Ribose-Induced Maillard Reaction as a Quality Index in Frozen Minced Chicken and Pork Meats

RIBOSE-INDUCED MAILLARD REACTION AS A QUALITY INDEXIN FROZEN MINCED CHICKEN AND PORK MEATSTHUAN-CHEW TAN1, ABBAS F.M. ALKARKHI2 and AZHAR MAT EASA1,3

1Food Technology Division, School of Industrial Technology, Universiti Sains Malaysia, 11800 Penang, Malaysia2Environmental Technology Division, School of Industrial Technology, Universiti Sains Malaysia, Penang, Malaysia

3Corresponding author.TEL: +60-4-653 2216;FAX: +60-4-657 3678;EMAIL: [email protected]

Received for Publication August 1, 2012Accepted for Publication July 31, 2013

10.1111/jfq.12041

ABSTRACT

Minced meats underwent denaturation, oxidation and aggregation during frozenstorage, causing decrease in denaturation enthalpy, sulfhydryl contents andprotein solubility, as well as increase in protein carbonyl and disulfide contents.These changes were more evident in samples stored at −4C than those stored at−20C, as reactions occurred at faster rates at higher temperature. The addition ofribose on thawed minced meats followed by heating at 95C initiated Maillardreaction browning. Browning index ( A420* ) was significantly higher (P < 0.05) infresh minced meats than in frozen ones, decreased with storage time and werelower in minced meat stored at −4C than those stored at −20C. The biochemicalchanges in meat proteins during frozen storage reduced the availability of basicamino acids to take part in the Maillard reaction. Hence, A420* could be used asfreshness index in minced chicken and minced pork.

PRACTICAL APPLICATIONS

Long-term frozen storage of meat causes degradative changes which leads tothe loss of functionality, flavor, texture and organoleptic properties of the meat.Due to these losses, fresh meat is often perceived to be of a better quality andoften valued at a higher market price compared to those of frozen meat. Thisstudy proposed an alternative, simpler, quicker and cheaper method to identifybetween fresh versus frozen/thawed meat. The proposed method was based on theMaillard reaction between proteins of meat and ribose, yielding brown pigments(melanoidins) that can be measured as absorbance and converted into a freshnessindex. It was demonstrated that the biochemical degradation during frozenstorage reduces the availability of meat proteins to react with ribose, yieldinglower freshness index as compared to those of fresh meat. Thus, freshness indexcould be used as a quality control method to ascertain meat freshness and toevaluate supplier’s reliability.

INTRODUCTION

Unlike in the past, when meats were mostly purchased fromwet markets (fresh food markets), there is a shift in con-sumer shopping habits where consumers purchased meatsfrom general merchandise stores (or hypermarket). Thisshift is led by a whole range of factors, which for the con-sumers includes, for example: array of choices, convenience,better comfort, hygiene, lower price and better service. Eventhough frozen meats are widely accepted by consumers,fresh (unfrozen) meats are often perceived as better qualityand often valued at a higher market price. Due to these

reasons, there are incidents where retailers sold frozen/thawed meats as fresh chilled meats. Frozen/thawed meatsand chilled fresh meats can be very similar in their appear-ance, which makes their differentiation challenging. Differ-entiation can be even more challenging when it comes todifferentiate frozen/thawed minced meat and chilled freshminced meat.

Analytical methods proposed by various studies evolvearound the use of sensory, enzymatic, spectroscopic andchromatographic techniques. Sensory assessment (such asouter appearance, odor and color) has always played a keyrole (Abbas et al. 2008); however, these techniques require

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Journal of Food Quality ISSN 1745-4557

351Journal of Food Quality 36 (2013) 351–360 © 2013 Wiley Periodicals, Inc.

experience and extensive training before the assessor iscapable of carrying out the inspection duties. Widelyused enzymatic approach (such as β-hydroxyacyl-CoA-dehydrogenase method [Hoz et al. 1993] and α-glucosidasemethod [Duflos et al. 2002]) does provide a simple method-ology to distinguish fresh from thawed meat. Unfortunately,enzymatic approach is impossible to be applied in mincedmeat (Ballin and Lametsch 2008). Chromatographicapproach was popular in determination of biogenic amine(Vinci and Antonelli 2002; Saccani et al. 2005) and hypo-xanthine (Hernández-Cázares et al. 2010) to determine thefreshness index. However, this approach requires expensiveequipment, elaborate sample preparation and high technicalexpertise. Therefore, there is a need for a simple and reliabletest to differentiate between fresh versus frozen meats,especially for minced meats.

The Maillard reaction (nonenzymatic browning) is thecondensation reaction of a reducing sugar with aminoacids or proteins. Upon heating with the reducing sugar, dif-ferent Maillard reaction products are generated dependingon the types of reactants and the heating conditions used(Davies and Labuza 1997). Ultimately, brown nitrogenouspolymers and copolymers, known as melanoidins, thatcan be measured at a wavelength of 420 nm are formed(Carabasa-Giribet and Ibarz-Ribas 2000). The applicationof the Maillard reaction in minced meat has been shown byMeinert et al. (2009), with the aim of studying flavor forma-tion. Because the Maillard reaction yields various physico-chemical changes in meat, such as browning, pH and color,it could provide a novel method for meat characterization.

The absorbance and color values of the ribose-inducedMaillard reaction has been suggested as an index for differ-entiation between fresh minced chicken and pork meats(Tan et al. 2012b), and bovine and porcine gelatines (Tanet al. 2012a); however, there is no report on the use of theMaillard reaction as a means to indicate meat freshness.Pork and chicken meats have different chemical composi-tions and structures, and they might show different behav-ior during frozen storage. As meat proteins are denatured,oxidized and aggregated during frozen storage, only aportion of the proteins will be available for chemical inter-actions. This could lead to a different extent of the Maillardreaction when a reactive reducing sugar is introduced andheated with these meats. The aim of this paper was to assessthe potential of using the ribose-induced Maillard reactionas an alternative method to indicate meat freshness.

MATERIAL AND METHODS

Materials

D-ribose was purchased from Sigma-Aldrich Co., St. Louis,MO. Chemicals for Bradford method were purchased from

Bio-Rad Laboratories, Hercules, CA. 2,4-dinitrophenyl-hydrazine (DNPH) was purchased from Cayman ChemicalCo., Ann Arbor, MI. Other chemicals (analytical grade)used were obtained from Sigma-Aldrich.

Methods

Preparation of Frozen Samples and Storage. Fivebatches of fresh cuts of chicken breast (from Ross chickens)and five batches of fresh blade shoulder cuts of pork (fromLarge White pigs) were purchased from a local wet marketin Gelugor, Penang, Malaysia. Each batch (around 500 g)was purchased on different days. The meat samples werecleaned and visible fats were removed before cutting intocubes (2 cm3). The meat cubes were kept in a refrigerator(Toshiba, GR-M48MP refrigerator, Minato-ku, Japan) at 4Cfor 2 h before homogenization in a meat mincer (Kenwood,MG470 meat grinder, Hampshire, UK) using a 4.5-mmplate to produce minced chicken or minced pork.

Sampling. For each batch of minced meat samples,samples were divided into two groups, one to be frozen at−4C and the other at −20C. For each group, the mincedmeat samples were divided into smaller groups of 10 g andstored in individual small plastic containers. A total of 40containers were prepared for each batch of minced meatsamples. Since there are five batches of samples, a grandtotal of 200 containers (100 containers to be frozen at −4C,and another 100 containers to be frozen at −20C) were pre-pared. Storage at −4C was carried out in the freezing com-partment of a refrigerator, while storage at −20C was carriedout in an upright freezer (Ardo, CV 382 freezer, Fabriano,Italy).

Samples for Maillard reaction induction and proteinstability tests were taken before freezing (day 0) and atstorage intervals of 1, 4, 8, 12, 16, 20, 24, 28, 30 and 60 days.For each storage sampling time, a total of 20 containerswere analyzed; 10 containers from frozen storage at −4C,and another 10 containers from frozen storage at −20C. Allfrozen samples were thawed at 4C for 4 h before analyses.

Maillard Reaction. Induction of Maillard reaction onminced meat was carried out according to the method asdescribed by Tan et al. (2012b). Ribose was used to inducethe Maillard reaction to the minced meat samples. Ribose(10%, w/w) and minced meat were manually mixed. Sevengrams of the ribose-minced meat mixtures were transferredinto a 30-mL universal bottle and heated in a water bath(Memmert, WB22 water bath, Schwabach, Germany) at 95Cfor 60 min. The heated samples were immediately cooled inice water. The samples were left at room temperature for upto 1 h before analysis. Control was carried out in a similar

QUALITY INDEX T. TAN, A.F.M. ALKARKHIB and A.M. EASA

352 Journal of Food Quality 36 (2013) 351–360 © 2013 Wiley Periodicals, Inc.

manner with substitution of ribose with a nonreducingsugar, sucrose.

Extraction of Water-Soluble Brown Polymers (Maillard Reac-tion Products). Water-soluble melanoidins from ribose-induced Maillard meat system were extracted according tothe method of Tan et al. (2012b). Cooled heated mincedmeat sample from universal bottle was removed individu-ally and comminuted using a mortar and pestle. Approxi-mately 1 g of the comminuted samples was transferred into15-mL polypropylene test tubes, and 5 mL of distilled waterwas added. The mixture was mixed by vortexing (Gilson,GVLab Vortex, Middleton, WI) for 15 s prior to mixing on aplatform shaker (Janke & Kunkel, KS 501 D shaker, Staufen,Germany) at a speed of 300×/min for 1 h. After mixing for1 h, the mixture was centrifuged (Kubota Corp., 2100 cen-trifuge, Bunkyo-ku, Japan) at 3,000 g for 15 min at roomtemperature. The supernatants were transferred into new15-mL polypropylene test tubes and 1 mL of n-hexane wasadded. The mixture was vortexed for 15 s and mixed on aplatform shaker at a speed of 300×/min for 10 min, andcentrifuged at 3,000 g for 15 min. The aqueous layers werestored in new 15-mL polypropylene test tubes at 4C prior tospectrophotometric analysis.

Measurement of Maillard Extract Absorbance. The Maillardreaction products were extracted from heated samplesobtained by heating ribose with thawed minced meats.Browning index ( A420* ) has been widely used as an indicatorfor melanoidins (browning polymers) formation inMaillard systems (Carabasa-Giribet and Ibarz-Ribas 2000).The absorption of the brown polymers formed in theribose-minced meat system was measured at 420 nm usingspectrophotometer (Konica Minolta, SpectrophotometerCM-3500d, Chiyoda, Japan). Water-soluble extracts weretransferred into a glass cuvette to measure their absorbanceat least twice, from the front and back of the cuvette. Theabsorbance at 550 nm was also measured to correct for anyturbidity in the extracts (Morales and van Boekel 1998).Extracts from sucrose-minced meat system were used asblank.

A A A B420 420 550* = − − (1)

where, A420* is the browning index; A420 is the absorptionat 420 for samples; A550 is the absorption at 550 for samplesand B is the absorption at 420 nm for blank. A420* valuecalculated was taken as freshness index.

Protein Stability during Storage. Thermal Dena-turation. Thermal denaturation of minced chicken andminced pork proteins was analyzed using differentialscanning calorimeter (DSC) (TA Instruments, DSC Q200,

New Castle, DE) based on the method described by Amakoand Xiong (2001) with some slight modification. Calibra-tion was done with indium prior to analysis. Small amount(15–25 mg) of minced meat was hermetically sealed inaluminum pan. The sealed pan with sample were cooled to5C and equilibrated for 10 min. After equilibration, it wasscanned at a heating rate of 5C/min from 5 to 95C. A sealedempty pan was used as the reference and for baseline cor-rection. Each thermogram was analyzed for the denatur-ation endotherm (endothermic peak and total enthalpyof denaturation) using TA Instruments Universal Analysis2000 software. All samples were analyzed in triplicate.

Protein Oxidation Measurement. Meat homogenate filtrates(2 mg/mL) were prepared for protein oxidation measure-ment (protein carbonyl content, sulfhydryl content anddisulfide content). One gram of minced meat samples washomogenized (IKA, T25 Digital Ultra Turrax, Staufen,Germany) with 50 mL of cold distilled water for 3 min at aspeed of 12,000 rpm. The homogenate was diluted to2 mg/mL (10 times dilution) with 0.1 M phosphate buffer(pH 7.4) before the protein content of the homogenate wasdetermined using Bradford method (Bradford 1976).Bovine serum albumin was used as the standard.

Protein Carbonyl Content. Protein carbonyl content wasdetermined based on the method described by Levine et al.(1990) and Srinivasan and Hultin (1995). Meat homo-genate filtrates (containing about 1 mg of protein) wereallowed to incubate with 2 mL of 10 mM DNPH in 2.0 NHCl for 1 h at room temperature. After incubation, 2 mLof 20% trichloroacetic acid was added to precipitate theproteins. The solutions were decanted before washingsolution (ethanol : ethyl acetate at ratio 1:1) was added toremove excess DNPH. Washing of the precipitate wasdone twice before the blow-dried remaining pellets weredissolved with 1.5 mL of 6 M guanidine hydrochloridewith 20 mM potassium phosphate buffer (pH 2.3). Absor-bance was measured at 370 nm. Control was prepared inthe same manner as the samples with the substitution ofDNPH with 2 N HCl. The protein carbonyls content wasexpressed as total carbonyl (μgmol/g protein) using thefollowing equation:

Total Carbonyl = × −( ) ××

106 A A D

M CS C (2)

where AS is the absorbance at 370 nm for samples; AC is theabsorbance at 370 nm for control; D is the dilution factor;M is the molar absorptivity (2.2 × 104 M−1/cm) and C is thesample protein concentration (mg/mL). Incorporation of106 into the equation is for conversion from the molar basisto the μmol/mL basis and from mg protein to g protein.

T. TAN, A.F.M. ALKARKHIB and A.M. EASA QUALITY INDEX


Sulfhydryl and Disulfide Contents. Sulfhydryl and disulfidecontents were determined based on the method describedby Hu et al. (2010). For free sulfhydryl (SHF) determination,1 mL of the meat homogenate filtrates was mixed with 4 mLof urea buffer (8 M urea with 5 M guanidine hydrochloridein Tris-Gly buffer [pH 8, containing 0.086 M Tris, 0.09 Mglycine and 4 mM ethylenediaminetetraacetic acid]) and0.05 mL of Ellman’s reagent (4 mg of Ellman’s reagent in1 mL of Tris-Gly buffer). After incubation at room tempera-ture for 1 h, absorbance was measured at 412 nm.

For total sulfhydryl (SHT) determination, 1 mL of meathomogenate filtrates was mixed with 4 mL of urea bufferand 0.05 mL of β-mercaptoethanol. After incubation atroom temperature for 1 h, 10 mL of 12% trichloroaceticacid in Tris-Gly buffer were added prior to another 1 hincubation at room temperature. After the second incuba-tion, the mixture was centrifuged at 3,000 g for 10 min. Theprecipitate was then dissolved in 10 mL of 8 M urea in Tris-Gly buffer and 0.04 mL of Ellman’s reagent. Absorbancewas measured at 412 nm.

The SHF and SHT contents (μgmol/g protein) were calcu-lated according to the following equation:

SH or SH ContentF T( ) = × − −( ) ××

1061 2A A A D

M CS (3)

where AS is the absorbance at 412 nm for samples; A1 is theabsorbance at 412 nm for samples without Ellman’sreagent; A2 is the absorbance at 412 nm for Ellman’s reagentin the sample buffer; D is the dilution factor; M is themolar absorptivity (1.36 × 104 M−1/cm) and C is the sampleprotein concentration (mg/mL). Incorporation of 106 intothe equation is for conversion from the molar basis to theμmol/mL basis and from mg protein to g protein.

Disulfide content (μgmol/g protein) was calculated basedSHF and SHT determination using the following equation:

Disulfide ContentSH SHT F= −

2(4)

where, SHT is the total sulfhydryl content and SHF is freesulfhydryl content.

Protein Solubility. Soluble proteins were extracted byhomogenizing 2 g of minced meat samples with 20 mL of0.6 M NaCl (pH 7.4) for 3 min at a speed of 12,000 rpm.The homogenate was centrifuged at 20,000 g for 30 minat 4C. The protein concentration in the supernatant wasdetermined by Bradford method and expressed as mg/mL.Bovine serum albumin was used as the standard.

Statistical Analysis

Two experiments (one for minced chicken and the other onefor minced pork) with a factorial design of type 2 × 11 were

carried out to study the effect of two factors, the storage tem-perature (X1) and frozen storage duration (X2) on ribose-induced Maillard browning index and meat protein stabilityduring frozen storage assessment (total enthalpy of dena-turation, protein carbonyl contents, sulfhydryl contents,disulfide contents and protein solubility) obtained fromribose-fresh/frozen meat Maillard system. Eleven storageintervals (0, 1, 4, 8, 12, 16, 20, 24, 28, 30 and 60 days) and twostorage temperatures (−4 and −20C) were tested. Five repli-cates were tested for each type of minced meat, except forDSC, which was carried out in three replicates. Analysis ofvariance and Tukey’s test for multiple comparisons were usedfor analyzing the data. SPSS version 16 (SPSS Inc., Chicago,IL) was used to complete the statistical analysis.

RESULTS AND DISCUSSION

Assessing Protein Quality Changes duringFrozen Storage

The stability of major protein components in mincedchicken and minced pork during frozen storage was evalu-ated. A typical DSC thermogram exhibits three major tran-sitions attributed to the denaturation of myosin (peak 1),sarcoplasmic proteins (peak 2) and actin (peak 3) (Amakoand Xiong, 2001) (Fig. 1). The total enthalpy of denatur-ation decreased significantly (P < 0.05) during the 2 monthsof frozen storage, with the decrease being more prominentin minced meat stored at −4C compared to those stored at−20C (Tables 1 and 2). Saeed and Howell (2002) reportedcold denaturation occurred at greater intensity at higherfrozen storage temperature during frozen storage on muscleproteins. An increase of unordered protein structure wasreported by Careche et al. (1999) to be presence in hakefillets stored at higher frozen storage temperature (−10C) ascompared to those stored at −30C.

A similar trend was observed for endothermic peak tem-perature for all the three transitions that shifted to lowervalues during the frozen storage (Tables 1 and 2). The shiftin endothermic peak temperature was possibly related tothe pH of the samples during storage (Jensen and Jørgensen2003). Atayeter and Ercoskun, (2011) reported that pHlevels of squid mantles and tentacles increased duringfrozen storage, with pH increases in higher frozen tempera-tures were greater than in lower frozen temperatures. Thesephenomenon changes on the endothermic peak tempera-tures and total enthalpy of denaturation of frozen storageminced chicken and minced pork were in agreement withthat reported in the literatures (Jensen and Jørgensen 2003;Xia et al. 2010; Kumar et al. 2011).

The oxidative deterioration of proteins of mincedchicken and minced pork during frozen storage can also beillustrated through the increase in the carbonyl (Fig. 2a)



and decrease in sulfhydryl contents (Fig. 2b) of the proteins.Both figures show that freezing had a strong impact onprotein oxidation in both types of minced meat. Proteinoxidation during frozen storage was previously reported bySoyer et al. (2010) on chicken meat, Chan et al. (2011)on turkey meat and Nopianti et al. (2013) on surimi. Theamount of protein carbonyls in minced chicken meat storedat −4 and −20C increased significantly (P < 0.05), with theincrease being more prominent in minced meat storedat −4C. Meanwhile, the amount of sulfhydryl contents inminced chicken meat stored at −4 and −20C decreased sig-nificantly (P < 0.05), and the decrease was also more promi-nent in minced meat stored at −4C. Similar changes in thecarbonyl and sulfhydryl contents of proteins were observedin minced pork. The more damaging effect on protein at−4C compared to −20C was mainly due to the lipid oxida-tion that occurred more readily at higher storage tempera-tures (Atayeter and Ercoskun, 2011; Hansen et al. 2004). Itis also noted that at a particular storage temperature theprotein oxidation occurred more extensively in mincedpork compared to the minced chicken samples. The car-bonyl content indicates that the minced chicken stored at−20C was the most stable, while minced pork stored at −4Cwas the least stable. This difference could mainly be due tothe higher fat content in pork meat (Rhee et al. 1996) thatled to greater lipid oxidation and caused more deleteriouseffect on protein structure and functions (Saeed and Howell2002; Estévez et al. 2007).

Oxidation of sulfhydryl groups led to the formation ofdisulfide cross-linkages and this can be observed from thesignificant increase (P < 0.05) in disulfide bonds content inminced chicken stored at −4 and −20C, and the increase wasmore prominent in minced chicken stored at −4C (Fig. 2c).

A similar trend was observed in minced pork. As storageprogressed, some proteins underwent denaturation-causingreduction in the number of available sulfhydryl groups. Aportion of the sulfhydryl groups also underwent oxidationprocesses yielding disulfides. These trends were similar tothose reported in the processing of Cantonese sausage (Sunet al. 2011a).

Changes on the sulfhydryl and disulfides contents(Fig. 2c) coincided with the decrease in protein solubility at−4 and −20C (Fig. 2d). For a particular type of mincedmeat, a higher decrease in solubility occurred in mincedmeat stored at −4C. It is evident that minced pork was moreprone to loss in solubility with storage time. The relationbetween disulfide content and protein solubility was similarto those reported in muscle proteins of Atlantic mackerel(Saeed and Howell 2002) and Cantonese sausage (Sun et al.2011a,b). The reasons for the decrease in solubility mayinclude the formation of aggregates (such as formation ofdisulfide cross linkages), increased exposure of hydrophobicregions and lipid oxidation (Howell et al. 2001; Sun et al.2011b). Protein denaturation may also exert effects on theprotein solubility (Mignino and Paredi 2006; Chan et al.2011). The higher decrease in the denaturation enthalpy forsamples stored at −4C (Tables 1 and 2) was in agreementwith greater decrease in protein solubility (Fig. 2d).

The Use of Ribose-Induced Maillard Reactionin Thawed Meat

Results from this study indicate that frozen storage (tem-perature and time) has a strong impact on minced meatdeterioration due mostly to aggregation of myofibrillar pro-teins (Benjakul et al. 1997). Even though it is possible to use

FIG. 1. TYPICAL DIFFERENTIAL SCANNINGCALORIMETRY THERMOGRAMS OF FRESH(DAY 0) MINCED CHICKEN (DOTTED LINE)AND MINCED PORK (SOLID LINE)(A) Peak 1: myosin; (B) Peak 2: sarcoplasmicproteins; (C) Peak 3: actin.



denaturation, oxidation and solubility analyses to indicatethe effect of frozen storage on meat protein, these methodsrequire long preparation time, are tedious and can beexpensive. Therefore, one idea is to use ribose to react withthe proteins from the frozen minced meats. Ribose, themost reactive reducing sugar was added into the thawedminced meat that was then heated to initiate the Maillardreaction. Following heating, all minced meat samples thatwere treated with ribose had a noticeable browning, whileno such change was observed in minced meats containingsucrose (control).

Changes in A420* value of the minced meats as a functionof storage time at −4 and −20C are shown in Fig. 2e. TheA420* value of minced chicken stored for up to 2 months at−4 and −20C significantly (P < 0.05) decreased from 0.2109to 0.1546 (∼27% reduction in absorbance), and 0.2109 to0.1745 (∼17% reduction in absorbance), respectively. Mean-while, the A420* value of frozen minced pork stored up to

2 months at −4 and −20C significantly (P < 0.05) decreasedfrom 0.1443 to 0.0695 (∼51% reduction in absorbance),and 0.1443 to 0.0944 (∼34% reduction), respectively. Thedecrease of A420* value in frozen minced meat was moreprominent in minced meat samples stored at −4C as com-pared to those stored at −20C. This difference could bea result of more rapid changes on meat protein andbiochemical reactions in samples stored at higher storagetemperatures. The % reduction in A420* value was higherin minced pork than in minced chicken as the extent ofprotein and lipid oxidation occurred more extensively inpork than in chicken samples (Rhee et al. 1996).

High A420* value indicates high availability of basic aminoacids to react with ribose, while low A420* value might implyreduced availability of the amino acids. The reduction inA420* value with storage time occurred because a portion ofminced meat proteins were oxidized, denatured and cross-linked during frozen storage (Decker et al. 1993). A study by

TABLE 1. INFLUENCE OF STORAGE ON THEENDOTHERMIC PEAK TEMPERATURES ANDTOTAL ENTHALPY OF DENATURATION OFMINCED CHICKEN STORED AT (A) −4 AND(B) −20C

(A) Minced chicken stored at −4C

Chicken stored at −4C Endothermic peak temperatures (C)

ΔH (J/G)Frozen storage duration (Day) Tmyosin Tsacroplasmic Tactin

0 63.5 ± 0.65 68.0 ± 0.41 78.5 ± 0.35 1.17 ± 0.05a

1 63.4 ± 0.63 68.0 ± 0.38 78.3 ± 0.43 1.15 ± 0.07a

4 62.5 ± 0.75 67.9 ± 0.52 77.4 ± 0.29 1.11 ± 0.10b

8 61.8 ± 0.78 67.6 ± 0.40 75.9 ± 0.87 1.05 ± 0.07b

12 60.5 ± 0.91 67.4 ± 0.78 74.1 ± 0.77 0.98 ± 0.11bc

16 60.1 ± 0.34 67.2 ± 0.45 73.9 ± 0.54 0.93 ± 0.01c

20 59.9 ± 0.45 67.0 ± 0.39 73.5 ± 0.83 0.86 ± 0.06d

24 59.5 ± 0.47 66.8 ± 0.59 73.1 ± 0.34 0.81 ± 0.06d

28 58.7 ± 0.47 66.3 ± 0.47 72.9 ± 0.45 0.70 ± 0.12de

30 58.2 ± 0.67 66.1 ± 0.56 72.6 ± 0.71 0.69 ± 0.15e

60 57.9 ± 0.52 65.8 ± 0.68 70.7 ± 0.82 0.57 ± 0.13f

(B) Minced chicken stored at −20C

Chicken stored at −20C Endothermic peak temperatures (C)

ΔH (J/G)Frozen storage duration (Day) Tmyosin Tsacroplasmic Tactin

0 63.5 ± 0.65 68.0 ± 0.41 78.5 ± 0.35 1.17 ± 0.05a

1 63.4 ± 0.75 68.1 ± 0.67 78.2 ± 0.41 1.17 ± 0.13a

4 63.1 ± 0.77 68.1 ± 0.81 78.1 ± 0.62 1.16 ± 0.04a

8 62.8 ± 0.64 67.9 ± 0.43 77.6 ± 0.88 1.12 ± 0.07a

12 62.5 ± 0.83 67.6 ± 0.91 77.0 ± 0.56 1.10 ± 0.09ab

16 62.3 ± 0.91 67.5 ± 0.90 76.5 ± 0.84 1.08 ± 0.11ab

20 62.1 ± 0.38 67.3 ± 0.53 76.3 ± 0.73 1.05 ± 0.07b

24 61.9 ± 0.63 67.1 ± 0.55 75.9 ± 0.74 1.02 ± 0.14b

28 61.5 ± 0.74 67.0 ± 0.62 75.4 ± 0.74 1.02 ± 0.15b

30 61.4 ± 0.88 67.0 ± 0.67 74.9 ± 0.10 0.95 ± 0.11c

60 60.5 ± 0.50 66.8 ± 0.39 73.0 ± 0.34 0.87 ± 0.08d

Notes: Tmyosin, Tsarcoplasmic, Tactin, peak temperature maximum of endothermic peak of denaturationfor myosin, sarcoplasmic proteins and actin, respectively; ΔH, total enthalpy of denaturation formyosin, sarcoplasmic proteins and actin. Data reported are mean ± standard deviation (n = 3).Comparison within the total enthalpy column (a–d) is shown in the table with means not followedby the same letter are significantly different at P < 0.05 level of significance according to Tukey’smultiple-range test.



Saeed and Howell (2002) indicated the formation of highmolecular weight polymers on mackerel protein stored at−10C for 4 weeks, but no such changes occurred in samplesstored at lower frozen storage of −20 and −30C, even after3 months of storage thus illustrating the importance oftemperature monitoring of the frozen mackerel. The highmolecular weight polymers formed at higher frozen storagetemperature could be present in greater quantity in mincedmeats stored at −4C compared to those stored at −20C. Inaddition to polymerization, certain amino acids (such ascysteine, histidine, methionine, lysine and tryptophan) werevery susceptible to oxidation (Xiong 2000). The free NH2

groups may interact with aldehydic products of lipid oxida-tion (Sun et al. 2011b). These changes led to a reduction inthe availability of the protein and amino acids to chemicallyreact with ribose during heating, thus reducing the A420*

value.

Spectrophotometry and Maillard Reaction asa Simple and Fast Meat Freshness Indicator

The decrease in A420* value with storage was due to theadverse effects of frozen storage on minced meat proteins.There are good indications that the A420* value is related tostorage duration in both the minced chicken and mincedpork. Extensively denatured and oxidized proteins yieldedlower A420* values, thus minced meat that was frozen fora long time will yield lower A420* and that A420* value offresh minced meat is expected to be higher than that hasbeen frozen. The observation that A420* value was lowerin samples stored at higher temperature (−4C) implies thepossible use of the Maillard reaction to indicate tempera-ture abuse during frozen storage. The possible use of ribose-induced Maillard reaction as a meat freshness indicator issuggested. Compared to the current analytical techniques,

TABLE 2. INFLUENCE OF STORAGE ON THEENDOTHERMIC PEAK TEMPERATURES ANDTOTAL ENTHALPY OF DENATURATION OFMINCED PORK STORED AT (A) −4 AND(B) −20C

(A) Minced pork stored at −4C

Pork stored at −4C Endothermic peak temperatures (C)

ΔH (J/G)Frozen storage duration (day) Tmyosin Tsacroplasmic Tactin

0 59.4 ± 0.65 68.1 ± 0.72 83.1 ± 0.58 1.07 ± 0.06a

1 59.0 ± 0.57 68.0 ± 0.65 82.9 ± 0.34 1.01 ± 0.07b

4 58.4 ± 0.51 67.8 ± 0.40 82.0 ± 0.45 0.96 ± 0.04c

8 58.1 ± 0.64 67.6 ± 0.82 81.5 ± 0.81 0.91 ± 0.05d

12 57.5 ± 0.79 67.5 ± 0.81 80.2 ± 0.75 0.87 ± 0.08d

16 56.9 ± 0.93 67.3 ± 0.54 79.6 ± 0.40 0.83 ± 0.04e

20 56.4 ± 0.51 67.0 ± 0.91 78.3 ± 0.52 0.79 ± 0.12ef

24 55.3 ± 0.70 66.9 ± 0.45 77.7 ± 0.34 0.73 ± 0.10f

28 54.9 ± 0.33 66.6 ± 0.62 76.9 ± 0.12 0.69 ± 0.09fg

30 54.7 ± 0.48 66.3 ± 0.71 76.7 ± 0.43 0.61 ± 0.15g

60 51.8 ± 0.98 65.1 ± 0.82 74.6 ± 0.15 0.35 ± 0.14h

(B) Minced pork stored at −20C

Pork stored at −20C Endothermic peak temperatures (C)

ΔH (J/G)Frozen storage duration (day) Tmyosin Tsacroplasmic Tactin

0 59.4 ± 0.65 68.1 ± 0.72 83.1 ± 0.58 1.07 ± 0.06a

1 59.1 ± 0.56 68.2 ± 0.66 83.1 ± 0.67 1.05 ± 0.06a

4 68.4 ± 0.72 67.9 ± 0.60 82.6 ± 0.73 0.97 ± 0.04b

8 57.7 ± 0.93 67.8 ± 0.83 81.5 ± 0.23 0.91 ± 0.01c

12 56.7 ± 0.53 67.5 ± 0.53 80.5 ± 0.17 0.87 ± 0.06cd

16 56.1 ± 0.18 67.5 ± 0.38 79.8 ± 0.77 0.81 ± 0.08d

20 55.8 ± 0.28 67.1 ± 0.72 78.4 ± 0.41 0.75 ± 0.05e

24 55.2 ± 0.48 67.2 ± 0.70 77.9 ± 0.97 0.69 ± 0.10ef

28 54.9 ± 0.59 67.1 ± 0.68 77.2 ± 0.66 0.61 ± 0.12f

30 54.8 ± 0.55 67.1 ± 0.57 76.9 ± 0.50 0.64 ± 0.09f

60 53.6 ± 0.84 66.4 ± 0.88 75.1 ± 0.48 0.46 ± 0.04g

Notes: Tmyosin, Tsarcoplasmic, Tactin, peak temperature maximum of endothermic peak of denaturationfor myosin, sarcoplasmic proteins and actin, respectively; ΔH, total enthalpy of denaturation formyosin, sarcoplasmic proteins and actin. Data reported are mean ± standard deviation (n = 3).Comparison within the total enthalpy column (a–h) is shown in the table with means not followedby the same letter are significantly different at P < 0.05 level of significance according to Tukey’smultiple-range test.



the proposed analysis involves straightforward samplepreparation and a short analysis time. Other Maillard reac-tion parameters such as color and pH values could also beevaluated to perform similar tests.

CONCLUSIONS

Frozen storage had an impact on protein quality of mincedchicken and minced pork, thus affecting their functional

properties. The A420* value obtained from ribose-inducedMaillard reaction on frozen/thawed minced meats wassignificantly (P < 0.05) lower than those of fresh mincedmeats. This approach showed potential as a simple and fastmethod to characterize and differentiate between fresh andfrozen minced meats and could be used as a freshness indexfor frozen minced chicken and pork meats. Further investi-gations using meats from different species, cuts of meat, ageof meat, and range of protein, moisture, and fat percentages

A B

C D

E

FIG. 2. CHEMICAL CHANGES IN MINCED CHICKEN (SOLID LINE) AND MINCED PORK (DOTTED LINE) AS A FUNCTION OF STORAGE −4 (◊) AND−20C (□). (A) TOTAL CARBONYL CONTENT; (B) SULFHYDRYL CONTENT; (C) DISULFIDES CONTENT; (D) PROTEIN SOLUBILITY; (E) A420* AS FRESH-

NESS INDEX

Data points are mean ± standard deviation (n = 5).



for different cuts and species need to be carried out toexamine the use of the proposed method.

ACKNOWLEDGMENTS

This work was supported by e-Science Fund Grant no.05-01-05-SF0405 which was awarded by the Malaysian Min-istry of Agriculture and Agro-Based Industry. The financialsupport of Post-Doctoral Fellowship from Universiti SainsMalaysia for Dr. Tan Thuan Chew was gratefully acknow-ledged. We gratefully acknowledge and are indebted to theanonymous referees for comments and constructive sugges-tions provided for improving the manuscript.

REFERENCES

ABBAS, K.A., MOHAMED, A., JAMILAH, B. andEBRAHIMIAN, M. 2008. A review on correlations betweenfish freshness and pH during cold storage. Am. J. Biochem.Biotechnol. 4, 416–421.

AMAKO, D.E.N. and XIONG, Y.L. 2001. Effects of carrageenanon thermal stability of proteins from chicken thigh and breastmuscles. Food Res. Int. 34, 247–253.

ATAYETER, S. and ERCOSKUN, H. 2011. Chemicalcomposition of European squid and effects of different frozenstorage temperatures on oxidative stability and fatty acidcomposition. J. Food Sci. Technol. 48, 83–89.

BALLIN, N.Z. and LAMETSCH, R. 2008. Analytical methods forauthentication of fresh vs. thawed meat – a review. Meat Sci.80, 151–158.

BENJAKUL, S., SEYMOUR, T.A., MORRISSEY, M.T. and AN,H. 1997. Physicochemical changes in Pacific whiting muscleproteins during iced storage. J. Food Sci. 62, 729–733.

BRADFORD, M.M. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal. Biochem. 72, 248–254.

CARABASA-GIRIBET, M. and IBARZ-RIBAS, A. 2000. Kineticsof colour development in aqueous glucose systems at hightemperatures. J. Food Eng. 44, 181–189.

CARECHE, M., HERRERO, A.M., RODRIGUEZ-CASADO, A.,DEL MAZO, M.L. and CARMONA, P. 1999. Structuralchanges of hake (Merluccius merluccius L.) fillets: Effects offreezing and frozen storage. J. Agric. Food Chem. 47, 952–959.

CHAN, J.T.Y., OMANA, D.A. and BETTI, M. 2011. Effect ofultimate pH and freezing on the biochemical properties ofproteins in turkey breast meat. Food Chem. 127, 109–117.

DAVIES, K. and LABUZA, T.P. 1997. The Maillard Reaction:Application to confectionery products. In ConfectioneryScience (G. Zeigler, ed.) pp. 35–66, Pennsylvania StateUniversity Press, Philadelphia, PA.

DECKER, E.A., XIONG, Y.L., CALVERT, J.T., CRUM, A.D. andBLANCHARD, S.P. 1993. Chemical, physical and functionalproperties of oxidized turkey white muscle myofibrillarproteins. J. Agric. Food Chem. 41, 186–189.

DUFLOS, G., FUR, B.L., MULAK, V., BECEL, P. and MALLE, P.2002. Comparison of methods of differentiating betweenfresh and frozen-thawed fish or fillets. J. Sci. Food Agric. 82,1341–1345.

ESTÉVEZ, M., VENTANAS, S. and CAVA, R. 2007. Oxidation oflipids and proteins in frankfurters with different fatty acidcompositions and tocopherol and phenolic contents. FoodChem. 100, 55–63.

HANSEN, E., LAURIDSEN, L., SKIBSTED, L.H., MOAWAD,R.K. and ANDERSEN, M.L. 2004. Oxidative stability of frozenpork patties: Effect of fluctuating temperature on lipidoxidation. Meat Sci. 68, 185–191.

HERNÁNDEZ-CÁZARES, A.S., ARISTOY, M.C. and TOLDRÁ,F. 2010. Hypoxanthine-based enzymatic sensor fordetermination of pork meat freshness. Food Chem. 123,949–954.

HOWELL, N.K., HERMAN, H. and LI-CHAN, E.C.Y. 2001.Elucidation of protein-lipid interactions in lysozyme-corn oilsystem by Fourier transform Raman spectroscopy. J. Agric.Food Chem. 49, 1529–1533.

HOZ, L., FERNÁNDEZ, M., DÍAZ, O., ORDÓÑEZ, J.A.,PAVLOV, A. and GARCÍA DE FERNANDO, G.D. 1993.Differentiation of unfrozen and frozen-thawed kurumaprawn (Penaeus japonicus) from the activity ofβ-hydroxyacyl-CoA-dehydrogenase (HADH) in aqueousextracts. Food Chem. 48, 127–129.

HU, X.Z., CHENG, Y.Q., FAN, J.F., LU, Z.H., YAMAKI, K. andLI, L.T. 2010. Effects of drying method on physicochemicaland functional properties of soy protein isolates. J. FoodProcess. Preserv. 34, 520–540.

JENSEN, K.N. and JØRGENSEN, B.M. 2003. Effect of storageconditions on differential scanning calorimetry profiles fromthawed cod muscle. Food Sci. Technol. 36, 807–812.

KUMAR, V., BISWAS, A.K., SAHOO, J., CHATLI, M.K. andSIVAKUMAR, S. 2011. Quality and storability of chickennuggets formulated with green banana and soybeanhulls flours. J. Food Sci. Technol. 1–11. DOI: 10.1007/s13197-011-0442-9.

LEVINE, R.L., GARAND, D., OLIVER, C.N., AMICI, A.,CLIMENT, I., LENZ, A.G., AHN, B.W., SHALTIEL, S. andSTADTMAN, E.R. 1990. Determination of carbonyl contentin oxidatively modified proteins. Methods Enzymol. 186,464–477.

MEINERT, L., SCHÄFER, A., BJERGEGAARD, C., AASLYNG,M.D. and BREDIE, W.L.P. 2009. Comparison of glucose,glucose 6-phosphate, ribose, and mannose as flavourprecursors in pork; the effect of monosaccharide additionon flavour generation. Meat Sci. 81, 419–425.

MIGNINO, L.A. and PAREDI, M.E. 2006. Physico-chemicaland functional properties of myofibrillar proteins fromdifferent species of molluscus. Food Sci. Technol. 39,35–42.

MORALES, F.J. and VAN BOEKEL, M.A.J. 1998. A study onadvanced Maillard reaction in heated casein/sugar solutions:Colour formation. Int. Dairy J. 8, 907–915.



NOPIANTI, R., HUDA, N., ISMAIL, N., ARIFFIN, F. and EASA,A.M. 2013. Effect of polydextrose on physicochemicalproperties of threadfin bream (Nemipterus spp) surimi duringfrozen storage. J. Food Sci. Technol. 50, 739–746.

RHEE, K.S., ANDERSON, L.M. and SAMS, A.R. 1996. Lipidoxidation potential of beef, chicken and pork. J. Food Sci.61, 8–12.

SACCANI, G., TANZI, E., PASTORE, P., CAVALLI, S. and REY,M. 2005. Determination of biogenic amines in fresh andprocessed meat by suppressed ion chromatography-massspectrometry using a cation-exchange column. J. Chromatogr.A 1082, 43–50.

SAEED, S. and HOWELL, N.K. 2002. Effect of lipid oxidationand frozen storage on muscle proteins of Atlantic mackerel(Scomber scombrus). J. Sci. Food Agric. 82, 579–586.

SOYER, A., ÖZALP, B., DALMI?, Ü. and BILGIN, V. 2010.Effects of freezing temperature and duration of frozen storageon lipid and protein oxidation in chicken meat. Food Chem.120, 1025–1030.

SRINIVASAN, S. and HULTIN, H.O. 1995. Hydroxyl radicalmodification of fish muscle proteins. J. Food Biochem. 18,405–425.

SUN, W., CUI, C., ZHAO, M., ZHAO, Q. and YANG, B. 2011a.Effects of composition and oxidation of proteins on theirsolubility, aggregation and proteolytic susceptibility duringprocessing of Cantonese sausage. Food Chem. 124, 336–341.

SUN, W., ZHAO, M., YANG, B., ZHAO, H. and CUI, C. 2011b.Oxidation of sarcoplasmic proteins during processingof Cantonese sausage in relation to their aggregationbehaviour and in vitro digestibility. Meat Sci. 88,462–467.

TAN, T.C., ALKARKHI, A.F.M. and EASA, A.M. 2012a.Assessment of the ribose-induced Maillard reaction as ameans of gelatine powder identification and quality control.Food Chem. 134, 2430–2436.

TAN, T.C., ALKARKHI, A.F.M. and EASA, A.M. 2012b.Characterization of the ribose-induced Maillardreaction in minced chicken and minced pork: A potentialmeans of species differentiation. Int. Food Res. J. 19,481–489.

VINCI, G. and ANTONELLI, M.L. 2002. Biogenic amines:Quality index of freshness in red and white meat. FoodControl 13, 519–524.

XIA, X., KONG, B., XIONG, Y. and REN, Y. 2010. Decreasedgelling and emulsifying properties of myofibrillar proteinfrom repeatedly frozen-thawed porcine longissimus muscleare due to protein denaturation and susceptibility toaggregation. Meat Sci. 85, 481–486.

XIONG, Y.L. 2000. Protein oxidation and implications formuscle food quality. In Antioxidants in Muscle Foods(E.A. Decker, C. Faustman and C.J. Lopez-Bote, eds.)pp. 85–111, Wiley, New York, NY.



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Ribose-Induced Maillard Reaction as a Quality Index in Frozen Minced Chicken and Pork Meats