1663 1250 100 60 42 25 2177 1450 115 70 52 31 26

1575 130 80 67 38 311700 145 82 45 362300 9724002550

Figure 1 Plots of Ln (CCo) versus time A 5oC B 20oC C 45oC D 60oC

62 Al-Habsi et al

Figure 3 Arrhenius plot Ln(k1) versus 1T

The composition of fresh broccoli was deter-mined and presented in our preliminary study(Suresh et al 2017) Moisture protein fatcrude fibre ash and carbohydrate content ofbroccoli was 9020 208 033 075 074 and 590g100 g sample respectively and pH was deter-mined as 68 The vitamin C in the fresh broc-coli varied from 536 to 643 mg100 g sampleIn order to study vitamin C stability with widevariations in temperature different time frameexperiments were used as also identified earlierby Hal et al (2012) El-Ishaq and Obirinakem(2015) and Polinati et al (2010) The heatingtime frame at different temperatures is shown inTable 1 Different time frames could be used tocompare vitamin C stability For example thevitamin C reduced to 404 mg100 g sample (ie628 loss) after 35 days of storage when storedin chilled conditions at 5oC whereas vitamin Creduced to 450 mg100 g sample (ie 699 loss)after 72 h when stored at 45oC Similarly vita-min C reduced to 384 and 360 mg100 g sample(ie 597 and 566 loss respectively) after 27and 16 min when stored at 60 and 120oC respec-tively Therefore time to a specific reductionof vitamin C (for example above 36-45 mg100g sample) could be used to compare data whenstored at different temperatures However ki-netics modelling with rate constants (sminus1) bet-ter standardize the processes when different timeframes are usedThe vitamin C (ascorbic acid AA) losses inbroccoli cauliflower and cabbage were stud-ied at different stages (delivery cooked for 30min and blast chilled 0-3oC) (Charlton PatrickDowling amp Jensen 2004) The AA content ofbroccoli cauliflower and cabbage from deliveryto blast chilling varied from 29-240 mg100 gsample The dramatic AA losses occurred dur-ing cooking (33-81) with broccoli showing thegreat loss (81) During the 4-day chill stor-age a steady decline of AA was observed with alossday of 42 The loss of AA levels in broc-coli during chilled storage was 36 per day Thelossday for cauliflower and cabbage was 76and 23 respectivelyGoncalves Abreu Brandao and Silva (2011)studied the vitamin C loss in frozen broccoli

(Brassica oleracea L ssp Italica) during stor-age The vitamin C content significantly de-creased after 121 days of isothermal storage by80 60 and 29 respectively at -7 -15 and-25oC The major loss of vitamin C occurred dur-ing the first 55 days of storage Shobham Mud-havath and Sukumaran (2017) studied the effectof microwave and pressure cooking on the stabil-ity of vitamin C in vegetables (carrot potatospinach brinjal cauliflower green chilli bittergourd and cabbage) The total content of vi-tamin C in raw vegetables ranged from 355 to9127 mg100 g Microwave and pressure cook-ing for 2 to 3 min resulted in considerable loss ofvitamin C The percentage loss of vitamin C con-tent in cabbage subjected to microwave and pres-sure cooking was 99 and 495 respectivelywhereas it was 207 for cauliflower subjected tomicrowave and 462 loss during pressure cook-ing A greater loss was observed for potato andspinach Shams El-Din Abdel-Kader Makhloufand Mohamed (2013) studied the effect of cook-ing methods on natural antioxidants in Brassicavegetables The result showed that boiling had agreater loss of vitamin C compared to microwavecooking Boiling for 6 min caused a loss of 645in broccoli 707 loss in white cabbage and668 in cauliflowerFigures 1 and 2 show the plot of Ln (CCo) ver-sus time according to first-order reaction kinet-ics (Equation 2) The high regression coefficientfrom 088 to 099 indicates that vitamin C decaycan be predicted according to the first-order reac-tion Similarly first-order reaction was used forthe degradation of vitamin C in cabbage and let-tuce (Awagu Ekanem Kolo amp Adamu 2017)cherry juice (Jirasatid amp Noipant 2015) andorange juice (Calligaris Manzocco amp Lagazio2012) The first-order reaction rate constant wasdetermined from the slope and these increasedfrom 903Ouml10minus8 sminus1 to 565Ouml10minus3 sminus1 whenstorage temperature varied from 5oC to 120oCVitamin losses in fresh capsicum at 5oC and 20oCwere observed as 822Ouml10minus7 and 115Ouml10minus6 sminus1

as compared to the results found in this study of903Ouml10minus8 (at 5oC) and 403Ouml10minus6 sminus1 (at 20oC)(Rahman et al 2015) At refrigerated tempera-tures the loss of vitamin C in broccoli was muchhigher than capsicum whereas comparable losseswere observed at 20oC In the case of orange juice

64 Al-Habsi et al

stored at 10oC the rate constant was observedas 463Ouml10minus7 sminus1 (Calligaris et al 2012) In thecase hot water blanching of pumpkin at pH 65the rate constant increased from 810Ouml10minus4 to137Ouml10minus3 sminus1 when blanching temperature in-creased from 60oC to 90oC (Ariahu et al 2011)The reaction rate constant of cherry juice in-creased from 312Ouml10-4 to 100Ouml10-3 s-1 withincreasing temperatures (75 80 85 90 and 95oC) (Jirasatid amp Noipant 2015) The rate con-stant observed in this study was similar to ear-lier reported values The degradation of ascorbicacid in broccoli could be mainly due to the oxida-tion of ascorbic acid by oxidizing enzymes egascorbic acid oxidase peroxidase catalase andpolyphenol oxidase (Mapson 1970 Venkatesh ampPark 2014) Two types of vitamin C degrada-tion could occur aerobic and anaerobic degrada-tion In aerobic degradation the AA is oxidisedto L-dehydro-ascorbic acid (L-DA) followed byhydrolysis and further oxidation whereas anaer-obic degradation has not been clearly studied andreported (Wang Law Mujumdar amp Xiao 2017)During processing matrix disruption could oc-cur thus facilitating the oxidation of L-AA to L-DA by the enzyme AAO The L-DA can thenbe further hydrolysed to 23-diketogulonic acidthus losing its antiscorbutic activity Other pos-sible chemical reactions associated with changesin flavour colour and odour could have oc-curred with time due to interactions among thecomponents and this resulted in changes in pH(El-Ishaq amp Obirinakem 2015 Munyaka et al2010) In this study low temperature likelycaused minimal destruction of structure thus en-zymatic degradation reaction was observed at aslower rate as compared to high temperature Athigh temperature structural damage could en-hance enzymatic degradation as well as increaseinteraction with other released componentsFigure 3 shows the Arrhenius plot of Ln (k1) ver-sus 1T and activation energy was estimatedfrom the slope as 742 kJmol Jirasatid andNoipant (2015) observed a similar activation en-ergy of 694 kJmol for cherry within the tem-perature range 75-95oC The higher activationenergy indicated that temperature could affectdegradation at a faster rate The activation en-ergy was observed as 580 390 and 290 kJmolrespectively for guava mango and marula pulps

within the temperature range of 80-150oC (Hal etal 2012) The high activation energy of vitaminC in broccoli implied that the deterioration wasmore sensitive to temperature as compared tothe vitamin C degradation in guava mango andmarula pulps The activation energy of ascorbicacid loss during pumpkin blanching (60-90oC)varied from 169 to 412 kJmol while pH in-creased from 50 to 65 respectively (Ariahu etal 2011) The pH of the fresh broccoli was mea-sured as 68 (Suresh et al 2017) Therefore theactivation energy of broccoli could be expectedto be higher than the reported values for pump-kin In the case of heat treatment (80-90oC) ofapple the activation energy was observed as 889kJmol The activation energy varied from 107to 992 depending on the type of fruits and veg-etables (Courtois Vedrenne amp George 2009)Therefore the activation energy of vitamin C lossdepended on the types of fruits and vegetablesas well as their physicochemical properties andpH

4 Conclusion

The loss of vitamin C increased with storage timeand temperature (5-120oC) Vitamin C degrada-tion in broccoli followed a first-order reaction andthe temperature dependence of rate constantswas described using the Arrhenius model Theactivation energy was estimated as 742 kJmolData on the retention of vitamin C could be usedwhen broccoli is stored as raw and when it iscooked at different temperatures The rate con-stants at different temperatures could be usedin simulation and optimization of thermal pro-cesses

Acknowledgements

Ms Sithara Suresh would like to appreciate Sul-tan Qaboos University for awarding her a PhDscholarship and all authors would like to ac-knowledge the supports of the Sultan QaboosUniversity (SQU) towards this research in thearea of food stability

References

AOAC (1990) In Helrich K (ed) Association ofofficial analytical chemists Official meth-ods of analysis of the AOAC AOAC Ar-lington

Ariahu C C Abashi D K amp Chinma C E(2011) Kinetics of ascorbic acid loss dur-ing hot water blanching of fluted pump-kin (telfairia occidentalis) leaves Journalof Food Science and Technology-mysore48 (4) 454ndash459 doi101007s13197- 010-0123-0

Awagu E F Ekanem E O Kolo A M ampAdamu M M (2017) Kinetic modelingof vitamin c (ascorbic acid) degradation inblanched commonly consumed salad veg-etables using computer simulation analysisJournal of Applied Chemistry 10 59ndash66

Bergquist S A M Gertsson U E amp Ols-son M E (2006) Influence of growth stageand postharvest storage on ascorbic acidand carotenoid content and visual qualityof baby spinach (spinacia oleracea l) Jour-nal of the Science of Food and Agriculture86 (3) 346ndash355 doi101002jsfa2373

Calligaris S Manzocco L amp Lagazio C(2012) Modeling shelf life using chemicalphysical and sensory indicators Shelf lifeassessment of food 75ndash126

Charlton K Patrick P Dowling L amp JensenE (2004) Ascorbic acid losses in vegeta-bles associated with cook-chill food prepa-ration South African Journal of Clini-cal Nutrition 17 (2) 56ndash63 doi10 108016070658200411734015 eprint httpsdoiorg10108016070658200411734015

Courtois F Vedrenne L amp George S (2009)Mathematical modelling of some nutrientlosses during heat treatment of stewedapples Czech Journal of Food Sciences27 (SI) S23ndashS26 6th Chemical Reactionsin Food Conferene 2009 Prague CZECHREPUBLIC MAY 13-15 2009 doi10 17221966-CJFS

Dominguez-Perles R Carmen Martinez-Ballesta M Carvajal M Garcia-Viguera C amp Moreno D A (2010)Broccoli-derived by-products-a promisingsource of bioactive ingredients Journal

of Food Science 75 (4) C383ndashC392doi101111j1750-3841201001606x

Favell D J (1998) A comparison of the vita-min c content of fresh and frozen vegeta-bles Food Chemistry 62 (1) 59ndash64 doi101016S0308-8146(97)00165-9

Goncalves E M Abreu M Brandao T R Samp Silva C L M (2011) Degrada-tion kinetics of colour vitamin c anddrip loss in frozen broccoli (brassica ol-eracea l ssp italica) during storage atisothermal and non-isothermal conditionsInternational Journal of Refrigeration-revue Internationale Du Froid 34 (8)2136ndash2144 23rd International-Institute-of-Refrigeration(IIR) International Congressof Refrigeration Prague CZECH REPUB-LIC AUG 21-26 2011 doi10 1016 j ijrefrig201106006

Hal P H Bosschaart C Twisk C V Verk-erk R amp Dekker M (2012) Kinetics ofthermal degradation of vitamin c in marulafruit (sclerocarya birrea subsp caffra) ascompared to other selected tropical fruitsLWT-Food Science and Technology 49 (2SI) 188ndash191 doi101016jlwt201112038

Hamad Q Y M M (2009) Spectrophotomet-ric determination of total vitamin c insome fruits and vegetables at koya areandashkurdistan regioniraq Kirkuk UniversityJournal for Scientific Studies 4 (2) 46ndash54

El-Ishaq A amp Obirinakem S (2015) Effect oftemperature and storage on vitamin c con-tent in fruits juice 1 17ndash21

Ismail F (2013) Determination of water solublevitamin in fruits and vegetables marketedin sindh pakistan

Jain H amp Mulay S (2014) A review on biolog-ical functions and sources of anti- scorbuticfactor Vitamin c

Jirasatid S amp Noipant P (2015) Ther-mal degradation kinetics of vitamin cin jamaican cherry (muntingia calabural) juice In Burapha University Interna-tional Conference 10-12 July BangsaenChonburi Thailand Retrieved from http www buuconference buu ac th registration getdownload php name =paper116ampfile=fileBUU2015paper116 pdf

66 Al-Habsi et al

Kumar G V Ajay Kumar K Raghu P G Ramp Manjappa S (2013) Determination ofvitamin c in some fruits and vegetables indavanagere city(karanataka)-india Inter-national Journal of Pharmacy amp Life Sci-ences 4 (3)

Mapson L W (1970) Vitamin in fruits Thebiochemistry of fruits and their products 1369ndash386

Masamba K G amp Mndalira K (2013) Vi-tamin c stability in pineapple guava andbaobab juices under different storage con-ditions using different levels of sodium ben-zoate and metabisulphite African Journalof Biotechnology 12 (2) 186ndash191

Munyaka A W Makule E E Oey I VanLoey A amp Hendrickx M (2010) Ther-mal stability of l-ascorbic acid and ascor-bic acid oxidase in broccoli (brassica oler-acea var italica) Journal of Food Science75 (4) C336ndashC340 doi10 1111 j 1750 -3841201001573x

Nath A Bagchi B Misra L K amp DekaB C (2011) Changes in post-harvest phy-tochemical qualities of broccoli florets dur-ing ambient and refrigerated storage FoodChemistry 127 (4) 1510ndash1514

Nisha P Singhal R S amp Pandit A B (2005)A study on degradation kinetics of ri-boflavin in green gram whole (vigna ra-diata l) Food Chemistry 89 (4) 577ndash582doi101016jfoodchem200403014

Oyetade O A (2012) Stability studies on ascor-bic acid (vitamin c) from different sources2 20ndash24

Phillips K M Council-Troche M McGintyR C Rasor A S amp Teresa Tarrago-Trani M (2016) Stability of vitamin cin fruit and vegetable homogenates storedat different temperatures Journal of FoodComposition and Analysis 45 147ndash162doi101016jjfca201509008

Polinati R M Kremer Faller A L amp FialhoE (2010) The effect of freezing at-18 de-grees c and-70 degrees c with and withoutascorbic acid on the stability of antioxi-dant in extracts of apple and orange fruitsInternational Journal of Food Science andTechnology 45 (9) 1814ndash1820 doi101111j1365-2621201002333x

Rahman M S Al-Rizeiqi M H GuizaniN Al-Ruzaiqi M S Al-Aamri A Hamp Zainab S (2015) Stability of vita-min c in fresh and freeze-dried capsicumstored at different temperatures Journalof Food Science and Technology-mysore52 (3) 1691ndash1697 doi101007s13197-013-1173-x

Shams El-Din M H A Abdel-Kader M MMakhlouf S K amp Mohamed O S S(2013) Effect of some cooking methods onnatural antioxidants and their activities insome brassica vegetables 26 697ndash703

Shobham K R Mudhavath M amp SukumaranM K (2017) Effect of Microwave andPressure Cooking on Stability of VitaminC in Some Selected Vegetables Interna-tional Journal of Current Microbiology andApplied Sciences 6 (9) 2391ndash2397 doi1020546ijcmas2017609293

Spinola V Mendes B Camara J S ampCastilho P C (2012) An improved andfast uhplc-pda methodology for determi-nation of l-ascorbic and dehydroascorbicacids in fruits and vegetables evaluationof degradation rate during storage Analyt-ical and Bioanalytical Chemistry 403 (4)1049ndash1058 doi101007s00216-011-5668-x

Spinola V Mendes B Camara J S ampCastilho P C (2013) Effect of time andtemperature on vitamin c stability in hor-ticultural extracts uhplc-pda vs iodomet-ric titration as analytical methods LWT-Food Science and Technology 50 (2) 489ndash495 doi101016jlwt201208020

Suresh S Al-Habsi N Guizani N amp Rah-man M S (2017) Thermal character-istics and state diagram of freeze-driedbroccoli Freezing curve maximal-freeze-concentration condition glass line andsolids-melting Thermochimica Acta 655129ndash136 doi101016jtca201706015

Venkatesh J amp Park S W (2014) Role of l-ascorbate in alleviating abiotic stresses incrop plants Botanical Studies 55 doi1011861999-3110-55-38

Wang J Law C Mujumdar A amp Xiao H-W(2017) The degradation mechanism andkinetics of vitamin c in fruits and vegeta-bles during thermal processing

Wootton-Beard P C amp Ryan L (2011)Improving public health The role ofantioxidant-rich fruit and vegetable bever-ages Food Research International 44 (10)3135ndash3148 doi101016jfoodres201109015

Physico-Chemical Composition and Antimicrobial ProteinContent of Early Lactation Donkey Milk

Maria Aspria Kallis Souroullasa Christina Ioannoua and Photis Papademasa

a Department of Agricultural Sciences Biotechnology and Food Science Cyprus University of TechnologyLimassol Cyprus

Corresponding authorphotispapademascutaccy

Tel +357 25002581Fax +357 25002562

Abstract

The influence of early lactation on chemical composition and the concentration of antimicrobialproteins of donkeyrsquos milk produced in Cyprus were investigated Milk samples from 10 female donkeysin their first season of lactation were collected at 7 15 and 30d postpartum The average contents ofdonkey milk gross composition were 140 protein 016 fat and 874 total solids Results showedthat lactation had a significant negative effect on protein concentration while total solid concentrationshowed an increased followed by a decrease Composition of antimicrobial proteins also showed asignificant decreased during lactation period except from lactoferrin which showed an increase On theother hand throughout the lactation pH and fat were constant

Keywords Donkey milk Antimicrobial proteins Immunoglobulin-A Lactation

1 Introduction

Milks from non traditional animal species (iedonkey camel and buffalo) are gaining interestdue to the fact that they are considered suit-able to supplement the needs of special popu-lation groups such as infants and elderly Don-key milk has been studied less compared to thatof ruminant milks but in recent years inter-est in donkeyrsquos milk production and commer-cialization have significantly increased This in-crease in interest is due to its unique nutritionaland physicochemical characteristics as well as itsfunctional properties such as antimicrobial im-munomodulatory anti-inflammatory and anti-hypertensive properties (Aspri Economou ampPapademas 2017 Brumini et al 2013 Jirillo ampMagrone 2014 Mao et al 2009 Zhang ZhaoJiang Dong amp Ren 2008)

The nutritional composition of donkey milk isvery similar to that of human milk and it hasbeen reported to be an adequate replacement forchildren with cow milk protein allergy mainlydue to its tolerability nutritional contents andgood taste (Aspri et al 2017 Monti et al 2012)Donkey milk is characterized by low fat and pro-tein content and high lactose content It is alsocharacterized by low casein content and a par-ticularly high whey protein concentration richin lysozyme which is around 6000 times morethan lysozyme content of bovine milk (Guo etal 2007 Salimei et al 2004)Previous studies have shown that the strong an-timicrobial activity of donkey milk arise from itshigh concentrations of antimicrobial agents suchas lysozyme and lactoferrin (Tidona et al 2011Vincenzetti et al 2008) Lysozyme is the mainprotein in donkeyrsquos milk that plays an impor-

Early Lactation Donkeyrsquos Milk Characteristics 69

tant role of fighting infections in breastfeedinginfants while lactoferrin is the second antimi-crobial protein in donkey milk (Uniacke-LoweHuppertz amp Fox 2010) In addition these twoantimicrobial proteins work synergistically withother proteins such as lactoperoxidase and im-munoglobulins (Polidori amp Vincenzetti 2010)The high content of these antimicrobial proteinsare considered to be the reason for the low micro-bial counts in donkey milk (Chiavari ColorettiNanni Sorrentino amp Grazia 2005 Salimei et al2004 Saric et al 2012 Vincenzetti et al 2008)As some donkey milk producers are market-ingldquoearly lactation milkrdquo the aim of this researchwas to study the influence of early lactation stageon some physicochemical parameters of donkeymilk (TS protein fat and pH) and also on theconcentration of antimicrobial proteins of donkeymilk

21 Animals and samplingprocedure

This study was carried out at ldquoGolden donkeyrsquosfarmrdquo located in Skarinou Cyprus The farmhad three breeds of donkeys (Asia Cyprus andIsrael) and their crosses Raw milk samples wereobtained by hand milked of 10 female donkeysafter parturition on 1st 15th and 30th day of lac-tation always at the same time of the day Theanimals were maintained within the same condi-tions and receiving the same feeding level andcomposition Their diet was composed of haywheat and silage The milk samples of an aver-age volume of 100mL were collected in standardcontainers and transported same day to the lab-oratory and kept at 4oC for further analysis

22 Physicochemical analysis

Determination of pH

The pH of the donkey milk samples was deter-mined by potentiometry The pH meter glassprobe was first calibrated using standard buffersat pH 4 and 7 before being used to measure the

sample pH Sample pH was measured by sub-merging the tip of the probe into the sample forsim1-2 min until a stable reading was registered onthe pH meter scale Measurements were done intriplicates and average values were reported

Protein content

The protein content of milk samples was deter-mined using the Bradford Protein assay (Brad-ford 1976) Bovine serum albumin (BSA) wasprepared for standard curve using the followingconcentrations 50 microgml to 300 microgml Afterincubated for 5 minutes at room temperatureabsorbance measurements were determined spec-trophotometrically (Infinite PRO 200 TecanSwitzerland) at 595 nm The Bradford reagentwas used as a blank Measurements were done intriplicates and average values were reported

Fat content

Milk fat content was determined by a butyromet-ric method according to Gerber (IDF ISO 4882008) Milk samples (11 ml) were mixed withsulfuric acid (10 ml) and 1 ml of isoamyl alcoholin butyrometer and closed with rubber cork Themixture was mixed and placed in a water bathat 65oC The sample was centrifuged in a Gerbercentrifuge for 5 min at 1000 rpm and the buty-rometer was placed back into the water bath foranother 5 min The fat content is expressed asa percentage on the butyrometer Measurementswere done in triplicates and average values werereported

Total Solids Content

Total solids were determined by the oven methodin accordance with ISO 6731 (IDF standard 212010a) 2g of fresh donkey milk samples weretransferred to a pre-weighed round flat bottomaluminum dish Then the dishes were trans-ferred to a hot air oven at 102 oC for 2 hoursThen the dish was transferred to a desiccator for30 min to cool down and weighted Total solidscontent was calculated by the following formula

Total solids () =m2 minusm0

m1 minusm0times 100 (1)

70 Aspri et al

where m0 is the mass (g) of the dish m1 is themass (g) of the dish and the test portion andm2 is the mass (g) of the dish and the dried testportion Measurements were done in triplicatesand average values were reported

23 Antimicrobial proteins andIgA

Lysozyme content of donkey milk

Lysozyme was quantified according to a sensitivefluorescence-based method using EnzChekreg kit(Life Technologies Carlsbad CA USA) The as-say is based on the assessment of the lytic activ-ity of lysozyme in the cell walls of Micrococcuslysodeikticus which have been labeled with thefluorescent dye fluorescein The test was per-formed according to the manufacturerrsquos instruc-tions and the absorbance was determined spec-trophotometrically (Infinite PRO 200 TecanSwitzerland) at 497nm and 518 nm All sam-ples were analyzed in triplicate

Lactoferrin content of donkey milk

Quantitative determination of LF in the skimmilk samples was performed using a commer-cial ELISA kit the Bovine Lactoferrin ELISAQuantification Kit (Bethyl Laboratories Mont-gomery TX) The procedures were performed ac-cording to manufacturerrsquos instructions A stan-dard curve was prepared using the supplied lacto-ferrin standard diluted to concentrations of 312microgml 625 microgml 125 microgml 250 microgml 500microgml kal 1000 microgml using the sample diluentbuffer (50 185 mM Tris 014 M NaCl 1 bovineserum albumin 005 Tween 20 pH 80) Thefinal absorbance of the samples and the stan-dards was measured at 450 nm using an ELISAplate-reader (Infinite F200 Tecan MannedorfSwitzerland) and the concentration of lactofer-rin calculated by extrapolating from a standardcurve All samples were analysed in triplicate

Immunoglobulin A content of donkeymilk

Total IgA in milk was determined by using HorseIgA ELISA Quantitation Kit (Bethyl Labora-

tories Inc Montgomery TX USA) accordingto the manufacturersrsquo instructions A stan-dard curve was prepared using the suppliedlactoferrin standard diluted to concentrationsof 1000 nmml 500 nmml 250 nmml 125nmml 625 nmml 3125 nmml 156 nmml0 nmml using the sample diluent buffer Thefinal absorbance of the samples and the stan-dards was measured at 450 nm using an ELISAplate-reader (Infinite F200 Tecan MannedorfSwitzerland) and the concentration of IgA cal-culated by extrapolating from a standard curveAll samples were analyzed in triplicate

All experiments were carried out in triplicatesThe data are expressed as the mean plusmn stan-dard deviation (SD) Statistical analysis wasperformed using SPSS version 170 (SPSS IncChicago IL USA) The data were subjected toone-way analysis of variance (ANOVA) to deter-mine the differences of samples Significant dif-ferences were compared by Tuckey test on thelevel of P lt 005

The changes in the physico-chemical parametersand composition (protein fat pH TS) of donkeymilk during the early lactation period (30 days)are presented in Table 1The pH value of donkey milk ranging from 703to 706 did not vary significantly throughout thelactation period (30 days) which was consistentwith the findings of Salimei et al (2004) Ac-cording to the literature the pH of donkey milkafter milking is 719plusmn 010 (Curadi GiampietroLucenti amp M 2001) This suggests that the pHvalue was not influenced by breed or stage of lac-tation (Chiavari et al 2005 Guo et al 2007Malacarne Martuzzi Summer amp Mariani 2002Polidori Beghelli Mariani amp Vincenzetti 2009)The average pH value (703plusmn002) of donkey milkwas higher than that of cow milk which is in therange of 660 to 680 According to Salimei et al(2004) this may be due to the lower casein (CN)

Table 1 Chemical composition of donkey milk at different stages during the lactation period

Day 7 Day 15 Day 30Mean Min Max Mean Min Max Mean Min Max

Protein Content155 plusmn 041b 102 219 175 plusmn 038c 130 264 090 plusmn 021a 102 194(g100ml)

Fat () 015 plusmn 008b 005 030 018 plusmn 022c 005 080 014 plusmn 007a 005 030TS () 873 plusmn 057a 813 1002 910 plusmn 040b 842 969 840 plusmn 040a 829 922pH 703 plusmn 013a 683 727 701 plusmn 011a 681 719 706 plusmn 007a 698 721

Values are means plusmn standard deviation values within the same row followed by different superscript letters significantly differ (plt005)

TS = Total Solids

and phosphate contents in donkey milk than incow milk (Salimei et al 2004) The phosphatecontent of cow milk is 1000 mgL while the phos-phate content of donkey milk is very low 638 42mgL (Fantuz et al 2012)The total protein content of donkey milk (ap-proximately 14 plusmn 029) was similar to that ofhuman milk and much lower than that of cowsrsquogoatsrsquo or sheeprsquos milk (Guo et al 2007) The ob-served average milk protein content (14) waslower (172 189 ) than reported by Salimeiet al (2004) and Giosue Alabiso Russo Alicataand Torrisi (2008) The protein content of don-key milk at 15 days after lactation is statisticallysignificant higher compared to the first day andthe 30th day of lactation (175plusmn038 155plusmn041and 090 plusmn 021 respectively plt005) The re-sults are consistent with the findings of Salimeiet al (2004) which have reported a falling trendin the protein level of donkey milk during lac-tation This may be due to the differential ex-pression of milk protein synthesis genes duringthe lactation period The levels of early lacta-tion protein mRNA increased after parturitionthey reach the maximum after a few weeks andthen they start decreasing (Demmer Ross Gin-ger Piotte amp Grigor 1998)In contrast to the protein content the fat contentof donkey milk does not show a statistically sig-nificant difference between the different samplingdays However other studies showed a significantvariation of donkey milk fat content throughoutthe lactation period (Cosentino Paolino Freschiamp Calluso 2012 Martemucci amp DrsquoAlessandro2012 Salimei et al 2004) These differences in-dicate that fat content could be affected by breed

breeding area and forage milking technique andinterval between milkings as also reported byFox (2003) The average fat content of donkeymilk was 016 plusmn 0 02 which is consistent withthat reported in the literature for donkeyrsquos milk(Ivankovic et al 2009 Salimei et al 2004)The total solids content of donkey milk wassignificantly higher on the 15th day of lacta-tion compared to the 1st and 30th of lactation(914 plusmn 038 873 plusmn 056 and 835 plusmn 039 respec-tively plt005) The observed average dry mat-ter content (874 plusmn 025 ) was consistent withthe data for dry matter content (884 ) reportedby Salimei et al (2004) and are not affected bybreed lactation stage or milking conditions Themean dry matter observed in current donkey milkstudy was of 873 + 011 which is consistentwith the values reported in the literature for don-key milk (Chiavari et al 2005 Guo et al 2007Salimei et al 2004) The total solids contentin the donkey milk according to Polidori et al(2009) can be up to 880 g 100g which is lowerthan the solid residue in cowrsquos milk (125 - 1300g 100 g) sheep (175 - 195 g 100 g) andhuman milk (1170 - 1290 g 100 g)

32 Antimicrobial Proteins

The amount of lysozyme lactoferrin and IgAconcentrations (mgml) at different stages ofdonkey lactation are presented in Table 2

72 Aspri et al

Table 2 Lysozyme lactoferrin and IgA concentrations in donkey milk at different stages of lactation

Day 7 Day 15 Day 30

Lysozyme (Uml) 22508 plusmn 705b 22411 plusmn 600a 20194 plusmn 1096a

Lactoferrin (microgml) 11103 plusmn 4104b Nd 13508 plusmn 2768c

IgA (ngml) 267440 plusmn 13372c 257647 plusmn 14304a 226732 plusmn 14512a

Values are means plusmn standard deviation values within the same row followed by different superscript letters significantly

differ (plt005)

Nd=Not determined

Lysozyme

The EnzChek fluorescence-based assay measureslysozyme activity on Micrococcus lysodeikticuscells which are labeled to such a degree thatthe fluorescence is quenched Lysozyme actioncan relieve this quenching and the increase influorescence that is proportional to lysozyme ac-tivity The mean lysozyme activity value was21704 plusmn 1309 Uml Our results were in accor-dance with other studies carried out by Gubic etal (2014) Guo et al (2007) Pilla Dapra Zec-coni and Piccinini (2010) Saric et al (2012)Saric et al (2014) Vincenzetti et al (2011)The lysozyme content at 30th day was signifi-cantly lower compared to the lysozyme contentat 1st and 15th day of lactation (20194 plusmn 109622508 plusmn 705 and 22411 plusmn 599 respectivelyplt005)The high lysozyme content of donkey milk playsan important role in the prevention or reductionof intestine infections in infants and is also re-sponsible for the low bacteria count of donkeymilk reported in literature (Salimei et al 2004)Moreover the high lysozyme content does notaffect the growth or the acidification activity ofprobiotic strains making donkey milk a goodbase for the production of probiotic fermentedmilk beverages (Chiavari et al 2005 Coppolaet al 2002) Finally the high lysozyme ac-tivity could explain the low incidences of mas-titis in donkeys (Conte et al 2006) Donkeymilk lysozyme belongs to C-type calcium-bindinglysozyme and is able to bind calcium ions thisbinding leads to more stable complex with anenhanced antimicrobial activity (Wilhelm et al2009)

Lactoferrin

Results of lactoferrin content in donkey milkshowed that lactoferrin content at 30th day oflactation was significantly higher compared tothe 1st day of lactation (13508 plusmn 27 68 and11103 plusmn 4104 respectively plt005) which is inagreement with literature A study carried outby Gubic et al (2015) showed that the concentra-tion of lactoferrin is increased over the lactationperiod Moreover according to Adlerova Bar-toskova and Faldyna (2008) there is an impor-tant correlation between lactation and lactofer-rin levels In general the content of lactoferrinin donkey milk is higher than in ruminant milkbut much lower than in mare and in human milk(Kanyshkova Buneva amp Nevinsky 2001)Lactoferrin is an iron-binding protein that dis-plays many biological functions such as antioxi-dant antiviral anti-inflammatory immunomod-ulatory and anti-carcinogenic activity (Kuwataet al 1998 Ward Paz amp Conneely 2005)Furthermore it controls the proper compositionof the intestinal microflora by suppressing thegrowth of pathogenic bacteria while promotingthe growth of beneficial bacteria such as Lacto-bacillus and Bifidobacterium (Madhusudan Ra-machandra Udaykumar Nagraj amp Jagjivan2017) Different studies suggest that lactofer-rin and lysozyme work synergistically to inhibitthe growth of pathogenic bacteria and contributeto donkey milkrsquos strong overall antibacterial ac-tivity against both Gram-positive and Gram-negative bacteria (Saric et al 2012 Tidona etal 2011) In fact Lf can bind different com-ponents in the outer membrane thereby open-ing ldquoporesrdquo to enhance susceptibility of Gram-negative bacteria to the lysozyme by increas-

ing in membrane permeability (Ellison amp Giehl1991 Leitch amp Willcox 1999)

Immunoglobulin-A (IgA)

The concentration of IgA of donkey milk wassignificantly lower at the 30th day of lactationcompared to the 1st and 15th day of lacta-tion (226732 plusmn 15991 267447 plusmn 17814 and257647 plusmn 16734 respectively plt005) The av-erage value of IgA was 250 gLImmunoglobulins (Igs) are a defence family ofglobular proteins with antimicrobial and otherprotective bioactivities They play an importantrole in transferring immunity to the newborn byestablishing an optimal microfloral population inthe gut of the newborn inactivate bacteria bybinding to specific sites on the bacterial surfacewhile its own immune system is developing (Gap-per Copestake Otter amp Indyk 2007)

4 Conclusion

In conclusion lactation stage affected the grosscomposition of donkey milk but had no signif-icant effect on pH and fat content It can beconcluded that the chemical composition of milkin terms of fat protein pH total solid residuelysozyme lactoferrin and IgA showed significantdifferences throughout the different stages of lac-tation except for fat and pH However furtherresearch is l needed to establish how donkeyrsquosnutrition affects the quality of milk as it is atype of milk of particular interest with uniquecomposition

Acknowledgements

The authors wish to thank the Golden DonkeysFarm Larnaca Cyprus for providing the samples

References

Adlerova L Bartoskova A amp Faldyna M(2008) Lactoferrin A review VeterinarniMedicina 53 (9) 457ndash468

Aspri M Economou N amp Papademas P(2017) Donkey milk An overview on func-tionality technology and future prospectsFood Reviews International 33 (3) 316ndash333 doi1010808755912920161175014

Bradford M M (1976) A rapid and sensitivemethod for the quantitation of microgramquantities of protein utilizing the princi-ple of protein-dye binding Analytical Bio-chemistry 72 (1) 248ndash254 doi10 1016 0003-2697(76)90527-3

Brumini D Furlund C B Comi I DevoldT G Marletta D Vegarud G E ampJonassen C M (2013) Antiviral activ-ity of donkey milk protein fractions onechovirus type 5 International Dairy Jour-nal 28 (2) 109ndash111 doi101016jidairyj201208010

Chiavari C Coloretti F Nanni M Sor-rentino E amp Grazia L (2005) Useof donkeyrsquos milk for a fermented bever-age with lactobacilli Lait 85 (6) 481ndash490doi101051lait2005031

Conte F Scatassa M L Monsu G Lo VerdeV Finocchiaro A amp De Fino M (2006)Monitoring of safety and quality of don-keyrsquos milk Food Safety Assurance and Vet-erinary Public Health Towards a Risk-based Chain Control 265ndash268

Coppola R Salimei E Succi M SorrentinoE Nanni M Ranieri P Grazia L(2002) Behaviour of lactobacillus rhamno-sus strains in assrsquos milk Annals of Micro-biology 52 (1) 55ndash60

Cosentino C Paolino R Freschi P amp CallusoA M (2012) Short communication Jennymilk production and qualitative charac-teristics Journal of Dairy Science 95 (6)2910ndash2915 doi103168jds2011-5232

Curadi M C Giampietro P G Lucenti Pamp M O (2001) Use of mare milk in pedi-atric allergology In Proceeding of the Asso-ciazione Scientifica di Produzione AnimaleXIV Congress Firenze 14 (Vol 2 pp 647ndash649)

Demmer J Ross I K Ginger M R PiotteC K amp Grigor M R (1998) Differen-tial expression of milk protein genes duringlactation in the common brushtail possum(trichosurus vulpecula) Journal of Molec-

74 Aspri et al

ular Endocrinology 20 (1) 37ndash44 doi10 1677jme00200037

Ellison R T amp Giehl T J (1991) Killing ofgram-negative bacteria by lactoferrin andlysozyme Journal of Clinical Investigation88 (4) 1080ndash1091 doi101172JCI115407

Fantuz F Ferraro S Todini L Piloni RMariani P amp Salimei E (2012) Donkeymilk concentration of calcium phosphoruspotassium sodium and magnesium Inter-national Dairy Journal 24 (2) 143ndash145 In-ternational Dairy Federation (IDF) Sym-posium on Sheep Goat and other Non-CowMilk Athens doi101016jidairyj201110013

Fox P F (2003) Dairy processing Improvingquality CRC Press and Woodhead Publish-ing Limited Boca Raton

Gapper L W Copestake D E J Otter D Eamp Indyk H E (2007) Analysis of bovineimmunoglobulin g in milk colostrum anddietary supplements A review Analyticaland Bioanalytical Chemistry 389 (1) 93ndash109 doi101007s00216-007-1391-z

Giosue C Alabiso M Russo G AlicataM L amp Torrisi C (2008) Jennet milkproduction during the lactation in a sicil-ian farming system Animal 2 (10) 1491ndash1495 doi101017S1751731108002231

Gubic J M Saric L C Saric B M MandicA I Jovanov P T Plavsic D V ampOkanovic D G (2014) Microbiologicalchemical and sensory properties of domes-tic donkeyrsquos milk from autochthones ser-bian breed Journal of Food and NutritionResearch 2 (9) 633ndash637

Gubic J Milovanovic I Ilicic M Tomic JTorbica A Saric L amp Ilic N (2015)Comparison of the protein and fatty acidfraction of balkan donkey and human milkMljekarstvo 65 (3) 168ndash176 doi1015567mljekarstvo20150303

Guo H Y Pang K Zhang X Y Zhao LChen S W Dong M L amp Ren F Z(2007) Composition physiochemical prop-erties nitrogen fraction distribution andamino acid profile of donkey milk Journalof Dairy Science 90 (4) 1635ndash1643 doi103168jds2006-600

IDF ISO 488 (2008) International dairy federa-tion milk - determination of fat content -gerber butyrometers brussels belgium

IDF standard 21 (2010a) Milk cream and evap-orated milk- determination of total solidscontent brussels belgium

Ivankovic A Ramljak J Stulina I An-tunac N Basic I Kelava N amp Kon-jacic M (2009) Characteristics of the lac-tation chemical composition and milk hy-giene quality of the littoral-dinaric ass Ml-jekarstvo 59 (2) 107ndash113

Jirillo F amp Magrone T (2014) Anti-inflammatory and anti-allergic proper-ties of donkeyrsquos and goatrsquos milk En-docrine Metabolic amp Immune Disorders-drug Targets 14 (1) 27ndash37 doi10 2174 1871530314666140121143747

Kanyshkova T G Buneva V N amp NevinskyG A (2001) Lactoferrin and its biologicalfunctions Biochemistry (Moscow) 66 (1)1ndash7 doi101023A1002817226110

Kuwata H Yip T T Yamauchi K Ter-aguchi S Hayasawa H Tomita M ampHutchens T W (1998) The survival ofingested lactoferrin in the gastrointestinaltract of adult mice Biochemical Journal334 (2) 321ndash323 doi101042bj3340321

Leitch E C amp Willcox M D P (1999) Elu-cidation of the antistaphylococcal action oflactoferrin and lysozyme Journal of Med-ical Microbiology 48 (9) 867ndash871 doi10 109900222615-48-9-867

Madhusudan N C Ramachandra C DUdaykumar H D N Da nd Shar-nagouda Nagraj N D amp Jagjivan R D(2017) Composition characteristics nutri-tional value and health benefits of donkeymilk-a review Dairy Science amp TechnologyEDP sciencesSpringer

Malacarne M Martuzzi F Summer A ampMariani P (2002) Protein and fat com-position of marersquos milk Some nutritionalremarks with reference to human and cowrsquosmilk International Dairy Journal 12 (11)869ndash877 doi10 1016 S0958 - 6946(02 )00120-6

Mao X Gu J Sun Y Xu S ZhangX Yang H amp Ren F (2009) Anti-proliferative and anti-tumour effect of ac-

tive components in donkey milk on a549human lung cancer cells InternationalDairy Journal 19 (11) 703ndash708 doi10 1016jidairyj200905007

Martemucci G amp DrsquoAlessandro A G (2012)Fat content energy value and fatty acidprofile of donkey milk during lactation andimplications for human nutrition Lipids inHealth and Disease 11 doi1011861476-511X-11-113

Monti G Viola S Baro C Cresi F TovoP A Moro G Bertino E (2012)Tolerability of donkeysrsquomilk in 92 highly-problematic cowsrsquo milk allergic childrenJournal of Biological Regulators amp Homeo-static Agents 26 75ndash82

Pilla R Dapra V Zecconi A amp Piccinini R(2010) Hygienic and health characteristicsof donkey milk during a follow-up studyJournal of Dairy Research 77 (4) 392ndash397doi101017S0022029910000221

Polidori P Beghelli D Mariani P amp Vin-cenzetti S (2009) Donkey milk pro-duction State of the art Italian Jour-nal of Animal Science 8 (2) 677ndash68318th Congress of the Scientific-Association-of-Animal-Production (ASPA) Palermodoi104081ijas2009s2677

Polidori P amp Vincenzetti S (2010) Differencesof protein fractions among fresh frozen andpowdered donkey milk Recent Patents onFood Nutrition amp Agriculture 2 (1) 56ndash60

Salimei E Fantuz F Coppola R Chiofalo BPolidori P amp Varisco G (2004) Compo-sition and characteristics of assrsquos milk An-imal Research 53 (1) 67ndash78 doi101051animres2003049

Saric L C Saric B M Kravic S Z PlavsicD V Milovanovic I L Gubic J Mamp Nedeljkovic N M (2014) Antibacterialactivity of domestic balkan donkey milk to-ward listeria monocytogenes and staphy-lococcus aureus Food and Feed Research41 (1) 47ndash54

Saric L C Saric B M Mandic A I Tor-bica A M Tomic J M CvetkovicD D amp Okanovic D G (2012) Antibac-terial properties of domestic balkan don-keysrsquo milk International Dairy Journal

25 (2) 142ndash146 doi101016jidairyj201203007

Tidona F Sekse C Criscione A Jacob-sen M Bordonaro S Marletta D ampVegarud G E (2011) Antimicrobial ef-fect of donkeysrsquo milk digested in vitrowith human gastrointestinal enzymes In-ternational Dairy Journal 21 (3) 158ndash165doi101016jidairyj201010008

Uniacke-Lowe T Huppertz T amp Fox P F(2010) Equine milk proteins Chemistrystructure and nutritional significance In-ternational Dairy Journal 20 (9 SI) 609ndash629 6th NIZO Dairy Conference Papen-dal doi101016jidairyj201002007

Vincenzetti S Polidori P Mariani P Cam-mertoni N Fantuz F amp Vita A (2008)Donkeyrsquos milk protein fractions character-ization Food Chemistry 106 (2) 640ndash649doi101016jfoodchem200706026

Vincenzetti S Savini M Cecchini C Mi-cozzi D Carpi F Vita A amp Poli-dori P (2011) Effects of lyophilization anduse of probiotics on donkeyrsquos milk nutri-tional characteristics International Jour-nal of Food Engineering 7 (5) doi1022021556-37582032

Ward P P Paz E amp Conneely O M (2005)Multifunctional roles of lactoferrin A crit-ical overview Cellular and Molecular LifeSciences 62 (22) 2540ndash2548 doi101007s00018-005-5369-8

Wilhelm K Darinskas A Noppe WDuchardt E Mok K H Vukojevic V Morozova-Roche L A (2009) Proteinoligomerization induced by oleic acid at thesolid-liquid interface-equine lysozyme cy-totoxic complexes Febs Journal 276 (15)3975ndash3989 doi101111j1742-4658200907107x

Zhang X-Y Zhao L Jiang L Dong M-Lamp Ren F-Z (2008) The antimicrobial ac-tivity of donkey milk and its microflorachanges during storage Food Control19 (12) 1191ndash1195 doi101016jfoodcont200801005

Study of the Self-Stabilization Ability of Tzatziki (a TraditionalGreek Ready-to-Eat Deli Salad)

Stavros Lalasa Vassilis Athanasiadisa Ioanna Karageorgoua Eleni Bozinouband Vassilis G Dourtoglouc

a Department of Food Technology Technological Educational Institution of Thessaly Terma N Temponerastr GR-43100 Karditsa Greece

b Department of Agricultural Technology Technological Educational Institution of Western Macedonia TermaKontopoulou str GR-53100 Florina Greece

c Department of Wine Vine and Beverage Sciences University of Western Attica Agiou Spyridonos strGR-12210 Egaleo Athens Greece

Corresponding authorvsathanasteilargr

Tel +30-24410-64783Fax +30-24410-64781

Abstract

Traditional Greek yogurt-based salad Tzatziki is one of the most popular ready-to-eat deli saladsin Greece The objective of this study was to estimate the microbial stability of Tzatziki with andwithout chemical preservatives using a rapid method Determination of the microbial count was carriedout using the bioluminescence method (ATP) and traditional microbiological analysis plate-countingmethod (CFU) in various batches of the final product of Tzatziki The results showed that the Tzatzikisalad without preservatives initially gave higher relative light units (RLU) values (79532) than the samesalad with preservatives (43198) because the potassium sorbate and the sodium benzoate used the inrecipe appeared to suspend the action of microorganisms After incubation in two different substratesMacConkey and Sabouraud the Tzatziki salad without preservatives gave higher RLU values (9488and 16176 respectively) than the salad with preservatives (12780 and 12005 respectively) In the twoselective substrates differences appeared between the two methods of microbial count (RLU and CFU)While RLU values were roughly at the same level the CFU values presented significant differences (p lt005) It was also shown that there was a strong correlation (R2 = 093-095) between bacterial countsestimated by traditional CFU and ATP methods As expected the dominant microbial populationin Tzatziki was Lactobacillus spp originated from yogurt Coliforms and yeasts were not able tosurvive in this environment Generally according to the results Greek traditional Tzatziki salad wasa microbial stable product and the bioluminescence method could be a rapid method to determine itsmicrobial state

Keywords Bioluminescence Microbial stability Tzatziki Greek deli salad

1 Introduction

Tzatziki one of the most popular Greek readyto eat deli salads is widely consumed in Greecethe East Mediterranean region and Balkan

countries as well as in other parts of the worldincluding the rest of Europe and USA Tzatzikiis made of strained yogurt (usually from sheep orgoat milk) mixed with fresh chopped cucumbergarlic salt olive oil vinegar and dill

Traditional Greek Tzatziki Microbial stability 77

Tzatziki is a low pH food due to the presence ofyogurt and the added vinegar and it has a lowsalt percentage 2-3 This dressing has shownto be resistant to microbial spoilage by pathogenmicroorganisms (Skandamis Davies McClureKoutsoumanis amp Tassou 2002) Tzatziki isgenerally recognized for its health propertiesand for more than a hundred years peopleaccompany meals rich in fat with this saladAs far as yogurt is concerned there is a tremen-dous scientific work related to its microbialstability The same goes for garlic and garlicextract with an emphasis on the effect of garlicon microorganisms Garlic is considered agermicide (Harris Cottrell Plummer amp Lloyd2001) Although there is little documentationabout cucumberrsquos activity on microorganismsit has been reported that the volatile oils ofcucumber (Cucumis sativus) show antibacte-rial activity as well as a minimum inhibitoryconcentration (MIC) 009-050 mg mLminus1against both gram-positive and gram-negativebacteria and anti-fungal activity against humanpathogen fungi (Candida albicans C tropicalisand C glabrata) (MIC = 134-290 mg mLminus1)(Sotiroudis Melliou Sotiroudis amp Chinou2010) (EZ )-26-nonadienal and (E )-2-nonenalhave been found in cucumber and have shownstrong activities to several human and foodbornepathogen bacteria such as Bacillus cereus Es-cherichia coli O157H7 Listeria monocytogenesand Salmonella typhimurium (Cho BuescherJohnson amp Janes 2004 Croft Juttner ampSlusarenko 1993)Conventional approaches to the determinationof microbial stability rely on fermentation testsand cell counting methods Among the relativelyrapid methods for detection of microorganismsin food adenosine triphosphate (ATP) biolumi-nescence is very suitable for on-line monitoring ofbacterial contamination in food and beveragesThis method does not require a culturing stepor large equipment to fulfil the measurementand it is fast and sensitive (Bottari Santarelliamp Neviani 2015) Bioluminescence is proposedto be the technique giving results in the shortesttime (Hawronskyj amp Holah 1997) The ATPbioluminescence sensing assay relies on the factthat ATP is a major biological energy sourceexisting in various microbes and therefore

reflects the existence of living microbes (Luoet al 2009)The Celsis Lumac IMC kit which was usedin this research was designed to detect thepresence or absence of microorganisms in indus-trial samples andor screen for microbial levelcontamination Results are obtained withinminutes when more than 104 microorganismsper mL are present in the sample tested Belowthis limit or when samples have high quenchinglevels either a filtration step or an incubationstep is required for the microbial enrichment ofthe sampleThe assay procedure is based on the ATPbioluminescence technique which employs thelight-producing system of the firefly to detectmicroorganisms The test procedure consistsof the selective extraction of non-microbial (so-matic) ATP by a specific somatic cell extractant(NRS) The extractant makes the cell membranepermeable for molecules like ATP which leakout Microbial cells are not affected becausethey have cell walls Extracted non-microbialATP is hydrolysed by an ATPase (Somase)Microbial ATP is then measured in a CelsisLumac Biocounter after the addition of NRBand Lumit-PM The amount of light producedcorrelates with the amount of ATP in thesample shown as Relative Light Units (RLU)The light measurement is based on the followingreactionLuciferin + ATP + O2

Luciferase

Oxyluciferin + AMP+ PPi + CO2 + Light

To our knowledge Tzatziki has not beensubjected to any study of its microbial floraexcept for some recent works on its stabilityagainst pathogen microorganisms (Tsiraki ampSavvaidis 2014)The aim of the current study is to develop arapid and low cost ATP bioluminescence-sensingapproach to determine the microbial stability ofindustrial Tzatziki samples

78 Lalas et al

21 Material

The reagents (Lumit-PM Lumit-Buffer NRSNRB Somase) for the determination of themicrobial population with the bioluminescencemethod were obtained from Lumac BV (Land-graaf Netherlands) The reagents used for thedetermination of Total microbial count Lacto-bacillus spp yeast and coliforms (Total PlateCount agar MRS agar Malt Extract agar andViolet Red Bile agar) were obtained from MerckLtd (Darmstad Germany) The substratesused for the determination of Lactobacillus sppyeasts and coliforms (Ringer solution Sabouraudbroth and MacConkey broth) were obtained fromMerck Ltd (Darmstad Germany)

22 Tzatziki samples

Tzatziki salad was made using a simple home-made recipe with ingredients purchased fromthe local market Strained yogurt cucumbers(peeled and diced) olive oil cloves of garlic(peeled) vinegar salt and chopped fresh dill (at60 20 9 45 3 25 and 1 of the total weight ofthe product respectively) were mixed in a foodprocessor until well-combined The mixture wasdivided in two equal batches In the first batchsodium benzoate 005 and potassium sorbate005 were added as preservatives No preserva-tives were added to the second batch Each batchwas then divided into 100 g sub-batches whichwere then packed in sealed plastic containers Allthe containers were stored at 5 oC

23 Determination of themicrobial population usingfirefly luciferase for ATPmeasurement

The method used for the inoculation of salad isdescribed in Fig 1 The ATP assay was car-ried out as follows 200 microL of every sample wasaseptically transferred to a cuvette 100 microL ofSomaseNRS reagent was added to each sampleand the samples were left for a 45 min periodfor incubation Then 100 microL from each mixture

were taken and placed into cuvettes The cu-vettes were inserted into the Celsis Lumac Bio-counter 1800 (Landgraaf Netherland) and theassay started The instrument automaticallyadded 100 microL of NRB solution and the sampleswere left for 10 sec in order for the extraction totake place Finally 100 microL of Lumit-PM reagentwas added to every sample

24 Determination of ColonyForming Units (CFU)

The determination of CFU was carried out ac-cording to ISO 4833-1 (International StandardOrganization 2003a) standard methodology forthe Total Plate Counts ISO 21527-1 (Interna-tional Standard Organization 2008) for the enu-meration of yeasts and moulds ISO 5541-1 (In-ternational Standard Organization 1986) for theenumeration of coliforms and ISO 9232 (Inter-national Standard Organization 2003b) for theidentification of yogurt Lactobacilli Subsamplesof 25 g of each Tzatziki sample were diluted in225 mL of Ringer solution homogenized with astomacher (Bioscience International 11607 Ma-gruder Lane Rockville MD 20852-4365 USA)and then incubated for 1 h at 25 oC Suitable110 dilutions were prepared The counting ofTotal Mesophilic Flora and Lactobacillus sppwas performed by pouring aliquots of the dec-imal dilutions in Total Plate Count agar andMRS agar respectively For yeast counting 25g of each sample was homogenized in 225 mL ofSabouraud broth and incubated at 35 oC for 24h Decimal dilutions of Sabouraud broth wereprepared and poured in plates for total viableyeast counting using Malt Extract agar Forcoliform counting 25 g of each sample was ho-mogenized in 225 mL of MacConkey broth andincubated at 30 oC for 24 h Decimal dilutions ofMacConkey broth were also prepared and pouredin plates for coliform counting using Violet RedBile agar The incubation of Violet Red Bile agarsamples was carried out at 30 oC for 48 h and ofMalt Extract agar at 25 oC for 5 days After theincubation the results were expressed in colonyforming units (CFU) gminus1 based on the averagecount of a triplicate determination

Figure 1 Method for the inoculation of salad

Results represent the average of three simulta-neous assays Statistical significance of the dif-ferences between mean values was assessed byANOVA p lt 005 was considered as statisticallysignificant

The microbiological analyses were carried outon various batches of Tzatziki using each timethree samples from each batch for each measure-ment in order to evaluate the reproducibility ofthe method There was no significant differencebetween the different batches The curves pre-sented a similar behavior in all cases (batches)As expected the Tzatziki salad without preser-vatives initially gave RLU values higher (79532)than the same salad with preservatives (43198)The potassium sorbate and the sodium benzoateused in the recipe appeared to suspend the ac-tion of microorganisms In both cases the RLUvalues decreased day after day and 10 days latertended to zero (Fig 2)

The same procedure took place after incuba-tion for the two selective mediums in orderto have information about spoilage microorgan-isms Incubation was carried out because theinitial microbial count of yeasts and coliformswas lower than 104 microorganisms per mL ofsample As expected the Tzatziki salad with-out preservatives after incubation in two differentsubstrates (Fig 3) MacConkey and Sabouraudgave RLU values higher (9488 and 16176) thanthe salad with preservatives (12780 and 12005)respectively The microbial count in Sabouraudmedium which is selective for the growth ofyeasts compared to MacConkey (selective for co-liforms) was higher because the cells of yeasts arebigger than those of bacteria and contain moreATP per cell and hence produce higher RLU val-ues (Deligaris 1981) The amount of ATP in mi-crobial cells may differ depending on their phys-iological state and types of cells present Forexample injured or starved microbial cells maycontain approximately 10-30 of ATP presentin healthy cells and yeast cells contain approxi-mately 100 times more ATP than bacterial cells(Mendonca Juneja amp Daraba 2014) Gener-

80 Lalas et al

Figure 2 RLU values of Tzatziki with and without preservatives (Ringer solution)

ally coliforms yeasts and fungi that existed inthe system were given small possibility to growdue to the higher number of Lactobacilli presentthat function competitively against them Thus(Fig 2) the decrease in population is progressivewhile the population of coliforms (Fig 3a) andyeasts (Fig 3b) decreases abruptly The corre-lation between CFU and RLU measurements ofTzatziki with and without preservatives appearin Fig 4In the two selective substrates differences ap-peared between the two methods of microbialcount (RLU and CFU) (Fig 5) While RLU val-ues were roughly at the same level the CFU val-ues presented significant differences (p lt 005)Particularly the CFU values in the Sabouraudbroth were initially lower than those in the Mac-Conkey broth This is explained by the size of thecells of yeasts that are bigger than those of thebacteria and contain more ATP per cell (Men-donca et al 2014)

The correlation between RLU and CFU gminus1

of salad in Ringer Solution MacConkey andSabouraud was almost linear in all casesIn yogurt generally because of the low pH valueand the high number of Lactobacilli the growthof other bacteria (proteolytical coliforms) oryeasts is not being allowed However yogurtis sensitive to alterations from fungi especiallyin the areas that are in contact with air Themicrobial flora of Tzatziki is mainly composedof Lactobacilli originated from yogurt (Tsir-aki amp Savvaidis 2014) and the results showedthat Tzatziki salad presented an extreme sta-bility from the 2nd day of its preparation un-til the last day (15th) of experiments Lacto-bacilli and other microorganisms were decreasedand this was possibly due to the combinationof ingredients (garlic cucumber) Allicin andits derivative products (diallyl disulfide dial-lyl trisulfide) which are found in garlic essen-tial oils have shown good antimicrobial activities

Figure 3 RLU values of Tzatziki with and without preservatives after incubation in (a) MacConkeyand in (b) Sabouraud for 24 h at 35 oC

82 Lalas et al

Figure 4 RLU and CFU gminus1 counts of Tzatziki (a) with and (b) without preservatives (Ringer solution)

Figure 5 RLU and CFU gminus1 counts of Tzatziki with preservatives incubated in (a) microacConkey and in(b) Sabouraud for 24 h at 35 oC

84 Lalas et al

(Kim Huh Kyung amp Kyung 2004) Cucum-ber is mainly responsible for the ldquofresh-greenrdquoaroma of fresh vegetables which characterizesTzatziki Aroma compounds are plant secondaryvolatiles which are produced when cucumber isfinely chopped in order to be incorporated intothe yogurt Secondary volatiles apart from theirsensory contribution can influence the micro-bial stability depending on their concentrationcomposition and type of plant material of ori-gin In some cases the enzymatically formedsecondary volatiles can accumulate to concentra-tions which are sufficient to decrease the micro-bial population There are examples such asthe case of cucumber where the main charac-teristic aroma predominates only when the en-zymes are allowed to react on decompartmenta-tion of the plant tissue by cutting and homog-enizing (Schrodter 1984) Green aldehydes andgreen alcohols are the compounds responsible forthe green flavor note They are produced fromlinolenic and linoleic acid in wounded plant tis-sues (Galliard 1978 Tressl amp Drawert 1973) Alarge number of enzymes are released after cellmembrane damage (Galliard 1978) Autolysisgives free linolenic and linoleic acid Lipoxyge-nase action on the above-mentioned acids givesrise to linolenic and linoleic hydroperoxides Thetwo hydroperoxides have antimicrobial activity(Brooks amp Watson 1985) Lyase action on thehydroperoxides is responsible for the formationof (E )-2-Hexenal (E )-2-Nonenal and(EZ )-26-Nonadienal These compounds are the basisof green natural aroma at low thresholds (007microg Lminus1) It has been proved that the above-mentioned aldehydes also possess fungistatic ac-tion in the gas phase (Gueldner Wilson amp Heidt1985 Huhtanen amp Guy 1984)Sulfur-containing volatiles are normally formedduring crushing of the plant material In Al-lium plants essential oils possess antimicrobialactivity This anti-yeast activity (Wills 1956) isprobably due to the high allicin content The ef-fect of allicin on triose-phosphate-dehydrogenasein microbial cell metabolism has been demon-strated by Barone and Tansey (1977) Inhibi-tion of several sulfhydryl metabolic enzymes wasobserved The biogenesis of allicin was summa-rized by Dourtoglou (1986) The precursors andthe products possess antifungal activity (Conner

amp Beuchat 1984 Huhtanen amp Guy 1984)Generally the preservation of such productsis based mainly on their high acidity and onchemical preservatives employed to inhibit thegrowth of spoilage and pathogenic microorgan-isms However consumers demand reduced lev-els of additives in foods and consider their useundesirable Although the safety of these itemsis supposed to be ensured primarily by their lowpH several pathogens namely E coli O157H7L monocytogenes and Salmonella spp havebeen reported to survive or even grow in thesefoods Usually the pathogens in such productscome from the raw ingredients as well as due tocontamination from the processing environmentand packaging operation (Panagou Nychas ampSofos 2013)That is why good manufacturing practice mustbe followed in order to minimize any possi-ble cross-contamination In the marketplaceand consumersrsquo homes Tzatziki salad must bekept refrigerated and handled with stringent hy-gienic practices to prevent cross-contaminationby other food products or the environmentAs indicated by the results chemical preser-vatives are not essential for the microbial sta-bilization of the product Specifically afterthe 2nd day the Tzatziki salad showed its self-stabilization ability even without the use ofpreservatives Also it was shown that an ini-tial population of Lactobacillus spp of 100E+08CFU gminus1 and RLU measurements lower than400E+04 RLU in Greek traditional Tzatzikisalad are considered as a minimum specificationfor the microbial stability and safety of the prod-uctBecause of the good correlation that is pre-sented between CFU gminus1 measurements andRLU values the bioluminescence method ap-pears to be an appropriate rapid method to de-termine Tzatziki microbial stability during in-dustrial production practices

4 Conclusions

In conclusion Greek traditional Tzatziki saladis a self-stable product due to its high acidityplant derived natural preservatives and storageunder refrigeration and the rapid biolumines-cence method can be used to define the microbialstability of the product from the beginning onan industrial scale The method requires littleto no sample pretreatment as compared to con-ventional approaches and it can detect the totalbacterial viability quickly and give an excellentaid in nonspecific detection of bacterial contam-inants

Acknowledgements

The authors would like to thank Mrs MariaAlexandraki technical personnel at Departmentof Food Technology for her guidance on the anal-ysis by the bioluminescence method

References

Barone F E amp Tansey M R (1977) Isolationpurification identification synthesis andkinetics of activity of anti-candidal compo-nent of allium-sativum and a hypothesisfor its mode of action Mycologia 69 (4)793ndash825 doi1023073758870

Bottari B Santarelli M amp Neviani E (2015)Determination of microbial load for differ-ent beverages and foodstuff by assessmentof intracellular atp Trends in Food Scienceamp Technology 44 (1) 36ndash48 doi101016jtifs201502012

Brooks C J W amp Watson D G (1985) Phy-toalexins Natural Product Reports 2 (5)427ndash459 doi101039np9850200427

Cho M J Buescher R W Johnson Mamp Janes M (2004) Inactivation ofpathogenic bacteria by cucumber volatiles(ez)-26-nonadienal and (e)-2-nonenalJournal of Food Protection 67 (5) 1014ndash1016 doi1043150362-028X-6751014

Conner D E amp Beuchat L R (1984) Effectsof essential oils from plants on growth offood spoilage yeasts Journal of Food Sci-

ence 49 (2) 429ndash434 doi101111j1365-26211984tb12437x

Croft K P C Juttner F amp SlusarenkoA J (1993) Volatile products of thelipoxygenase pathway evolved fromphaseolus-vulgaris (l) leaves inocu-lated with pseudomonas-syringae pv-phaseolicola Plant Physiology 101 (1)13ndash24 doi101104pp101113

Dourtoglou V (1986) Production of naturalflavourings perfumes and other fine chem-icals by infected plants Symbiosis (USA)2 189ndash200

Galliard T (1978) Lipolytic and lipoxygenaseenzymes in plants and their action inwounded tissues Biochemistry of WoundedPlant Tissues 155ndash201

Gueldner R C Wilson D M amp HeidtA R (1985) Volatile compounds inhibit-ing aspergillus-flavus Journal of Agricul-tural and Food Chemistry 33 (3) 411ndash413doi101021jf00063a022

Harris J C Cottrell S L Plummer S ampLloyd D (2001) Antimicrobial propertiesof allium sativum (garlic) Applied Micro-biology and Biotechnology 57 (3) 282ndash286doi101007s002530100722

Hawronskyj J M amp Holah J (1997) Atp Auniversal hygiene monitor Trends in FoodScience amp Technology 8 (3) 79ndash84 doi101016S0924-2244(97)01009-1

Huhtanen C N amp Guy E J (1984) Antifungalproperties of esters of alkenoic and alkynoicacids Journal of Food Science 49 (1) 281ndashamp doi101111j1365-26211984tb13726x

International Standard Organization (1986)5541-1 - Milk and milk products - Enumer-ation of coliforms - Part 1 Colony counttechnique at 30 degrees C Geneva BSI

International Standard Organization (2003a)4833-1 - Microbiology of food and animalfeeding stuffs - Horizontal method for theenumeration of microorganisms - Colony-count technique at 30 degrees C GenevaBSI

International Standard Organization (2003b)9232 - Yogurt - Identification of charac-teristic microorganisms (Lactobacillus del-brueckii subsp bulgaricus and Streptococ-cus thermophilus) Geneva BSI

86 Lalas et al

International Standard Organization (2008)21527-1 - Microbiology of food and animalfeeding stuffs - Horizontal method for theenumeration of yeasts and moulds - Part 1Colony count technique in products withwater activity greater than 095 GenevaBSI

Kim J W Huh J E Kyung S H amp KyungK H (2004) Antimicrobial activity ofalk(en)yl sulfides found in essential oils ofgarlic and onion Food Science and Biotech-nology 13 (2) 235ndash239

Luo J Liu X Tian Q Yue W Zeng JChen G amp Cai X (2009) Disposablebioluminescence-based biosensor for detec-tion of bacterial count in food AnalyticalBiochemistry 394 (1) 1ndash6 doi101016jab200905021

Mendonca A F Juneja V K amp Daraba A(2014) Total viable counts mdash metabolic ac-tivity tests Elsevier

Panagou E Z Nychas G-J E amp Sofos J N(2013) Types of traditional greek foodsand their safety Food Control 29 (1) 32ndash41 doi101016jfoodcont201205050

Schrodter R (1984) Chromatographic studies ofbiogenesis of plant volatiles (H HeidelbergEd)

Skandamis P N Davies K W McClure P JKoutsoumanis K amp Tassou C (2002)A vitalistic approach for non-thermal inac-tivation of pathogens in traditional greeksalads Food Microbiology 19 (5) 405ndash421doi101006yfmic495

Sotiroudis G Melliou E Sotiroudis T Gamp Chinou I (2010) Chemical analysisantioxidant and antimicrobial activity ofthree greek cucumber (cucumis sativus)cultivars Journal of Food Biochemistry34 (s1) 61ndash78 doi10 1111 j 1745 - 4514 200900296x

Tressl R amp Drawert F (1973) Biogenesisof banana volatiles Journal of Agricul-tural and Food Chemistry 21 (4) 560ndash565doi101021jf60188a031

Tsiraki M I amp Savvaidis I N (2014) Cit-rus extract or natamycin treatments onldquotzatzikirdquo-a traditional greek salad FoodChemistry 142 416ndash422 doi10 1016 j foodchem201307087

Wills E D (1956) Enzyme inhibition by al-licin the active principle of garlic Bio-chemical Journal 63 (3) 514ndash520 doi101042bj0630514

Moisture Sorption Isotherm and Thermal Characteristics ofFreeze-Dried Tuna

Mohammad Shafiur Rahmana Mohammed Khalfan Al-Khusaibia Kutaila AbbasAL-Farsia Ismail Mohamed Al-Bulushia Aisha Abushelaibib and Nasser

Al-Habsia

a Department of Food Science and Nutrition College of Agricultural and Marine Sciences Sultan QaboosUniversity P O Box 34-123 Al-Khod 123 Oman

b Food Science Department College of Food and Agriculture UAE UniversityCorresponding authormohamedksqueduomTel +968 2414 3682Fax +968 2441 3418

Abstract

Water activity is considered an important factor in assessing the stability of food Understandingthe relationship between water activity and equilibrium moisture content (moisture sorption isotherm)benefits food processing in terms of modeling of drying and estimation of shelf life In addition glasstransition helps to quantify molecular mobility which helps in determining the stability of food Theaim of this study was to determine the moisture sorption isotherm and thermal characteristics of freeze-dried tuna These characteristics will help in determining the monolayer moisture and glassy state ofthe product at which food is considered most stable Moisture sorption isotherm at 20 oC and thermalcharacteristics (over a wide temperature range ie from -90 to 250 oC) of freeze-dried tuna flesh weremeasured Isotherm data were modeled by BET (Brunauer-Emmett-Teller) and GAB (Guggenheim-AndersonndashDe Boer) models The GAB and BET monolayer water values were determined as 0052and 0089 g gminus1 dry-solids (dry-basis) respectively In the case of samples at moisture contents above010 g gminus1 (wet basis) DSC (Differential Scanning Calorimetry) thermograms showed two-step statechanges (ie two glass transitions) one exothermic peak (ie molecular ordering) and another en-dothermic peak (ie solids-melting) However the sample at moisture content of 0046 g gminus1 showedthree-step state changes (ie three glass transitions) The multiple glass transition could be explainedby the natural heterogeneity of tuna flesh and inhomogeneity due to molecular incompatibility of thedifferent compositions The moisture content did not affect the first glass transition temperature northe exothermic peak (pgt005) whereas the third glass transition temperature decreased (ie plasti-cized) with increasing moisture content (plt005) The solids-melting peak temperature decreased andenthalpy increased with decreasing moisture content (plt005)

Keywords Dried fish Sorption isotherm Tuna Thermal transition Solids-melting

1 Introduction

Water interacts with different ingredients presentin foods This interaction is important in de-termining the physical chemical and microbial

stability of the foods (Bhandari amp Howes 1999Rahman 2006 Sablani Kasapis Rahman Al-Jabri amp Al-Habsi 2004) Water activity is con-sidered an important property to determine the

88 Rahman et al

stability of foods compared to the total amountof water (Labuza Mcnally Gallagher Hawkes ampHurtado 1972 Roos 1993 Scott 1953) There-fore the water activity concept is commonly usedto determine the stability of foods in relation tomicrobial growth lipid oxidation non-enzymaticand enzymatic activities and the texture of foods(Labrousse Roos amp Karel 1992 Shi ZhaoChen Li amp Xue 2009 Shimada Roos amp Karel1991) In addition it is used as a tool in foodprocessing operations such as modeling of dry-ing processes estimating shelf-life and choos-ing suitable packaging material for a food prod-uct (Orlien Risbo Andersen amp Skibsted 2003)Water activity is related to the moisture contentin the sorption isotherm (ie the relationship ofwater activity and equilibrium moisture content)(Delgado amp Sun 2002)Recently it has been observed that the water ac-tivity concept is not sufficient to determine sta-bility of food thus glass transition has been pro-posed to quantify molecular mobility (Delgadoamp Sun 2002 Karel Buera amp Roos 1993 Kar-mas Buera amp Karel 1992 Sablani et al 2004Slade amp Levine 1988 1991) Foods are consid-ered most stable when they are stored at or belowtheir glass transition because of the low molecu-lar mobility of the reactants in a highly viscousmedium This reduced mobility could signifi-cantly retard microbial and chemical reactions(Meste Champion Roudaut Blond amp Simatos2002 Rahman 2006) As the storage tempera-ture increases above the glass transition temper-ature foods become less stable with increasedmolecular mobility (Goula Karapantsios Achil-ias amp Adamopoulos 2008 Sablani et al 2007Syamaladevi Sablani Tang Powers amp Swanson2010 Tonon et al 2009) The water activity andglass transition concepts could be combined todetermine the stability of foods (Rahman 2009Rahman amp Al-Saidi 2017 Shi et al 2009)Fresh tuna fish has a short shelf life unless it isstored at very low temperatures or dried (Or-lien et al 2003) It is important to measurethe thermal characteristics and sorption isothermin order to determine efficient processing andstorage stability of dried tuna (Roos amp Karel1991) Glass transition of different types of fishand other muscle tissues like chicken and beefare reported in the literature (Brake amp Fennema

1999 Sablani et al 2007 Tolstorebrov Eike-vik amp Bantle 2014) Harnkarnsujarit Kawaiand Suzuki (2015) measured the glass transi-tion and water sorption isotherm of water extractfrom freeze-dried tuna Most probably they at-tempted to simplify the complex intact struc-ture into a sample of only soluble componentsMoreover their temperature range in Differen-tial Scanning Calorimetry (DSC) was within -80 to 80 oC without measuring solids-melting orsolids-decomposition However there are neg-ligible data available on the thermal character-istics of dried fish (ie whole sample) over awide range of temperature (ie including solids-melting or solids-decomposition) (Suresh Al-Habsi Guizani amp Rahman 2017) The struc-tural collapse during drying processes influencedwater uptake during rehydration which couldbe related to a glassy state of dried fish (Mar-ques Prado amp Freire 2009) The hygroscopic-ity was also affected by different drying methodsand affected the sorption isotherm (Caparino etal 2012) The aim of this study was to deter-mine the moisture sorption isotherm and thermalcharacteristics (ie -90 to 250oC) of freeze-driedtuna These characteristics could be used todetermine monolayer moisture and glassy statewhich is considered the best stability during pro-cessing and storage

21 Sample Preparation

Fresh yellowfin tuna (Thunnus albacares) waspurchased from a local fish market and brought(iced) within 30 min to the laboratory Tunawas harvested overnight by the fisherman andice-stored in the boat Tuna was landed in themorning and displayed at the beach fish mar-ket (ie around 10 hr after harvest) The fil-lets were prepared and diced at room tempera-ture (ie 20 oC) into 3 cm cubes and placedinto several 50 ml plastic containers The dicedcubes were then frozen at ndash 40 oC for 24 h andfreeze-dried at room temperature (20 oC) (iesample enter at -40 oC and dried at 20 oC) un-der vacuum of 200 Pa for 96 h using an EdwardsK4 Freeze Dryer (Corawky Crawley England)

Stability of freeze-dried tuna 89

Freeze-dried tuna was then ground into powderusing a KMF grinder (KIKA Werke Wilming-ton USA) running at 6000 rpm and stored at-20 oC until used for the DSC experiments

22 Moisture Sorption Isotherm

The isopiestic method was used to developthe moisture sorption isotherm (Rahman ampHamed Al-Belushi 2006) In this method 1 g offreeze-dried tuna powder was placed in an openbottle and stored in an air-sealed glass jar con-taining a saturated salt solution (for examplelithium chloride) in a 50 ml beaker to maintaina specific relative humidity environment in thejar Thymol in a 5 ml beaker was also placedinside the jar to prevent the growth of moldsduring storage The salts used were lithiumchloride potassium acetate magnesium chlo-ride potassium carbonate magnesium nitratesodium nitrate sodium chloride potassiumchloride and potassium nitrate (water activity01 - 09) and the water activity values weretaken from Rahman (1995) The air-sealed glassjars were then stored at 20oC for 3 to 4 weeksuntil equilibrium was reached This temperaturewas close to commonly used room temperaturestorage Equilibrium conditions were achievedwhen there was no loss or gain of mass (plusmn001 g)for two consecutive days Weight was monitoredusing an analytical balance (OHAUS modelAS200 Florham Park USA) The moisturecontent was determined by an oven methodaccording to AOAC (2005) by drying 1 g of thesample in a conventional hot-air oven at 105 oCfor at least 20 h until a constant weight wasachieved The moisture content in the sorptionisotherm data was expressed in dry basis ascommonly presented in the literature Eachwater activity data collection was replicated 3timesThe moisture sorption isotherms were mod-eled using BrunauerndashEmmendashTeller (BET)(Brunauer Emmet amp Teller 1938) andGuggenheimndashAndersenndashde Boer (GAB) equa-tions (Bizot 1983) BET and GAB equationsare as follows

Mw =MbmBaw

(1 minus aw)[1 + (B minus 1)aw](1)

Mw =MgmCKaw

(1 minusKaw)(1 minusKaw + CKaw)(2)

where Mw Mbm and Mgm are the moisture con-tent at any water activity BET-monolayer watercontent and GAB monolayer water content re-spectively (all in g gminus1 dry-solids) aw is the wa-ter activity B is the parameter of BET modeland C and K are the parameters of the GABmodel The BET equation has been widely usedand been found to give a good fit when water ac-tivities are less than 045 The GAB model hasbeen found to be successful up to water activi-ties as high as 09 (Basu Shivhare amp Mujumdar2006 Al-Muhtaseb McMinn amp Magee 2002)

23 Differential ScanningCalorimetry

Differential Scanning Calorimetry (DSC) wasused to determine state and phase transitionsin freeze-dried tuna (Rahman Al-Saidi Guizaniamp Abdullah 2010) The samples containingdifferent moisture (ie 010 015 and 020 ggminus1 sample wet basis) were prepared from thefreeze-dried sample (initial moisture of 0046 g100gminus1 sample wet basis) by spraying the de-sired amount of distilled water and mixing thor-oughly with a spatula The samples were thenstored at 4 oC for 24 h for equilibration Afterequilibration samples were stored at - 20 oC for24 hours until they were used for DSC measure-ments The samples were analyzed thermally ina DSC (Q200 TA Instrument New Castle DEUSA) and the thermal characteristics were de-termined The instrument was calibrated usingindium with melting temperature of 1565 oC andenthalpy of 285 J gminus1Samples of 3 to 5 mg of freeze-dried tuna wereplaced in Tzero aluminum hermetic pans andsealed The pans containing the samples werecooled from 25 to -90 oC at a rate of 5oC minminus1

and then heated to 250 oC at 10 oC minminus1 undernitrogen (carrier gas) purged at 50 mL minminus1An empty pan was used as a reference Thermo-grams were obtained in triplicates for the sam-ples with different moisture contents and thesewere analyzed for exothermic (ie ordering inmolecules) and endothermic (ie solids-melting)peaks and shifts in the thermogram (ie glass

90 Rahman et al

transition) The moisture content of samplesused for the DSC analysis is expressed in wetbasis as commonly presented in the literature

Each value is presented as mean plusmn standard devi-ation A non-linear regression analysis was usedto estimate the parameters of the isotherm mod-els (ie Guggenheim-Anderson-de Boer GABand Brunauer-Emmett-Teller BET) The signif-icant effect of moisture on the thermal character-istics was determined using ANOVA (MicrosoftExcel 2010)

Figure 1 shows the moisture sorption isothermof tuna flesh at 20 oC The data were fitted withBET and GAB models and the parameters arepresented in Table 1 with their prediction accu-racy The BET and GAB monolayer water val-ues were 0089 and 0052 g gminus1 dry-solids re-spectively and samples at BET monolayer watercould be considered most stable since deteriora-tive chemical reactions are minimal (Rahman ampHamed Al-Belushi 2006 Sablani et al 2007)However the GAB model is useful for predictingwater activity over the complete range of mois-ture up to 090 In the case of king fish flesh (fatcontent 0039 g gminus1 sample) BET and GABmonolayer water content were found to be 0036and 0052 g gminus1 dry-solids respectively (Sablaniet al 2007) The same authors analysed fat-free king fish and they found values of 0048and 0086 for the BET and GAB models respec-tively This indicated that fat content marginallyincreased the monolayer values Delgado andSun (2002) reported values of 0073 and 0068g gminus1 dry-solids respectively for chicken meat(fat content 0004 g gminus1 sample) The mainvariations of the monolayer values depended onthe types of proteins It was reported that fatcontent had very small or negligible effects as aplasticizer when added to protein (KalichevskyJaroszkiewicz amp Blanshard 1992 Shaw Mona-han OrsquoRiordan amp OrsquoSullivan 2002) Fat free

protein such as gelatine and collagen showedhigh BET and GAB monolayers 012 and 010 ggminus1 dry-solid respectively (Timmermann Chir-ife amp Iglesias 2001)

Figure 1 Experimental moisture sorptionisotherm of freeze-dried tuna and predicted GABmodel

32 Thermal Characteristics

A typical DSC thermogram for freeze dried tunais shown in Figure 2 (moisture content 0046 ggminus1 sample wet basis) Three letters A B andC represent three transitions however they couldnot be visualized clearly due to the high exother-mic and endothermic enthalpy (ie high heatflow) Therefore these regions were expandedwith the appropriate scales and are shown in Fig-ure 3 Figure 2 also shows an exothermic peak(ie molecular ordering) and an endothermicpeak (ie solids-melting) The sample at mois-ture 0046 g gminus1 showed three glass transitionswhile other samples showed only two glass tran-sitions This additional shift (ie Tgi 976plusmn57oC Tgp 1032plusmn82 oC Tge 1047plusmn82 oC ∆Cp400plusmn120 J kgminus1 Kminus1) was observed between thefirst and the third glass transition Table 2 showstwo glass transitions (first and third) as a func-tion of moisture content At moisture content0046 (g 100gminus1 sample) the first and third glasstransitions were observed at 283 and 1485 oCrespectively (Table 2) Similarly Tolstorebrov etal (2014) observed variation in the glass tran-sition onset temperature from 366 to 403 oC

in the case of vacuum dried cod salmon troutherring and mackerel at a moisture of 0055 ggminus1 sample This transition was close to the val-ues of the first transition observed in the caseof tuna In the case of king fish muscle samples(moisture content 0035 g gminus1 sample) Sablaniet al (2007) observed two glass transitions oneat -110 oC and the other at 747 oC In the caseof air-dried tuna (moisture 0046 g gminus1 sam-ple) (Rahman Kasapis Guizani amp Al-Amri2003) observed a glass transition at 671 oC asmeasured by mechanical thermal analysis Theirvalue was relatively close to the second glasstransition 976 oC In the case of water-solubleextract of freeze-dried tuna one glass transitionwas observed and it was plasticized with the in-crease of water content (ie more hydrophiliccomponents) (Harnkarnsujarit et al 2015) Thewhole freeze-dried sample was more rigid andamorphous and the plasticization process washindered although water content increased Ear-lier it was identified that multicomponent foodsystems could show multi-glass transitions (Bil-iaderis 1991 Hashimoto Hagiwara Suzuki ampTakai 2003)

Table 1 BET and GAB model parameters formoisture sorption isotherm of dried tuna

Model Parameter

GAB Mbm 0052 g gminus1 dry solidK 0926C 1936r2 099SE 0013p-value lt005

BET Mgm 0089 g gminus1 dry solidB 4038r2 095SE 00056p-value lt005

Moisture content in freeze-dried tuna did notaffect the first glass transition temperature (p gt005) and specific heat change at that transition(p gt 005) whereas the third glass transitiontemperature decreased (p lt 005) and specific

Figure 2 A typical DSC thermogram of freeze-dried tuna at a moisture content 46 g 100g-1sample A first glass transition B second glasstransition C third glass transition D exother-mic peak (ie molecular ordering E endother-mic peak (ie solids-melting)

heat change increased with the increase of mois-ture content (p lt 005) (Table 2) Similarly inthe case of king fish Sablani et al (2007) did notobserve any plasticization (ie decrease of glasstransition) with water for the first glass transi-tion In the case of the third glass transition (iein tuna) higher water contents decreased glasstransition due to plasticization and increasedspecific heat change which suggested the forma-tion of hydrogen bonding causing the develop-ment of disordered structure (ie more amor-phous domain) (Rahman amp Al-Saidi 2017) Inthe case of sugar-based foods containing freezablewater two glass transitions were commonly ob-served one at low temperature and another justbelow ice-melting temperature (Rahman 2004)In the case of tuna the low glass transition tem-perature did not plasticize with increasing wa-ter content However glass transition tempera-ture before melting decreased with the increaseof water content (ie plasticized) In the caseof rice Cao Nishiyama and Koide (2004) ob-served three transitions and the second transi-tion showed a significant decrease with increas-ing moisture while first and third transitions re-mained stable with increasing moisture Theyhypothesized that the second transition could bethe glass transition as plasticization (ie de-crease of glass transition) occurred at this tran-sition The multiple glass transitions could be

92 Rahman et al

Figure 3 Expansion of the glass transitions as marked in Figure 2 A first glass transition B secondglass transition C third glass transition

Table 2 First and third glass transitions of freeze-dried tuna

First Glass Transition Third Glass Transition

Xw Tgi Tgp Tge ∆Cp Tgi Tgp Tge ∆Cp

(oC) (oC) (oC) (J kg-1 oCminus1) (oC) (oC) (oC) (J kgminus1 oCminus1)0046 283plusmn05 299plusmn04 341plusmn11 300plusmn50 1485plusmn12 1486plusmn11 1488plusmn12 510plusmn1700100 309plusmn07 321plusmn06 355plusmn09 184plusmn3 1382plusmn01 1382plusmn01 1384plusmn01 530plusmn1800150 306plusmn08 325plusmn20 357plusmn13 207plusmn8 1341plusmn23 1341plusmn23 1342plusmn24 630plusmn2300200 319plusmn21 338plusmn27 357plusmn16 192plusmn12 1317plusmn25 1318plusmn26 1320plusmn26 760plusmn240

pgt005 pgt005 plt005 plt005

NoteEach data point is presented as mean plusmn Standard deviation and p-value was set at 005 for determiningsignificanceXw water content (g waterg sample) Tgi initial glass transition temperature Tgp peak glass transitiontemperature Tge end of glass transition temperature ∆Cp change in specific heat capacity

Table 3 Characteristics of exothermic and endothermic (ie solids-melting) peaks of freeze-dried tuna

Xw Tea Tep Tee ∆He Tmi Tmp Tme ∆Hm

(oC) (oC) (oC) (kJ kgminus1) (oC) (oC) (oC) (kJ kgminus1)

0046 1555plusmn04 1570plusmn05 1614plusmn04 361plusmn11 2022plusmn14 2032plusmn16 2179plusmn12 85plusmn70100 1555plusmn02 1570plusmn02 1617plusmn02 371plusmn23 1884plusmn36 1884plusmn35 2027plusmn25 151plusmn90150 1554plusmn02 1569plusmn02 1609plusmn14 313plusmn34 1758plusmn38 1816plusmn50 1921plusmn36 166plusmn60200 1555plusmn04 1569plusmn05 1611plusmn08 346plusmn26 1768plusmn08 1767plusmn18 1896plusmn30 299plusmn5

NoteEach data point is presented as mean plusmn Standard deviation and p-value was set at 005 for determiningsignificanceTmi peak onset temperature Tmp peak maximum temperature Tme peak end temperature ∆H changein enthalpy Other variables are explained in Table 2

explained by the natural heterogeneity of biolog-ical materials such as meat and fish muscle andthe inhomogeneity due to molecular incompati-bility in complex foods (Orlien et al 2003)Table 3 shows the exothermic and endothermicpeaks as a function of moisture content It wasobserved that the moisture content did not af-fect the exothermic peak and enthalpy (p gt005) The average values of the peak tem-perature and enthalpy were 1555plusmn01 oC and348plusmn25 kJ kgminus1 respectively Similarly in thecase of freeze-dried broccoli Suresh et al (2017)observed an exothermic peak before the solids-melting endothermic The exothermic peak in-dicated that there was an order in the moleculesat the higher temperature before melting Theendothermic peak can be considered as solids-melting (ie softening of the solid phase as pro-teins denature with the release of heat) Thepeak temperature decreased and enthalpy in-creased with the decrease of moisture (p lt 005)Protein denaturation can be noticed as an en-dothermic peak in the DSC curve in the tem-perature range of 40-80 oC in case of fresh foodsamples (ie high moisture content) (Hashimotoet al 2003) The peak temperature increased(eg 120 oC) as sample moisture content de-creased It was suggested that protein denatu-ration significantly affected the glass transitionof protein-rich food (Hashimoto Suzuki Hagi-wara amp Takai 2004 Sochava amp Smirnova 1993Wright Leach amp Wilding 1977) The increaseof enthalpy during solids melting could be ex-plained by the formation of more hydrogen bonds

at higher water content A similar trend was ob-served in the case of freeze-dried broccoli (Sureshet al 2017)Considering the water activity concept stabilityof freeze dried samples could be achieved if storedat 0089 g gminus1 dry-solids at room temperature(ie 20 oC) The two or three state changes (ieglass transitions) indicated that different types ofphysico-chemical changes could occur in the tunasample The lower one is important if freeze-dried fish is to be stored at room temperatureand higher glass transition is relevant in roastingand frying processes Further studies need to beconducted for determining the types of chemicalreactions that could occur at the different stagesof glass transitions

4 Conclusions

Moisture sorption isotherm of freeze-dried tunawas measured and modeled by BET and GABequations The BET and GAB monolayer watercontent were 0052 and 0089 g gminus1 dry-solids re-spectively In this work isotherm was measuredat 20 oC (ie close to usual sale display around18-20 oC) However further work at extreme con-ditions such as 10 oor 30 oC could provide en-riched information on the effects of temperatureon isotherm For a sample with moisture con-tent of 0046 g gminus1 three glass transitions wereobserved at different temperatures first one on-set at 283 oC second one at 976 oC and thirdat 1485 oC However samples at higher moisture

94 Rahman et al

content showed only two glass transitions one atlow and the other at higher temperature Thelower glass transition was un-affected by mois-ture content whereas higher temperature glasstransitions decreased with increasing moisturecontent Similarly solids-melting (ie proteindenaturation) decreased with increasing mois-ture content In addition specific heat changeat the glass transition and enthalpy at solids-melting increased with the increase of water con-tent The sample used in this study was powderto simplify sample handling and measurementThe thermal transitions may not be changed forpowder or intact solid samples however kinet-ics of moisture sorption would be faster in thecase of powder samples although final equilib-rium conditions would be unaffected This pointneeds to be considered when freeze-dried samplesare stored

Acknowledgements

This project was funded by SQU-UAEU Project(CLSQU-UAEU1503) and authors would liketo thank Sultan Qaboos University and UnitedArab Emirates University for their support dur-ing this collaborative work

References

AOAC (2005) Solids (Total) and Moisture inFlour Method 92510 Official Methodsof Analysis 18th Edition AOAC Interna-tional Gaithersburg

Basu S Shivhare amp Mujumdar A (2006)Models for sorption isotherms for foods Areview Drying Technology 24 (8) 917ndash930doi10108007373930600775979

Bhandari B amp Howes T (1999) Implicationof glass transition for the drying and sta-bility of foods Journal of Food Engineer-ing 40 (1-2) 71ndash79 doi10 1016 S0260 -8774(99)00039-4

Biliaderis C G (1991) Non-equilibrium phasetransitions of aqueous starch systems InWater relationships in foods (pp 251ndash273)Springer

Bizot H (1983) Using the rsquogabrsquo model to con-struct sorption isotherms Physical Proper-ties of Foods 43ndash54

Brake N C amp Fennema O R (1999) Glasstransition values of muscle tissue Journalof Food Science 64 (1) 10ndash15 doi101111j1365-26211999tb09851x

Brunauer S Emmet P H amp Teller E (1938)Adsorption of gases in multimolecular lay-ers Journal of the American ChemicalSociety 60 (2) 309ndash319 doi10 1021 ja01269a023

Cao W Nishiyama Y amp Koide S (2004)Physicochemical mechanical and thermalproperties of brown rice grain with variousmoisture contents International Journal ofFood Science and Technology 39 899ndash906doi101111j1365-2621200400849x

Caparino O Tang J Nindo C Sablani SPowers J amp Fellman J (2012) Effect ofdrying methods on the physical propertiesand microstructures of mango (philippinersquocarabaorsquo var) powder Journal of FoodEngineering 111 (1) 135ndash148 doi101016jjfoodeng201201010

Delgado A amp Sun D-W (2002) Desorptionisotherms and glass transition temperaturefor chicken meat Journal of Food Engi-neering 55 (1) 1ndash8 doi10 1016 S0260 -8774(01)00222-9

Goula A Karapantsios T Achilias D ampAdamopoulos K (2008) Water sorptionisotherms and glass transition temperatureof spray dried tomato pulp Journal of FoodEngineering 85 (1) 73ndash83 doi101016jjfoodeng200707015

Harnkarnsujarit N Kawai K amp Suzuki T(2015) Effects of freezing temperature andwater activity on microstructure color andprotein conformation of freeze-dried bluefintuna (thunnus orientalis) Food and Bio-process Technology 8 (4) 916ndash925 doi101007s11947-014-1460-1

Hashimoto T Hagiwara T Suzuki T ampTakai R (2003) Study on the glass tran-sition of katsuobushi (boiled and driedbonito fish stick) by differential scanningcalorimetry and dynamic mechanical anal-ysis Fisheries Science 69 (6) 1290ndash1297doi101111j0919-9268200300757x

Hashimoto T Suzuki T Hagiwara T ampTakai R (2004) Study on the transitionfor several processed fish muscles and itsprotein fractions using differential scan-ning calorimetry Fisheries Science 70 (6)1144ndash1152 doi101111j1444-2906200400916x

Kalichevsky M Jaroszkiewicz E amp BlanshardJ (1992) Glass transition of gluten 2The effect of lipids and emulsifiers In-ternational Journal of Biological Macro-molecules 14 (5) 267ndash273 doi10 1016 S0141-8130(05)80039-X

Karel K Buera M P amp Roos Y (1993) Thescience and technology of the glassy statein foods In J M V Blanshard amp P J Lill-ford (Eds) (Chap Effects of glass transi-tions on processing and storage pp 13ndash34)Nottingham University Press

Karmas R Buera P amp Karel M (1992) Effectof glass transition on rates of nonenzymaticbrowning in food Journal of Agriculturaland Food Chemistry 40 873 doi101021jf00017a035

Labrousse S Roos Y amp Karel M (1992) Col-lapse and crystallization in amorphous ma-trices with encapsulated compounds Jour-nal of Food Science 12 757ndash769

Labuza T Mcnally L Gallagher D HawkesJ amp Hurtado F (1972) Stability of in-termediate moisture foods 1 lipid oxida-tion Journal of Food Science 37 154ndash159doi101111j1365-26211972tb03408x

Marques L G Prado M M amp FreireJ T (2009) Rehydration characteristicsof freeze-dried tropical fruits LWT - FoodScience and Technology 42 (7) 1232ndash1237doi101016jlwt200902012

Meste M L Champion D Roudaut GBlond G amp Simatos D (2002) Glasstransition and food technology A criticalappraisal Journal of Food Science 67 (7)2444ndash2458 doi101111j1365-26212002tb08758x

Al-Muhtaseb A McMinn W A M ampMagee T R A (2002) Moisture sorp-tion isotherm characteristics of food prod-ucts A review Food and Bioproducts Pro-cessing 80 (2) 118ndash128 doi10 1205 09603080252938753

Orlien V Risbo J Andersen M amp Skib-sted L (2003) The question of high- orlow-temperature glass transition in frozenfish construction of the supplemented statediagram for tuna muscle by differentialscanning calorimetry Journal of Agricul-tural and Food Chemistry 51 (1) 211ndash217doi101021jf020355x

Rahman M (1995) Water activity and sorptionproperties of food chapter 1 Food Proper-ties Handbook 1ndash65

Rahman M (2004) State diagram of dateflesh using differential scanning calorimetry(dsc) International Journal of Food Prop-erties 7 (3) 407ndash428 doi10 1081 JFP -200032930

Rahman M (2006) State diagram of foods Itspotential use in food processing and prod-uct stability Trends in Food Science andTechnology 17 (3) 129ndash141 doi101016jtifs200509009

Rahman M (2009) Food stability beyond wateractivity and glass transtion Macro-microregion concept in the state diagram Inter-national Journal of Food Properties 12 (4)726ndash740 doi10108010942910802628107

Rahman M amp Hamed Al-Belushi R (2006)Dynamic isopiestic method (dim) Measur-ing moisture sorption isotherm of freeze-dried garlic powder and other potentialuses of dim International Journal of FoodProperties 9 (3) 421ndash437 doi10 1080 10942910600596134

Rahman M amp Al-Saidi G (2017) Exploringvalidity of the macro-micro region conceptin the state diagram Browning of raw andfreeze-dried banana slices as a function ofmoisture content and storage temperatureJournal of Food Engineering 203 32ndash40doi101016jjfoodeng201701017

Rahman M Al-Saidi G Guizani N amp Ab-dullah A (2010) Development of state di-agram of bovine gelatin by measuring ther-mal characteristics using differential scan-ning calorimetry (dsc) and cooling curvemethod Thermochimica Acta 509 (1-2)111ndash119 doi101016jtca201006011

Rahman S M Kasapis S Guizani N amp Al-Amri O S (2003) State diagram of tunameat Freezing curve and glass transition

96 Rahman et al

Journal of Food Engineering 57 (4) 321ndash326 doi101016S0260-8774(02)00346-1

Roos Y H (1993) Water activity and physicalstate effects on amorphous food stabilityJournal of Food processing and Preserva-tion 16 (6) 433ndash447 doi101111j1745-45491993tb00221x

Roos Y amp Karel M (1991) Applying state dia-grams to food processing and developmentFood technology 45 (12) 66 68ndash71 107

Sablani S Kasapis S Rahman M Al-Jabri A amp Al-Habsi N (2004) Sorptionisotherm and the state diagram for eval-uating stability criteria of abalone FoodResearch International 37 (10) 915ndash924doi101016jfoodres200405006

Sablani S Rahman M Al-Busaidi S GuizaniN Al-Habsi N Al-Belushi R amp SoussiB (2007) Thermal transitions of kingfish whole muscle fat and fat-free muscleby differential scanning calorimetry Ther-mochimica Acta 462 (1-2) 56ndash63 doi10 1016jtca200706008

Scott W J (1953) Water relations of S au-reus at 30ordmC Australian Journal of Biolog-ical Sciences 6 (4) 549ndash64 doi10 1071 BI9530549

Shaw N B Monahan F J OrsquoRiordan E Damp OrsquoSullivan M (2002) Effect of soya oiland glycerol on physical properties of com-posite wpi films Journal of Food Engineer-ing 51 (4) 299ndash304 doi10 1016 S0260 -8774(01)00071-1

Shi Q Zhao Y Chen H-H Li J-Z amp XueC-H (2009) Glass transition and state di-agram for freeze-dried horse mackerel mus-cle Thermochimica Acta 493 (1-2) 55ndash60doi101016jtca200904006

Shimada Y Roos Y amp Karel M (1991)Oxidation of methyl linoleate in amor-phous lactose based food model Journal ofAgricultural and Food Chemistry 39 637doi101021jf00004a001

Slade L amp Levine H (1988) Non-equilibriumbehavior of small carbohydrate-water sys-tems Pure and Applied Chemistry 601841ndash1864 doi101351pac198860121841

Slade L amp Levine H (1991) Beyond water ac-tivity Recent advances based on an alter-native approach to the assessment of food

quality and safety Critical Reviews in FoodScience and Nutrition 30 115ndash360 doi10108010408399109527543

Sochava I V amp Smirnova O I (1993) Heat ca-pacity of hydrated and dehydrated globularproteins denaturation increment of heatcapacity Food Hydrocolloids 6 (6) 513ndash524 doi101016S0268-005X(09)80075-1

Suresh S Al-Habsi N Guizani N amp Rah-man M (2017) Thermal characteris-tics and state diagram of freeze-driedbroccoli Freezing curve maximal-freeze-concentration condition glass line andsolids-melting Thermochimica Acta 655129ndash136 doi101016jtca201706015

Syamaladevi M R Sablani S Tang J Pow-ers J amp Swanson B (2010) Water sorp-tion and glass transition temperatures inred raspberry (rubus idaeus) Thermochim-ica Acta 503 90ndash96 doi10 1016 j tca 201003013

Timmermann E O Chirife J amp Iglesias H A(2001) Water sorption isotherms of foodsand foodstuffs Bet or gab parametersJournal of Food Engineering 48 (1) 19ndash31doi101016S0260-8774(00)00139-4

Tolstorebrov I Eikevik T amp Bantle M(2014) A DSC study of phase transition inmuscle and oil of the main commercial fishspecies from the North-Atlantic Food Re-search International 55 303ndash310 doi10 1016jfoodres201311026

Tonon R Baroni A Brabet C Gibert OPallet D amp Hubinger M (2009) Watersorption and glass transition temperatureof spray dried acai (euterpe oleracea mart)juice Journal of Food Engineering 94 (3-4) 215ndash221 doi101016jjfoodeng200903009

Wright D J Leach I B amp Wilding P (1977)Differential scanning calorimetric studies ofmuscle and its constituent proteins Jour-nal of the Science of Food and Agricul-ture 28 (6) 557ndash564 doi10 1002 jsfa 2740280614

Antioxidant Indices and Amino Acid Composition of PhenolicContaining Lima Beans (Phaseolus lunatus) After Simulated

Human Gastrointestinal Digestion

Sule O Salawua b Oluwaseun M Folorunsoa Akintunde A Akindahunsia andAline A Boligonc

a Department of Biochemistry Federal University of Technology PMB 704 Akure Nigeriab Department of Biochemistry Osun State University Osogbo Nigeria

c Universidade Federal de Santa Maria Centro de Ciencias da Saude Departamento de Farmacia IndustrialSala 1115 Campus Camobi Santa Maria Rio Grande do Sul CEP 97105-900 Brasil

Corresponding authorsosalawufutaedung

Tel +2348132595807

Received 30 June 2018 Published online 18 April 2019

Abstract

The present investigation was designed to characterize the phenolic profile of Lima beans (PhaseolusLunatus) and also to evaluate the antioxidant indices total phenolic content (TPC) total flavonoidcontent (TFC) ferric reducing antioxidant power (FRAP) 22-Diphenyl-1-picrylhydrazyl (DPPH) rad-ical scavenging activity and amino acid composition at different stages of simulated gastrointestinaldigestion (oral gastric intestinal) High Performance Liquid Chromatography (HPLC-DAD) analysisrevealed the presence of some phenolic compounds (gallic acid catechin caffeic acid rutin quercitrinquercetin kaempferol and apigenin) with a reduced amount (mgg) after cooking gallic acid (raw196 plusmn 002 cooked 182 plusmn 001) catechin (raw 083 plusmn 001 cooked 073 plusmn 001) rutin (raw 261plusmn 003 cooked174 plusmn 003) quercitrin (raw 573 plusmn 001 cooked 568 plusmn 001) apigenin (raw 209plusmn 001 cooked 179 plusmn 002) with exception of quercetin (raw 211 plusmn 002 cooked 573 plusmn 002) andcaffeic acid (raw 208plusmn004 cooked 295 plusmn 004) The results of the antioxidant indices of in vitroenzyme digested lima beans revealed higher values for cooked Lima beans compared to the raw coun-terpart with a stepwise increase at the different stages of in vitro digestion with the exception of ferricreducing antioxidant power TPC (oral digestion 6544 plusmn 096 gastric digestion13487 plusmn 046 intesti-nal digestion 51772 plusmn 470 mgg tannic acid equivalent) TFC (oral digestion 19930 plusmn 643 gastricdigestion 106597plusmn 122 intestinal digestion 369187 plusmn 42 mgg quercetin equivalent) DPPH (oraldigestion 8580000 plusmn 30550 gastric digestion 9906666 plusmn 11547 intestinal digestion 21135420 plusmn36084 micromol TEg sample) The results also revealed a progressive increase in the antioxidant indicesand amino acid composition (mgkg) for both raw and processed lima beans at various stages of thein vitro digestion with the intestinal phase of simulated digestion ranking higher This implied thatthe Lima beans contained some essential amino acids and antioxidant molecules that would be readilyavailable after passing through the gastrointestinal tract and could therefore be explored as functionalfood in the management of free radical mediated diseases

Keywords Antioxidant Activities Amino Acid Composition Phenolic Compounds P Lunatus invitro Digestion

98 Salawu et al

1 Introduction

Plants are the most predominant harvesters ofsolar energy and they are valuable primary re-sources of essential nutrients for human food pro-duction (Bain et al 2013 Fanzo 2015) In com-mon with most developing nations of the worldthe state of nutrition in Nigeria is still charac-terized by inadequate calorie and protein sup-plies (Babatunde Adejobi amp Fakayode 2010FAO 2011) Protein foods and particularly an-imal protein have continued to be in short sup-ply Most experts consider protein deficiency asthe commonest form of malnutrition in the devel-oping countries especially in regions where dietsare based mainly on roots and tuber crops (FAO2013 Trehan amp Manary 2015) The productionof protein-rich foods (leguminous seeds and par-ticularly animal products) has been much lessefficient Therefore in an attempt to widen thenarrow food base food and agricultural scientistsare screening lesser known and under-exploitednative plants for possible potential sources offood nutrients (Baldermann et al 2016)Grain legumes are grouped into two classesmajor and minor species The major speciesinclude the industrial legumes such as soy-bean groundnut common beans (Phaseolus vul-garis) chickenpea (Cicer arietinum) and pea(Pisum sativum) Minor species exist in awide range of diversity either as cultivated orwild species across various regions of the worldand are usually cultivated by the traditionalfarmers they are also referred to as neglectedunderutilized under-cultivated or lesser-knownlegumes (Pastor-Cavada Juan Pastor Alaiz ampVioque 2016) The wild species of the minorgrain legumes include kersting groundnut (Ker-stingiella geocarpa) and marama bean (Tylosemaesculentum) The miscellaneous legumes are theminor grain legumes that have received very littleresearch attention when compared with the ma-jor grain legumes such as cowpea and soybeanLeguminous seeds are an important source ofproteins energy and other nutrients in the di-ets of large population of people around theworld they form an excellent source of lysinemethionine and tryptophan water-soluble vita-mins (riboflavin niacin and folacin) and of min-erals phosphorus iron and magnesium (FAO

2012) Legume seed coats commonly referred toas hulls are rich sources of polyphenolics and an-tioxidants (Iriti amp Varoni 2017 Pastor-Cavadaet al 2016) A number of studies have shownthat legumes are rich in antioxidants some ofthese legumes include peanut lentils and soy-bean (Zou Chang Gu amp Qian 2011) Bam-bara groundnut (Nyau Prakash Rodrigues ampFarrant 2015) African yam bean Lima bean(Oboh Ademiluyi amp Akindahunsi 2009) Pi-geon pea (Oboh et al 2009)Lima bean P Lunatus belongs to the familyleguminosae and is mostly cultivated in SouthAmerica (Yellavila Agbenorhevi Asibuo ampSampson 2015) Lima beans are sometimes re-ferred to as haba beans sugar beans butterbeans Guffin beans civet beans Hibbert beansPallar beans Sieva beans Madagascar beansand Burma beans It is one of the underuti-lized legumes found in Nigeria though a mi-nor crop they have been an important sourceof plant protein to millions of Nigerians and arewidely known for their fibre mineral and pro-tein contents but with a lesser attention to theirnutraceutical value (Saleem Ahmed amp Hasan2016 Yellavila et al 2015)The role of medicinal food plants in diseaseprevention has been attributed to the antiox-idant properties of their constituents usuallyassociated with a wide range of amphipathicmolecules broadly termed polyphenolic com-pounds (Miguel 2010 Ozcan akpınar Yilmaz-Ersan amp Delikanli 2014) The protective ef-fects of the dry beans in disease prevention maynot be entirely associated with dietary fibre butto phenolic compounds and other non-nutritivecompounds (Miguel 2010 Oomah Tiger Ol-son amp Balasubramanian 2006) Polyphenolsfrom Lima beans will possibly act as antioxi-dants hindering the formation of free radicalsthat eventually lead to the deterioration of bio-logical molecules These naturally occurring phe-nolic compounds are predominantly present inthe seed coat and possess anti-mutagenic and an-tioxidant activities (Attree Du amp Xu 2015 Iritiamp Varoni 2017)The antioxidant compounds and other phyto-nutrients present in most legumes have beenextracted and measured by different methods(Khang Dung Elzaawely amp Xuan 2016 Nyau

Antioxidant Indices and Amino Acid Composition of Lima Beans After in vitro Digestion 99

et al 2015 Oboh et al 2009 Salawu Besteramp Duodu 2014) The results of such exper-iments clearly demonstrate a range of phyto-constituents with potential antioxidant actionbut that cannot be translated to be availablephyto-constituents when the legumes are de-graded by the enzymes and juices of the gastro-intestinal tract during passage through it Somestudies have compared the content of bioactivecompounds and antioxidant activities in undi-gested and digested extracts (Bouayed Hoff-mann amp Bohn 2011 Faller Fialho amp Liu2012 Gimenez Moreno Lopez-Caballero Mon-tero amp Gomez-Guillen 2013 Tavares et al2012) Hence the present investigation sought toevaluate the antioxidant activities and the aminoacid profile of Lima beans after a stepwise simu-lated human gastrointestinal digestion

21 Chemicals

Follin-Ciocalteursquos reagent tannic acid sodiumcarbonate iron (iii) chloride potassium fer-ricyanide trichloroacetic acid aluminiumchloride potassium acetate 22- diphenyl-1-picrylhydrazyl radical and 6-hydroxy-2578-tetramethylchroman-2-carboxylic acid(Trolox) were obtained from Sigma chemicalcompany USA All other chemicals were ob-tained from standard chemical suppliers andwere of analytical grade while the water usedwas glass distilled

22 Sample collection

Lima beans (P Lunatus) were obtained fromOba market in Akure Nigeria They were iden-tified and authenticated in the Department ofCrop Soil and Pest Management School of Agri-culture and Agricultural Technology FederalUniversity of Technology Akure Ondo stateNigeria

23 Sample treatment andpreparation

The sample was divided into two portions thefirst portion of the sample (raw) was sortedwashed air-dried and milled into a powderedform prior to enzyme treatments of in vitro di-gestion While the second portion was sortedwashed and cooked at 100 oC for 4 h cooledair-dried and then milled prior to the enzymetreatments of in vitro digestion All the milledsamples were stored in plastic containers at roomtemperature in the Department of BiochemistryFederal University of Technology Akure Nigeriauntil used

24 in vitro enzyme digestion

The in vitro digestion using sequential enzymaticsteps was based on a slightly modified method asreported by Delgado-Andrade Alberto Conde-Aguilera Haro Pastoriza de la Cueva and An-gel Rufian-Henares (2010) Various stages of thegastrointestinal digestion (oral gastric and totalgastrointestinal digestion) were carried out

Oral digestion

Ten grams of the milled Lima beans were weighedand dispersed in 200 mL of distilled water 325mg of alpha amylase was dissolved in 25 mL 1mMcalcium chloride at pH 70 and 1500 microl of thealpha amylase was added to each test tube (sim-ulating pH conditions in the mouth) The tubeswere incubated in a shaking water bath set at 37oC for 40 min and at 80 strokes min The sam-ple was incubated in boiling water at 100 oC for 4min for enzyme inactivation and centrifuged for60 min at 4000 x g then the soluble fraction waskept in the refrigerator for subsequent analysisand the insoluble fraction was discarded A non-enzymic digest (control) was also done for bothraw and cooked samples with the same proce-dure used for the in vitro enzyme digestion butwithout the addition of enzymes

Oral-Gastric digestion

Ten grams of the milled lima beans were weighedand dispersed in 200 mL of distilled water 325

100 Salawu et al

mg of alpha amylase was dissolved in 25 mL 1mMcalcium chloride at pH 70 and 1500 microl of thealpha amylase was added to each test tube (sim-ulating pH conditions in the mouth) The tubeswere incubated in a shaking water bath set at37 oC for 40 min and at 80 strokes min ThepH was adjusted to 2 after 40 min using concen-trated HCl 10 mg pepsin which was dissolvedin 5 mL of 005 M HCl was added to each tube(simulating pH conditions in the stomach) Thetubes were incubated in a shaking water bath setat 37 oC for 40 min and at 80 strokes min Thesample was incubated in boiling water at 100 oCfor 4 min for enzyme inactivation and centrifugedfor 30 min at 4000 x g then the soluble fractionwas kept in the refrigerator for subsequent analy-sis the insoluble fraction was discarded A non-enzymic digest (control) (without enzyme) wasalso done for both raw and cooked samples withthe same procedure used for the in vitro diges-tion

Total gastrointestinal digestion

Ten grams of the milled lima beans were weighedand dispersed in 200 mL of distilled water 325mg of alpha amylase were dissolved in 25 mL 1mM calcium chloride at pH 70 and 1500 microl ofthe alpha amylase was added to each test tube(simulating pH conditions in the mouth) Thetubes were incubated in a shaking water bath setat 37 oC for 40 min and at 80 strokes min ThepH was adjusted to 2 after 40 min using concen-trated HCl 10 mg pepsin which was dissolvedin 5 mL of 005 M HCL was added to each tube(simulating pH conditions in the stomach) Thetubes were incubated in a shaking water bath setat 37 oC for 40 min and at 80 strokes min After40 min shaking the pH was adjusted to 6 usingNaOH After 20 min 50 mL of pancreatin solu-tion (9 gram of pancreatin in 60 mL of distilledwater) was added in each test tube and the pHwas adjusted finally to 75 using NaOH (simulat-ing pH conditions in the small small intestine)The sample was incubated in boiling water at100 oC for 4 min for enzyme inactivation andcentrifuged for 60 min at 4000 x gThe solublefraction was kept in the refrigerator for subse-quent analysis and the insoluble fraction was dis-carded A non-enzymic digest (control) (without

enzyme) was also made for both raw and cookedsamples with the same procedure used for the invitro digestion

25 Quantification of phenoliccompounds by HPLC-DAD

For analysis of the Lima bean extracts 10 mL ofthe each extract was injected into a PhenomenexC18 column (46 mm x 250 mm) packed with 5microm diameter pore size Mobile phases A and Bwere Milli-Q water acidified to pH 20 with 1 of phosphoric acid and methanol correspond-ingly solvent gradient was used as follows 0-10min 5 B 10-25 min 15 B 25-40 min 30 40-55 min 50 B 50-65 min 70 B 65-80min 100 B following the method described byOmoba Obafaye Salawu Boligon and Athayde(2015) with slight modifications The extractsand mobile phase were filtered through a 045 micrommembrane filter (Millipore) and then degassedby ultrasonic bath prior to use The extract wasanalyzed at a concentration of 10 mgmL Theflow rate was 06 mLmin and the injection vol-ume was 40 microL The sample and mobile phasewere filtered through a 045 microm membrane filter(Millipore) and then degassed by ultrasonic bathprior to use Stock solutions of reference stan-dards were prepared in the HPLC mobile phaseat a concentration range of 0050 ndash 0300 mgmLQuantifications were carried out by integration ofthe peaks using the external standard methodat 254 nm for gallic acid 280 nm for catechin327 nm for caffeic acid and 365 nm for rutinquercitrin quercetin kaempferol and apigeninThe chromatography peaks were confirmed bycomparing the retention time with those of thereference standards and by DAD spectra (200 to700 nm) All chromatography operations werecarried out at ambient temperature and in trip-licate The limit of detection (LOD) and limit ofquantification (LOQ) were calculated based onthe standard deviation of the responses and theslope using three independent analytical curvesLOD and LOQ were calculated as 33 and 10 δSrespectively where δ is the standard deviation ofthe response and S is the slope of the calibrationcurve (Boligon et al 2015)

26 Determination of totalphenolic content

The total phenolic content of the in vitro enzymedigested Lima bean and undigested control sam-ples was determined by the Folin-Ciocalteu as-say as described by Mole and Waterman (1994)500microl of Folin-Ciocalteu reagent was added andmixed with a solution containing 100microL of the ex-tract and 2 mL of distilled water 15 mL of 75 sodium carbonate was then added to the solu-tion and the volume was made up to 10 mL withdistilled water The mixture was left to standfor 2 h after addition of the sodium carbonateThe absorbance of the mixture was measured at760 nm using a Lambda EZ150 spectrophotome-ter (Perkin Elmer USA) The standard used wastannic acid and the results were expressed as mgtannic acid equivalents per gram of the sample

27 Determination of totalflavonoid content

The total flavonoid content of the in vitro en-zyme digested Lima bean and undigested controlsamples were determined using a slightly modi-fied method reported by Meda Lamien RomitoMillogo and Nacoulma (2005) Briefly 05 mL ofenzyme digested sample was mixed with 05 mLdistilled H2O 50 microL of 10 AlCl3 50microl of 1molLminus1 potassium acetate and 14 mL water and in-cubated at room temperature for 30 min There-after the absorbance of each reaction mixturewas measured at 415 nm using a Lambda EZ150spectrophotometer (Perkin Elmer USA) The to-tal flavonoid was calculated using quercetin asstandard by making use of a seven point stan-dard curve (0-100 microgmL) The total flavonoidcontent of samples was determined in triplicateand the results were expressed as mg quercetinequivalent per gram of the sample

28 Reducing power assay

The reducing power of the in vitro enzyme di-gested Lima bean samples and undigested controlwere determined by assessing the ability of eachextract to reduce FeCl3 solution as described byOyaizu (1986) Briefly 1 mL of the sample was

mixed with 1 mL 200 mM sodium phosphatebuffer (pH 66) and 1 mL 1 potassium ferri-cyanide The mixture was incubated at 50oC for20 min and then 1 mL 10 trichloroacetic acid(TCA) was added This mixture was centrifugedat 353 x g for 10 min 2 mL of the supernatantwas mixed with an equal volume of water and 04mL of 01 ferric chloride The absorbance wasmeasured at 700 nm using a Lambda EZ150 spec-trophotometer (Perkin Elmer USA) The ferricreducing antioxidant power was expressed as mgascorbic acid equivalentg of the sample

29 DPPH radical scavengingassay

DPPH radical scavenging activity was measuredaccording to the method of Brand-Williams Cu-velier and Berset (1995) with some modifica-tions The stock solution was prepared by dis-solving 24 mg DPPH with 100 mL methanol andthen stored at -20oC until needed The workingDPPH solution was obtained by mixing 10 mLstock solution with 45 mL methanol to obtainan absorbance of 11 units at 515 nm using thespectrophotometer 05 mL of the in vitro en-zyme digested sample was diluted with 2 mL ofmethanol to obtain a mother solution 150 microL ofthe mother solution were allowed to react with2850 microL of the DPPH working solution for 6 hin the dark Then the absorbance was taken at515 nm using a Lambda EZ150 spectrophotome-ter (Perkin Elmer USA) Result was expressedin micromol Trolox equivalentg sample

210 Amino acid profile assay

Amino acid profile was determined accordingto the method of Obreshkova Tsvetkova andIvanov (2012) In order to ensure that the driedand pulverized undigested (cooked and raw) sam-ples were completely dry they were dried to con-stant weight for a period of time in the labora-tory The sample of 100 g was weighed into a 250mL conical flask The sample was defatted with30 mL of the petroleum spirit three times withsoxhlet extractor that was equipped with thim-ble The sample was hydrolyzed three times toensure complete hydrolysis to achieve total recov-

102 Salawu et al

ery of amino acids The pulverized and defattedsample was soaked with 30 mL of 1 M potas-sium hydroxide solution and incubated for 48 hat 110 oC in a hermetically sealed borosilicateglass container After the alkaline hydrolysisthe hydrolysate was neutralized to get the pHin the range 25-50 The solution was purifiedby cation-exchange solid-phase extraction Theamino acids in the purified solution were deriva-tised with ethylchloroformate by the establishedmechanism and subsequently injected into thegas chromatograph (GC) for amino acid analy-sis The deactivated enzyme digests of raw andcooked samples (digest incubated in boiling wa-ter at 100 oC for 4 min) obtained after the totalintestinal phase of the in vitro digestion were alsoinjected into the GC for amino acid analysis

All the analyses were run in triplicate Resultswere then computed using Microsoft Excel soft-ware (Microsoft Corporation Redmond WA)and followed by one-way Anova with DuncanrsquosMultiple Range Test (DMRT) to compare themeans that showed significant variation by us-ing SPSS 1109 for Windows The significancelevel was set at p lt 005

31 Results

HPLC-DAD PhenolicCharacterization

The HPLC-DAD analysis of phenolic compoundsin processed Lima beans (cooked and raw) asshown in Fig 1 revealed the presence of gal-lic acid catechin caffeic acid rutin quercitrinquercetin kaempferol and apigenin The quan-titative estimates of these phenolic compoundsin Lima beans (Table 1) revealed a slight reduc-tion in the level of some of them (gallic acidcatechin rutin quercitrin apigenin) after cook-ing with exception of caffeic acid and quercetinwhich were higher in the raw Lima beans

Total Phenolic and Flavoid Content

The phenolic contents of raw and processed Limabeans at various stages of in vitro digestion wereas shown in Fig 2 The results revealed aslightly higher phenolic content in the enzyme di-gested cooked beans compared to the raw coun-terpart The results also showed a progressiveincrease in the phenolic content (mgg) of thedigested Lima beans at each stage of in vitro di-gestion with the oral phase of digestion showingthe least phenolic content (raw 4567 cooked6544) followed by the oral-gastric phase of di-gestion (raw 6176 cooked 13487) The high-est phenolic content was recorded after the totalgastrointestinal digestion (raw 43918 cooked51772) The total flavonoid content of raw andprocessed Lima beans at various stages of invitro digestion is shown in Fig 3 Similarlythe results revealed higher flavonoid content inthe enzyme digested cooked beans compared tothe raw sample The results also showed a pro-gressive increase in the total flavonoid contentof the digested Lima beans at each stage of invitro digestion with the oral phase of digestionshowing the least total flavonoid content Thehigher flavonoid content was recorded after thetotal gastrointestinal digestion in digested rawand digested cooked Lima beans respectively

Antioxidant Properties

The results of the ferric reducing antioxidantpower of raw and processed lima beans at var-ious stages of in vitro digestion were as shownin Fig 4 Conversely the ferric reducing an-tioxidant power of the digested raw lima beanswas higher than that of the digested cooked limabeans The DPPH radical scavenging activity ofraw and cooked lima beans at various stages ofin vitro digestion was as shown in Fig 5 The re-sults also revealed higher DPPH radical scaveng-ing activity in the enzyme digested cooked beanscompared to the raw counterpart Similarly theresults also showed a progressive increase in theferric reducing power and DPPH radical scaveng-ing activity of the digested Lima beans at eachstage of in vitro digestion with the total phase ofthe simulated gastro intestinal digestion rankinghigher

Table 1 Phenolic and flavonoid composition of Lima Bean extracts

CompoundsRaw Lima beans Cooked Lima beans

mgg mgg

Gallic acid 196 plusmn 002a 182 plusmn 001a

Catechin 083 plusmn 001b 073 plusmn 001b

Caffeic acid 208 plusmn 004a 295 plusmn 004c

Rutin 261 plusmn 003d 174 plusmn 003a

Quercitrin 573 plusmn 001e 568 plusmn 001d

Quercetin 211 plusmn 002a 573 plusmn 002d

Kaempferol - 081 plusmn 003b

Apigenin 209 plusmn 001a 179 plusmn 002a

Results are expressed as mean plusmn standard deviations (SD) of three determinations

Means followed by different letters differ significantly by Tukey test at p lt 005

Figure 1 Representative high performance liquid chromatography profile of Lima bean raw (A) andcooked (B) extracts Gallic acid (peak 1) catechin (peak 2) caffeic acid (peak 3) rutin (peak 4)quercitrin (peak 5) quercetin (peak 6) kaempferol (peak 7) and apigenin (peak 8)

Amino acid Profile

The amino acid profiles of raw and processedLima beans at various stages of in vitro diges-tion were as shown in Table 2 The results re-vealed a varied level of glycine alanine serineproline valine threonine isoleucine leucine as-partate lysine methionine glutamate pheny-lalanine histidine arginine tyrosine tryptophanand cysteine in the raw and cooked Lima beansrespectively High levels of these amino acidswere found after the total intestinal phase ofthe simulated gastrointestinal digestion in bothcooked and raw in vitro digested Lima beansThe results also showed that the in vitro en-

zyme digested cooked sample had higher aminoacid values than the raw counterpart It was ob-served that the process of cooking gave signifi-cantly higher amino acid contents compared withthe raw counterpart

Discussion

Legumes are one of the most important classesof food that have been used as a stable food toprovide the proteins and energy requirements ofman (Brand Brandt amp Cruywagen 2004) Inaddition to the provision of nutrients legumesalso contain valuable bioactive compounds such

104 Salawu et al

Table 2 The Amino Acid Profile of Raw and Cooked in vitro Enzyme Digested and Undigested LimaBeans (mgkg)

Amino Acid RP ORD OGRD TDR CP ODC OGCD TCD

Glycine 310plusmn324f 0083plusmn0002a 015plusmn003c 290plusmn192e 330plusmn194h 014plusmn002b 029plusmn003d 320plusmn342g

Alanine 290plusmn125f 0096plusmn0004a 015plusmn005c 270plusmn151e 320plusmn172h 011plusmn004b 023plusmn005d 310plusmn125g

Serine 500plusmn212g 010plusmn005a 023plusmn004c 440plusmn213e 520plusmn223h 017plusmn004b 046plusmn003d 490plusmn433f

Proline 120plusmn143f 0012plusmn0006a 0037plusmn002c 82plusmn41e 120plusmn114g 0029plusmn0001b 0059plusmn0002d 87plusmn64gf

Valine 500plusmn215f 015plusmn003a 024plusmn005c 460plusmn213e 510plusmn174g 021plusmn003b 027plusmn004d 500plusmn243f

Threonine 340plusmn133g 012plusmn004a 021plusmn001c 320plusmn142e 350plusmn152h 018plusmn002b 024plusmn003d 330plusmn313f

Isoleucine 410plusmn141f 014plusmn002a 019plusmn003c 400plusmn153e 500plusmn213h 016plusmn006b 023plusmn006d 480plusmn324g

Leucine 600plusmn152f 017plusmn003a 023plusmn001c 570plusmn204e 650plusmn314h 019plusmn006b 026plusmn005d 640plusmn184g

Aspartate 850plusmn236g 035plusmn001a 044plusmn004c 710plusmn181e 870plusmn285h 039plusmn003b 050plusmn007d 780plusmn335f

Lysine 520plusmn213g 017plusmn005a 019plusmn003c 410plusmn123e 570plusmn214h 019plusmn007b 022plusmn004d 500plusmn412f

Methionine 75plusmn47f 0014plusmn0003a 0015plusmn0006ab 73plusmn41e 79plusmn55g 0015plusmn0001ab 015plusmn003c 70plusmn85d

Glutamate 960plusmn156f 038plusmn004a 042plusmn003c 940plusmn14e 1000plusmn297h 040plusmn005b 053plusmn002d 930plusmn273g

Phenylalanine 420plusmn173g 015plusmn003a 022plusmn004c 370plusmn214e 440plusmn123h 017plusmn003b 025plusmn004d 400plusmn334f

Histidine 270plusmn124f 025plusmn002a 031plusmn003c 250plusmn142e 280plusmn214g 029plusmn002b 036plusmn006d 250plusmn145e

Arginine 400plusmn253e 013plusmn004a 017plusmn005c 400plusmn136e 530plusmn252h 015plusmn001b 022plusmn003d 470plusmn234g

Tyrosine 250plusmn13g 0055plusmn0003a 026plusmn003c 230plusmn102e 240plusmn224h 008plusmn0001b 032plusmn006d 210plusmn194f

Tryptophan 94plusmn75g 011plusmn002a 017plusmn001c 73plusmn51e 110plusmn97h 012plusmn003ab 018plusmn003cd 82plusmn37f

Cysteine 100plusmn97g 0034plusmn0005a 013plusmn003c 80plusmn43e 100plusmn85g 0047plusmn0002b 014plusmn001cd 99plusmn29f

Abbreviations RP = Raw dried and powdered ORD = Oral raw digested OGRD = Oral and gastric raw digested TDR = Totaldigested raw CP = Cooked dried and powdered ODC = Oral cooked digested OGDC = Oral and gastric cooked digestedTCD = Total cooked digested Values are given as mean plusmn SE of independent experiments performed in triplicateValues are given as mean plusmn SE of independent experiments performed in triplicate Values with different superscriptsin the same row differ significantly (Plt005)

Figure 2 Total phenolic content of P Lunatusextract The results are expressed as milligramsof tannic acid equivalent (mg TA) per 100 g ofdry weight sample Value are given as meanplusmn SEof independent experiments performed in tripli-cate TA= Tannic Acid Oral= Oral digestionGastric= Gastric digestion Total=Total intesti-nal digestion

Figure 3 Total flavonoid content of P Lunatusextract expressed as mg of quercetin equivalents(QE) per 100 g of dry weight of the sample Valueare given as mean plusmn SE of independent experi-ments performed in triplicate Oral= Oral di-gestion Gastric= Gastric digestion Total=Totalintestinal digestion

Figure 4 Ferric reducing antioxidant power of PLunatus extract expressed as milligrams ascorbicacid equivalent per 100 g of dry weight sampleValue are given as mean plusmn SE of independent ex-periments performed in triplicate AA= Ascor-bic Acid Oral= Oral digestion Gastric= Gastricdigestion Total=Total intestinal digestion

Figure 5 DPPH radical scavenging activity ofP Lunatus extract expressed as micro mole Troloxequivalents per 100 g of dry weight of the sam-ple Value are given as mean plusmn SE of indepen-dent experiments performed in triplicate Oral=Oral digestion Gastric= Gastric digestion To-tal=Total intestinal digestion

as enzyme inhibitors lectins phytates oxalatespolyphenols phytosterols and saponins that mayhelp prevent humans developing various diseasesPhenolic compounds constitute an importantgroup of natural products contributing signifi-cantly to the medicinal value of a number of foodplants (Huang Cai amp Zhang 2010) Studieshave focused on the biological activities of pheno-lic compounds which have potential antioxidantand free radical scavenging abilities (Lisanti etal 2016)Investigations on the phenolic composition oflegumes have revealed that legumes are goodsources of phenolic compounds with antioxidantactivities that can be harnessed in the manage-ment of free radical related diseases (Attree etal 2015 Iriti amp Varoni 2017) A previous re-port on the phenolic composition of three vari-eties of the common bean (Phaseolus vulgarisL) soybeans (Glycine max L) and peas (Pisumsativum L) from Rwanda revealed the pres-ence of 11 different phenolic compounds in thesecommon beans gallic acid (+)-catechin (ndash)-epicatechin caffeic acid o-coumaric acid chloro-genic acid quercetin 4-hydrobenzoic acid sy-ringic acid ferulic acid and vanillic acid (Josephet al 2014) Catechin was also reported in greenbeens (Escarpa amp Gonzalez 2000) In the samevein a study on the phenolic acidsrsquo content offifteen dry edible bean varieties revealed a highamount of caffeic acid in black beans (Luthriaamp Pastor-Corrales 2006) The phenolic profilesof Phaseolus vulgaris revealed the presence of13 quercetin-3-O-glycosides eight kaempferol-3-O-glycosides two myricetin glycosides and fourphenolic acids with potential antioxidant activi-ties (Reyes-Martinez et al 2014)The reduction in phenolic contents of the Limabeans during cooking could be due to leaching ofphenolic compounds in the soaking water Theslight reduction in the level of some of the phe-nolic compounds after cooking was in agree-ment with previous reports where cooking lowersthe total phenolic content of the food materialsthat were studied (Gujral Angurala Sharma ampSingh 2011 Han amp Xu 2014)Several investigations on common beans high-light their antioxidant potential (Akond et al2011 Karamac amp Amarowicz 2004) Phe-nolic compounds are widely distributed in the

106 Salawu et al

plant kingdom and are important in the antiox-idant capacity in vitro because of their abil-ity to donate hydrogen and form stable radi-cal intermediates (Scalbert Manach MorandRemesy amp Jimenez 2005) The higher to-tal phenolic content of the in vitro digestedcooked beans compared with those digested inthe raw form may have been due to alterationsin the chemical structure and composition as aresult of heat during boiling (Han amp Xu 2014)Thus cooking might have enhanced the break-down of the insoluble fibre matrix of the beansthereby making its polyphenols more accessiblefor further breakdown by the enzymes of thegastro-intestinal tract (Tagliazucchi VerzelloniBertolini amp Conte 2010)The higher phenolic content at the intestinalphase of gastrointestinal digestion was in agree-ment with previous reports (Ryan amp Prescott2010 Wootton-Beard Moran amp Ryan 2011)where a higher phenolic content and antioxi-dant activity was reported for commercial avail-able juices after the gastric and intestinal phasesof gastrointestinal digestion The higher totalflavonoid content of the in vitro digested cookedbeans compared with those digested in the rawform may have also been due to alterations inthe chemical structure and composition as a re-sult of heat during boiling (Han amp Xu 2014)thereby making its constituent flavonoids moreaccessible for further breakdown by the enzymesof the gastro-intestinal tract (Tagliazucchi et al2010) The higher total flavonoid content atthe intestinal phase of the gastrointestinal di-gestion was in agreement with previous reports(Ryan amp Prescott 2010 Wootton-Beard et al2011) where a higher polyphenolic content andantioxidant activity was reported for commer-cially available juices after the gastric and in-testinal phases of gastrointestinal digestionThe decreasing value of the ferric reducing an-tioxidant power in the cooked lima beans com-pared to the raw lima beans might have beenassociated with a quantitative loss of someflavonoids during the heat treatment (Jeong etal 2004 Salawu Innocenti Giaccherini Akin-dahunsi amp Mulinacci 2008) The higher DPPHradical scavenging activity in the cooked beansof the enzyme digested Lima beans compared tothe raw counterpart was in agreement with pre-

vious report (Han amp Xu 2014)The observed progressive increase in the ferric re-ducing power and DPPH radical scavenging ac-tivity of the digested Lima beans at each stageof in vitro digestion may have been associatedwith a significantly positive correlation that usu-ally exists between the polyphenolic content andantioxidant activities (Rahman Khan Das ampHossain 2016 Ryan amp Prescott 2010 SadeghiValizadeh Shermeh amp Akaberi 2015 Wootton-Beard et al 2011)High levels of amino acids found at the to-tal intestinal phase of the simulated gastroin-testinal digestion in both cooked and raw invitro digested lima beans was in agreement withprevious reports (Jin Zhou Li Lai amp Li2015) where it was reported that pepsin di-gestion brings about a marked increase in thefree amino acid content of a coconut meat pro-tein hydrolysate by 303 compared to the con-trol and that after digestion with pancreatin thefree amino acid content of a coconut meat pro-tein hydrolysate increased dramatically by 867 compared to the control Thus the maxi-mum amounts of amino acids were released dur-ing the total gastrointestinal phase of the sim-ulated in vitro digestion process as a result ofthe action of the digestive enzymes (α-amylasepepsin and pancreatin) of the GIT This obser-vation was in agreement with a previous report(Tagliazucchi et al 2010) that suggested thatdigestion might be a determinant factor in therelease of nutritionally relevant compounds fromthe food matrix The higher amino acid con-tents in the cooked Lima beans compared withthe raw counterpart was in agreement with previ-ous reports (Lombardi-Boccia Schlemmer Cap-pelloni amp Di Lullo 1998 Oluwaniyi Dosumu ampAwolola 2010) where it was reported that theprocess of boiling generally gives slight but sig-nificantly higher amino acid contents respectivelycompared with the fresh samples

4 Conclusions

The results of this study revealed that Limabeans have a number of health promoting pheno-lic compounds with antioxidant potential Theresults also revealed higher antioxidant indices

after the intestinal phase of the simulated hu-man digestion Furthermore the results also es-tablished the availability of a number of essen-tial amino acids after the simulated in vitro di-gestion with that of the total intestinal phaseof simulated in vitro digestion ranking higherTherefore Lima beans could be considered afunctional food with consumption providing asource of nutritionally important amino acidsand aiding the prevention of free radical medi-ated diseases

Acknowledgements

Authors would like to acknowledge UniversidadeFederal de Santa Maria Centro de Ciencias daSaude Departamento de Farmacia IndustrialSala 1115 Campus Camobi Santa Maria RioGrande do Sul CEP 97105-900 Brasil for theirgoodwill to use their facilities to characterize thephenolic compounds present in Lima beans

References

Akond A S M G M Khandaker L BertholdJ Gates L Peters K Delong H ampHossain K (2011) Anthocyanin totalpolyphenols and antioxidant activity ofcommon bean American Journal of FoodTechnology 6 (5) 385ndash394 doi10 3923 ajft2011385394

Attree R Du B amp Xu B (2015) Distributionof phenolic compounds in seed coat andcotyledon and their contribution to antiox-idant capacities of red and black seed coatpeanuts (arachis hypogaea l) IndustrialCrops and Products 67 448ndash456 doi10 1016jindcrop201501080

Babatunde R O Adejobi A O amp FakayodeS B (2010) Income and calorie intakeamong farming households in rural Nige-ria results of parametric and nonparamet-ric analysis Journal of Agricultural Sci-ence 2 (2) 135 doi105539jasv2n2p135

Bain L E Awah P K Geraldine N KindongN P Siga Y Bernard N amp TanjekoA T (2013) Malnutrition in Sub-SaharanAfrica burden causes and prospects Pan

African Medical Journal 15 (1) doi10 11604pamj2013151202535

Baldermann S Blagojevic L Frede K Klop-sch R Neugart S Neumann A Schreiner M (2016) Are neglected plantsthe food for the future Critical Reviewsin Plant Sciences 35 (2) 106ndash119 doi1010800735268920161201399

Boligon A A Piana M Kubica T F MarioD N Dalmolin T V Bonez P C Athayde M L (2015) HPLC anal-ysis and antimicrobial antimycobacterialand antiviral activities of Tabernaemon-tana catharinensis A DC Journal of Ap-plied Biomedicine 13 (1) 7ndash18 doi10 1016jjab201401004

Bouayed J Hoffmann L amp Bohn T (2011)Total phenolics flavonoids anthocyaninsand antioxidant activity following simu-lated gastro-intestinal digestion and dial-ysis of apple varieties Bioaccessibility andpotential uptake Food Chemistry 128 (1)14ndash21 doi101016jfoodchem201102052

Brand-Williams W Cuvelier M E ampBerset C (1995) Use of a free-radicalmethod to evaluate antioxidant activityFood Science and Technology-lebensmittel-wissenschaft amp Technologie 28 (1) 25ndash30

Brand T S Brandt D A amp CruywagenC W (2004) Chemical composition truemetabolisable energy content and aminoacid availability of grain legumes for poul-try South African Journal of Animal Sci-ence 34 (2) 116ndash122 doi10 4314 sajas v34i23815

Delgado-Andrade C Alberto Conde-AguileraJ Haro A Pastoriza de la Cueva S ampAngel Rufian-Henares J (2010) A com-bined procedure to evaluate the global an-tioxidant response of bread Journal of Ce-real Science 52 (2) 239ndash246 doi101016jjcs201005013

Escarpa A amp Gonzalez M C (2000) Identifi-cation and quantitation of phenolics fromgreen beans by high-performance liquidchromatography Chromatographia 52 (1-2) 33ndash38 doi101007BF02490789

Faller A L K Fialho E amp Liu R H (2012)Cellular antioxidant activity of feijoadawhole meal coupled with an in vitro diges-

108 Salawu et al

tion Journal of agricultural and food chem-istry 60 (19) 4826ndash4832

Fanzo J (2015) Ethical issues for human nutri-tion in the context of global food securityand sustainable development Global FoodSecurity-agriculture Policy Economics andEnvironment 7 15ndash23 doi101016jgfs201511001

FAO (2011) The state of food insecurity in theworld Retrieved from wwwfaoorg3a-i2330epdf

FAO (2013) Women men and nutritionGimenez B Moreno S Lopez-Caballero

M E Montero P amp Gomez-GuillenM C (2013) Antioxidant properties ofgreen tea extract incorporated to fishgelatin films after simulated gastrointesti-nal enzymatic digestion LWT - FoodScience and Technology 53 (2) 445ndash451doi101016jlwt201303020

Gujral H S Angurala M Sharma P amp SinghJ (2011) Phenolic content and antioxi-dant activity of germinated and cookedpulses International Journal of Food Prop-erties 14 (6) 1366ndash1374 doi10 1080 10942911003672167

Han S amp Xu B (2014) Bioactive compo-nents of leafy vegetable edible amaranth(Amaranthus mangostanus l) as affectedby home cooking manners American Jour-nal of Food Science and Technology 2 (4)122ndash127

Huang W-Y Cai Y-Z amp Zhang Y (2010)Natural phenolic compounds from medic-inal herbs and dietary plants Potentialuse for cancer prevention Nutrition andCancer-an International Journal 62 (1) 1ndash20 doi10108001635580903191585

Iriti M amp Varoni E M (2017) Pulses healthyand sustainable food sources for feeding theplanet International Journal of MolecularSciences 18 (2) doi103390ijms18020255

Jeong S M Kim S Y Kim D R JoS C Nam K C Ahn D U amp LeeS C (2004) Effect of heat treatment onthe antioxidant activity of extracts fromcitrus peels Journal of Agricultural and

Food Chemistry 52 (11) 3389ndash3393 doi101021jf049899k

Jin B Zhou X Li B Lai W amp Li X(2015) Influence of in vitro digestion on an-tioxidative activity of coconut meat proteinhydrolysates Tropical Journal of Pharma-ceutical Research 14 (3) 441ndash447 doi10 4314tjprv14i312

Joseph O Phelomene M Helene N ValensH Patrick O M Thavarajah D ampThavarajah P (2014) Phenolic compoundprofiles of two common beans consumed byrwandans American Journal of Plant Sci-ences 5 (20) 2943ndash2947 doi104236ajps2014520310

Karamac M amp Amarowicz R (2004) Antioxi-dant activity of phenolic fractions of whitebean (phaseolus vulgaris) Journal of FoodLipids 11 (2) 165ndash177 doi101111j1745-45222004tb00268x

Khang D T Dung T N Elzaawely A A ampXuan T D (2016) Phenolic profiles andantioxidant activity of germinated legumesFoods 5 (2) doi103390foods5020027

Lisanti A Formica V Ianni F Albertini BMarinozzi M Sardella R amp NataliniB (2016) Antioxidant activity of pheno-lic extracts from different cultivars of ital-ian onion (Allium cepa) and relative humanimmune cell proliferative induction Phar-maceutical Biology 54 (5) 799ndash806

Lombardi-Boccia G Schlemmer U Cappel-loni M amp Di Lullo G (1998) The in-hibitory effect of albumin extracts fromwhite beans (phaseolus vulgaris l) on invitro iron and zinc dialysability Role ofphytic acid Food Chemistry 63 (1) 1ndash7doi101016S0308-8146(97)00244-6

Luthria D L amp Pastor-Corrales M A (2006)Phenolic acids content of fifteen dry edi-ble bean (phaseolus vulgaris l) varietiesJournal of Food Composition and Analysis19 (2-3) 205ndash211 doi101016jjfca200509003

Meda A Lamien C E Romito M MillogoJ amp Nacoulma O G (2005) Determina-tion of the total phenolic flavonoid andproline contents in burkina fasan honeyas well as their radical scavenging activity

Food Chemistry 91 (3) 571ndash577 doi10 1016jfoochem200410006

Miguel M G (2010) Antioxidant activity ofmedicinal and aromatic plants a reviewFlavour and Fragrance Journal 25 (5)291ndash312 doi101002ffj1961

Mole S amp Waterman S (1994) Analysis ofphenolic plant metabolites Blackwell Scien-tific Publications

Nyau V Prakash S Rodrigues J amp FarrantJ (2015) Antioxidant activities of bam-bara groundnuts as assessed by frap anddpph assays American Journal of Food andNutrition 3 7ndash11

Oboh G Ademiluyi A O amp AkindahunsiA A (2009) Changes in polyphenols dis-tribution and antioxidant activity dur-ing fermentation of some underutilizedlegumes Food Science and Technology In-ternational 15 (1) 41ndash46 doi10 1177 1082013208101022

Obreshkova D Tsvetkova D D amp Ivanov K(2012) Simultaneous identification and de-termination of total content of aminoacidsin food supplements-tablets by gas chro-matography 5 57ndash68

Oluwaniyi O O Dosumu O O amp AwololaG V (2010) Effect of local processingmethods (boiling frying and roasting) onthe amino acid composition of four ma-rine fishes commonly consumed in nigeriaFood Chemistry 123 (4) 1000ndash1006 doi101016jfoodchem201005051

Omoba O S Obafaye R O Salawu S OBoligon A A amp Athayde M L (2015)Hplc-dad phenolic characterization and an-tioxidant activities of ripe and unripe sweetorange peels Antioxidants 4 (3) 498ndash512doi103390antiox4030498

Oomah B D Tiger N Olson M amp Balasub-ramanian P (2006) Phenolics and antiox-idative activities in narrow-leafed lupins(lupinus angustifolius l) Plant Foods forHuman Nutrition 61 (2) 91ndash97 doi10 1007s11130-006-0021-9

Oyaizu M (1986) Studies on products of brown-ing reaction Antioxidative activity of prod-ucts of browning reaction prepared fromglucosamine Japanese Journal of Nutri-tion 40 307ndash316

Ozcan T akpınar A Yilmaz-Ersan L ampDelikanli B (2014) Phenolics in humanhealth 5 393ndash396

Pastor-Cavada E Juan R Pastor J E AlaizM amp Vioque J (2016) A comprehen-sive approach to antioxidant activity inthe seeds of wild legume species of tribefabeae Journal of Botany 1ndash6 Retrievedfrom httpsearchebscohostcomloginaspx direct = true amp AuthType = ip uid ampdb=a9hampAN=114319915ampsite=eds-liveampscope=siteampauthtype=ipcookieuid

Rahman M M Khan F E Das R amp Hos-sain M A (2016) Antioxidant activityand total phenolic content of some indige-nous fruits of bangladesh InternationalFood Research Journal 23 (6) 2399ndash2404

Reyes-Martinez A Almaraz-Abarca NGallardo-Velazquez T del SocorroGonzalez-Elizondo M Herrera-ArrietaY Pajarito-Ravelero A IsabelTorres-Moran M (2014) Evaluation offoliar phenols of 25 mexican varieties ofcommon bean (phaseolus vulgaris l) asantioxidants and varietal markers NaturalProduct Research 28 (23) 2158ndash2162doi101080147864192014930855

Ryan L amp Prescott S L (2010) Stability ofthe antioxidant activity of twenty-five com-mercially available fruit juices subjected toan in vitro digestion International Journalof Food Science amp Technology 45 (6) 1191ndash1197 doi101111j1365-2621201002254x eprint httpsonlinelibrarywileycomdoipdf101111j1365-2621201002254x

Sadeghi Z Valizadeh J Shermeh O A amp Ak-aberi M (2015) Antioxidant activity andtotal phenolic content of boerhavia elegans(choisy) grown in baluchestan iran Avi-cenna Journal of Phytomedicine 5 (1) 1ndash9

Salawu S O Bester M J amp Duodu K G(2014) Phenolic composition and bioac-tive properties of cell wall preparationsand whole grains of selected cereals andlegumes Journal of Food Biochemistry38 (1) 62ndash72 doi101111jfbc12026

Salawu S O Innocenti M Giaccherini CAkindahunsi A A amp Mulinacci N(2008) Phenolic profiles of four processed

110 Salawu et al

tropical green leafy vegetables commonlyused as food Natural Product Communi-cations 3 (12) 2043ndash2048

Saleem Z M Ahmed S amp Hasan M M(2016) Phaseolus lunatus linn Botanymedicinal uses phytochemistry and phar-macology World Journal of Pharmacy andPharmaceutical Sciences 5 (11) 87ndash93

Scalbert A Manach C Morand C RemesyC amp Jimenez L (2005) Dietary polyphe-nols and the prevention of diseases Crit-ical Reviews in Food Science and Nu-trition 45 (4) 287ndash306 doi10 1080 1040869059096

Tagliazucchi D Verzelloni E Bertolini D ampConte A (2010) In vitro bio-accessibilityand antioxidant activity of grape polyphe-nols Food Chemistry 120 (2) 599ndash606doi101016jfoodchem200910030

Tavares L Figueira I Macedo D McDougallG J Leirao M C Vieira H L A Santos C N (2012) Neuroprotectiveeffect of blackberry (rubus sp) polyphe-nols is potentiated after simulated gastroin-testinal digestion Food Chemistry 131 (4)1443ndash1452 doi101016jfoodchem201110025

Trehan I amp Manary M J (2015) Man-agement of severe acute malnutrition inlow-income and middle-income countriesArchives of Disease in Childhood 100 (3)283ndash287 doi10 1136 archdischild - 2014 -306026

Wootton-Beard P C Moran A amp RyanL (2011) Stability of the total antioxi-dant capacity and total polyphenol con-tent of 23 commercially available veg-etable juices before and after in vitro di-gestion measured by frap dpph abts andfolinndashciocalteu methods Food Research In-ternational 44 (1) 217ndash224 doihttps doiorg101016jfoodres201010033

Yellavila S B Agbenorhevi J K AsibuoJ Y amp Sampson G Y (2015) Prox-imate composition minerals content andfunctional properties of five lima bean ac-cessions Journal of Food Security

Zou Y Chang S K C Gu Y amp Qian S Y(2011) Antioxidant activity and phenoliccompositions of lentil (lens culinaris var

morton) extract and its fractions Journalof Agricultural and Food Chemistry 59 (6)2268ndash2276 doi101021jf104640k

Moisture Sorption Isotherm and Thermodynamic Properties ofJamun (Syzygium cumini L) Powder made from Jamun Pulp

and Seed

Indira Dey Paula and Madhusweta Dasa

a Agricultural and Food Engineering Department Indian Institute of Technology Kharagpur Pincode-721302West Bengal India

Corresponding authormadhuagfeiitkgpacinTel +91 8777359497

Fax +91 3222 282244 255303

Received 10 July 2018 Published online 18 April 2019

Abstract

The present work aimed to i) find the suitable proportion based on sensory evaluation ofmicrowave-convective hot air dried jamun (Syzygium cumini L) pulp and seed kernel powder to bemixed for the preparation of jamun powder (JP) ii) generate and model the moisture sorption isotherm(MSI) of JP and iii) estimate net isosteric heat of sorption (qst) spreading pressure (Φ) net integralenthalpy (Qin) and net integral entropy (Sin) To formulate JP the proportion (ww db) comprising2 kernel and 98 pulp powder was the most desirable The Peleg model was the best fit to MSI ofJP The qst decreased following linear relationship from 1102 kJ molminus1 at 5 equilibrium moisturecontent (EMC) to 027 kJ molminus1 at 30 EMC The Φ increased with increase in water activity anddecreased with increase in temperature from 25 oC to 35 oC and the values of Φ at 45 oC were evenhigher than at 25 oC Net integral enthalpy (Qin) initially decreased till 6 moisture content in JP anddisplayed an increasing trend with further increase in moisture content On the contrary Sin kept ondecreasing continually with increasing moisture content The moisture zone of 7-11 was consideredsafe for storage of JP within the temperature range of 45-25 oC

Keywords Jamun powder Moisture sorption isotherm Net isosteric heat of sorption Spreadingpressure Net integral enthalpy Net integral entropy

1 Introduction

Jamun (Syzygium cumini L) a type of berrywith an attractive sweet-sour taste and purplecolour is a fruit native to India and East Indiesbut is also found in other countries like ThailandPhilippines and Madagascar All the fractionsof whole jamun fruit besides their nutritionalconstituents contain a lot of phytochemicalsand hence are widely popular for their medic-inal values Sehwag and Das (2015) reported abroad classification of the phytochemicals such

as anthocyanins phenolic acids flavonols fla-vanonols carotenoids and terpenes in the pulpand skin and alkaloids flavonoids glycosidesphytosterols saponins tannins and triterpenoidsin the seed Recent studies on jamun pulp andskin have established many therapeutic proper-ties such as antidiabetic antioxidant hepatopro-tective antibacterial and anticancerous Jamunseed kernel is richer in medicinal properties thanpulp and skin and possesses a range of pharma-cological actions (Sehwag amp Das 2015 2016)However due to its short seasonal availability

112 Dey Paul and Das

Nomenclature

am Surface area of a water molecule (m2)

aw Water activity

Φ Spreading pressure (J mminus2)

alowastw Geometric mean water activity at con-stant Φ

EMC Equilibrium moisture content ( db)

GMS Glycerol monostearate

JP Jamun powder

Kβ Boltzmann constant (J Kminus1)

LDPE Low density polyethylene

LSD Least significant difference

M EMC ( db) of the sample

MCD Microwave-convective hot air drying

MD Maltodextrin

MRE Mean residual error ()

MSI Moisture sorption isotherm

Qin Net integral enthalpy (kJ molminus1)

qst Net isosteric heat of sorption (kJmolminus1)

R Universal gas constant (kJ molminus1Kminus1)

r2 Co-efficient of determination

RH Relative humidity ()

RMSE Root mean square error

Sin Net integral entropy (kJ molminus1 Kminus1)

SD Standard deviation

T Absolute temperature (K)

TCP Tricalcium phosphate

Z Integration constant

and highly perishable nature jamun remains un-derutilizedRemoval of moisture by quick and efficient dry-ing is the most promising way for seasonal andperishable fruits to be made available through-out the year Dehydrated fruit in powderform can be incorporated within a variety ofrecipes like desserts biscuits and spreads toadd case specific flavor and functionalities with-out the moisture and volume of fresh fruitAmong different methods a hybrid techniquemicrowave-convective hot air drying (MCD) hasbeen used effectively for drying various food ma-terials such as apricot halves (Horuz BozkurtKaratas amp Maskan 2017) button mushrooms(Das amp Arora 2018) oyster mushrooms (Bhat-tacharya Srivastav amp Mishra 2015) okra (Ku-mar Prasad amp Murthy 2014) and garlic cloves(Sharma amp Prasad 2001) Prolonged exposurein conventional hot air drying leads to unde-sirable changes in product quality Combiningmicrowave with convective hot air can signifi-

cantly reduce the drying time and improve prod-uct quality (Schiffmann 1992) Although jamunseed powder (no mention of the presence or ab-sence of seed coat) is available as an ayurvedicmedicine for control of diabetic mellitus techni-cal information on drying of jamun seed and pulpis scanty in the literature Recently Dey Pauland Das (2017) produced free flowing jamun pulp(along with skin) powder containing maltodex-trin (MD 122) tricalcium phosphate (TCP04) and glycerol monostearate (GMS 14)on a dry basis (db) of pulp-skin using MCD at70 oC 1 watt gminus1 (W gminus1 power density) 05m sminus1 air velocity to maximally maintain itsantioxidant activity Monica Dey Paul and Das(2016) also observed that MCD (60 oC 2 Wgminus1 05 m sminus1) of jamun seed retained max-imum antioxidant activity of its kernel Thusif pulp powder is mixed with seed kernel pow-der (bitter in taste probably due to the presenceof saponins and tannins) (Kamal 2014 Kapooramp Iqbal 2013) in a sensorially acceptable ratio

Moisture Sorption and Thermodynamic Properties of Syzygium cumini Powder 113

the mixture could be used as jamun powder (JP)for food purpose with more health benefits thanthat of pulp powder aloneMoisture sorption tendency which depends onrelative humidity (RH) and temperature is amajor criterion in maintaining quality of fruitpowders during handling and storage Mois-ture sorption isotherm (MSI) provides the re-lationship between equilibrium moisture content(EMC) and its water activity (aw) when the foodequilibrates in air with different RHs at a certaintemperature On equilibration the aw of food(relates to stability) equals the RH of environ-ment (Al-Muhtaseb McMinn amp Magee 2002)Moisture sorption isotherm (MSI) is an essen-tial tool in design of drying packaging and stor-age of food Mathematical modelling enablesgeneration of MSI at any unknown temperaturewithin the temperature range for which the MSIshave already been developed empirically de San-tana et al (2015) reported MSIs of freeze driedjamun pulp (without skin) containing no addi-tives Biswal Mohapatra Panda and Dash(2017) modelled the desorption isotherm of freshjamun fruits Ferrari Marconi Germer AlvimVissotto and de Aguirre (2012) modelled theMSI of spray dried blackberry (Rubus species)powder containing maltodextrin and gum ara-bic Information on the MSI of powder contain-ing pulp skin and kernel of jamun together withany drying aid (to make the powder form) is notavailableMoisture sorption isotherms at different tem-peratures are used to evaluate thermodynamicparameters like net isosteric heat of sorption(qst) spreading pressure (Φ) net integral en-thalpy (Qin) and net integral entropy (Sin) (Al-Muhtaseb McMinn amp Magee 2004) The qst(total heat of sorption for binding water vaporon sorbent less than the heat of vaporization ofpure water at the system moisture content) de-termines moisture-sorbent binding strength andis required in designing equipment for dehydra-tion processes as well as prediction of aw whenthe food is stored at fluctuating temperature(Chowdhury amp Das 2010) Spreading pressure(Φ) the difference in surface tension betweenbare sorption sites of the sorbent and sites withadsorbed molecules indicates the free surface en-ergy of adsorption (Al-Muhtaseb et al 2004)

Net integral enthalpy (Qin) at constant Φ ameasure of the strength of moisture-solid bind-ing is used to determine Sin (McMinn amp Magee2003) Net integral entropy (Sin) explains thedegree of disorder and randomness of the ad-sorbed water molecules (Arslan amp Togrul 2005Igathinathane Womac Sokhansanj amp Pordes-imo 2007) and helps to determine the moistureat which the sorbent is stableBased on the above knowledge the objectives ofthe present work were to i) find the suitable pro-portion of pulp (with skin) and kernel powderto be mixed for preparation of JP ii) generateand model the MSI of JP and iii) estimate thethermodynamic parameters including net isos-teric heat of sorption (qst) spreading pressure(Φ) net integral enthalpy (Qin) and net integralentropy (Sin) for the JP

21 Material

Fully matured jamun fruit (raw material) ap-parently free from any damage was procuredfrom the local market of IIT Kharagpur IndiaAbout 75 of the fruit was comprised of pulpwith adherent skin (hereunder called pulp) withthe remaining 25 being the seed The mois-ture protein fat ash crude fiber and carbohy-drate content of pulp were analysed and foundto be 8304plusmn010 (wet basis wb) 859plusmn007323plusmn039 4plusmn0001 249plusmn000 and 8171plusmn045(dry basis db) respectively For seed kernelthe respective values were 1512plusmn004 (wb)590plusmn014 101plusmn001 234plusmn002 233plusmn037 and8841plusmn053 (db) (Sehwag amp Das 2016) MD(dextrose equivalent lt 20) and TCP both fromHiMedia Laboratories Pvt Ltd Mumbai In-dia and GMS from Alfa Aesar MassachusettsUSA were used as additives for production ofpulp powder Analytical grade lithium chloride(LiCl) potassium acetate (CH3COOK) mag-nesium chloride (MgCl2) potassium carbonate(K2CO3) magnesium nitrate Mg(NO3)2 sodiumnitrate (NaNO3) sodium chloride (NaCl) andpotassium chloride (KCl) were used to adjust RH(described below) All the chemicals were usedas received Glass distilled water was used for

preparation of salt solutions

22 Preparation of raw materials

The fruits were washed with potable water andair dried at room temperature The pulp wasseparated from the seed with a knife and storedin low density polyethylene (LDPE) pouches(about 250 g pouchminus1 pouch film thickness sim005 mm) at -30 oC The seeds were washed withwater to remove any adhering pulp and similarlypacked and stored

23 Preparation of jamun pulpand seed kernel powder

The frozen pulp was put in an empty glassbeaker which was then thawed in a water bathat room temperature (28 oC) The thawed masswas then ground into paste (at same tempera-ture) using domestic grinder (Sumeet Researchand Holdings Limited Chennai India) Thepaste was mixed with 122 MD 04 TCP and14 GMS on dry basis (db) of the pulp Themixed paste was dried using a fully controlledminiature conveyer type industrial microwave-convective hot air dryer (Enerzi Microwave Sys-tems Pvt Ltd Bangalore Karnataka India)having 20-100 kg hminus1 drying capacity 2450MHz frequency and 250-3000 W power An in-built heater of 6 kW provided hot air (25 oC to200 oC) circulation The drying chamber 2 mx 300 mm x 200 mm was made of 2 mm thickstainless steel Drying was carried out at 70 oC1 W gminus1 and 05 m sminus1 air velocity The com-position of the mixture and drying method andconditions were described earlier (Dey Paul ampDas 2017 Paul amp Das 2018) The dried pulpobtained in flake form was ground into fine pow-der (212 microm passing)The frozen jamun seeds were thawed dried usingthe same dryer but at 60 oC 2 W gminus1 and 05 msminus1 air velocity and the seed coat was manuallyremoved (Monica et al 2016) The dried kernelswere ground into fine powder (212 microm passing)To judge the functionality in separate work (notincluded here) total phenolic content (TPC)in mg gallic acid equivalent (GAE)g DPPHantioxidant activity (AA) in mg butylated hy-

droxyanisole (BHA)g db both in 50 aque-ous ethanolic extract and monomeric antho-cyanin content (MAC) in mg malvidin3glucoside(M3G) g db were measured for different por-tions (triplicate) of the powder before andafter mixing The pulp powder contained2619plusmn045 TPC 2379plusmn035 AA and 996plusmn016MAC whereas for kernel powder the values were5395plusmn026 TPC and 5797plusmn031 AA

24 Mixing of jamun pulp andseed kernel powder

Pulp powder and kernel powder in different pro-portions were mixed by tumbling (10 min) fol-lowed by wire whipping (10 min) using a handblender (Anjali Marketing amp Research CentreMumbai India) Based on a nine-point hedo-nic rating of appearance aroma taste mouthfeeland overall acceptability of the mixtures by 27semi-trained panellists a suitable proportion wasselected (Ranganna 1986) The selected mix-ture referred to as JP was double packaged inLDPE pouches and stored at -30 oC for furtherstudies

25 Determination and modellingof EMC

The isopiestic vapour transfer technique was usedto determine EMC (Chowdhury amp Das 2010)Around one gram of JP was placed in a weigh-ing bottle (in triplicate) and the bottles placed ineight vacuum desiccators each containing a sat-urated solution of a salt (section 32) to maintainRH (10-90) The desiccators were placed in anincubator at 25 oC After equilibration (aboutfive days based on preliminary experiments) themass of the bottles plus samples were notedThese were then dried overnight at 105plusmn1 oC todetermine the dry mass of JP The EMC ( db)was calculated using Eq (1)

EMC() =Weq minusWdry

Wdrytimes 100 (1)

where Weq and Wdry are the masses of equili-brated and dried powderThe experiment was similarly carried out at 35

oC and 45 oCFive MSI models (Table 1) were used to fit theexperimental EMCs using non-linear regressionanalysis (Microsoft Excel 2013) In the modelsM represents EMC ( db) of the sample and awis its water activity A B and C are the modelspecific coefficients In the GAB model A B andC respectively represent the monolayer moisturecontent heat of the first layer sorption and heatof the multilayer sorptionModel fitting parameters including co-efficientof determination (r2) root mean square error(RMSE) (Eq 2) mean residual error (MRE) (Eq 3) and residual plot (a plot of (Mi -Mpi) versus Mi for respective aws) were evalu-ated The r2 was evaluated through regressionanalysis

RMSE =

ntimes

nsumi=1

(Mi minusMpi)2

MRE() =100

nsumi=1

∣∣∣Mi minusMpi

∣∣∣ (3)

where Mi and Mpi are the experimental and pre-dicted values of EMC and n is the number ofexperimental runsTo be a good fit the model should have a r2

close to 1 RMSE close to 0 MRE below 10and randomness in the residual plot where thedata points are distributed in a horizontal bandaround zero (Lomauro Bakshi amp Chen 1985)

26 Net isosteric heat of sorption(qst)

The qst (kJ molminus1) was estimated using theClausius-Clapeyron equation (Eq 4)

ln aw = minus qstRT

+ Z (4)

where R is the universal gas constant(8314Ouml10minus3 kJ molminus1 Kminus1) T is theabsolute temperature in Kelvin (K) and Z is theintegration constantUsing the best fit model aw values were eval-uated at any particular moisture content fordifferent temperatures The slope of the plot ofln aw versus 1T gave the qst at that moisture

content This was repeated for different mois-ture contents to evaluate the effect of moisturecontent on qst The absolute value of qst wasconsidered as the sign of qst doesnrsquot bear anyphysical interpretation (Chowdhury amp Das2010)

27 Spreading pressure (Φ)

According to Igathinathane et al (2007) spread-ing pressure Φ (J mminus2) was calculated usingEq (5) as shown below

Φ =KβT

int aw

Aawdaw (5)

where Kβ is the Boltzmann constant (1380 Ouml10minus23 J Kminus1) Am is the surface area of a watermolecule (106 Ouml 10minus19 m2) M is the EMC andA is the monolayer moisture contentSince the integral cannot be evaluated at aw =00 the lower limit in Eq (5) was considered as005 After substituting the GAB equation (Ta-ble 1) in Eq (5) and integrating spreading pres-sure for the range of aw from 005 to aw was cal-culated using Eq (6) (Lago Liendo-Cardenasamp Zapata Norena 2013)

Φ =KβT

[1 +BCaw minus Caw

1minus Caw

]aw005

The integral covering the aw range from 000 to005 was evaluated (Eq 7) assuming a linearrelationship (Henryrsquos law) between MA and aw(Fasina Ajibola amp Tyler 1999 Igathinathaneet al 2007)

Φ =KβTM

AmA(7)

Totalling the values of Eq (6) and Eq (7) the Φcorresponding to any temperature and aw combi-nation was produced Spreading pressure versusaw was plotted for different temperatures

28 Net integral enthalpy (Qin)

Net integral enthalpy (Qin kJ molminus1) was eval-uated (Eq 8) in a comparable methodology tothat of the qst but at constant Φ instead of con-stant moisture content∣∣∣d[lnaw]

d[1T ]

∣∣∣Φasymp minusQin

Table 1 Sorption models used for fitting experimental EMC data of JP

Model Equation Reference

Peleg M = A(aw)B + C(aw)D Chowdhury and Das (2010)Guggenheim-Anderson-de-Boer (GAB) M = ABCaw[(1-Caw)(1-Caw + BCaw)] Ibanoglu Kaya and Kaya (1999)Oswin M = A [aw(1-aw)B ] de Santana et al (2015)Henderson M = [-ln(1-aw)A]1B de Santana et al (2015)Halsey M = [-Aln(aw)]1B Chowdhury and Das (2010)

From the plot of Φ versus aw at various temper-atures aw values at any particular Φ were ob-tained through interpolation (Igathinathane etal 2007) The slope of the plot of ln aw ver-sus 1T at constant Φ produced the Qin Nextmoisture contents at the interpolated aw valueswere calculated using the best fit MSI equationThus at three temperatures there were threemoisture contents for each Φ and the geometricmean moisture content was derived Qin versusgeometric mean moisture content was then plot-ted

29 Net integral entropy (Sin)

Net integral entropy (Sin kJ molminus1 Kminus1) wascalculated using the following equation (Eq 9)(Igathinathane et al 2007)

Sin =minusQinTminusRln(alowastW ) (9)

where alowastw is the geometric mean water activity atconstant Φ The temperature term T of Eq (9)was interpolated linearly for alowastw from the T ver-sus aw data for a given Φ Sin was then plottedagainst moisture content

210 Statistical analyses

The arithmetic mean of the replicated values ofdifferent observations and the respective stan-dard deviation (SD) were evaluated Geometricmeans in calculations of thermodynamic param-eters were also evaluated The mean values wereused in analysis of variance (F test one way) re-lating to parametric variation (treatment effect)followed by evaluation of least significant differ-

ence (LSD at Plt005 in cases of positive F test)paired t-test (Plt005) and model fitting

3 Results and discussion

31 Mixing of pulp and kernelpowder

In JP the sensory quality of pulp is most desir-able as the kernel powder possesses bitter tasteThe results of sensory evaluation on mixing ofpulp and kernel powder are shown in Table 2It is seen that aroma taste and overall accept-ability of the pulp powder were negatively af-fected (F test positive) by addition of kernel pow-der whereas appearance and mouthfeel remainedunaffected The corresponding LSD value (in-cluded in the same table) indicates that deteri-oration of the pulp powder starts at 6 addi-tion of kernel powder for aroma and 4 addi-tion of kernel powder for both taste and over-all acceptability At 2 addition none of thesequalities were affected Therefore 2 kernel ad-dition was finally selected for the production ofJP The produced powder (JP) is shown in Fig-ure 1 which contained TPC AA and MAC val-ues of 2725plusmn019 (mg GAEg db) 2504plusmn007(mg BHAg db) and 977plusmn022 (mg M3Gg db)respectively These values are statistically differ-ent (t-test) from those of pulp powder Thusaddition of 2 kernel powder led to an increasein TPC and AA values of the pulp powder by 4-5 while the MAC value was less (sim 2) Fur-thermore the standard deviation of these func-tionalities was within 02-2 Since low standarddeviation is an indication of powder mixing theJP can be considered to have good overall uni-formity

Table 2 Sensory evaluation of the mixed powder containing different proportions of pulp and kernelpowder

Mixed powder constituents MeanplusmnSD( ww db)

AppearanceΨ Aroma Taste MouthfeelΨOverall

Pulp powder Kernel powder acceptability

100 0 756plusmn070 763plusmn097 743plusmn072 663plusmn142 767plusmn064

98 2 756plusmn070 774plusmn082lowast 746plusmn080 680plusmn094 756plusmn066

96 4 752plusmn080 726plusmn132lowast 639plusmn075 652plusmn153 650plusmn07194 6 720plusmn111 691plusmn158lowast$ 600plusmn077 652plusmn142 582plusmn08492 8 713plusmn089 683plusmn139$ 556plusmn074 622plusmn131 539plusmn08390 10 704plusmn129 674plusmn168$ 493plusmn084 606plusmn130 474plusmn090

LSD - 071 041 - 041Ψ P gt005 in F-test $ The same superscript indicates no significant difference between the mean values

Figure 1 Jamun powder (JP) having particlesize below 212 microm

32 Equilibrium moisture content(EMC) of JP

The mean EMCsplusmnSD of JP for varying RH (withdifferent salts) at 25 oC 35 oC and 45 oC are pre-sented in Table 3 One-way ANOVA at any tem-perature resulted in a positive F-test (p lt005)indicating that EMC is significantly dependent

on the RH of the environment (aw of the sam-ple) LSD values further confirm that at all threetemperatures EMC increased with increase inRHaw except for the range of aw from 0113 to0215 at 35 oC Thus the sorption characteristicsshow a direct relationship between aw and EMC

33 Fitting of sorption model

Table 4 summarizes the results of regression anal-ysis for fitting the EMC data to various modelsand the model fitting criteria All five modelsproduced r2 close to 1 at all three temperaturesConsidering MRE RMSE and residual plot re-quirements the Peleg model was found to be themost suitable Except for a RMSE value at 25oC the GAB model satisfied all the selection cri-teria at all three temperatures The Oswin modelsuited all criteria better at 35 oC At 25 oC it in-dicated a MRE gt15 and at 45 oC there wasa residual plot pattern The Halsey model wasmore satisfactory at 45 oC At 25 oC it indicateda pattern shape and at 35 oC the MRE was asymp10 The Henderson model produced a patternplot at 35 oC and 45 oC and a gt10 MRE at allthe temperatures Hence the Peleg model can beconsidered as the best fit followed by the GABmodel for EMCs of microwave-convective hot airdried JP within the temperature range of 25-45oC The Peleg model was found to be suitable byde Santana et al (2015) for freeze dried jamun

Table 3 The mean equilibrium moisture content (EMC) along with standard deviation (SD) of jamunpowder (JP) for varying aw at 25 oC 35 oC and 45 oC

Salt solutions used 25 oC 35 oC 45 oCfor generation of aw aw EMC aw EMC aw EMC

( db)plusmnSDΨ ( db)plusmnSDΨ ( db)plusmnSDΨ

Lithium chloride (LiCl) 0114 443plusmn036 0113 338plusmn037 0112 282plusmn031Potassium acetate (CH3COOK) 0237 609plusmn027 0215 473plusmn064 0197 457plusmn027Magnesium chloride (MgCl2) 0327 998plusmn041 0320 751plusmn044 0311 673plusmn011Potassium carbonate (K2CO3) 0443 1284plusmn024 0436 1072plusmn094 0429 1051plusmn027Magnesium nitrate Mg(NO3)2 0536 1652plusmn047 0515 1425plusmn249 0497 1207plusmn026Sodium nitrate (NaNO3) 0742 3059plusmn053 0720 2712plusmn165 0699 2489plusmn042Sodium chloride (NaCl) 0752 3220plusmn024 0748 2964plusmn137 0745 3116plusmn084Potassium chloride (KCl) 0855 4317plusmn019 0822 4119plusmn064 0791 4009plusmn008

LSD 062 LSD 219 LSD 068Ψ Average of three replications plusmn SD indicates no significant difference between the values

pulp isotherms

34 Moisture sorption isotherms

Figure 2 illustrates the MSIs of JP at 25 oC35 oC and 45 oC as fitted by the Peleg modelDepending on slope the MSIs can be dividedinto three zones At any temperature the EMCof JP increased relatively slowly at aws withinasymp 01-05 compared to the region beyond 05aw where sharp increases were observed Thusshape of the isotherms indicates Type III be-haviour which is a characteristic of foods rich insoluble components (Rizvi 2014) JP containsabout 45 reducing sugars (db) with glucose it-self amounting to about 19 (db) (obtained indifferent experiments not included here) Theobserved trend of moisture sorption can be at-tributed to the dissolution tendency of fruit sug-ars in the absorbed moisture (Maroulis TsamiMarinos-Kouris amp Saravacos 1988) A similarkind of sorption trend was also noticed for sugar-rich foods such as tomato pulp powder (GoulaKarapantsios Achilias amp Adamopoulos 2008)grapes apples (Kaymak-Ertekin amp Gedik 2004)and orange juice powder (Sormoli amp Langrish2015) Since EMC underwent a sharp increasebeyond 05 aw it can be said that at RH higherthan 50 JP needs special storage conditionsto inhibit several possible degradation reactionsda Silva Meller da Silva and Pena (2008) alsovoiced a similar opinion in their study

In Figure 2 the EMC decreased with increas-ing temperature (25 oC gt35 oC gt45 oC) at anyvalue of aw up to about 075 with slopes be-ing higher than that exhibited below 05 awThe decrease in the total number of activesites as a consequence of temperature inducedphysicalchemical changes may result in thefall of EMC (Chowdhury amp Das 2012 Muzaf-far amp Kumar 2016) At increased tempera-tures water molecules because of their activa-tion at higher energy levels gain higher mo-bility become unstable and break away fromthe binding sites thereby causing a reductionin EMC as explained by Menkov and Du-rakova (2007) According to de Santana et al(2015) and Al-Muhtaseb et al (2002) the wa-ter vapour pressure (WVP) on powder surfacesincreases at higher temperatures allowing thewater molecules to get detached from the bind-ing sites Interestingly for aw higher than 075the increase in EMC with aw is even higher thanfor the previously displayed regions Moreovera crossover is seen so that the EMCs follow theorder 45 oC gt35 oC gt25 oC As described byBasu Shivhare and Muley (2013) and Labuzaand Altunakar (2007) such crossover at high wa-ter activity is mainly due to increased solubilityof sugar and dissolution of new solutes at highertemperatures Also at high water activity thesoluble components absorb more water which isfurther accentuated by the temperature (Basu etal 2013)

Table 4 Model coefficients and error terms for different sorption models

Temp (oC) EMC Model A B C D r2 RMSE MRE () Residual Plot

Peleg 303963 09989 368762 50221 09946 08827 78715 RandomGAB 111246 52765 08781 - 09874 14263 91236 RandomOswin 164801 05272 - - 09844 18427 154206 RandomHenderson 00198 12322 - - 09960 18681 124555 RandomHalsey 380501 14649 - - 09740 24035 195301 Pattern

Peleg 239963 09999 678792 59221 09875 04133 51785 RandomGAB 95502 29603 09584 - 09848 03629 48470 RandomOswin 134154 07336 - - 09858 03076 40173 RandomHenderson 00740 08542 - - 09956 09373 106109 PatternHalsey 104390 10626 - - 09765 08959 99953 Random

Peleg 230082 09978 1233597 73802 09802 02312 25248 RandomGAB 73339 39545 10452 - 09798 02416 20775 RandomOswin 127807 08417 - - 09905 06595 68383 PatternHenderson 00478 10028 - - 09984 30425 105552 PatternHalsey 66982 09085 - - 09837 03622 44701 Random

Figure 2 Effect of temperature on moisture sorption isotherm of JP as expressed by the Peleg model

Figure 3 Variation in net isosteric heat of sorption (qst) of jamun powder (JP) as a function of moisturecontent

The absolute value of qst (Eq 4) of JP as a func-tion of moisture content is represented in Figure3 Mathematically the trend is represented bythe following linear relation (Eq 10) and theresulting r2 justifies the appropriateness of thefit

qst = minus04743M + 14337 r2 = 09728 (10)

where M is the moisture content in db Thusthe qst decreases with the increase in moisturecontent from 1102 kJ molminus1 at 5 moisture to027 kJmolminus1 at 30 thus approaching the heatof vaporization of pure water The linear decreas-ing trend of qst was also observed by de Santanaet al (2015) for freeze dried jamun pulp and byBasu et al (2013) for pectin It may be worthmentioning that the maximum qst in this study is

comparable with the value for orange juice pow-der (905 kJ molminus1) calculated by Sormoli andLangrish (2015)Since higher the value of qst means higher is thedegree of binding the water molecules becometightly bound to the active sites on the surface ofthe JP at low moisture contents (Sormoli amp Lan-grish 2015) As explained by Iglesias and Chir-ife (1982) sorption occurs initially at the mostactive sites involving higher interaction energyAs active sites get exhausted moisture bindingstarts at less active sites producing less heat ofsorption

The value of Φ increases almost linearly withincrease of aw (Figure 4) At any aw Φ de-creases for an increase of temperature from 25oC to 35 oC However for further increase to

Figure 4 Variation in spreading pressure (Φ) with water activity (aw) at 25 oC 35 oC and 45 oC

45 oC Φ increased to levels even higher thanthat at 25 oC and the differences in Φ fur-ther increased for aw from 03 onwards Itmay be stated that the effect of temperatureon Φ is case specific depending on composi-tion of the sorbent Depending on temperatureΦ may be increasingdecreasingdecreasing thenincreasingincreasing then decreasing (Aviara ampAjibola 2002 Lago et al 2013 Al-Muhtaseb etal 2004 Togrul amp Arslan 2007) Similar obser-vations as for the present study were reportedfor high amylose starch powder (Al-Muhtaseb etal 2004) at 30-60 oC and walnut kernel (Togrulamp Arslan 2007) at 25-45 oCSpreading pressure is the driving force for dif-fusion of moisture in a porous solid (Skaar ampBabiak 1982) Torres Moreira Chenlo andVazquez (2012) explained that high spreadingpressure values indicate a high affinity of water

molecules for binding sites This may lead toswelling in a high RH environment (discussed insection 37)

Net integral enthalpy (Qin) of bound watermolecules initially decreases up to about 6moisture content and then increases for highermoisture content (Figure 5) The decreasing por-tion probably arises from the monolayer cover-ing of JP (Fasina et al 1999) At low moisturecontents water is preferentially adsorbed on themost accessible locations on the exterior surfaceof the solid Following this as less favourablesites are filled up the Qin then gradually de-clines till there is formation of multi layers of wa-ter (Al-Muhtaseb et al 2004) With still highermoisture content gained in a higher RH atmo-

Figure 5 Variation in net integral enthalpy (Qin) of jamun powder (JP) with moisture content

sphere there is probably swelling of the pow-der with concomitant exposition of higher andhigher energy binding sites that require increas-ingly higher Qin involvement Such a decreas-ing followed by increasing profile has been doc-umented for mango skin (Ferreira de Souza etal 2015) and orange peel (Villa-Velez Ferreirade Souza Ramos Polachini amp Telis-Romero2015) An increase in Qin with increasing mois-ture content was also reported in studies on cas-sava (Aviara amp Ajibola 2002) and potato andsweet potato flakes (Lago et al 2013) In thecase of walnuts kernels the Qin increased slightlywith increasing moisture content and then re-mained constant (Togrul amp Arslan 2007)

38 Net integral enthalpy (Sin)

Net integral entropy (Sin) of the adsorbed wa-ter molecules was found to decrease continuallywith moisture content of JP (Figure 6) The lossin entropy in general indicates the loss of ro-

tational freedom or randomness of the adsorbatemolecules On exposure to low RHaw the easilyavailable sites on the surface become filled wherein addition to ligandndashsubstrate binding lateralinteractions in the adsorbed molecules also con-tribute to reduction in randomness (Aviara ampAjibola 2002) At increasingly higher RH thereis attainment of a higher EMC leading to struc-tural transformation arising from solubilisationof ingredients and swelling of the powder whichultimately opens more and more sites to bind wa-ter molecules (McMinn amp Magee 2003 ZhangBai Zhao amp Duan 2016) In Figure 6 the max-imum values of Sin were found at the moisturecontent of around 1 (db) and can be consideredas the zone of most energetic water molecules inthe material Following this up to about 8 dbmoisture content Sin decreased but remained ina positive zone With gt8 moisture the valueswere all negative Similar profiles of Sin coveringboth positive and negative zones have been re-ported for moisture sorption of mango skin (Fer-

Figure 6 Variation in net integral entropy (Sin) of jamun powder (JP) with moisture content

reira de Souza et al 2015) and gari (Aviara ampAjibola 2002) For mango skin adsorption thenegative value was also comparable to JP Igle-sias Chirife and Viollaz (1976) explained thatnegative values of Sin might be attributed to theexistence of chemical adsorption andor struc-tural modifications of the adsorbent while Rizvi(2014) attributed it to the fact that the prod-ucts contain more polar groups which bind wa-ter more stronglyThe minimum integral entropy zone correspondsto maximum stability of food products since inthis zone the water molecules are well accommo-dated and less available to take part in deterio-rating reactions In the case of JP where Sin isgradually decreasing probably due to structuralmodification (as mentioned above) the mini-mum integral entropy zone can be consideredcorresponding to the monolayer moisture content

(Ferreira de Souza et al 2015) Thus from Ta-ble 4 the safe moisture content may be in thezone of 7-11 (GAB monolayer value) for thetemperature range of 45ndash25 oC

4 Conclusions

Jamun seed kernel powder could be added to ja-mun pulp powder at a level of 2 (db) with-out affecting the sensorial acceptance of the pulppowder in the mixture finally named as jamunpowder (JP) The EMC of JP decreased with in-creasing temperature (25 oC gt35 oC gt45 oC)at any constant value of aw up to about 075and the Peleg model could satisfactorily describethe moisture sorption isotherms within 25-45 oCOver the range of EMC from 5-30 the qst de-creased linearly from 1102 kJ molminus1 to 027 kJmolminus1 The Φ increased with increasing aw and

with respect to temperature it followed the or-der 45 oC gt25 oC gt35 oC Net integral en-thalpy (Qin) displayed a decreasing trend till 6moisture content of JP followed by an increasingpattern thereafter for higher moisture contentsNet integral entropy (Sin) was observed to de-crease continuously with the increase in moisturecontent and the monolayer moisture content 7-11 may be regarded as the safe moisture con-tent zone for JP within the temperature range of45-25 oC

References

Arslan N amp Togrul H (2005) Modelling of wa-ter sorption isotherms of macaroni storedin a chamber under controlled humidityand thermodynamic approach Journal ofFood Engineering 69 (2) 133ndash145 doi101016jjfoodeng200408004

Aviara N A amp Ajibola O O (2002) Ther-modynamics of moisture sorption in melonseed and cassava Journal of Food En-gineering 55 (2) 107ndash113 doi10 1016 S0260-8774(02)00023-7

Basu S Shivhare U S amp Muley S (2013)Moisture adsorption isotherms and glasstransition temperature of pectin Journalof Food Science and Technology-mysore50 (3) 585ndash589 doi101007s13197- 011-0327-y

Bhattacharya M Srivastav P P amp MishraH N (2015) Thin-layer modeling of con-vective and microwave-convective dryingof oyster mushroom (Pleurotus ostreatus)Journal of Food Science and Technology-mysore 52 (4) 2013ndash2022 doi10 1007 s13197-013-1209-2

Biswal S Mohapatra M Panda M Kamp Dash S K (2017) Moisture desorp-tion isotherms of fresh jamun (Syzygiumcumini) fruit Indian Journal of Agricul-tural Research 51 (3) 267ndash271

Chowdhury T amp Das M (2010) Moisture sorp-tion isotherm and isosteric heat of sorptioncharacteristics of starch based edible filmscontaining antimicrobial preservative In-ternational Food Research Journal 17 (3)601ndash614 Retrieved from httpwwwifrj

upm edu my 17 5C 20(03 ) 5C 202010IFRJ-2010-601-6145C20Indiapdf

Chowdhury T amp Das M (2012) Moisture sorp-tion isotherm and isosteric heat of sorptionof edible films made from blends of starchamylose and methyl cellulose InternationalFood Research Journal 19 (4) 1669ndash1678Retrieved from httpwwwifrjupmedumy

da Silva A E Meller da Silva L H ampPena R d S (2008) Hygroscopic behav-ior of acai and cupuacu powders Ciencia ETecnologia De Alimentos 28 (4) 895ndash901doi101590S0101-20612008000400020

Das I amp Arora A (2018) Alternate microwaveand convective hot air application for rapidmushroom drying Journal of Food En-gineering 223 208ndash219 doi10 1016 j jfoodeng201710018

de Santana R F de Oliveira Neto E R San-tos A V Faria Soares C M Lima A Samp Cardoso J C (2015) Water sorptionisotherm and glass transition temperatureof freeze-dried Syzygium cumini fruit (jam-bolan) Journal of Thermal Analysis andCalorimetry 120 (1) 519ndash524 doi101007s10973-014-4014-x

Dey Paul I amp Das M (2017) Effect of mal-todextrin tricalcium phosphate and glyc-erol monostearate on moisture sorptioncharacteristics of jamun (Syzygium cuminiL) pulp powder In Proceedings of Inter-national Food Research Conference heldat Universiti Putra Malaysia Malaysia(p 87) 25-27 July

Fasina O O Ajibola O O amp Tyler R T(1999) Thermodynamics of moisture sorp-tion in winged bean seed and gari Jour-nal of Food Process Engineering 22 (6)405ndash418 doi10 1111 j 1745 - 4530 1999 tb00496x

Ferrari C C Marconi Germer S P AlvimI D Vissotto F Z amp de Aguirre J M(2012) Influence of carrier agents on thephysicochemical properties of blackberrypowder produced by spray drying Interna-tional Journal of Food Science and Tech-nology 47 (6) 1237ndash1245 doi10 1111 j 1365-2621201202964x

Ferreira de Souza S J Alves A I RufinoVieira E N Gomez Vieira J A RamosA M amp Telis-Romero J (2015) Study ofthermodynamic water properties and mois-ture sorption hysteresis of mango skin FoodScience and Technology 35 (1) 157ndash166doi1015901678-457X6557

Goula A M Karapantsios T D AchiliasD S amp Adamopoulos K G (2008) Wa-ter sorption isotherms and glass transitiontemperature of spray dried tomato pulpJournal of Food Engineering 85 (1) 73ndash83doi101016jjfoodeng200707015

Horuz E Bozkurt H Karatas H amp MaskanM (2017) Drying kinetics of apricothalves in a microwave-hot air hybrid ovenHeat and Mass Transfer 53 (6) 2117ndash2127doi101007s00231-017-1973-z

Ibanoglu S Kaya S amp Kaya A (1999)Evaluation of sorption properties of Turk-ish tarhana powder Food Nahrung43 (2) 122ndash125 doi101002(SICI)1521-3803(19990301)432〈122AID-FOOD122〉30CO2-3

Igathinathane C Womac A R SokhansanjS amp Pordesimo L O (2007) Mois-ture sorption thermodynamic properties ofcorn stover fractions Transactions of theAsabe 50 (6) 2151ndash2160 Annual Meetingof the American-Society-of-Agricultural-and-Biological-Engineers Univ Wisconsin-Madison Madison WI NOV 09-12 2005

Iglesias H A amp Chirife J (1982) Hand-book of food isotherms Water sorptionparameters for food and food compo-nents (Chap Mathematical description ofisotherms pp 262ndash335) New York Aca-demic Press

Iglesias H A Chirife J amp Viollaz P (1976)Thermodynamics of water vapour sorptionby sugar beet root International Journalof Food Science amp Technology 11 (1) 91ndash101 doi10 1111 j 1365 - 2621 1976 tb00705 x eprint https onlinelibrarywileycomdoipdf101111j1365-26211976tb00705x

Kamal A (2014) Phytochemical screening ofSyzygium cumini seeds Indian Journal ofPlant Sciences 3 (4) 1ndash4

Kapoor A amp Iqbal H (2013) Efficiency of tan-nase produced by Trichoderma harzianumMTCC 10841 in pomegranate juice clar-ification and natural tannin degradationInternational Journal of Biotechnology andBioengineering Research 4 (6) 641ndash650

Kaymak-Ertekin F amp Gedik A (2004) Sorp-tion isotherms and isosteric heat of sorp-tion for grapes apricots apples and pota-toes LWT - Food Science and Technology37 (4) 429ndash438 doi101016jlwt200310012

Kumar D Prasad S amp Murthy G S (2014)Optimization of microwave-assisted hot airdrying conditions of okra using responsesurface methodology Journal of Food Sci-ence and Technology-mysore 51 (2) 221ndash232 doi101007s13197-011-0487-9

Labuza T P amp Altunakar B (2007) Wateractivity in foods Fundamentals and ap-plications In G V Barbosa-Canovas JA J Fontana S J Schmidt amp T PLabuza (Eds) (Chap Water activity pre-diction and moisture sorption isothermspp 109ndash154) Blackwell Publishing Ox-ford UK

Lago C C Liendo-Cardenas M amp Zap-ata Norena C P (2013) Thermodynamicsorption properties of potato and sweetpotato flakes Food and Bioproducts Pro-cessing 91 (C4) 389ndash395 doi10 1016 j fbp201302005

Lomauro C J Bakshi A S amp Chen J Y(1985) Evaluation of food moisture sorp-tion isotherm equations Part I Fruit veg-etable and meat products LWT - Food Sci-ence and Technology 18 111ndash117

Maroulis Z B Tsami E Marinos-Kouris Damp Saravacos G D (1988) Application ofthe GAB model to the moisture sorptionisotherms for dried fruits Journal of FoodEngineering 7 (1) 63ndash78 doi10 1016 0260-8774(88)90069-6

McMinn W A M amp Magee T R A (2003)Thermodynamic properties of moisturesorption of potato Journal of Food En-gineering 60 (2) 157ndash165 doi10 1016 S0260-8774(03)00036-0

Menkov N D amp Durakova A G (2007) Mois-ture sorption isotherms of sesame flour at

several temperatures Food Technology andBiotechnology 45 (1) 96ndash100

Monica N Dey Paul I amp Das M (2016)Microwave-convective hot air and vacuumdrying of jamun seed powder In H NMishra S L Srivastava P P Srivas-tava amp P P Tripathy (Eds) Food pro-cess engineering and technology (Vol Pa-pers presented at International Conferenceon Emerging Technologies in Agriculturaland Food Engineering held at Indian Insti-tute of Technology Kharagpur India 27-30December pp 311ndash323) New Delhi ExcelIndia Publishers

Al-Muhtaseb A H McMinn W A M ampMagee T R A (2002) Moisture sorp-tion isotherm characteristics of food prod-ucts A review Food and Bioproducts Pro-cessing 80 (2) 118ndash128 doi10 1205 09603080252938753

Al-Muhtaseb A H McMinn W A M ampMagee T R A (2004) Water sorp-tion isotherms of starch powders part 2Thermodynamic characteristics Journal ofFood Engineering 62 (2) 135ndash142 doi101016S0260-8774(03)00202-4

Muzaffar K amp Kumar P (2016) Moisturesorption isotherms and storage study ofspray dried tamarind pulp powder Pow-der Technology 291 322ndash327 doi101016jpowtec201512046

Paul I D amp Das M (2018) Effect of freezemicrowave-convective hot air vacuum anddehumidified air drying on total phe-nolics content anthocyanin content andantioxidant activity of jamun (Syzygiumcumini L) pulp Journal of Food Scienceand Technology-mysore 55 (7) 2410ndash2419doi101007s13197-018-3158-2

Ranganna S (1986) Handbook of analysis andquality control for fruit and vegetable prod-ucts (Chap Sensory Evaluation pp 594ndash645) Tata McGraw-Hill Education

Rizvi S S H (2014) Thermodynamic proper-ties of foods in dehydration In Engineeringproperties of foods third edition (pp 359ndash435) CRC Press

Schiffmann R F (1992) Microwave processingin the US food industry Food Technology

Sehwag S amp Das M (2015) Nutritive ther-apeutic and processing aspects of jamunSyzygium cuminii (L) skeels-an overviewNatural Product Radiance 5 295ndash307

Sehwag S amp Das M (2016) Composition andantioxidant potential of jamun (Syzygiumcumini L) from eastern india Asian Jour-nal of Biochemical and Pharmaceutical Re-search 6 106ndash121

Sharma G P amp Prasad S (2001) Dry-ing of garlic (allium sativum) cloves bymicrowave-hot air combination Journal ofFood Engineering 50 (2) 99ndash105 doi10 1016S0260-8774(00)00200-4

Skaar C amp Babiak M (1982) A modelfor bound-water transport in wood WoodScience and Technology 16 (2) 123ndash138doi101007BF00351098

Sormoli M E amp Langrish T A G (2015)Moisture sorption isotherms and net isos-teric heat of sorption for spray-dried pureorange juice powder LWT - Food Sci-ence and Technology 62 (1 2 SI) 875ndash882doi101016jlwt201409064

Togrul H amp Arslan N (2007) Moisture sorp-tion isotherms and thermodynamic prop-erties of walnut kernels Journal of StoredProducts Research 43 (3) 252ndash264 doi101016jjspr200606006

Torres M D Moreira R Chenlo F ampVazquez M J (2012) Water adsorptionisotherms of carboxymethyl cellulose guarlocust bean tragacanth and xanthan gumsCarbohydrate Polymers 89 (2) 592ndash598doi101016jcarbpol201203055

Villa-Velez H A Ferreira de Souza S JRamos A P Polachini T amp Telis-Romero J (2015) Thermodynamic prop-erties of water adsorption from orangepeels Journal of Bioenergy and Food Sci-ence 2 (2) 72ndash81 doi1018607jbfsv2i232

Zhang H Bai Y Zhao X amp Duan R(2016) Water desorption isotherm and itsthermodynamic analysis of glutinous riceflour American Journal of Food Technol-ogy 11 (4) 115ndash124 Retrieved from 10 3923ajft2016115124

wwwiseki-food-ejournalcom

Introduction

Materials and Methods

Materials

Preparation of cashew flour

Chemical analyses

Statistical analysis

Results and Discussions

Conclusion

References

Introduction


Experimental design and Extrusion Cooking

Moisture content determination

Bulk density (BD) and Expansion Index (EI)

Color characteristics determination


Results and Discussion

Experimental results of extrusion exercise

Effects of individual variables and interaction on responses

Fitting and validating RSM models for optimizing production processes

Conclusions

References

Introduction


Materials

Preparation of Flours

Experimental design for the development of flour blends

Proximate composition determination of the composite flour

Minerals analysis

Amino acid profile determination

Evaluation of Antioxidants Properties

Determination of Antinutrients

In-vitro protein (IVPD) determination

In-vitro carbohydrate digestibility determination (IVCD)


Proximate composition and functional properties of the composite flour

Minerals composition of optimized composite flour

Antioxidant properties of the composite flour

Conclusions

References

Introduction


Wheat and barley flour

Non-traditional flour samples

Analytical tests

Rheological behaviour

Bread baking trial



Effects of non-traditional plant materials on properties of wheat polysaccharides

Effect of non-traditional plant materials on properties of wheat proteins

Effect of non-traditional plant materials on wheat dough during kneading

Effect of non-traditional plant materials on baking test results

Sensory analysis of bread

Correlation analysis

Principal component analysis

Conclusion

References

Introduction


Sample Collection and Preparation

Measurement of Ascorbic Acid in Broccoli

Reaction Kinetics

Statistical Analysis


Conclusion

References

Introduction


Animals and sampling procedure

Physicochemical analysis

Antimicrobial proteins and IgA




Antimicrobial Proteins

Conclusion

References

Introduction


Material

Tzatziki samples

Determination of the microbial population using firefly luciferase for ATP measurement

Determination of Colony Forming Units (CFU)



Conclusions

References

Introduction


Sample Preparation

Moisture Sorption Isotherm

Differential Scanning Calorimetry




Thermal Characteristics

Conclusions

References

Introduction


Chemicals

Sample collection

Sample treatment and preparation

in vitro enzyme digestion

Quantification of phenolic compounds by HPLC-DAD

Determination of total phenolic content

Determination of total flavonoid content

Reducing power assay

DPPH radical scavenging assay

Amino acid profile assay



Results

Conclusions

References

Introduction


Material

Preparation of raw materials

Preparation of jamun pulp and seed kernel powder

Mixing of jamun pulp and seed kernel powder

Determination and modelling of EMC

Net isosteric heat of sorption (qst)

Spreading pressure ()

Net integral enthalpy (Qin)

Net integral entropy (Sin)

Statistical analyses

Results and discussion

Mixing of pulp and kernel powder

Equilibrium moisture content (EMC) of JP

Fitting of sorption model

Moisture sorption isotherms




Net integral enthalpy (Sin)

Conclusions

References

International Journal of Food Studies

The International Journal of Food Studies (IJFS) a journal of the ISEKI_Food Association is an international peer-

reviewed open-access journal featuring scientific articles on the world of Food in Education Research and Industry

This journal is a forum created specifically to improve the dissemination of Food Science and Technology

knowledge between Education Research and Industry stakeholders Manuscripts focusing on Food related

Education topics are particularly welcome The IJFS also accepts original research works dealing with food processing

design storage and distribution including effects on productrsquos safety and quality and food chain sustainability The

journal is also open to other food-related topics such as food security and food policy

Editor-in-Chief

PROFESSOR CRISTINA L M SILVA

Catholic University of Portugal - College of Biotechnology

Rua Arquiteto Lobatildeo Vital 172 4200-374 Porto Portugal

Vice Editors-in-Chief

High Institute of Engineering of University of Algarve

Estrada da Penha 139 8005-139 Faro Portugal

University of Satildeo Paulo - Faculty of Animal Science and

Food Engineering Av Duque de Caxias Norte 225

Campus Fernando Costa ndash USP CEP 13635-900

Pirassununga Satildeo Paulo Brazil

Associate Editors

Professor Liliana Tudoreanu

University of Agronomic Sciences and Veterinary

Medicine Bucharest Romania

University of Satildeo Paulo - Faculty of Animal

Science and Food Engineering Brazil

Professor Petras Rimantas Venskutonis

Kaunas University of Technology Lithuania

Professor Rui Costa

College of Agriculture - Polytechnic Institute of

Coimbra Portugal

Dr Rui Cruz

Professor Tanaboon Sajjaanantakul

Kasetsart University Thailand

Professor Victoria Jideani

Cape Peninsula University of Technology South

Africa

Advisory Board

Afam Jideani

University of Venda South Africa

Antoacutenio Vicente

Brian McKenna

University College Dublin Ireland

Elisabeth Dumoulin

Paris Institute of Technology for

Life France

Ferruh Erdogdu

Ankara University Turkey

Gerhard Schleining

BOKU Austria

Gustavo V Barbosa-Canovas

Washington State University

United States of America

Gustavo Gutieacuterrez-Loacutepez

National School of Biological

Sciences Mexico

Joseacute Antoacutenio Teixeira

Kristberg Kristbergsson

University of Iceland Iceland

Mustapha El Idrissi

Mohamed Premier University

Morocco

Pablo Ribotta

School of Exact Physics and

Natural Sciences Argentina

Paul Singh

University of California - Davis

United States

Paola Pittia

University of Teramo Italy

Peter Ho

University of Leeds United

Kingdom

Pilar Buera

School of Exact and Natural

Sciences Argentina

Sam Saguy

Hebrew University of Jerusalem

Israel

Teresa Brandatildeo

Catholic University of Portugal

Portugal

V Prakash

Central Food Technological

Research Institute India

Venkatesh Meda

University of Saskatchewan

Canada

OF FOOD STUDIES

CONTENTS

Lemon Pomace

DOURTOGLOU

and Seed

Abstract

1 Introduction

2 Maria and Hannah

21 Materials

Soaking

Autoclaving

Germination

4 Maria and Hannah

6 Maria and Hannah

8 Maria and Hannah

nd - not detected

10 Maria and Hannah

4 Conclusion

References

12 Maria and Hannah

Abstract

1 Introduction

Nomenclature

EI Expansion index

BD Bulk density

L Lightness

a Redness

b Yellowness

D Deviations

16 Nahemiah et al

Y = b0+

nsumi=1

biix2i +

nminus1sumi=1

nsumj=i+1

bijxixj+ei

xi =xi minus x0δx

Υtimes 100 (4)

πd2L(5)

18 Nahemiah et al

20 Nahemiah et al

Bulk density

Expansion index

22 Nahemiah et al

Y D RD Y D RD

1 0089 0092 10337 1278 1282 100312 0058 0041 6897 1311 1307 99693 0035 0021 6000 1257 1258 100084 0156 0162 10385 1309 1308 99925 0358 0329 9190 1307 1308 100086 0072 0083 11528 1299 1298 99927 0075 0081 10800 1317 1316 99928 0039 0028 7179 1327 1329 100159 0142 0146 10282 1288 1283 996110 0059 0059 10000 1315 1315 1000011 0147 0151 10272 1289 1289 1000012 0048 0045 9375 1295 1295 1000013 0058 0062 10690 1298 1298 1000014 0154 0148 9610 1338 1338 1000015 0059 0058 9831 1335 1335 1000016 0058 0057 9828 1337 1338 1000717 0059 0056 9492 1336 1338 10015Mean 0098 0095 9511 1308 1308 10000SD 0076 0073 1433 222 224 016LSD 0012 - - 212 - -

βo + β1X1 + β2X2+ β3X3+ β11X21 + β22X2

2 + β33X23 + β12 X1X2 + β13 X1X3

1 X22and X2

1 minus 0007X22+

0043X2X3

(R2 = 0957 R2adj = 0946)

0014X3 minus 0008X21 minus 0004X2

2 minus 0004X23+

0004X1X2 minus 0007X1X3 + 0012X2X3

(R2 = 0889 R2adj = 0860)

1 + 0173X22 minus 0330X2

0625X1X2 + 0649X1X3 minus 1282X2X3

(R2 = 0973 R2adj = 0966)

24 Nahemiah et al

1 1031 1032 10010 09 097 10778 1601 1591 99382 367 355 9673 521 513 9846 2326 2341 100643 713 692 9705 157 154 9809 210 2097 99864 223 242 10852 23 187 8130 221 2198 99465 551 545 9891 075 092 12267 237 23497 991436 139 173 12446 364 341 9368 2649 2667 100687 673 698 10371 32 302 9438 1717 1717 100008 54 552 10222 201 168 8358 1313 1338 101909 69 697 10101 263 249 9468 255 2549 999610 031 006 1935 435 486 11172 288 286 993111 716 712 9944 261 253 9693 237 2348 990712 76 746 9816 11 155 14091 1655 1656 1000613 642 656 10218 102 117 11471 1478 1492 1009514 538 506 9405 073 095 13014 1481 1447 977015 841 844 10036 136 13 9559 2312 2316 1001716 1031 1032 10010 09 097 10778 1601 1591 993817 367 355 9673 521 513 9846 2326 2341 10064Mean 580 579 9665 232 232 10417 2055 3297 15239SD 284 - - 153 - - 475 - -LSD 104 - - 021 - - 202 - -

2 + β33X23 + β12 X1X2 +

0051X3 minus 1135X21 + 0368X2

2 + 0154X23+

(R2 = 0954 R2adj = 0942)

1770X3 minus 0720X21 + 0602X2

2ndash1652X23+

(R2 = 0952 R2adj = 0939)

R2adj = 0870 (L) R2 = 0954 R2

adj = 0942

b22 -0004 0000 -0007 0004 0173 0012 0154 0003 -0720 0000

b32 -0004 0001 0001 0008 -0330 0017 0001 0021 -0602 0016

ajusted 0860 - 0946 - 0966 - 0942 - 0939 -

Lack-of-fit 0710 - 0071 - 0319 - 0082 - 0982 -

1 + b22 X2

2 + b23 X2

3 +b12 X1 X2 + b13 X1 X3 + b23 X2 X3 + ε

26 Nahemiah et al

4 Conclusions

Acknowledgements

References

28 Nahemiah et al

Lemon Pomace

Abstract

1 Introduction

21 Materials

32 Awolu and Osigwe

Total flavonoids

C = 000255 timesAb (R2 = 09812) (2)

WSample(4)

200times 100 (5)

34 Awolu and Osigwe

1 7984 1016 1000 640 181 751 371 400 66572 7500 2000 500 620 125 1299 289 428 67393 8350 1150 500 650 105 856 280 400 72094 7030 2000 970 630 176 1257 334 430 62035 7698 1575 725 646 170 912 321 427 67986 7984 1016 1000 647 130 843 362 400 66187 8228 1000 771 630 148 841 370 400 68408 7500 2000 500 681 130 128 290 444 66759 8500 1000 500 640 098 841 268 401 725210 7316 1907 776 662 168 1272 318 436 636711 8228 1000 771 680 160 846 372 411 676012 8500 1000 500 641 106 799 266 400 728813 8031 1468 500 643 132 955 252 420 709814 7744 1255 1000 641 180 900 378 410 649115 7356 1643 999 630 174 1162 309 419 631016 7030 2000 970 630 176 1263 330 424 6207

36 Awolu and Osigwe

(mg100g) () (mg100g) (mg100g) ()

38 Awolu and Osigwe

4 Conclusions

References

40 Awolu and Osigwe

Abstract

1 Introduction

42 Hruskova et al

23 Analytical tests

44 Hruskova et al

Analytical tests

Pasting behaviour

Analytical tests

46 Hruskova et al

(mL) (1) (cm2)

48 Hruskova et al

() (min) (min) (BU)

50 Hruskova et al

develo

taddit

(degre

record

52 Hruskova et al

54 Hruskova et al

4 Conclusion

Acknowledgements

References

56 Hruskova et al

Abstract

1 Introduction

60 Al-Habsi et al

)= minusk1t (1)

)= minusk1t+ a (2)

Ln(k1) = minus(Ea

)+ b (3)

Broccoli

Time 0 0 0 0 0 0 0 01 5 525 10 5 4 3 23 195 725 27 15 9 6 521 255 875 40 25 14 9 835 425 1000 55 40 22 13 1149 1125 70 50 32 19 1663 1250 100 60 42 25 2177 1450 115 70 52 31 26

1575 130 80 67 38 311700 145 82 45 362300 9724002550

62 Al-Habsi et al

64 Al-Habsi et al

4 Conclusion

Acknowledgements

References

66 Al-Habsi et al

Tel +357 25002581Fax +357 25002562

Abstract

1 Introduction

Determination of pH

Protein content

Fat content

70 Aspri et al

TS = Total Solids

72 Aspri et al

Day 7 Day 15 Day 30

differ (plt005)

Nd=Not determined

Lysozyme

Lactoferrin

4 Conclusion

Acknowledgements

References

74 Aspri et al

Tel +30-24410-64783Fax +30-24410-64781

Abstract

1 Introduction

Luciferase

78 Lalas et al

21 Material

22 Tzatziki samples

80 Lalas et al

82 Lalas et al

84 Lalas et al

4 Conclusions

Acknowledgements

References

86 Lalas et al

Al-Habsia

Abstract

1 Introduction

88 Rahman et al

Mw =MbmBaw

Mw =MgmCKaw

90 Rahman et al

Model Parameter

92 Rahman et al

4 Conclusions

94 Rahman et al

Acknowledgements

References

96 Rahman et al

Tel +2348132595807

Abstract

98 Salawu et al

1 Introduction

21 Chemicals

Oral digestion

100 Salawu et al

102 Salawu et al

31 Results

mgg mgg

Amino acid Profile

Discussion

104 Salawu et al

106 Salawu et al

4 Conclusions

Acknowledgements

References

108 Salawu et al

110 Salawu et al

and Seed

Fax +91 3222 282244 255303

Abstract

1 Introduction

Nomenclature

aw Water activity

JP Jamun powder

MD Maltodextrin

21 Material

Wdrytimes 100 (1)

RMSE =

ntimes

nsumi=1

(Mi minusMpi)2

MRE() =100

nsumi=1

∣∣∣ (3)

ln aw = minus qstRT

+ Z (4)

Φ =KβT

int aw

Aawdaw (5)

Φ =KβT

[1 +BCaw minus Caw

1minus Caw

]aw005

Φ =KβTM

AmA(7)

d[1T ]

pulp isotherms

qst = minus04743M + 14337 r2 = 09728 (10)

4 Conclusions

References

Introduction

Materials


Chemical analyses



Conclusion

References

Introduction











Conclusions

References

Introduction


Materials




Minerals analysis










Conclusions

References

Introduction




Analytical tests


Bread baking trial










Conclusion

References

Introduction




Reaction Kinetics



Conclusion

References

Introduction









Conclusion

References

Introduction


Material

Tzatziki samples





Conclusions

References

Introduction


Sample Preparation







Conclusions

References

Introduction


Chemicals

Sample collection











Results

Conclusions

References

Introduction


Material



















Conclusions

References

OF FOOD STUDIES

CONTENTS

Lemon Pomace

DOURTOGLOU

and Seed

Abstract

1 Introduction

2 Maria and Hannah

21 Materials

Soaking

Autoclaving

Germination

4 Maria and Hannah

6 Maria and Hannah

8 Maria and Hannah

nd - not detected

10 Maria and Hannah

4 Conclusion

References

12 Maria and Hannah

Abstract

1 Introduction

Nomenclature

EI Expansion index

BD Bulk density

L Lightness

a Redness

b Yellowness

D Deviations

16 Nahemiah et al

Y = b0+

nsumi=1

biix2i +

nminus1sumi=1

nsumj=i+1

bijxixj+ei

xi =xi minus x0δx

Υtimes 100 (4)

πd2L(5)

18 Nahemiah et al

20 Nahemiah et al

Bulk density

Expansion index

22 Nahemiah et al

Y D RD Y D RD

1 0089 0092 10337 1278 1282 100312 0058 0041 6897 1311 1307 99693 0035 0021 6000 1257 1258 100084 0156 0162 10385 1309 1308 99925 0358 0329 9190 1307 1308 100086 0072 0083 11528 1299 1298 99927 0075 0081 10800 1317 1316 99928 0039 0028 7179 1327 1329 100159 0142 0146 10282 1288 1283 996110 0059 0059 10000 1315 1315 1000011 0147 0151 10272 1289 1289 1000012 0048 0045 9375 1295 1295 1000013 0058 0062 10690 1298 1298 1000014 0154 0148 9610 1338 1338 1000015 0059 0058 9831 1335 1335 1000016 0058 0057 9828 1337 1338 1000717 0059 0056 9492 1336 1338 10015Mean 0098 0095 9511 1308 1308 10000SD 0076 0073 1433 222 224 016LSD 0012 - - 212 - -

βo + β1X1 + β2X2+ β3X3+ β11X21 + β22X2

2 + β33X23 + β12 X1X2 + β13 X1X3

1 X22and X2

1 minus 0007X22+

0043X2X3

(R2 = 0957 R2adj = 0946)

0014X3 minus 0008X21 minus 0004X2

2 minus 0004X23+

0004X1X2 minus 0007X1X3 + 0012X2X3

(R2 = 0889 R2adj = 0860)

1 + 0173X22 minus 0330X2

0625X1X2 + 0649X1X3 minus 1282X2X3

(R2 = 0973 R2adj = 0966)

24 Nahemiah et al

1 1031 1032 10010 09 097 10778 1601 1591 99382 367 355 9673 521 513 9846 2326 2341 100643 713 692 9705 157 154 9809 210 2097 99864 223 242 10852 23 187 8130 221 2198 99465 551 545 9891 075 092 12267 237 23497 991436 139 173 12446 364 341 9368 2649 2667 100687 673 698 10371 32 302 9438 1717 1717 100008 54 552 10222 201 168 8358 1313 1338 101909 69 697 10101 263 249 9468 255 2549 999610 031 006 1935 435 486 11172 288 286 993111 716 712 9944 261 253 9693 237 2348 990712 76 746 9816 11 155 14091 1655 1656 1000613 642 656 10218 102 117 11471 1478 1492 1009514 538 506 9405 073 095 13014 1481 1447 977015 841 844 10036 136 13 9559 2312 2316 1001716 1031 1032 10010 09 097 10778 1601 1591 993817 367 355 9673 521 513 9846 2326 2341 10064Mean 580 579 9665 232 232 10417 2055 3297 15239SD 284 - - 153 - - 475 - -LSD 104 - - 021 - - 202 - -

2 + β33X23 + β12 X1X2 +

0051X3 minus 1135X21 + 0368X2

2 + 0154X23+

(R2 = 0954 R2adj = 0942)

1770X3 minus 0720X21 + 0602X2

2ndash1652X23+

(R2 = 0952 R2adj = 0939)

R2adj = 0870 (L) R2 = 0954 R2

adj = 0942

b22 -0004 0000 -0007 0004 0173 0012 0154 0003 -0720 0000

b32 -0004 0001 0001 0008 -0330 0017 0001 0021 -0602 0016

ajusted 0860 - 0946 - 0966 - 0942 - 0939 -

Lack-of-fit 0710 - 0071 - 0319 - 0082 - 0982 -

1 + b22 X2

2 + b23 X2

3 +b12 X1 X2 + b13 X1 X3 + b23 X2 X3 + ε

26 Nahemiah et al

4 Conclusions

Acknowledgements

References

28 Nahemiah et al

Lemon Pomace

Abstract

1 Introduction

21 Materials

32 Awolu and Osigwe

Total flavonoids

C = 000255 timesAb (R2 = 09812) (2)

WSample(4)

200times 100 (5)

34 Awolu and Osigwe

1 7984 1016 1000 640 181 751 371 400 66572 7500 2000 500 620 125 1299 289 428 67393 8350 1150 500 650 105 856 280 400 72094 7030 2000 970 630 176 1257 334 430 62035 7698 1575 725 646 170 912 321 427 67986 7984 1016 1000 647 130 843 362 400 66187 8228 1000 771 630 148 841 370 400 68408 7500 2000 500 681 130 128 290 444 66759 8500 1000 500 640 098 841 268 401 725210 7316 1907 776 662 168 1272 318 436 636711 8228 1000 771 680 160 846 372 411 676012 8500 1000 500 641 106 799 266 400 728813 8031 1468 500 643 132 955 252 420 709814 7744 1255 1000 641 180 900 378 410 649115 7356 1643 999 630 174 1162 309 419 631016 7030 2000 970 630 176 1263 330 424 6207

36 Awolu and Osigwe

(mg100g) () (mg100g) (mg100g) ()

38 Awolu and Osigwe

4 Conclusions

References

40 Awolu and Osigwe

Abstract

1 Introduction

42 Hruskova et al

23 Analytical tests

44 Hruskova et al

Analytical tests

Pasting behaviour

Analytical tests

46 Hruskova et al

(mL) (1) (cm2)

48 Hruskova et al

() (min) (min) (BU)

50 Hruskova et al

develo

taddit

(degre

record

52 Hruskova et al

54 Hruskova et al

4 Conclusion

Acknowledgements

References

56 Hruskova et al

Abstract

1 Introduction

60 Al-Habsi et al

)= minusk1t (1)

)= minusk1t+ a (2)

Ln(k1) = minus(Ea

)+ b (3)

Broccoli

Time 0 0 0 0 0 0 0 01 5 525 10 5 4 3 23 195 725 27 15 9 6 521 255 875 40 25 14 9 835 425 1000 55 40 22 13 1149 1125 70 50 32 19 1663 1250 100 60 42 25 2177 1450 115 70 52 31 26

1575 130 80 67 38 311700 145 82 45 362300 9724002550

62 Al-Habsi et al

64 Al-Habsi et al

4 Conclusion

Acknowledgements

References

66 Al-Habsi et al

Tel +357 25002581Fax +357 25002562

Abstract

1 Introduction

Determination of pH

Protein content

Fat content

70 Aspri et al

TS = Total Solids

72 Aspri et al

Day 7 Day 15 Day 30

differ (plt005)

Nd=Not determined

Lysozyme

Lactoferrin

4 Conclusion

Acknowledgements

References

74 Aspri et al

Tel +30-24410-64783Fax +30-24410-64781

Abstract

1 Introduction

Luciferase

78 Lalas et al

21 Material

22 Tzatziki samples

80 Lalas et al

82 Lalas et al

84 Lalas et al

4 Conclusions

Acknowledgements

References

86 Lalas et al

Al-Habsia

Abstract

1 Introduction

88 Rahman et al

Mw =MbmBaw

Mw =MgmCKaw

90 Rahman et al

Model Parameter

92 Rahman et al

4 Conclusions

94 Rahman et al

Acknowledgements

References

96 Rahman et al

Tel +2348132595807

Abstract

98 Salawu et al

1 Introduction

21 Chemicals

Oral digestion

100 Salawu et al

102 Salawu et al

31 Results

mgg mgg

Amino acid Profile

Discussion

104 Salawu et al

106 Salawu et al

4 Conclusions

Acknowledgements

References

108 Salawu et al

110 Salawu et al

and Seed

Fax +91 3222 282244 255303

Abstract

1 Introduction

Nomenclature

aw Water activity

JP Jamun powder

MD Maltodextrin

21 Material

Wdrytimes 100 (1)

RMSE =

ntimes

nsumi=1

(Mi minusMpi)2

MRE() =100

nsumi=1

∣∣∣ (3)

ln aw = minus qstRT

+ Z (4)

Φ =KβT

int aw

Aawdaw (5)

Φ =KβT

[1 +BCaw minus Caw

1minus Caw

]aw005

Φ =KβTM

AmA(7)

d[1T ]

pulp isotherms

qst = minus04743M + 14337 r2 = 09728 (10)

4 Conclusions

References

Introduction

Materials


Chemical analyses



Conclusion

References

Introduction











Conclusions

References

Introduction


Materials




Minerals analysis










Conclusions

References

Introduction




Analytical tests


Bread baking trial










Conclusion

References

Introduction




Reaction Kinetics



Conclusion

References

Introduction









Conclusion

References

Introduction


Material

Tzatziki samples





Conclusions

References

Introduction


Sample Preparation







Conclusions

References

Introduction


Chemicals

Sample collection











Results

Conclusions

References

Introduction


Material



















Conclusions

References

Abstract

1 Introduction

2 Maria and Hannah

21 Materials

Soaking

Autoclaving

Germination

4 Maria and Hannah

6 Maria and Hannah

8 Maria and Hannah

nd - not detected

10 Maria and Hannah

4 Conclusion

References

12 Maria and Hannah

Abstract

1 Introduction

Nomenclature

EI Expansion index

BD Bulk density

L Lightness

a Redness

b Yellowness

D Deviations

16 Nahemiah et al

Y = b0+

nsumi=1

biix2i +

nminus1sumi=1

nsumj=i+1

bijxixj+ei

xi =xi minus x0δx

Υtimes 100 (4)

πd2L(5)

18 Nahemiah et al

20 Nahemiah et al

Bulk density

Expansion index

22 Nahemiah et al

Y D RD Y D RD

1 0089 0092 10337 1278 1282 100312 0058 0041 6897 1311 1307 99693 0035 0021 6000 1257 1258 100084 0156 0162 10385 1309 1308 99925 0358 0329 9190 1307 1308 100086 0072 0083 11528 1299 1298 99927 0075 0081 10800 1317 1316 99928 0039 0028 7179 1327 1329 100159 0142 0146 10282 1288 1283 996110 0059 0059 10000 1315 1315 1000011 0147 0151 10272 1289 1289 1000012 0048 0045 9375 1295 1295 1000013 0058 0062 10690 1298 1298 1000014 0154 0148 9610 1338 1338 1000015 0059 0058 9831 1335 1335 1000016 0058 0057 9828 1337 1338 1000717 0059 0056 9492 1336 1338 10015Mean 0098 0095 9511 1308 1308 10000SD 0076 0073 1433 222 224 016LSD 0012 - - 212 - -

βo + β1X1 + β2X2+ β3X3+ β11X21 + β22X2

2 + β33X23 + β12 X1X2 + β13 X1X3

1 X22and X2

1 minus 0007X22+

0043X2X3

(R2 = 0957 R2adj = 0946)

0014X3 minus 0008X21 minus 0004X2

2 minus 0004X23+

0004X1X2 minus 0007X1X3 + 0012X2X3

(R2 = 0889 R2adj = 0860)

1 + 0173X22 minus 0330X2

0625X1X2 + 0649X1X3 minus 1282X2X3

(R2 = 0973 R2adj = 0966)

24 Nahemiah et al

1 1031 1032 10010 09 097 10778 1601 1591 99382 367 355 9673 521 513 9846 2326 2341 100643 713 692 9705 157 154 9809 210 2097 99864 223 242 10852 23 187 8130 221 2198 99465 551 545 9891 075 092 12267 237 23497 991436 139 173 12446 364 341 9368 2649 2667 100687 673 698 10371 32 302 9438 1717 1717 100008 54 552 10222 201 168 8358 1313 1338 101909 69 697 10101 263 249 9468 255 2549 999610 031 006 1935 435 486 11172 288 286 993111 716 712 9944 261 253 9693 237 2348 990712 76 746 9816 11 155 14091 1655 1656 1000613 642 656 10218 102 117 11471 1478 1492 1009514 538 506 9405 073 095 13014 1481 1447 977015 841 844 10036 136 13 9559 2312 2316 1001716 1031 1032 10010 09 097 10778 1601 1591 993817 367 355 9673 521 513 9846 2326 2341 10064Mean 580 579 9665 232 232 10417 2055 3297 15239SD 284 - - 153 - - 475 - -LSD 104 - - 021 - - 202 - -

2 + β33X23 + β12 X1X2 +

0051X3 minus 1135X21 + 0368X2

2 + 0154X23+

(R2 = 0954 R2adj = 0942)

1770X3 minus 0720X21 + 0602X2

2ndash1652X23+

(R2 = 0952 R2adj = 0939)

R2adj = 0870 (L) R2 = 0954 R2

adj = 0942

b22 -0004 0000 -0007 0004 0173 0012 0154 0003 -0720 0000

b32 -0004 0001 0001 0008 -0330 0017 0001 0021 -0602 0016

ajusted 0860 - 0946 - 0966 - 0942 - 0939 -

Lack-of-fit 0710 - 0071 - 0319 - 0082 - 0982 -

1 + b22 X2

2 + b23 X2

3 +b12 X1 X2 + b13 X1 X3 + b23 X2 X3 + ε

26 Nahemiah et al

4 Conclusions

Acknowledgements

References

28 Nahemiah et al

Lemon Pomace

Abstract

1 Introduction

21 Materials

32 Awolu and Osigwe

Total flavonoids

C = 000255 timesAb (R2 = 09812) (2)

WSample(4)

200times 100 (5)

34 Awolu and Osigwe

1 7984 1016 1000 640 181 751 371 400 66572 7500 2000 500 620 125 1299 289 428 67393 8350 1150 500 650 105 856 280 400 72094 7030 2000 970 630 176 1257 334 430 62035 7698 1575 725 646 170 912 321 427 67986 7984 1016 1000 647 130 843 362 400 66187 8228 1000 771 630 148 841 370 400 68408 7500 2000 500 681 130 128 290 444 66759 8500 1000 500 640 098 841 268 401 725210 7316 1907 776 662 168 1272 318 436 636711 8228 1000 771 680 160 846 372 411 676012 8500 1000 500 641 106 799 266 400 728813 8031 1468 500 643 132 955 252 420 709814 7744 1255 1000 641 180 900 378 410 649115 7356 1643 999 630 174 1162 309 419 631016 7030 2000 970 630 176 1263 330 424 6207

36 Awolu and Osigwe

(mg100g) () (mg100g) (mg100g) ()

38 Awolu and Osigwe

4 Conclusions

References

40 Awolu and Osigwe

Abstract

1 Introduction

42 Hruskova et al

23 Analytical tests

44 Hruskova et al

Analytical tests

Pasting behaviour

Analytical tests

46 Hruskova et al

(mL) (1) (cm2)

48 Hruskova et al

() (min) (min) (BU)

50 Hruskova et al

develo

taddit

(degre

record

52 Hruskova et al

54 Hruskova et al

4 Conclusion

Acknowledgements

References

56 Hruskova et al

Abstract

1 Introduction

60 Al-Habsi et al

)= minusk1t (1)

)= minusk1t+ a (2)

Ln(k1) = minus(Ea

)+ b (3)

Broccoli

Time 0 0 0 0 0 0 0 01 5 525 10 5 4 3 23 195 725 27 15 9 6 521 255 875 40 25 14 9 835 425 1000 55 40 22 13 1149 1125 70 50 32 19 1663 1250 100 60 42 25 2177 1450 115 70 52 31 26

1575 130 80 67 38 311700 145 82 45 362300 9724002550

62 Al-Habsi et al

64 Al-Habsi et al

4 Conclusion

Acknowledgements

References

66 Al-Habsi et al

Tel +357 25002581Fax +357 25002562

Abstract

1 Introduction

Determination of pH

Protein content

Fat content

70 Aspri et al

TS = Total Solids

72 Aspri et al

Day 7 Day 15 Day 30

differ (plt005)

Nd=Not determined

Lysozyme

Lactoferrin

4 Conclusion

Acknowledgements

References

74 Aspri et al

Tel +30-24410-64783Fax +30-24410-64781

Abstract

1 Introduction

Luciferase

78 Lalas et al

21 Material

22 Tzatziki samples

80 Lalas et al

82 Lalas et al

84 Lalas et al

4 Conclusions

Acknowledgements

References

86 Lalas et al

Al-Habsia

Abstract

1 Introduction

88 Rahman et al

Mw =MbmBaw

Mw =MgmCKaw

90 Rahman et al

Model Parameter

92 Rahman et al

4 Conclusions

94 Rahman et al

Acknowledgements

References

96 Rahman et al

Tel +2348132595807

Abstract

98 Salawu et al

1 Introduction

21 Chemicals

Oral digestion

100 Salawu et al

102 Salawu et al

31 Results

mgg mgg

Amino acid Profile

Discussion

104 Salawu et al

106 Salawu et al

4 Conclusions

Acknowledgements

References

108 Salawu et al

110 Salawu et al

and Seed

Fax +91 3222 282244 255303

Abstract

1 Introduction

Nomenclature

aw Water activity

JP Jamun powder

MD Maltodextrin

21 Material

Wdrytimes 100 (1)

RMSE =

ntimes

nsumi=1

(Mi minusMpi)2

MRE() =100

nsumi=1

∣∣∣ (3)

ln aw = minus qstRT

+ Z (4)

Φ =KβT

int aw

Aawdaw (5)

Φ =KβT

[1 +BCaw minus Caw

1minus Caw

]aw005

Φ =KβTM

AmA(7)

d[1T ]

pulp isotherms

qst = minus04743M + 14337 r2 = 09728 (10)

4 Conclusions

References

Introduction

Materials


Chemical analyses



Conclusion

References

Introduction











Conclusions

References

Introduction


Materials




Minerals analysis










Conclusions

References

Introduction




Analytical tests


Bread baking trial










Conclusion

References

Introduction




Reaction Kinetics



Conclusion

References

Introduction









Conclusion

References

Introduction


Material

Tzatziki samples





Conclusions

References

Introduction


Sample Preparation







Conclusions

References

Introduction


Chemicals

Sample collection











Results

Conclusions

References

Introduction


Material



















Conclusions

References

2 Maria and Hannah

21 Materials

Soaking

Autoclaving

Germination

4 Maria and Hannah

6 Maria and Hannah

8 Maria and Hannah

nd - not detected

10 Maria and Hannah

4 Conclusion

References

12 Maria and Hannah

Abstract

1 Introduction

Nomenclature

EI Expansion index

BD Bulk density

L Lightness

a Redness

b Yellowness

D Deviations

16 Nahemiah et al

Y = b0+

nsumi=1

biix2i +

nminus1sumi=1

nsumj=i+1

bijxixj+ei

xi =xi minus x0δx

Υtimes 100 (4)

πd2L(5)

18 Nahemiah et al

20 Nahemiah et al

Bulk density

Expansion index

22 Nahemiah et al

Y D RD Y D RD

1 0089 0092 10337 1278 1282 100312 0058 0041 6897 1311 1307 99693 0035 0021 6000 1257 1258 100084 0156 0162 10385 1309 1308 99925 0358 0329 9190 1307 1308 100086 0072 0083 11528 1299 1298 99927 0075 0081 10800 1317 1316 99928 0039 0028 7179 1327 1329 100159 0142 0146 10282 1288 1283 996110 0059 0059 10000 1315 1315 1000011 0147 0151 10272 1289 1289 1000012 0048 0045 9375 1295 1295 1000013 0058 0062 10690 1298 1298 1000014 0154 0148 9610 1338 1338 1000015 0059 0058 9831 1335 1335 1000016 0058 0057 9828 1337 1338 1000717 0059 0056 9492 1336 1338 10015Mean 0098 0095 9511 1308 1308 10000SD 0076 0073 1433 222 224 016LSD 0012 - - 212 - -

βo + β1X1 + β2X2+ β3X3+ β11X21 + β22X2

2 + β33X23 + β12 X1X2 + β13 X1X3

1 X22and X2

1 minus 0007X22+

0043X2X3

(R2 = 0957 R2adj = 0946)

0014X3 minus 0008X21 minus 0004X2

2 minus 0004X23+

0004X1X2 minus 0007X1X3 + 0012X2X3

(R2 = 0889 R2adj = 0860)

1 + 0173X22 minus 0330X2

0625X1X2 + 0649X1X3 minus 1282X2X3

(R2 = 0973 R2adj = 0966)

24 Nahemiah et al

1 1031 1032 10010 09 097 10778 1601 1591 99382 367 355 9673 521 513 9846 2326 2341 100643 713 692 9705 157 154 9809 210 2097 99864 223 242 10852 23 187 8130 221 2198 99465 551 545 9891 075 092 12267 237 23497 991436 139 173 12446 364 341 9368 2649 2667 100687 673 698 10371 32 302 9438 1717 1717 100008 54 552 10222 201 168 8358 1313 1338 101909 69 697 10101 263 249 9468 255 2549 999610 031 006 1935 435 486 11172 288 286 993111 716 712 9944 261 253 9693 237 2348 990712 76 746 9816 11 155 14091 1655 1656 1000613 642 656 10218 102 117 11471 1478 1492 1009514 538 506 9405 073 095 13014 1481 1447 977015 841 844 10036 136 13 9559 2312 2316 1001716 1031 1032 10010 09 097 10778 1601 1591 993817 367 355 9673 521 513 9846 2326 2341 10064Mean 580 579 9665 232 232 10417 2055 3297 15239SD 284 - - 153 - - 475 - -LSD 104 - - 021 - - 202 - -

2 + β33X23 + β12 X1X2 +

0051X3 minus 1135X21 + 0368X2

2 + 0154X23+

(R2 = 0954 R2adj = 0942)

1770X3 minus 0720X21 + 0602X2

2ndash1652X23+

(R2 = 0952 R2adj = 0939)

R2adj = 0870 (L) R2 = 0954 R2

adj = 0942

b22 -0004 0000 -0007 0004 0173 0012 0154 0003 -0720 0000

b32 -0004 0001 0001 0008 -0330 0017 0001 0021 -0602 0016

ajusted 0860 - 0946 - 0966 - 0942 - 0939 -

Lack-of-fit 0710 - 0071 - 0319 - 0082 - 0982 -

1 + b22 X2

2 + b23 X2

3 +b12 X1 X2 + b13 X1 X3 + b23 X2 X3 + ε

26 Nahemiah et al

4 Conclusions

Acknowledgements

References

28 Nahemiah et al

Lemon Pomace

Abstract

1 Introduction

21 Materials

32 Awolu and Osigwe

Total flavonoids

C = 000255 timesAb (R2 = 09812) (2)

WSample(4)

200times 100 (5)

34 Awolu and Osigwe

1 7984 1016 1000 640 181 751 371 400 66572 7500 2000 500 620 125 1299 289 428 67393 8350 1150 500 650 105 856 280 400 72094 7030 2000 970 630 176 1257 334 430 62035 7698 1575 725 646 170 912 321 427 67986 7984 1016 1000 647 130 843 362 400 66187 8228 1000 771 630 148 841 370 400 68408 7500 2000 500 681 130 128 290 444 66759 8500 1000 500 640 098 841 268 401 725210 7316 1907 776 662 168 1272 318 436 636711 8228 1000 771 680 160 846 372 411 676012 8500 1000 500 641 106 799 266 400 728813 8031 1468 500 643 132 955 252 420 709814 7744 1255 1000 641 180 900 378 410 649115 7356 1643 999 630 174 1162 309 419 631016 7030 2000 970 630 176 1263 330 424 6207

36 Awolu and Osigwe

(mg100g) () (mg100g) (mg100g) ()

38 Awolu and Osigwe

4 Conclusions

References

40 Awolu and Osigwe

Abstract

1 Introduction

42 Hruskova et al

23 Analytical tests

44 Hruskova et al

Analytical tests

Pasting behaviour

Analytical tests

46 Hruskova et al

(mL) (1) (cm2)

48 Hruskova et al

() (min) (min) (BU)

50 Hruskova et al

develo

taddit

(degre

record

52 Hruskova et al

54 Hruskova et al

4 Conclusion

Acknowledgements

References

56 Hruskova et al

Abstract

1 Introduction

60 Al-Habsi et al

)= minusk1t (1)

)= minusk1t+ a (2)

Ln(k1) = minus(Ea

)+ b (3)

Broccoli

Time 0 0 0 0 0 0 0 01 5 525 10 5 4 3 23 195 725 27 15 9 6 521 255 875 40 25 14 9 835 425 1000 55 40 22 13 1149 1125 70 50 32 19 1663 1250 100 60 42 25 2177 1450 115 70 52 31 26

1575 130 80 67 38 311700 145 82 45 362300 9724002550

62 Al-Habsi et al

64 Al-Habsi et al

4 Conclusion

Acknowledgements

References

66 Al-Habsi et al

Tel +357 25002581Fax +357 25002562

Abstract

1 Introduction

Determination of pH

Protein content

Fat content

70 Aspri et al

TS = Total Solids

72 Aspri et al

Day 7 Day 15 Day 30

differ (plt005)

Nd=Not determined

Lysozyme

Lactoferrin

4 Conclusion

Acknowledgements

References

74 Aspri et al

Tel +30-24410-64783Fax +30-24410-64781

Abstract

1 Introduction

Luciferase

78 Lalas et al

21 Material

22 Tzatziki samples

80 Lalas et al

82 Lalas et al

84 Lalas et al

4 Conclusions

Acknowledgements

References

86 Lalas et al

Al-Habsia

Abstract

1 Introduction

88 Rahman et al

Mw =MbmBaw

Mw =MgmCKaw

90 Rahman et al

Model Parameter

92 Rahman et al

4 Conclusions

94 Rahman et al

Acknowledgements

References

96 Rahman et al

Tel +2348132595807

Abstract

98 Salawu et al

1 Introduction

21 Chemicals

Oral digestion

100 Salawu et al

102 Salawu et al

31 Results

mgg mgg

Amino acid Profile

Discussion

104 Salawu et al

106 Salawu et al

4 Conclusions

Acknowledgements

References

108 Salawu et al

110 Salawu et al

and Seed

Fax +91 3222 282244 255303

Abstract

1 Introduction

Nomenclature

aw Water activity

JP Jamun powder

MD Maltodextrin

21 Material

Wdrytimes 100 (1)

RMSE =

ntimes

nsumi=1

(Mi minusMpi)2

MRE() =100

nsumi=1

∣∣∣ (3)

ln aw = minus qstRT

+ Z (4)

Φ =KβT

int aw

Aawdaw (5)

Φ =KβT

[1 +BCaw minus Caw

1minus Caw

]aw005

Φ =KβTM

AmA(7)

d[1T ]

pulp isotherms

qst = minus04743M + 14337 r2 = 09728 (10)

4 Conclusions

References

Introduction

Materials


Chemical analyses



Conclusion

References

Introduction











Conclusions

References

Introduction


Materials




Minerals analysis










Conclusions

References

Introduction




Analytical tests


Bread baking trial










Conclusion

References

Introduction




Reaction Kinetics



Conclusion

References

Introduction









Conclusion

References

Introduction


Material

Tzatziki samples





Conclusions

References

Introduction


Sample Preparation







Conclusions

References

Introduction


Chemicals

Sample collection











Results

Conclusions

References

Introduction


Material



















Conclusions

References

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$0 8 - Issue 1.pdf · reports the e ect of soaking, autoclaving, roast-ing or germination on the nutritional quality and functional properties of cashew our. 2 Materials and Methods