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Research ArticleDevelopment of a Potential Probiotic Fresh Cheese Using TwoLactobacillus salivarius Strains Isolated from Human Milk

Nivia Caacuterdenas12 Javier Calzada3 Aacutengela Peiroteacuten3 Esther Jimeacutenez12 Rosa Escudero1

Juan M Rodriacuteguez12 Margarita Medina3 and Leoacutenides Fernaacutendez12

1 Departamento de Nutricion Bromatologıa y Tecnologıa de los Alimentos Universidad Complutense de MadridCiudad Universitaria Avenida Puerta de Hierro sn 28040 Madrid Spain

2 Probisearch 28760 Tres Cantos Spain3 Instituto Nacional de Investigacion y Tecnologıa Agraria y Alimentaria INIA Carretera de La Coruna km 75 28040Madrid Spain

Correspondence should be addressed to Leonides Fernandez leonidesucmes

Received 28 February 2014 Revised 24 April 2014 Accepted 1 May 2014 Published 29 May 2014

Academic Editor Clara G de los Reyes-Gavilan

Copyright copy 2014 Nivia Cardenas et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Cheeses have been proposed as a good alternative to other fermented milk products for the delivery of probiotic bacteria to theconsumer The objective of this study was to assess the survival of two Lactobacillus salivarius strains (CECT5713 and PS2) isolatedfrom human milk during production and storage of fresh cheese for 28 days at 4∘C The effect of such strains on the volatilecompounds profile texture and other sensorial properties including an overall consumer acceptance was also investigated BothL salivarius strains remained viable in the cheeses throughout the storage period and a significant reduction in their viable countswas only observed after 21 days Globally the addition of the L salivarius strains did not change significantly neither the chemicalcomposition of the cheese nor texture parameters after the storage period although cheeses manufactured with L salivariusCECT5713 presented significantly higher values of hardness A total of 59 volatile compounds were identified in the headspaceof experimental cheeses and some L salivarius-associated differences could be identified All cheeses presented good results ofacceptance after the sensory evaluation Consequently our results indicated that fresh cheese can be a good vehicle for the two Lsalivarius strains analyzed in this study

1 Introduction

Among all dairy products cheese has the highest consump-tion rateworldwide because of its versatility Fresh cheeses areusually not or minimally aged have high moisture contentdo not have a rind and got very mild flavour and a softand smooth texture In this category milk coagulation isdue to rennet andor acid produced from a bacterial cultureor other sources such as lemon juice When bacteria areinvolved in theirmanufacture they also contribute to developtypical flavours to improve quality andor to promote healthbenefits if they display probiotic properties [1]

Probiotics are defined as ldquolive microorganisms whichwhen administered in adequate amounts confer a healthbenefit on the hostrdquo [2] being Lactobacillus and Bifidobac-terium the most frequently used genera [3] Yogurt andfermented milks are the most common foods for delivery

of probiotic bacteria but some studies have found thattheir characteristics may compromise the viability of theprobiotic strains [4ndash6] Cheese may offer several advantagesas a probiotic carrier due to its higher pH and fat contentand harder consistency compared to fermented milks [7]These features provide more protection to probiotics notonly during cheese production ripening and storage butalso during the passage through the gastrointestinal tractallowing bacteria to arrive in higher numbers at the target siteafter ingestion [7]

Several studies have confirmed that human milk is asource of live bacteriamainly staphylococci and streptococcibut also contains lactic acid bacteria and bifidobacteria [8ndash11]The lactobacilli species more frequently isolated from milksamples of healthy women are Lactobacillus casei Lactobacil-lus fermentum Lactobacillus gasseri Lactobacillus gastricusLactobacillus plantarum Lactobacillus reuteri Lactobacillus

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 801918 12 pageshttpdxdoiorg1011552014801918

2 BioMed Research International

salivarius and Lactobacillus vaginalis [12] Some lactobacilliisolated from human milk have been characterized andshown to have probiotic potential [13ndash15] Specifically Lsalivarius CECT5713 that was isolated from human milk andinfant feces of a healthy mother-child pair has been shownto have remarkable probiotic potential because it had highrate of survival in simulated gastrointestinal tract conditionsand strong adherence to mucus and intestinal cells in vitrostimulated the expression of mucin-encoding genes andproduced antimicrobial compounds [14ndash17] More recentlyits complete genome has been sequenced [18] and its geneticfeatures such as proteins potentially involved in humanmolecular mimetism may explain its immunomodulatoryanti-inflammatory and anti-infectious properties [19 20]Moreover its safety and health beneficial effects have beenproved in animal models and in human clinical assays [20ndash23] More recently L salivarius PS2 has also been isolatedfrom humanmilk and preliminary assays have shown similartraits and probiotic potential

The aim of this work was to evaluate the performanceof these two human milk L salivarius strains (CECT5713and PS2) in fresh cheese in order to develop a probioticcheese The survival of these two L salivarius strains inthe cheese has been studied as well as their impact onchemical composition volatile compounds texture and otherorganoleptic properties and overall consumer acceptance ofthe experimental cheeses

2 Materials and Methods

21 Starter and Probiotic Organisms Lactococcus lactisESI153 originally isolated from artisanal raw milk cheese[24] was selected to be used as starter culture Lc lactisESI153 cells were grown in M17 (Oxoid Basinstoke UK)broth supplemented with 05 (wtvol) glucose (GM17) at32∘C Before use Lc lactis ESI153 cells were subcultured (1)into reconstituted at 11 (wtvol) and heat-treated (121∘C5min) nonfat drymilk (HT-NFDM) and incubated overnightat 32∘C

Freeze-dried cultures of probiotic L salivariusCECT5713andPS2were prepared as follows A fully grown liquid cultureon de Man Rogosa and Sharpe (MRS) (Oxoid) broth wascentrifuged at 10000timesg for 10min at 4∘C The cell pelletwas washed with 085 (wtvol) NaCl and resuspended inHT-NFDM to one tenth of its original volume The cellsuspension was frozen at minus80∘C for 12 h inmetal trays Freezedrying was carried out at first 0∘C for 24 h and then at20∘C for 24 h under 13 Pa in a Lyph-Lock Stoppering TrayDryer model 77560 (Labconco Corporation Kansas CityMO USA) Freeze-dried cultures containing approximately103 log

10colony forming units (cfu)g were vacuum pack-

aged and stored at 4∘C before use

22 Experimental Cheese Manufacture Cheeses were madefrom commercial pasteurized (high temperature short timeHTST) cowrsquos milk (Ganaderıa Priegola SA Villanueva delPardillo Madrid Spain) following a laboratory-scale pro-cedure described previously by Rodrıguez et al [25] and

Pasteurized whole cowrsquos milk

Starter culture(Lactococcus lactis ESI 153

Acid production

Rennet(Fromase 44 IMCUL)

Coagulation

Cutting

Cooking

Whey drainage

Mixing

Probiotic culture(Lactobacillus salivarius CECT 5713 or

Lactobacillus salivarius PS2

Whey

Pressing

Brine salting

Vacuum packaging

Storage

(32∘C)

001 CaCl2

(32∘C 30min)

(30∘C 40min)

(curd 1 cm3)

(38∘C 40min)

(20∘C 18h)

(4∘C 28days)

(15 NaCl 20∘C 1h)

sim8 log10

cfug)

sim9 log10

cfumL)

Figure 1 Flowchart of the cheese making process

Reviriego et al [26] with some modifications (Figure 1)Briefly pasteurized milk (15 Lvat) at 32∘C with 001(wtvol) CaCl

2was inoculated with Lc lactis ESI153 (approx-

imately 9 log10cfumL) as starter culture Rennet (Fromase

44 IMCUL DSM Food Specialities Seclin Cedex France)was added to milk 30min after the inoculation of Lc lactisESI153 Curds were cut 40min after rennet addition andheated at 38∘C for 40min Whey was drained off and freeze-dried L salivarius CECT5713 or PS2 were added to the curdto reach a final concentration of sim8 log

10cfug before the

curds were distributed into the molds Control cheese wasmanufactured at the same conditions with the addition of theequivalent amount of HT-NFDM used as the excipient forfreeze drying the lactobacilli strains Cheeses were pressedfor 16 h at room temperature and salted in 15 brine (wtvol)during 3 hThe resulting cheeses (sim190 g)were cut into pieceswhich were individually vacuum-packed in Cryovac plasticbags and kept refrigerated at 4∘C during 28 days All cheesemanufacturing trials were made in triplicate

BioMed Research International 3

23 Gross Composition pH and Water Activity in CheeseCheese samples were analyzed for moisture (ISO 5534IDF0042004) fat (ISO 1735IDF 0052004) protein (ISO 8968-1IDF 020-12014) and ash content (AOAC 93542) ThepH value of a cheese slurry prepared by blending 20 g ofgrated cheese with 12mL of water [27] was measured witha pH meter (Crison Digit-501) The water activity (119886w) wasdetermined with a Decagon CX-1 hygrometer (DecagonPullman Washington USA) Determinations were made ontriplicate samples

24 Viable Bacterial Counts in Cheese Viability of the Lsalivarius strains was monitored in cheese samples at 0 714 21 and 28 days at 4∘C For this purpose portions (10 g)of cheese were blended with 100mL of 01 (wtvol) sterilepeptone water in a stomacher Serial dilutions weremade alsoin sterile peptone water and plated following the surface platetechnique in appropriate media L salivarius strains wereenumerated on de Man Rogosa and Sharpe (MRS Oxoid)agar containing 0002 (wtvol) of bromophenol blue after24 h at 37∘C under aerobic conditions Lc lactis ESI153 wasenumerated on M17 (Oxoid) agar supplemented with 05(wtvol) glucose (GM17) after 24 h at 32∘C under aerobicconditions To confirm their identity selected colonies wereobserved by optical microscopy to check their morphologyand Gram staining and typed by Random Amplification ofPolymorphic DNA (RAPD) using primer OPL5 (51015840-ACGCAG GCA C-31015840) as described by Ruiz-Barba et al [28] Tenrandomly chosen isolates sharing the same RAPD profile(only two different RAPD profiles were obtained) weresubjected to Pulse Field Gel Electrophoresis (PFGE) afterdigestion with SmaI following the procedure described byMartın et al [12] The absence of Enterobacteriaceae andBacillus cereus in cheese samples was assessed by pouringonto MacConkey and PEMBA agar (BioMerieux MarcylrsquoEtoile France) respectively and incubation in aerobicallyconditions at 37∘C for up to 48 h Bacterial counts wererecorded as the cfug of cheese and transformed to log

10

values before statistical analysis

25 Isolation of Bacterial DNA and PCR-DGGE AnalysisCheeses samples (5 g) were homogenized into sodium citrate(50mL) using a stomacher (260 rpm times 1min) Then analiquot of the mixture (10mL) was centrifuged at 19000timesgduring 5min The resulting pellet was used for the isolationof total bacterial DNA from each cheese sample followingthe protocol described previously by Moles et al [29]DNA yield was measured using a NanoDrop ND 1000 UVspectrophotometer (Nano-Drop Technologies WilmingtonDelawere USA) and was stored at minus20∘C until PCR DGGEanalysis

Primers U968-GC-f (51015840-CGC CCG GGG CGC GCCCCG GGC GGG GCG GGG GCA CGG GGG GAA CGCGAA GAA CCT TAC-31015840) and L1401-r (51015840-CGG TGT GTACAA GAC CC-31015840) [30] and Lab159f (51015840-GGA AAC AGGTGC TAA TAC CG-31015840) and Uni-515-GCr (51015840-CGC CCGGGG CGC GCC CCG GGC GGG GCG GGG GCA CGGGGG GAT CGT ATT ACC GCG CTG CTG GCA C-31015840) [31]

were used to amplify V6ndashV8 regions from 16S rRNA geneson bacterial DNAThe PCR reaction was performed in a totalreaction volume of 50 120583L containing 5timesMy Taq Red reactionbuffer (Bioline London UK) My Taq Red DNA polymerase(Bioline) and 10 gmL of the isolated DNAThe amplificationprogram was as follows 95∘C for 2min 35 cycles of 95∘C for30 s 56∘C for 40 s 72∘C for 60 s and then 72∘C for 5min PCRproducts were stored at minus20∘C until use

PCR fragments were separated by denaturing gradientgel electrophoresis (DGGE) using a DCode System (BioRadLaboratories Inc Hercules California USA) and gels witha linear denaturant gradient of 30 to 50 as described byMartın et al [32] A DNAmixture made with equal amountsof amplicons from L salivarius CECT5713 or PS2 and Lclactis ESI153 was used as a marker

26 Analysis of Cheese Texture Texture profile analysis (TPA)of the cheeses was performed in a texturometer TA-XT2i(Stable Micro Systems Ltd Surrey UK) The texturometerwas provided with a 02N load cell and a 20mm diameterprobe at a crosshead speed of 5mms to perform a uniaxialcompression test in two consecutive compressions Cheesesamples were prepared by cutting 2 cm3 cubes which werekept during 1 h at 25∘C before performing the assay Thecheese cube was placed between the two parallel platesand compressed to 50 of its original height sample TPAparameters (hardness cohesiveness adhesiveness chewi-ness gumminess and springiness) were determined from theTPA two-compression force-time curve with the aid of theTexture Expert for Windows software version 120 (StableMicro Systems) All measurements were made in triplicate

27 Colour Analysis The colour of cheese samples was deter-mined with a tristimulus colour analyzer (Minolta ChromaMeter CR300 Minolta Corporation Ramsey NJ USA) thatmeasures reflective coloursThemeasurementwasmade bothon the surface and the core of cheese samples and the resultswere expressed using the CIE 119871lowast119886lowast119887lowast (CIELAB) space Thisthree-dimensional model describes all the colours visible tothe human eye bymeans of three spatial coordinates a centralvertical axis that represents lightness (119871lowast) in which values runfrom 0 (black) to 100 (white) a second perpendicular axis(119886lowast) that represents the red-green channel where positivevalues indicate red and negative values indicate green andthe third perpendicular axis (119887lowast) that represents the opponentyellow-blue channelwhere positive values indicate yellow andnegative values indicate blue Therefore each colour can berepresented as a point in a three-dimensional space definedby its three parameters 119871lowast 119886lowast and 119887lowast Allmeasurements weremade in triplicate

28 Analysis of Volatile Compounds Cheese samples werewrapped in aluminium foil vacuum-packed in Cryovacplastic bags and frozen at minus80∘C until analysis Volatile com-pounds were extracted by headspace solid-phase microex-traction (SPME) and then analysed by gas chromatography-mass spectrometry GC-MS (HP6890-MSDHP 5973 AgilentPalo Alto CA USA) according to the procedure described by


Lee et al [33] Cheese samples (10 g) were homogenized withanhydrous sodium sulphate (20 g) and 20120583L of an aqueoussolution containing cyclohexanone (1058 ppm) and camphor(1040 ppm) as internal standards using a mechanical grinderThen 5 g of this mixture was weighed in a 40mL glass vialthat was sealed with a polytetrafluoroethylene (PTFE) facedsilicone septum Volatile compounds were isolated using aSPME manual holder equipped with a 2 cm times 5030 120583mStable Flex DivinylbenzeneCarboxenPolydimethylsiloxane(DVBCARPDMS) coated fibre (Supelco Bellefonte Penn-sylvania USA) Vials were equilibrated in a thermostatic bathat 37∘C for 20min before the fiber was inserted through thePTFE septum for headspace extractionThefiberwas exposedto the headspace for 30min and then it was inserted into theGC injection port for desorption (260∘C10min in splitlessmode)

Separation of volatile compounds was performed on aZebron ZB-WAX plus (60m times 025mm times 050 120583m) capillarycolumn coated with 100 polyethylene glycol (PhenomenexTorrance California USA) For chromatographic separationthe temperature was maintained at 40∘C for 7 min increasedfrom 40∘C to 90∘C at a rate of 2∘Cmin from 90∘C to150∘C at a rate of 3∘Cmin from 150∘C to 240∘C at a rate of9∘Cmin and finally held at 240∘C for 8min Detection wasperformed with the mass selective detector operating in thescanmode collecting data at a rate of 516 scanss over a rangeof 33ndash300mz at ionization energy of 70 eV Identificationof volatile compounds was based on comparison of spectrausing the Wiley 275 Library (Wiley and Sons Inc New YorkUSA) Relative abundances of compounds were expressed aspercentages of their peak areas to the cyclohexanone peakarea Samples were tested in duplicate

29 Sensory Evaluation The sensory evaluation of cheeseswas done by panellists (staff and students of the Departmentof Food Science and Nutrition Universidad Complutense deMadrid) who were familiar with sensory evaluation tech-niques The evaluation was performed in individual boothsunder controlled conditions of environment and light

Representative cheese samples (sim20 g) after 28 d of stor-age at 4∘C were equilibrated at room temperature andpresented to the tester in disposable plastic containers exceptfor odour assessment for which samples were presentedin closed glass flasks Samples were codified with random3 digit numbers following a completely randomized blockdesign Cheese portions from replications of the same batchwere mixed so a representative sample was presented to thepanellists

Initially 30 tester semitrained panellists participated ina triangle test to determine if potential probiotic cheesescontaining lactobacilli differ in any aspect from the controlcheese Significant differences were determined using themethod of Roessler et al [34] Later on 18 selected trainedpanellists were asked to perform a descriptive test for a num-ber of specific descriptors clustered in groups related to odour(buttery cow fermented milk floral fruity lawn rancidand vinegar) flavour and taste (aftertaste astringent bittercow fermented milk fruity salty and vinegar) and texture

10

8

6

4

2

0

0 7 14 21 28

Time (days)

lowast

lowast

Viab

le co

unts

(log 10

cfu

g)

Figure 2 Viable counts (log10

cfug) of starter Lc lactis ESI153 (I)and L salivarius CECT5713 (◻) and PS2 (◼) in fresh cheese duringstorage at 4∘C

and appearance (adhesive bright colour (white or yellow)creamy friable hardness moist springy and smooth) andto score for the overall impression using a 10-point intensityscale

210 Statistical Analysis The influence of the addition of Lsalivarius CECT5713 and PS2 to cheeses was analyzed byone way analysis of variance (ANOVA) Tukeyrsquos multiplerange tests were applied to determine differences amongthe cheeses Differences were considered significant at 119875 lt005 StatGraphics Centurion XVI version 16115 (StatpointTechnologies Inc Warrenton Virginia USA) was used toperform these analyses

3 Results

31 Gross Composition pH and Water Activity The meancomposition of control cheeses without the addition ofprobiotic bacteria was 245 (wtwt) fat 178 (wtwt)protein and 31 (wtwt) ash after 28 days of storage at 4∘C(Table 1) Chemical composition of cheeses manufacturedwith L salivarius CECT5713 or PS2 was similar slightlyhigher moisture and lower fat and protein contents werefound compared to control cheeses although the differencewas statistically significant only for the protein content Thepresence of lactobacilli in cheeses was associated with a lowerfinal pH compared to control cheeses but it did not modifythe 119886w of the final product (096-097)

32 Viability of L salivarius during Cheese Storage Thegrowth of L salivarius CECT5713 and PS2 in MRS agar con-taining bromophenol produced large and blue colonies whilein the same conditions Lc lactis ESI153 colonies were smalland white Initial viable counts in MRS agar of L salivariusCECT5713 and PS2 according to the morphology of thecolony were 81 and 79 log

10cfug respectively (Figure 2)

Both L salivarius strains remained viable in the cheeses after


Table 1 Chemical composition pH and water activity of control cheese and cheeses using L salivarius CECT5713 or PS2 after 28 days ofstorage at 4∘Clowast

Cheese119875 valuelowastlowast

Control L salivarius CECT5713 L salivarius PS2Moisture ( wtwt) 5248 plusmn 214 5617 plusmn 195 5662 plusmn 196 0086Fat ( wtwt) 2447 plusmn 130 2088 plusmn 184 2237 plusmn 127 0067Protein ( wtwt) 1775 plusmn 080

a1494 plusmn 079

b1543 plusmn 085

b 0012Ash ( wtwt) 310 plusmn 011 328 plusmn 045 288 plusmn 010 0278pH 486 plusmn 013 471 plusmn 003 475 plusmn 011 0287119886119908

096 plusmn 001 096 plusmn 001 097 plusmn 001 0398lowastResults are expressed as mean plusmn standard deviation values of triplicate samples lowastlowastone-way ANOVA to determine differences on chemical composition pHand 119886119908between cheeses abMean values within the same row followed by different letter were significantly different when compared using the Tukeyrsquos test

Table 2 Texture profile analysis of control and experimental cheeses using L salivarius CECT5713 or PS2 after 28 days of storage at 4∘Clowast

Parameter Cheese119875 valuelowastlowast

Control L salivarius CECT5713 L salivarius PS2Hardness (N) 2100 plusmn 071

a2442 plusmn 097

b2127 plusmn 065

a 0001Cohesiveness 015 plusmn 003 015 plusmn 001 014 plusmn 001 0662Adhesiveness (N s) minus057 plusmn 030 minus077 plusmn 009 minus057 plusmn 004 0236Springiness (m) 00048 plusmn 00015 00043 plusmn 00017 00048 plusmn 00010 0851Gumminess (N) 322 plusmn 064 361 plusmn 023 299 plusmn 029 0171Chewiness (Nm) 0015 plusmn 0004 0014 plusmn 0005 0013 plusmn 0001 0927lowastTexture parameters are expressed asmeanplusmn standard deviation values of quadruplicate measurements in triplicate samples lowastlowastone-way ANOVA to determinedifferences on texture parameters between cheeses abMean values within the same row followed by different letter were significantly different when comparedusing the Tukeyrsquos test

28 days at 4∘C (Figure 2) The probiotic counts decreasedduring storage but the reduction in viable counts was signif-icant only after 21 days Final concentrations of L salivariusCECT5713 andPS2 were 67 and 66 log

10cfug respectively

representing about a 13 log10-unit reduction at the end of

28-day storage The identity of selected colonies isolated atthe end of the storage period was confirmed by RAPD andPFGE (results not shown) On the other hand initial viablecounts of the starter culture Lc lactis ESI153 were higher(about 9 log

10cfug in all cheese samples) and remained

fairly constant along the storage period Bacterial growthwas not detected on PEMBA and MacConkey agar platesconfirming the absence of contamination with B cereus andEnterobacteriaceae

33 PCRDGGEAnalysis DGGEanalysiswas also performedin order to check the bacterial diversity and to confirm thepresence of the probiotic strainsrsquo DNA in the cheese samples(Figure 3) The amplification of the V6ndashV8 variable region ofthe 16S rRNA gene of L salivarius CECT5713 and Lc lactisESI153 using the primers U968-GC-f and L1401-r resulted ina single fragment differing in size for each bacterial species(Figure 3(a)) The same primer pair did not amplify anyfragment when L salivarius PS2 DNA was used as templateHowever amplification of one fragment corresponding tothe V6ndashV8 variable region of the 16S rRNA gene of Lsalivarius PS2 was successful when Lactobacillus-specificprimers (Lab159f and Uni-515-GCr) were used (Figure 3(b))The DGGE profile obtained from L salivarius PS2 using

this pair of primers comprised 3 dominant bands and it wasdifferent from the one obtained for Lc lactis ESI153

The DGGE profiles of cheese samples analyzed and theladders constructed in this study (with amplicons obtainedfrom pure cultures of Lc lactis ESI153 and the correspondingL salivarius strain) using universal or Lactobacillus-specificprimers were identical (Figure 3) This result indicates thatthe inoculated strains were the predominant in the respectivecheeses during storage

34 Textural Analysis of Cheeses Texture parameters at theend of the cheese storage were similar in control and Lsalivarius-containing cheeses (Table 2) No significant differ-ences were observed in cohesiveness adhesiveness springi-ness gumminess and chewiness values Globally cheeses hada crumbly and brittle texture requiring only a relatively smallload to fracture However cheeses manufactured with Lsalivarius CECT5713 presented significantly higher values ofhardness compared to control cheeses and cheeses containingL salivarius PS2

35 Colour Analysis The colour was measured both onthe surface and the core of cheese samples by tristimulusreflectance measurement (Table 3) In general all sampleshad high lightness (119871lowast sim 92 to 94) indicating no differencesin the mechanical openings exhibited by the three cheeseslow yellow (119887lowast sim 11 to 12) and very low green (119886lowast sim minus1 to minus2)colour Globally the lightness value was lower in the surfacethan in the interior possibly reflecting a closer structure in


Table 3 Colour parameters (Llowast alowast and blowast) of control and experimental cheeses manufactured with L salivarius CECT5713 or PS2 after 28days of storage at 4∘Cdagger

Parameter Cheese119875 valuedaggerdagger

Control L salivarius CECT5713 L salivarius PS2Surface119871lowast

9220 plusmn 090 9267 plusmn 056 9241 plusmn 012 0664119886lowast

minus158 plusmn 002a

minus116 plusmn 004b

minus133 plusmn 011c 0001

119887lowast

1166 plusmn 041a

1074 plusmn 016b

1192 plusmn 026a 0006

Core119871lowast



minus148 plusmn 012ab

minus170 plusmn 004b 0024

119887lowast

1075 plusmn 017 1069 plusmn 021 1092 plusmn 034 0521daggerColour parameters are expressed as mean plusmn standard deviation values of quadruplicate measurements in triplicate samples daggerdaggerone-way ANOVA to determinedifferences on colour parameters between cheeses abcMeans values within the same row followed by different letter were significantly different when comparedusing the Tukeyrsquos test

1 2 3 4 5 6 7 8

MarkersCheese

L salivarius CECT5713

Lc lactis ESI153

1-d 7-d 14-d 21-d 28-d

(a)

2 3 4 5 6 71 8

L salivarius PS2

Lc lactis ESI153

(b)

Figure 3 DGGE profiles of 16S rRNA gene V6ndashV8 regions obtained from samples of cheesemanufactured with L salivariusCECT5713 usinguniversal primers U968-GC-f and L1401-r (a) and L salivarius PS2 using Lactobacillus-specific primers Lab159f and Uni-515-GCr (b) duringstorage at 4∘C (a) lane 1 marker (L salivarius CECT5713 and Lc lactis ESI153) lane 2 Lc lactis ESI153 lane 3 L salivarius CECT5713 lane4 1-day cheese lane 5 7-day cheese lane 6 14-day cheese lane 7 21-day cheese lane 8 28-day cheese (b) Lane 1 marker (L salivarius PS2and Lc lactis ESI153) lane 2 Lc lactis ESI153 lane 3 L salivarius PS2 lane 4 1-day cheese lane 5 7-day cheese lane 6 14-day cheese lane 721-day cheese lane 8 28-day cheese

the surface and more open pores in the interiorThe oppositewas observed for 119887lowast parameter indicating more yellownessin the surface The surface of cheeses elaborated with Lsalivarius CECT5713 was whiter and had less intense greencolour than the others (Table 3)

36 Volatile Analysis A total of 59 volatile compounds wereidentified in the headspace of experimental cheeses includ-ing aldehydes ketones alcohols esters alkanes and car-boxylic and fatty acids (Table 4) All cheese samples presentedhigh relative abundance of the alcohols 3-methyl-1-butanol


Table 4 Volatile compounds in control cheese and cheeses containing L salivarius CECT5713 or PS2 after 28 days at 4∘Clowast

Volatile compound RTlowastlowast Cheese119875 valuelowastlowastlowast

Control L salivarius CECT5713 L salivarius PS2Aldehydes

3-Methylbutanal 1242 282 plusmn 015a

098 plusmn 014b

162 plusmn 025c 0000

Hexanal 2321 064 plusmn 024 050 plusmn 007 056 plusmn 006 0552Ketones

2-Butanone 801 366 plusmn 028 225 plusmn 039 264 plusmn 086 01012-Propanone 1158 1685 plusmn 199

a1500 plusmn 136

a2139 plusmn 108

b 00012-Heptanone 3078 402 plusmn 016

a189 plusmn 029

b209 plusmn 009

b 00003-Hydroxy-2-butanone 3808 214 plusmn 036 191 plusmn 146 098 plusmn 031 0413

AlcoholsEthanol 1376 4505 plusmn 286 4795 plusmn 396 5079 plusmn 301 02123-Methyl-1-butanol 3289 34848 plusmn 649 35880 plusmn 1298 36884 plusmn 2033 03752-Furanmethanol 5559 087 plusmn 006 011 plusmn 005 052 plusmn 049 02173-Methyl-3-buten-1-ol 3571 104 plusmn 003 104 plusmn 010 118 plusmn 014 0138

EstersEthyl acetate 1093 071 plusmn 001 065 plusmn 020 069 plusmn 015 0905Ethyl butanoate 2002 055 plusmn 019 100 plusmn 022 106 plusmn 021 0110Ethyl hexanoate 3441 034 plusmn 003 051 plusmn 011 054 plusmn 015 0221

Alkanes2-Methylpentane 433 031 plusmn 004 035 plusmn 006 050 plusmn 017 02003-Methylpentane 444 025 plusmn 002 034 plusmn 006 047 plusmn 015 0073Hexane 451 182 plusmn 036 182 plusmn 046 180 plusmn 064 0998Heptane 540 072 plusmn 001 065 plusmn 019 040 plusmn 009 006424-Dimethylheptane 755 132 plusmn 005

a040 plusmn 009

b040 plusmn 015

b 00014-Methyloctane 930 083 plusmn 004 020 plusmn 003 028 plusmn 018 0113

Carboxylic and fatty acidsAcetic acid 4678 2276 plusmn 102

a4261 plusmn 237

b3353 plusmn 862

ab 0016Propanoic acid 5093 007 plusmn 000 008 plusmn 000 008 plusmn 000

Butanoic acid 5447 2036 plusmn 180 2823 plusmn 299 2615 plusmn 626 0209Hexanoic acid 5958 1482 plusmn 099 2249 plusmn 351 2004 plusmn 414 0110Octanoic acid 6289 742 plusmn 230 1380 plusmn 386 1085 plusmn 204 0133Heptanoic acid 6133 048 plusmn 001 062 plusmn 009 108 plusmn 044 0083Nonanoic acid 6446 135 plusmn 019 073 plusmn 026 658 plusmn 890 0362Decanoic acid 6621 315 plusmn 189 566 plusmn 318 329 plusmn 127 0401

OthersD-Limonene 3178 074 plusmn 002 118 plusmn 013 072 plusmn 047 0163

lowastRelative abundance of compoundswas expressed as percentages of their peak areas to the cyclohexanone peak area lowastlowastRT retention time lowastlowastlowastone-wayANOVAto determine differences on the relative abundance of volatile compounds between cheeses abcMean values within the same row followed by different letterwere significantly different when compared using the Tukeyrsquos test

and ethanol as well as of acetic butanoic and hexanoic acidsalthough significant differences were observed only for aceticacid that had higher abundance in cheeses containing Lsalivarius CECT5713 Aldehyde 3-methylbutanal ketone 2-heptanone and alkane 24-dimethylheptane were present atstatistically significant lower levels in cheeses manufacturedwithL salivarius than in control cheese Level of 2-propanonewas higher in cheese made with L salivarius PS2 than in theother two cheeses

37 Sensory Evaluation The results of the triangle test toevaluate differences in sensory properties indicated thatsignificant differences were detected at the end of the storageThe panellists could appreciate significant variations betweencontrol cheese and cheese manufactured with L salivariusCECT5713 (119875 = 0018) or PS2 (119875 = 0002)

Trained panellists performed a quantitative descriptiveanalysis using attributes describing odour flavour taste tex-ture and appearance of cheeses after 28 days of storage at 4∘C


0

3

6

9Fermented milk

Lawn

Floral

Fruity

Cow

Buttery

Vinegar

Rancid

c

(a)

0

3

6

9Aftertaste

Astringent

Bitter

Cow

Fermented milk

Fruity

Salty

Vinegar

(b)

0

3

6

9

Bright

Friable

HardnessMoist

Springy

Smooth

Yellow

Adhesive lowastP = 0005 (CECT 5713 PS2)

Creamy lowastP = 0042 (CECT 5713 PS2)

(c)

Figure 4 Graphical charts of the sensory profile of control cheese (I) and cheeses containing L salivarius CECT5713 (◼) and PS2 (998771) after28 days at 4∘C (a) Odour related attributes (b) Flavour and taste related attributes (c) Texture and appearance descriptors

(Figure 4) Globally the descriptors that obtained the highestscores were fermented milk taste and smell and butter-likesmell Among all attributes only the intensity of adhesivenessand creaminess in control cheese was statistically higher thanthat of the cheeses containing L salivarius CECT5713 orPS2 However these differences did not have any statisticallysignificant effect on the overall quality of the cheeses (119875 lt005) and all cheese samples presented good results ofacceptance after 28 days of storage at 4∘C In average theacceptance level of odour flavour appearance and textureas well as the global score was up to 6 (on a 0ndash10 numericrating scale) in the three types of cheese

4 Discussion

Cheese has been considered as an excellent alternative tofermented milk and yogurts as a food vehicle for probioticdelivery Its buffering capacity is one of its advantages because

it protects probiotics against the highly acidic stomach envi-ronmentThe structure of the gel and its high fat content andsolid consistency also add to the probiotic protection [35 36]Several studies have demonstrated that cheese is an excellentcarrier for probiotic bacteria including fresh and Cheddarcheese varieties [5 27 36ndash38] However variable results havebeen obtained with different probiotic strains and each strainshould be tested individually Therefore the objective of thisstudy was to check the viability of two lactobacilli strainsisolated from human milk after their incorporation to cheesecurds and to test their impact in the final product

Theoretically the probiotic bacteria could be added eitherdirectly to milk andor incorporated at a later stage duringthe manufacture of cheese Ong et al [36] manufactured pro-biotic Cheddar cheese containing different combinations ofsix probiotic Lactobacillus and Bifidobacterium strains whichwere coculturedwith the cheese starter cultureThey reportedsome loss of probiotic lactobacilli and bifidobacteria in whey


(about 6-7 log10cfug) but final counts in all fresh cheeses

were high and acceptable (8-9 log10cfug) In preliminary

trials following our procedure L salivarius CECT5713 andPS2 were also cocultured with the starter culture but only asmall amount of probiotic was retained in the curd resultingin a high loss of lactobacilli in the cheese whey (results notshown) Notably the addition of the probiotic lactobacillito the curds after whey drainage when most of the wheyhad been removed and before molding resulted in improvedretention of lactobacilli in the cheese

In any probiotic food in order to have industrial appli-cation and to exert its health benefit to the host the incor-porated probiotic strain must maintain its viability duringthe manufacture through the shelf life of the product andup to the time of consumption In the present study both Lsalivarius strains remained viable in the experimental cheesesafter 28 days at 4∘C In addition the hygienic quality of thefinal product was adequate and growth of any other bacteriawas not detected in the culture media used Antibacterialproperties against pathogenic bacteria have been reportedfor L salivarius CECT5713 due to the production of antimi-crobial compounds such as lactate acetate and hydrogenperoxide [14] This strain also harbors a bacteriocin clusterlocated in a megaplasmid that contains several genes thatwould allow the biosynthesis of several bacteriocins but adeletion at the beginning of the regulatory system results inthe absence of any bacteriocin production [19]

A minimum probiotic daily dose of 108-109 cfu has beenrecommended in processed foods in order to exert theirbeneficial effects [35 39] This would be equivalent to a dailyintake of 100 g of product containing 106-107 cfugThe resultsobtained in this study show that the counts of L salivariusCECT5713 and PS2 in cheeses were always in the range of thisrecommended levelTherefore theywould satisfy this criteriaestablished for a probiotic food Furthermore the presence ofthese potentially probiotic bacteria did not interfere with theperformance of starter lactococci as it has been described byother authors [27]

Another challenge associated with the addition of pro-biotic bacteria during cheese manufacturing is to maintainthe characteristics of the cheese Actually consumers demandthe addition of probiotic cultures to many foods includingcheese but a primary consideration is that the sensoryproperties especially taste of any probiotic food should beappealing [40] The addition of certain levels of any viablebacteria and their enzymes to cheese most probably willcontribute to glycolysis proteolysis and lipolysis processesthat take place during manufacture and cheese ripening andcontribute to the organoleptic properties of the final product[41] In order to maintain an adequate organoleptic qualityprobiotic bacteria must not adversely affect cheese compo-sition texture flavour and final acceptance The addition ofprobiotic L salivarius CECT5713 and PS2 did not result ina substantial change of the fresh cheese composition whichwas within the gross chemical composition of this type ofcheeses

A slight (but not statistically significant) increase inmoisture content of cheeses containing lactobacilli compared

to control cheese was noted although 119886w values remainedunchanged These differences may be related at least in partto several factors during cheesemanufacture that exert a greatinfluence on moisture retention in the curd such as cuttingintensity final size of the curds or curd manipulation [42]On the other hand exopolysaccharide- (EPS-) producinglactic acid bacteria have been reported to increase moistureretention in cheese [43ndash45] and could improve the texture ofreduced-fat cheese that tends to be tough and rubbery Twogene clusters for EPS biosynthesis have been described in Lsalivarius strains although it has been reported that the levelof production of EPS does not correlate with the presence ofthese clusters depends on the available carbohydrate and ishighly strain-dependent [46] However at present it is notknown if L salivarius CECT5713 and PS2 are EPS-producingstrains Another tentative explanation may involve microbialdynamics and metabolism It has been reported that cheesemicrobiota and its metabolic activitymay confound the effectofmoisture on 119886w [47] Changes in the type and concentrationof low molecular weight soluble compounds such as anincrease in lactate free fatty acids amino acids and evenvery small peptides might decrease the value of 119886w althoughthis effect is usually more pronounced for ripened cheeses[47 48]

Among the texture parameters analyzed in this workinstrumental methods only detected differences in hard-ness which was higher in cheeses containing L salivariusCECT5713 than in control cheeses and those manufacturedwith L salivarius PS2 However this difference was notperceived by panellists during the descriptive test indicatingthat it did not have a relevant impact in the sensory qualityand acceptance of the cheese On the contrary the panellistsidentified both cheeses containing probiotic lactobacilli ashaving lower adhesiveness than the control cheese althoughthe texture profile analysis did not reveal a statistically signif-icant difference Reduction in adhesiveness in cheeses madewith an EPS-producing Streptococcus thermophilus culturehas been related to the production and liberation of EPS[49 50] Also a higher perception of creaminess in controlcheese was reported in contrast to cheeses manufacturedwith L salivarius CECT5713 and PS2 Creaminess is oftenrelated to a high fat level and the presence of fat globulesin agreement with the slightly higher fat content of controlcheese although sensorial discrimination of fat levels in solidfoods is more difficult than in liquid products [51] Regardingcolour small although statistically significant differenceswere also detected when using instrumental methods ofanalysis but they were not perceived by the trained panellistsThis indicates that the presence of probiotic lactobacilli didnot disturb the distinctive white colour of fresh cheese

Following the general component balance theory cheeseflavour is the result of a synergistic effect of the appropriateand balanced blend of various flavour compounds producedfrom proteins lipids and lactose through numerous bio-chemical reactions involving enzymes from milk rennetstarter cultures secondary cheese microbiota and evenspontaneous reactions [52] The volatile composition of thecheese made with both L salivarius strains was not qualita-tively different from that of the control cheese and only a


few quantitative differences were observed The main changedetected was a higher acetic acid concentration in the cheesescontaining L salivarius CECT5713 and PS2 probably relatedto higher lactose degradation during cheese storage whichwill explain a lower final pH in the probiotic cheese Howeverthese differences did not impact the sensory perception givenby the panellists or the global acceptance of the cheesesmanufactured with L salivarius CECT5713 and PS2 as it hasbeen described with other probiotic strains by other authors[36ndash38 49 50 53]

5 Conclusion

The results of the present study demonstrate that L salivariusCECT5713 and PS2 incorporated into fresh cheese survived atadequate levels during a 28-day storage at 4∘C The presenceof the lactobacilli did not interfere with normal growth ofstarter culture and did not modify significantly the compo-sition and organoleptic properties of the probiotic cheesescontaining L salivarius strains that had good acceptance bytrained panellists

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by CSD2007-00063 (FUN-C-FOOD Consolider-Ingenio 2010) and AGL2010-15420Projects from the Ministerio de Ciencia e Innovacion(Spain)

References

[1] A R Hill and P Kethireddipalli ldquoDairy products cheeseand yogurtinrdquo in Biochemistry of Foods N A M Eskin andF Shahidi Eds pp 319ndash362 Elsevier Science amp TechnologyBooks San Diego Calif USA 3rd edition 2012

[2] FAOWHO ldquoHealth and nutritional properties of probiotics infood including powdermilk with live lactic acid bacteria reportof a Joint FAOWHO Expert Consultation on Evaluation ofHealth and Nutritional Properties of Probiotics in Food Includ-ing Powder Milk with Live Lactic Acid Bacteriardquo TechnicalReport FAOWHO Geneva Switzerland 2001

[3] M Sharma andMDevi ldquoProbiotics a comprehensive approachtoward health foodsrdquo Critical Reviews in Food Science andNutrition vol 54 no 4 pp 537ndash552 2014

[4] M E Sanders and M L Marco ldquoFood formats for effectivedelivery of probioticsrdquo Annual Review of Food Science andTechnology vol 1 pp 65ndash85 2010

[5] C G VinderolaW Prosello D Ghiberto and J A ReinheimerldquoViability of probiotic (Bifidobacterium Lactobacillus aci-dophilus and Lactobacillus casei) and nonprobiotic microflorain Argentinian Fresco cheeserdquo Journal of Dairy Science vol 83no 9 pp 1905ndash1911 2000

[6] E W Ng M Yeung and P S Tong ldquoEffects of yogurt startercultures on the survival of Lactobacillus acidophilusrdquo Interna-tional Journal of Food Microbiology vol 145 no 1 pp 169ndash1752011

[7] C Stanton G Gardiner P B Lynch J K Collins G Fitzgeraldand R P Ross ldquoProbiotic cheeserdquo International Dairy Journalvol 8 no 5-6 pp 491ndash496 1998

[8] RMartın S Langa C Reviriego et al ldquoHumanmilk is a sourceof lactic acid bacteria for the infant gutrdquo Journal of Pediatricsvol 143 no 6 pp 754ndash758 2003

[9] M P Heikkila and P E J Saris ldquoInhibition of Staphylococcusaureus by the commensal bacteria of human milkrdquo Journal ofApplied Microbiology vol 95 no 3 pp 471ndash478 2003

[10] R Martın E Jimenez H Heilig et al ldquoIsolation of bifidobac-teria from breast milk and assessment of the bifidobacterialpopulation by PCR-denaturing gradient gel electrophoresisand quantitative real-time PCRrdquo Applied and EnvironmentalMicrobiology vol 75 no 4 pp 965ndash969 2009

[11] T Jost C Lacroix C Braegger and C Chassard ldquoAssessment ofbacterial diversity in breast milk using culture-dependent andculture-independent approachesrdquo British Journal of Nutritionvol 110 no 7 pp 1253ndash1262 2013

[12] V Martın A Maldonado L Moles et al ldquoSharing of bacterialstrains between breast milk and infant fecesrdquo Journal of HumanLactation vol 28 no 1 pp 36ndash44 2012

[13] R Martın M Olivares M L Marın L Fernandez J Xaus andJ M Rodrıguez ldquoProbiotic potential of 3 lactobacilli strainsisolated from breast milkrdquo Journal of Human Lactation vol 21no 1 pp 8ndash17 2005

[14] R Martın E Jimenez M Olivares et al ldquoLactobacillus salivar-ius CECT 5713 a potential probiotic strain isolated from infantfeces and breast milk of a mother-child pairrdquo InternationalJournal of Food Microbiology vol 112 no 1 pp 35ndash43 2006

[15] M Olivares M P Dıaz-Ropero RMartın J M Rodrıguez andJ Xaus ldquoAntimicrobial potential of four Lactobacillus strainsisolated from breast milkrdquo Journal of Applied Microbiology vol101 no 1 pp 72ndash79 2006

[16] M PDıaz-Ropero RMartın S Sierra et al ldquoTwoLactobacillusstrains isolated from breast milk differently modulate theimmune responserdquo Journal of AppliedMicrobiology vol 102 no2 pp 337ndash343 2007

[17] F J Perez-Cano H Dong and P Yaqoob ldquoIn vitro immun-omodulatory activity of Lactobacillus fermentum CECT5716and Lactobacillus salivarius CECT5713 two probiotic strainsisolated from human breast milkrdquo Immunobiology vol 215 no12 pp 996ndash1004 2010

[18] E Jimenez R Martın A Maldonado et al ldquoComplete genomesequence of Lactobacillus salivarius CECT 5713 a probioticstrain isolated from human milk and infant fecesrdquo Journal ofBacteriology vol 192 no 19 pp 5266ndash5267 2010

[19] S Langa AMaldonado-Barragan SDelgado et al ldquoCharacter-ization of Lactobacillus salivarius CECT 5713 a strain isolatedfrom human milk from genotype to phenotyperdquo AppliedMicrobiology and Biotechnology vol 94 no 5 pp 1279ndash12872012

[20] E Jimenez L Fernandez A Maldonado et al ldquoOral Admin-istration of Lactobacillus strains isolated from breast milk asan alternative for the treatment of infectious mastitis duringlactationrdquo Applied and Environmental Microbiology vol 74 no15 pp 4650ndash4655 2008

[21] F Lara-Villoslada S SierraM P Dıaz-RoperoMOlivares andJ Xaus ldquoSafety assessment of the humanmilk-isolated probiotic


Lactobacillus salivarius CECT5713rdquo Journal of Dairy Sciencevol 90 no 8 pp 3583ndash3589 2007

[22] R Arroyo V Martın A Maldonado E Jimenez L Fernandezand J M Rodrıguez ldquoTreatment of infectious mastitis duringlactation antibiotics versus oral administration of lactobacilliisolated from breast milkrdquo Clinical Infectious Diseases vol 50no 12 pp 1551ndash1558 2010

[23] J Maldonado F Lara-Villoslada S Sierra et al ldquoSafety andtolerance of the human milk probiotic strain Lactobacillussalivarius CECT5713 in 6-month-old childrenrdquo Nutrition vol26 no 11-12 pp 1082ndash1087 2010

[24] T M Cogan M Barbosa E Beuvier et al ldquoCharacterization ofthe lactic acid bacteria in artisanal dairy productsrdquo Journal ofDairy Research vol 64 no 3 pp 409ndash421 1997

[25] E Rodrıguez J Calzada J L Arques J M Rodrıguez MNunez and M Medina ldquoAntimicrobial activity of pediocin-producing Lactococcus lactis on Listeria monocytogenes Staphy-lococcus aureus andEscherichia coliO157H7 in cheeserdquo Interna-tional Dairy Journal vol 15 no 1 pp 51ndash57 2005

[26] C Reviriego L Fernandez and J M Rodrıguez ldquoA food-gradesystem for production of pediocin PA-1 in nisin-producing andnon-nisin-producing Lactococcus lactis strains application toinhibit Listeria growth in a cheese model systemrdquo Journal ofFood Protection vol 70 no 11 pp 2512ndash2517 2007

[27] L Ong A Henriksson and N P Shah ldquoProteolytic pattern andorganic acid profiles of probiotic Cheddar cheese as influencedby probiotic strains of Lactobacillus acidophilus Lb paracaseiLb casei or Bifidobacterium sprdquo International Dairy Journal vol17 no 1 pp 67ndash78 2007

[28] J L Ruiz-Barba A Maldonado and R Jimenez-Dıaz ldquoSmall-scale total DNA extraction from bacteria and yeast for PCRapplicationsrdquo Analytical Biochemistry vol 347 no 2 pp 333ndash335 2005

[29] L Moles M Gomez H Heilig et al ldquoBacterial diversity inmeconium of preterm neonates and evolution of their fecalmicrobiota during the first month of liferdquo PLoS One vol 8 no6 Article ID e66986 2013

[30] U Nubel B Engelen A Felske et al ldquoSequence heterogeneitiesof genes encoding 16S rRNAs in Paenibacillus polymyxadetected by temperature gradient gel electrophoresisrdquo Journalof Bacteriology vol 178 no 19 pp 5636ndash5643 1996

[31] H G Heilig E G Zoetendal E E Vaughan P Marteau AD L Akkermans and W M De Vos ldquoMolecular diversityof Lactobacillus spp and other lactic acid bacteria in thehuman intestine as determined by specific amplification of 16Sribosomal DNArdquo Applied and Environmental Microbiology vol68 no 1 pp 114ndash123 2002

[32] R Martın S Delgado A Maldonado et al ldquoIsolation oflactobacilli from sow milk and evaluation of their probioticpotentialrdquo Journal of Dairy Research vol 76 no 4 pp 418ndash4252009

[33] J H Lee R Diono G Y Kim and D B Min ldquoOptimization ofsolid phase microextraction analysis for the headspace volatilecompounds of Parmesan cheeserdquo Journal of Agricultural andFood Chemistry vol 51 no 5 pp 1136ndash1140 2003

[34] E B Roessler R M Pangborn J L Sidel and H StoneldquoExpanded statistical tables for estimating significance inpaired-preference paired-difference duo-trio and triangletestsrdquo Journal of Food Science vol 43 no 3 pp 940ndash943 1978

[35] A Gomes da Cruz F C A Buriti C H Batista de Souza JA Fonseca Faria and S M Isay Saad ldquoProbiotic cheese health

benefits technological and stability aspectsrdquo Trends in FoodScience and Technology vol 20 no 8 pp 344ndash354 2009

[36] L Ong A Henriksson and N P Shah ldquoDevelopment of pro-biotic Cheddar cheese containing Lactobacillus acidophilus Lbcasei Lb paracasei and Bifidobacterium spp and the influenceof these bacteria on proteolytic patterns and production oforganic acidrdquo International Dairy Journal vol 16 no 5 pp 446ndash456 2006

[37] F C A Buriti J S Da Rocha E G Assis and S M I SaadldquoProbiotic potential of Minas fresh cheese prepared with theaddition of Lactobacillus paracaseirdquo LWT-Food Science andTechnology vol 38 no 2 pp 173ndash180 2005

[38] F C A Buriti J S Da Rocha and S M I Saad ldquoIncorporationof Lactobacillus acidophilus in Minas fresh cheese and its impli-cations for textural and sensorial properties during storagerdquoInternational Dairy Journal vol 15 no 12 pp 1279ndash1288 2005

[39] A Talwalkar C W Miller K Kailasapathy and M H NguyenldquoEffect of packaging materials and dissolved oxygen on thesurvival of probiotic bacteria in yoghurtrdquo International Journalof Food Science and Technology vol 39 no 6 pp 605ndash611 2004

[40] C M Bruhn J C Bruhn A Cotter et al ldquoConsumer attitudestoward use of probiotic culturesrdquo Journal of Food Science vol67 no 5 pp 1969ndash1972 2002

[41] P L HMcSweeney ldquoBiochemistry of cheese ripeningrdquo Interna-tional Journal of Dairy Technology vol 57 no 2-3 pp 127ndash1442004

[42] C D Everard D J OrsquoCallaghan M J Mateo C P OrsquoDonnellM Castillo and F A Payne ldquoEffects of cutting intensity andstirring speed on syneresis and curd losses during cheesemanufacturerdquo Journal of Dairy Science vol 91 no 7 pp 2575ndash2582 2008

[43] D B Perry D J McMahon and C J Oberg ldquoEffect ofexopolysaccharide-producing cultures onmoisture retention inlow-fat Mozzarella cheeserdquo Journal of Dairy Science vol 80 no5 pp 799ndash805 1997

[44] D Low J A Ahlgren D Horne D J McMahon C J Obergand J R Broadbent ldquoRole of Streptococcus thermophilus MR-1C capsular exopolysaccharide in cheese moisture retentionrdquoApplied and Environmental Microbiology vol 64 no 6 pp2147ndash2151 1998

[45] W Wang L Zhang and Y Li ldquoEffect of specialized combinedstrains on reconstituted milk reduced-fat cheeserdquo African Jour-nal of Biotechnology vol 11 no 5 pp 1169ndash1176 2012

[46] E J Raftis E Salvetti S Torriani G E Felis and PW OrsquoTooleldquoGenomic diversity of Lactobacillus salivariusrdquo Applied andEnvironmental Microbiology vol 77 no 3 pp 954ndash965 2011

[47] D K Hickey T P Guinee J Hou andM GWilkinson ldquoEffectsof variation in cheese composition and maturation on wateractivity inCheddar cheese during ripeningrdquo InternationalDairyJournal vol 30 no 1 pp 53ndash68 2013

[48] R Saurel A Pajonk and J Andrieu ldquoModelling of frenchemmental cheese water activity during salting and ripeningperiodsrdquo Journal of Food Engineering vol 63 no 2 pp 163ndash1702004

[49] C H B De Souza F C A Buriti J H Behrens and S MI Saad ldquoSensory evaluation of probiotic Minas fresh cheesewith Lactobacillus acidophilus added solely or in co-culture witha thermophilic starter culturerdquo International Journal of FoodScience and Technology vol 43 no 5 pp 871ndash877 2008


[50] N H Ahmed M El Soda A N Hassan and J FrankldquoImproving the textural properties of an acid-coagulated (Kar-ish) cheese using exopolysaccharide producing culturesrdquo LWT-Food Science and Technology vol 38 no 8 pp 843ndash847 2005

[51] M B Froslashst and T Janhoslashj ldquoUnderstanding creaminessrdquo Interna-tional Dairy Journal vol 17 no 11 pp 1298ndash1311 2007

[52] G Smit B A Smit and W J M Engels ldquoFlavour formation bylactic acid bacteria and biochemical flavour profiling of cheeseproductsrdquo FEMS Microbiology Reviews vol 29 no 3 pp 591ndash610 2005

[53] C H B Souza and S M I Saad ldquoViability of Lactobacillusacidophilus La-5 added solely or in co-culture with a yoghurtstarter culture and implications on physico-chemical andrelated properties of Minas fresh cheese during storagerdquo LWT-Food Science and Technology vol 42 no 2 pp 633ndash640 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


salivarius and Lactobacillus vaginalis [12] Some lactobacilliisolated from human milk have been characterized andshown to have probiotic potential [13ndash15] Specifically Lsalivarius CECT5713 that was isolated from human milk andinfant feces of a healthy mother-child pair has been shownto have remarkable probiotic potential because it had highrate of survival in simulated gastrointestinal tract conditionsand strong adherence to mucus and intestinal cells in vitrostimulated the expression of mucin-encoding genes andproduced antimicrobial compounds [14ndash17] More recentlyits complete genome has been sequenced [18] and its geneticfeatures such as proteins potentially involved in humanmolecular mimetism may explain its immunomodulatoryanti-inflammatory and anti-infectious properties [19 20]Moreover its safety and health beneficial effects have beenproved in animal models and in human clinical assays [20ndash23] More recently L salivarius PS2 has also been isolatedfrom humanmilk and preliminary assays have shown similartraits and probiotic potential

The aim of this work was to evaluate the performanceof these two human milk L salivarius strains (CECT5713and PS2) in fresh cheese in order to develop a probioticcheese The survival of these two L salivarius strains inthe cheese has been studied as well as their impact onchemical composition volatile compounds texture and otherorganoleptic properties and overall consumer acceptance ofthe experimental cheeses

2 Materials and Methods

21 Starter and Probiotic Organisms Lactococcus lactisESI153 originally isolated from artisanal raw milk cheese[24] was selected to be used as starter culture Lc lactisESI153 cells were grown in M17 (Oxoid Basinstoke UK)broth supplemented with 05 (wtvol) glucose (GM17) at32∘C Before use Lc lactis ESI153 cells were subcultured (1)into reconstituted at 11 (wtvol) and heat-treated (121∘C5min) nonfat drymilk (HT-NFDM) and incubated overnightat 32∘C

Freeze-dried cultures of probiotic L salivariusCECT5713andPS2were prepared as follows A fully grown liquid cultureon de Man Rogosa and Sharpe (MRS) (Oxoid) broth wascentrifuged at 10000timesg for 10min at 4∘C The cell pelletwas washed with 085 (wtvol) NaCl and resuspended inHT-NFDM to one tenth of its original volume The cellsuspension was frozen at minus80∘C for 12 h inmetal trays Freezedrying was carried out at first 0∘C for 24 h and then at20∘C for 24 h under 13 Pa in a Lyph-Lock Stoppering TrayDryer model 77560 (Labconco Corporation Kansas CityMO USA) Freeze-dried cultures containing approximately103 log

10colony forming units (cfu)g were vacuum pack-

aged and stored at 4∘C before use

22 Experimental Cheese Manufacture Cheeses were madefrom commercial pasteurized (high temperature short timeHTST) cowrsquos milk (Ganaderıa Priegola SA Villanueva delPardillo Madrid Spain) following a laboratory-scale pro-cedure described previously by Rodrıguez et al [25] and

Pasteurized whole cowrsquos milk

Starter culture(Lactococcus lactis ESI 153

Acid production

Rennet(Fromase 44 IMCUL)

Coagulation

Cutting

Cooking

Whey drainage

Mixing

Probiotic culture(Lactobacillus salivarius CECT 5713 or

Lactobacillus salivarius PS2

Whey

Pressing

Brine salting

Vacuum packaging

Storage

(32∘C)

001 CaCl2

(32∘C 30min)

(30∘C 40min)

(curd 1 cm3)

(38∘C 40min)

(20∘C 18h)

(4∘C 28days)

(15 NaCl 20∘C 1h)

sim8 log10

cfug)

sim9 log10

cfumL)

Figure 1 Flowchart of the cheese making process

Reviriego et al [26] with some modifications (Figure 1)Briefly pasteurized milk (15 Lvat) at 32∘C with 001(wtvol) CaCl

2was inoculated with Lc lactis ESI153 (approx-

imately 9 log10cfumL) as starter culture Rennet (Fromase

44 IMCUL DSM Food Specialities Seclin Cedex France)was added to milk 30min after the inoculation of Lc lactisESI153 Curds were cut 40min after rennet addition andheated at 38∘C for 40min Whey was drained off and freeze-dried L salivarius CECT5713 or PS2 were added to the curdto reach a final concentration of sim8 log

10cfug before the

curds were distributed into the molds Control cheese wasmanufactured at the same conditions with the addition of theequivalent amount of HT-NFDM used as the excipient forfreeze drying the lactobacilli strains Cheeses were pressedfor 16 h at room temperature and salted in 15 brine (wtvol)during 3 hThe resulting cheeses (sim190 g)were cut into pieceswhich were individually vacuum-packed in Cryovac plasticbags and kept refrigerated at 4∘C during 28 days All cheesemanufacturing trials were made in triplicate




10















10

8

6

4

2

0

0 7 14 21 28

Time (days)

lowast

lowast

Viab

le co

unts

(log 10

cfu

g)





3 Results









a1494 plusmn 079

b1543 plusmn 085






a2442 plusmn 097

b2127 plusmn 065



10cfug respectively


















119887lowast

1166 plusmn 041a

1074 plusmn 016b

1192 plusmn 026a 0006

Core119871lowast





119887lowast


1 2 3 4 5 6 7 8

MarkersCheese


Lc lactis ESI153

1-d 7-d 14-d 21-d 28-d

(a)

2 3 4 5 6 71 8

L salivarius PS2

Lc lactis ESI153

(b)









098 plusmn 014b

162 plusmn 025c 0000



a1500 plusmn 136

a2139 plusmn 108


a189 plusmn 029

b209 plusmn 009





a040 plusmn 009

b040 plusmn 015



a4261 plusmn 237

b3353 plusmn 862









0

3

6

9Fermented milk

Lawn

Floral

Fruity

Cow

Buttery

Vinegar

Rancid

c

(a)

0

3

6

9Aftertaste

Astringent

Bitter

Cow

Fermented milk

Fruity

Salty

Vinegar

(b)

0

3

6

9

Bright

Friable

HardnessMoist

Springy

Smooth

Yellow



(c)



4 Discussion

















5 Conclusion




Acknowledgments


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




10















10

8

6

4

2

0

0 7 14 21 28

Time (days)

lowast

lowast

Viab

le co

unts

(log 10

cfu

g)





3 Results









a1494 plusmn 079

b1543 plusmn 085






a2442 plusmn 097

b2127 plusmn 065



10cfug respectively


















119887lowast

1166 plusmn 041a

1074 plusmn 016b

1192 plusmn 026a 0006

Core119871lowast





119887lowast


1 2 3 4 5 6 7 8

MarkersCheese


Lc lactis ESI153

1-d 7-d 14-d 21-d 28-d

(a)

2 3 4 5 6 71 8

L salivarius PS2

Lc lactis ESI153

(b)









098 plusmn 014b

162 plusmn 025c 0000



a1500 plusmn 136

a2139 plusmn 108


a189 plusmn 029

b209 plusmn 009





a040 plusmn 009

b040 plusmn 015



a4261 plusmn 237

b3353 plusmn 862









0

3

6

9Fermented milk

Lawn

Floral

Fruity

Cow

Buttery

Vinegar

Rancid

c

(a)

0

3

6

9Aftertaste

Astringent

Bitter

Cow

Fermented milk

Fruity

Salty

Vinegar

(b)

0

3

6

9

Bright

Friable

HardnessMoist

Springy

Smooth

Yellow



(c)



4 Discussion

















5 Conclusion




Acknowledgments


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







10

8

6

4

2

0

0 7 14 21 28

Time (days)

lowast

lowast

Viab

le co

unts

(log 10

cfu

g)





3 Results









a1494 plusmn 079

b1543 plusmn 085






a2442 plusmn 097

b2127 plusmn 065



10cfug respectively


















119887lowast

1166 plusmn 041a

1074 plusmn 016b

1192 plusmn 026a 0006

Core119871lowast





119887lowast


1 2 3 4 5 6 7 8

MarkersCheese


Lc lactis ESI153

1-d 7-d 14-d 21-d 28-d

(a)

2 3 4 5 6 71 8

L salivarius PS2

Lc lactis ESI153

(b)









098 plusmn 014b

162 plusmn 025c 0000



a1500 plusmn 136

a2139 plusmn 108


a189 plusmn 029

b209 plusmn 009





a040 plusmn 009

b040 plusmn 015



a4261 plusmn 237

b3353 plusmn 862









0

3

6

9Fermented milk

Lawn

Floral

Fruity

Cow

Buttery

Vinegar

Rancid

c

(a)

0

3

6

9Aftertaste

Astringent

Bitter

Cow

Fermented milk

Fruity

Salty

Vinegar

(b)

0

3

6

9

Bright

Friable

HardnessMoist

Springy

Smooth

Yellow



(c)



4 Discussion

















5 Conclusion




Acknowledgments


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





a1494 plusmn 079

b1543 plusmn 085






a2442 plusmn 097

b2127 plusmn 065



10cfug respectively


















119887lowast

1166 plusmn 041a

1074 plusmn 016b

1192 plusmn 026a 0006

Core119871lowast





119887lowast


1 2 3 4 5 6 7 8

MarkersCheese


Lc lactis ESI153

1-d 7-d 14-d 21-d 28-d

(a)

2 3 4 5 6 71 8

L salivarius PS2

Lc lactis ESI153

(b)









098 plusmn 014b

162 plusmn 025c 0000



a1500 plusmn 136

a2139 plusmn 108


a189 plusmn 029

b209 plusmn 009





a040 plusmn 009

b040 plusmn 015



a4261 plusmn 237

b3353 plusmn 862









0

3

6

9Fermented milk

Lawn

Floral

Fruity

Cow

Buttery

Vinegar

Rancid

c

(a)

0

3

6

9Aftertaste

Astringent

Bitter

Cow

Fermented milk

Fruity

Salty

Vinegar

(b)

0

3

6

9

Bright

Friable

HardnessMoist

Springy

Smooth

Yellow



(c)



4 Discussion

















5 Conclusion




Acknowledgments


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









119887lowast

1166 plusmn 041a

1074 plusmn 016b

1192 plusmn 026a 0006

Core119871lowast





119887lowast


1 2 3 4 5 6 7 8

MarkersCheese


Lc lactis ESI153

1-d 7-d 14-d 21-d 28-d

(a)

2 3 4 5 6 71 8

L salivarius PS2

Lc lactis ESI153

(b)









098 plusmn 014b

162 plusmn 025c 0000



a1500 plusmn 136

a2139 plusmn 108


a189 plusmn 029

b209 plusmn 009





a040 plusmn 009

b040 plusmn 015



a4261 plusmn 237

b3353 plusmn 862









0

3

6

9Fermented milk

Lawn

Floral

Fruity

Cow

Buttery

Vinegar

Rancid

c

(a)

0

3

6

9Aftertaste

Astringent

Bitter

Cow

Fermented milk

Fruity

Salty

Vinegar

(b)

0

3

6

9

Bright

Friable

HardnessMoist

Springy

Smooth

Yellow



(c)



4 Discussion

















5 Conclusion




Acknowledgments


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






098 plusmn 014b

162 plusmn 025c 0000



a1500 plusmn 136

a2139 plusmn 108


a189 plusmn 029

b209 plusmn 009





a040 plusmn 009

b040 plusmn 015



a4261 plusmn 237

b3353 plusmn 862









0

3

6

9Fermented milk

Lawn

Floral

Fruity

Cow

Buttery

Vinegar

Rancid

c

(a)

0

3

6

9Aftertaste

Astringent

Bitter

Cow

Fermented milk

Fruity

Salty

Vinegar

(b)

0

3

6

9

Bright

Friable

HardnessMoist

Springy

Smooth

Yellow



(c)



4 Discussion

















5 Conclusion




Acknowledgments


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

3

6

9Fermented milk

Lawn

Floral

Fruity

Cow

Buttery

Vinegar

Rancid

c

(a)

0

3

6

9Aftertaste

Astringent

Bitter

Cow

Fermented milk

Fruity

Salty

Vinegar

(b)

0

3

6

9

Bright

Friable

HardnessMoist

Springy

Smooth

Yellow



(c)



4 Discussion

















5 Conclusion




Acknowledgments


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














5 Conclusion




Acknowledgments


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



5 Conclusion




Acknowledgments


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Development of a Potential Probiotic ...downloads.hindawi.com/journals/bmri/2014/801918.pdf · Lactobacillus salivarius Strains Isolated from ... terium the most