effect of chitosan edible coating on the biochemical and ... · researcharticle effect of chitosan...

Research ArticleEffect of Chitosan Edible Coating on the Biochemicaland Physical Characteristics of Carp Fillet (Cyprinus carpio)Stored at minus18∘C

Ana Gabriela Morachis-Valdez12 LeobardoManuel Goacutemez-Olivaacuten1

Imelda Garciacutea-Argueta3 Mariacutea Dolores Hernaacutendez-Navarro1

Daniel Diacuteaz-Bandera2 and Octavio Dublaacuten-Garciacutea2

1Departamento de Toxicologıa Ambiental Facultad de Quımica Universidad Autonoma del Estado de Mexico Toluca MEX Mexico2Departamento de Alimentos Facultad de Quımica Universidad Autonoma del Estado de Mexico Toluca MEX Mexico3Departamento de Nutricion Facultad de Medicina Universidad Autonoma del Estado de Mexico Toluca MEX Mexico

Correspondence should be addressed to Octavio Dublan-Garcıa octavio_dublanyahoocommx

Received 29 December 2016 Revised 20 March 2017 Accepted 18 April 2017 Published 28 May 2017

Academic Editor Alejandro Castillo

Copyright copy 2017 Ana Gabriela Morachis-Valdez et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

The effect of an edible coating (EC) with 15 chitosan as an additive on common carp (Cyprinus carpio) fillet was determinedevaluating the biochemical physicochemical textural microbiological and nutritional characteristics periodically during itsstorage in the freezer (minus18∘C) observing a decrease in the rate of biochemical reactions related to degradation (119901 lt 005)hydroperoxides content (HPC) (08324 nM hydroperoxidesmg of protein versus 05540 nMmg with regard to the EC sample)as well as protein carbonyl content (PCC) (05860 nM versus 04743 nM of reactive carbonyl groupsmg of protein of noncoatedmaterial) keeping properties for a longer period of time and a lower protein solubility (78mg of supernatant proteinmg of totalprotein versus 68mgmg) and less loss of moisture (8 less with regard to EC) for the nutritional characteristics of the filletlysine is the limiting aminoacid in the sample without EC while leucine is the limiting aminoacid for the EC sample According tomicrobial growth the count was 22 times 105 CFUg of sample inmesophiles versus 47 times 104 in the EC sampleThe results indicate thatthe use of EC added with chitosan maintains the quality of the product regarding lipid and protein oxidation until fourth monthof storage maintaining moisture content without variation for at least 3 months and inhibits microbial growth up to 2 logarithmicunits during five months of frozen storage

1 Introduction

The quality of fish is a complex concept in which nutri-tional microbiological biochemical and physicochemicalattributes are involved The freshness of fish decreases afterits sacrifice this is due to microbiological contamination andvarious biochemical reactions which produce changes in theprotein fractions [1] Some investigations have emphasizedhow the lipid compounds are altered due to oxidative deteri-oration [2] Proteins including the sarcoplasmic myofibrillarand stromal proteins are susceptible to oxidative damage byintermediates of lipid oxidation [4-hydroxy-trans-2-nonenal(HNE) acrolein malondialdehyde (MDA) glyoxal and

4-oxo-trans-2-nonenal (ONE)] isoketals and metallic ions(such as the iron in the heme group or the copper andzinc found in enzymes and metalloenzymes) present in themuscles of animals and those originated through processing(exposure of meat to oxygen light and temperature coolinguse of additives irradiation and vacuum-packaging) thatinitiate oxidative damage generating changes in flavor colortexturestructure and nutritional value [1ndash5] The use oflow temperatures such as freezing is a general method usedfor the control and decrease of biochemical changes thatcan occur during storage time however this does not com-pletely inhibit the microbiological and chemical reactionsthat result in the deterioration of the quality of the fish

HindawiInternational Journal of Food ScienceVolume 2017 Article ID 2812483 10 pageshttpsdoiorg10115520172812483

2 International Journal of Food Science

for which the use of edible coatings (EC) as adjuvants ofpreservation have demonstrated to provide an increase inshelf-life due to their function as a barrier to oxygen besidesbeing employed as a vehicle of diverse components suchas essential oils bacteriocins organic acids and chitosanwhich help in the control of oxidation and diminish thedeterioration by microorganisms [6ndash8] Chitosan (poly-b-(1ndash4)-D-glucosamine) is a versatile biopolymer having abroad range of applications in the food industry It has beenreported to have a number of functional properties thatmake chitosan useful in food preservation these include itsantimicrobial activity [2] and antioxidant activity [9] and itsability to form protective films or coatings [10] Althoughstudies have been carried out concerning the use of chitosanas an antioxidant andor an antimicrobial agent in EC [3 9ndash11] none have thoroughly discussed its effect on nutritionalproperties The purpose of this study was the use of an ECcontaining 15 chitosan in order to reduce the speed ofdeterioration caused by oxidation andor microbial growthevaluating physicochemical textural and nutritional prop-erties during storage at commercial freezing temperatures(minus18∘C) in common carp

2 Materials and Methods

21 Preparation and Treatment of Fish Samples

211 Chemicals Chitosan medium molecular weight dea-cetylation value of 75ndash85 and viscosity of 200ndash800 cP waspurchased from Aldrich Chemical Co

Bovine serum albumin acrylamide NN1015840-methylene-bisacrylamide trichloroacetic acid (TCA) FeSO4 sulfu-ric acid cumene hydroperoxide (CHP) butylhydroxy-toluene methanol xylenol orange di-nitrophenylhydrazine(DNPH) guanidine ethanol ethyl acetate hydrochloric acidCoomassie Brilliant Blue R-250 thioglycolic acid NBD-Cland o-phthalaldehyde (OPA) were purchased from Sigma-Aldrich (St Louis Missouri USA)

Thiobarbituric acid was purchased (TBA) from Fluka(Sigma-Aldrich Toluca MX) sodium chloride EDTA dis-odium salt NN1015840N1015840- tetramethylethylenediamine (TEMED)Tris (base) urea 120573-mercaptoethanol glycine acetic acidglacial sodium phosphate monobasic sodium phosphatedibasic and copper sulfate pentahydrate were purchasedfrom JT Baker (Pennsylvania USA) sodium carbonate andlactic acid were purchased from Fermont (Monterrey MX)sodium dodecyl sulfate (SDS) and bromophenol blue wereobtained from Hycel (Mexico MX) and plate count agarwas purchased from Bioxon Becton and Dickinson (MexicoMX) All reagents used were of analytical grade

212 Fish Sample Preparation A total of 40 freshwater carps(Cyprinus carpio) with an average weight of 550ndash650 g werepurchased at Tiacaque Aquacultural Center in Toluca Stateof Mexico Mexico and were transferred to the Food ScienceLaboratory in the School of Chemistry at the UniversidadAutonoma del Estado de Mexico and were filleted by handusing knives and cutting boards sanitized in chlorine solution

and rinsed in sterile distilled water The fish were harvestedduring May 2016 Two fillets were obtained from each fishafter removing the head and bone and were then immersedin the coating solution

213 Preparation of Coating Solution and Treated FilletsChitosan solution was prepared with 15 (wv) chitosanin 1 vv lactic acid To achieve complete dispersion ofchitosan the solution was stirred at 40∘C for 15 h on ahotplatemagnetic stirrer the final coating forming solutionconsisted of 13 whey 6 gelatin 13 glycerol and 4inulin according to Garcia-Argueta et al [12] with a finalpH of 35 Fillet samples were randomly assigned to twotreatment batches consisting of one control batch (uncoated)and one batch treated with the coating solution For eachcoated batch approximately 20 carp fillets (12ndash15 cm) wereimmersed for 15 s in the coating solution and then allowedto stand for 1min Then the fish fillets were drained on asterile metal net and air-dried for 20min in order to formthe edible coatings placed on polyethylene containers andthen stored at minus18 plusmn 1∘C for subsequent quality assessmentin a commercial freezer (Torrey Mexico MX) Chemicaland microbiological analyses were performed at monthlyintervals to determine the overall quality of fish for fivemonths

22 Chemical Analyses

221 Moisture and Total Protein Analysis Moisture contentwas determined by difference in weight between the freshsample of minced fillet and the dried sample after drying inan oven at 105∘C until reaching constant weight The result isexpressed as a percentage of moisture The content of totalprotein was determined through the Kjeldahl method andresults are expressed as g of protein100 g of fish as describedin AOAC [13]

222 pH 10 g of fillet muscle was weighed and homoge-nized at high speed in mixerblender (Osterizer 450-20) for1min with 90mL of distilled water Connective tissue waseliminated by filtering with cloth in accordance with thatdescribed by Owen et al [14] pH was determined with adigital pH meter (Conductronic pH 120 New York USA)

223 Myofibrillar Protein Extraction Myofibrillar protein(MP) was obtained in accordance with the methodologydescribed byNgapo et al [15] with slightmodifications 100 gof common carp muscle was homogenized with a blenderfor 10min with a mixture of ice-cold water 1 1 1 (wwv)and was then placed in an ice bath with a magnetic stirrerThemyofibrillar suspensionwas filtered through two layers ofcloth in order to remove the connective tissue this procedurewas carried out twice The homogenized muscle was thencentrifuged at 3000timesg at 4∘C for 25min and the supernatantwas discarded The protein concentration of the myofibrillarprecipitate was determined using the Biuret method [16]25mgmL of MP was stored in a glass container with a lid forthe formation of the gel Gel forming was developed in two-step heating first incubation at 40∘C for 30min followed by

International Journal of Food Science 3

heating with a gradual increase until 90∘C was reached withconstant stirring and then maintaining it for 20min Finallyglass containers were removed and stored at 4∘C

224 Solubility According to Pilosof [17] 2 g of MP wascentrifuged at 2500timesg at 4∘C for 30minThe protein contentin the supernatant was determined as well as the total proteincontent in the MP sample prior to centrifugation Solubilitywas defined by following equation

Solubility = Protein content in supernatantProtein content in the sample

times 100 (1)

225 Total Sulfhydryl Content The total content ofsulfhydryls (SH) was determined according to the methoddescribed by Ellman [18] An aliquot of 1mL of MP solution(5mgmL) reacted with 9mL of Tris-glycine buffer (104 g ofTris-HCl 69 g of glycine 480 g of urea and 12 g of EDTALat pH 80) at room temperature for 30min 005mL of Ellmanreagent (4mg DTNBmL) was added to aliquots of 3mL andwas incubated in darkness for 30min The reaction mixturewas measured at 412 nm using a TU-1800 spectrophotometer(Beijing Purkinje General Instrument Co Ltd BeijingChina) The concentration of SH was expressed as total 120583MSHmg of protein

226 Determination of Total Volatile Base (TVB-N) ContentThe content of TVB-N was determined according to theConway and Byrne method [19] with slight modifications5 g of the homogenized sample was added to 4TCA in a 1 2(wv) ratioThen it was filtered throughWhatman Number 1paper (Schleicheramp SchuellMaidstone England) 1mLof thefiltrate obtained was placed in the outer ring of the ConwayCamara while in the inner ring a solution of 1 boric acidcontaining Shiro Tashiro indicator was added To initiate thereaction 2mL of K2CO3 was mixed with the filtrate Thecamera was incubated at 25∘C for 24 hr The solution of theinner ring was titrated using 001 N HCl until a change in thecolor to a pink tone

227 Determination of Hydroperoxides (HPC) The contentof HPC was determined by the Jiang et al method [20](FOXmdashferrous oxidation-xylenol orange) A 100 120583L aliquotof supernatant was obtained by the deproteinization of thesample with 10 TCA 900120583L of the reaction mixture wasadded consisting of 25mMH2SO4 025mM FeSO4 01mMxylenol orange and 4mMbutyl hydroxytoluene in 90 (vv)The mixture obtained was incubated for 60min at roomtemperature and absorbance wasmeasured at 560 nm againstthe reaction blank in the spectrophotometerThe results wereinterpolated in a normal curve previously elaborated andwere expressed as nM HPCmg protein

228 Determination of Lipoperoxides (LPX) For the deter-mination of LPX the technique described by Buege andAust thiobarbituric acid reactive substances (TBARS) wasemployed which consists of an aliquot of 100120583L of super-natant obtained with prior deproteinization that was added

until 1mL of Tris-HCl buffer solution pH 74 is reachedThe samples were incubated at 37∘C for 30min then 2mLof the TBA-TCA reagent (0375 TBA in 15 TCA) wasadded and thoroughly mixed using a vortex It was taken toboiling point in a hot water bath for 45min and was left tocool eliminating the precipitate formed by centrifugation at3000timesg for 10min Absorbance readings were carried out at awavelength of 535 nm against a reaction blankThe content ofmalondialdehyde (MDA) was calculated utilizing the molarextinction coefficient (MEC) of MDA (156 times 105Mcm)Theresults were expressed as mMMDAmg protein

229 Determination of Protein Carbonyl Content (PCC) Themethod is described by Levine et al [21] and modified byParvez and Raisuddin [22] and Burcham [23] To an aliquotof 100 120583L of supernatant obtained from the deproteinizedsample 150 120583L of 10mM DNPH dissolved in 2M HCl wasadded allowing for the reaction to be carried out in the darkfor an hour at room temperature placing 500120583L of 20TCA and placing the mixture at rest for 15min at 4∘C Thesample was centrifuged at 11000timesg for 5minThe precipitateobtained was washed at least three times with a solutionof ethyl acetate ethanol (1 1) Using a 6M guanidine (pH23) solution the button was dissolved and was incubatedfor 30min at 37∘C Absorbance readings were obtained at366 nm employing the corresponding MEC of 21000McmThe results were expressed as nM reactive carbonyls formed(C=O)mg protein

2210 SDS-PAGE SDS gel electrophoresis was carried outaccording to Laemmli [24] with slight modifications inelectrophoresis equipment which consists of Bio-Rad Mini-PROTEAN II Cell camera employing 10 acrylamide MPextracts were added to 10 urea and buffer sample [01MTris-HCl (pH 68) 04 SDS 10 glycerol and 0004bromophenol blue] The gel of 140 times 140 nm was preparedat a T = 10 in 12M Tris-HCl (pH 88) and 03 SDS theconcentration gel at a T = 4 was prepared with 025M Tris-HCl (pH 68) and 02 SDS The electrode buffer contained0025M Tris-HCl 0192M glycine and 015 SDS at pH 816An electrophoretic run was carried out with a current of 200volts once the run was concluded the gels were stained witha solution consisting of 40 methanol 15 acetic acid and01 Coomassie R-250 Brilliant Blue

2211 Amino Acid Composition 3mg of the dehydratedsimple was placed into tubes to carry out hydrolysis with6NHCl and thioglycolic acid as antioxidants Posteriorly testtubes were heated for 6 hr at 150∘C At the end of hydrolysisthe reagent was evaporated in a Buchi rotary evaporator(Buchi Flawil Switzerland) obtaining a concentrate whichwas resuspended in 2mL of 02 N sodium citrate buffer pH22

For the determination of primary aminoacids 250 120583L ofhydrolyzed extract was taken and mixed with 250120583L of o-phthalaldehyde (OPA) an aliquot of 20120583L was injected intothe HPLC chromatograph (Varian 9012) For the determina-tion of secondary aminoacids (proline and hydroxyproline)


125 120583L of the lyophilized extract was put in 05mL of 04Mborate buffer pH 104 From this solution 250120583L was takenand mixed with 250 120583L of the derivative solution (NBD-Cl2mgmL inMeOH) once filtered the solution was heated to60∘C during 5min in a dark vial with a lidThe derivatizationreaction was stopped with the addition of 50120583L of 1M HCland was cooled to 0∘C For the analysis 20mL of the finalextract was taken and injected into the HPLC a 10 cm times46mm times 3 120583m Varian Microsorb C18 column was utilizedfor this analysis as was HPLC-grade methanol (with 99purity) and sodium acetate buffer (pH 72) as mobile phase A430020-02 Fluorichrom fluorescence detector was used andthe quantification was carried out using external standards asreference

23 Total Viable Counts (TVC) A 10 g sample was homog-enized in 90mL of 01 peptone solution Decimal dilutionswere prepared from this solution and plated using plate countagarThe inoculated plates were incubated at 35∘C for 48 h fortotal viable counts (log 10CFUg) as described by IbrahimSallam [25]

24 Statistical Analysis All experiments were performed intriplicate and a completely randomized design was used Alldata were statistically analyzed by SPSSPC software (version17) One-way analysis of variance independent samplingand paired Studentrsquos 119905-tests were used for comparison of themeans

3 Results and Discussion

31 Physicochemical Analysis

311 Moisture During its storage under freezing a signif-icant (119901 lt 005) decrease in moisture in both treatmentswas observed (Figure 1(a)) For the carp with the coating thedecrease was observed until the fourth month this could bedue to the chitosan in the EC which might promote cross-linking in the gelatin thus diminishing the free volume ofthe polymeric matrix which reduces the diffusion rate ofthe water molecules through the coating filmThe aforemen-tioned results in a decrease in vapor permeability in the filletas well as with the coating itself [26] According to Dutta etal [8] this is a desirable characteristic in coatings and is notobserved in the control sample of this study since a loss inmoisture occurs (119901 lt 005) during all of its storage periodAfter fivemonths of storage the percentage of losswas similarin both the control sample and the sample with the treatmentthis could be due to the fact that both treatments were storedin polyethylene containers which act as protection where thecoatingwould be able tomaintain themoisture of the productduring the first four months of storage [27]

312 pH A significant decrease (p lt 005) in pH wasobserved (Figure 1(c)) at the end of both treatments whichcould be associatedwith the production of lactic acid throughanaerobic glycolysis and the liberation of inorganic phos-phate a product of ATP degradation An increase in pH isobserved by the second month for the control sample and by

the third month for the coated sample which could be due tothe accumulation of basic compounds such as ammonia andtrimethylamine a result of autolytic and microbial reactions[4 28ndash30] The greatest variation in percentage at the endof storage corresponds to the noncoated sample a lowerpH in the sample with the coating can bolster microbialinhibition and contribute to the preservation of the samplesinhibiting the endogenous proteases and this result suggeststhat during storage the coating diminished the decrease inpH [11 28]

313 Total Volatile Basic Nitrogen (TVB-N) TVB-N is agroup primarily composed of primary secondary and ter-tiary amines which are used as indicators of meat deteriora-tion the increase in these is related to the activity of endoge-nous enzymes and bacteria [28] According to Connell [31]and Gimenez et al [32] 25ndash40mg of N100 g of tissue is con-sidered as acceptable for consumption [9 11 28] Althoughboth samples have values below said limit (Figure 1(b)) theconcentration of TVB-N is greater in the control sample ineach stage of storage this could be due to the fact that thepresence of chitosan helps in reducing the capacity of bacteriafor oxidative deamination of nonprotein nitrogenated com-pounds [11 28] The storage time was not enough to identifywhen the acceptable threshold is exceeded due to the factthat enzymatic and microbial activity diminish at low tem-peratures The carp utilized in this study presented adequateconditions for human consumption at the end of the storageperiod coinciding with that reported by Soares et al [27]

32 Microbiological Changes

321 Total Viable Count Microbial activity is a limitingfactor in the quality of the fish and the total viable counthas been used as indicator of acceptability of the same [2933] The initial value for the carp without the coating was23 log 104 CFUg and 11 log 104 CFUg for the carp with thecoating these values depend on the environment fromwhichthe fish is obtained as well as postmortem conditions [11]Theevolution of the aforementioned is detailed in Figure 1(d)observing significant differences (119901 lt 005) by the secondmonth obtaining a time-dependent increase in both treat-ments however the limit recommended by the ICMSF [34]of 5 times 105 CFUg for quality fish is not exceeded [29]The properties of the chitosan added to the coating had aninhibitory effect thereby obtaining 47 log 104 CFUg whilefor the control a value of 22 log 106 CFUg was obtainedafter five months of storage The antimicrobial effect of thiscompound has been widely reported by Fernandez-Saiz etal [35] Jeon et al [36] and Lopez-Caballero et al [37]and its mechanism of action is related to the rupture of thelipopolysaccharide layer of the external membrane of Gram-negative bacteria and its function as a barrier against thetransfer of oxygen Another mechanism of action could bethe interaction with anionic groups on the cell surface due toits polycationic nature [3 9] this demonstrates that the ECinhibits microbial growth up to 2 logarithmic units decreas-ing reactions involved in deterioration by microorganismsduring its storage in the freezer


A B C D D E

A A A AB CD

CC + EC

0 1 2 3 4 5(Months)

30

40

50

60

70

80

90

m

oistu

re

(a)

A AAB BC

C C

A B C DE F

CC + EC

0

5

10

15

20

25

0 1 2 3 4 5(Months)

mg

N100

g

(b)

A

B CD

E D

A

BC D D

E

CC + EC

552545658

662646668

7

0 1 2 3 4 5

pH

(Months)

(c)

A A

B CD E

A A

B CD D

CC + EC

log 1

0U

FCg

sam

ple

0 1 2 3 4 5(Months)

107

106

105

104

103

(d)

Figure 1 Changes in moisture (a) total volatile basic nitrogen TVB-N (b) pH (c) and total viable count (TVC) (d) values of common carpfillets stored at minus18∘C for 5 monthsThe results are the mean of three replications C fillet carp without coating C + EC fillet carp with ediblecoating The different letters indicate significant differences between treatment times for the same treatment (119901 lt 005)

33 Lipid Oxidation Products The concentration of primaryproducts of oxidation can be measured by the content ofperoxides The carp with coating shows a significant increase(119901 lt 005) in peroxides by the fourth month and presentsa final value of 055 nM HPOxmg of protein (Figure 2(a))while the control sample presents an increase by the firstmonth having an increase of 59 with regard to the samplewith coating this demonstrates that the coating retards lipidoxidation in the carp fillet These results are in accordancewithOjagh et al [9] Nowzari et al [10] Jeon et al [36] and Liet al [38] and those that report that additional coatings withchitosan retard production of oxidated primary compoundsin herring trout cod and croaker in freezer storage as wellas in ice storage Lipid oxidation in fish is influenced byvarious factors like fat content the degree of microsomeassociated with the oxidation system heme group contentand the presence of ions [10 38] Chitosan-added coatingsact as excellent barriers to the permeability of oxygen oncethey are applied directly over the meatrsquos surface retarding thediffusion of oxygen [10]

In storage at freezing temperature (minus18∘C) oxidationis the most important factor in deterioration even over

microbial activity TBARS quantifies the compoundsresponsible for the loss of flavor and scent and is alsoimportant in the stages of deterioration of foods The valueof TBA is an indicator of lipid oxidation widely used whichquantifies the content of malondialdehyde (MDA) formedfrom hydroperoxides which are the initial products of theoxidation of unsaturated fatty acids by oxygen [29 39] In thepresent study the values of TBA of both treatment samplespresented a significant increase (119901 lt 005) by the secondand forth months however by the fourth month the controlsample presented a 39 increase with regard to a 6 increasein the sample with coating this is due to the absence ofchitosan in the control samplersquos coating This same behaviorwas observed by Jeon et al [36] in herring and cod with achitosan coating and Ojagh et al [9] in rainbow trout Theantioxidative mechanism of the chitosan is due to the factthat its primary amino groups form a stable fluorospherewith volatile aldehydes such as malondialdehyde derivedfrom the rupture of fats during oxidation [38]

The results indicate that the chitosan employed in a15 EC preserves the fish fillet through reduction of lipidoxidation


A

B

BC BC BC

D

A A AA A

B

03

04

05

06

07

08

09

0 1 2 3 4 5

nM H

POx

mg

prot

ein

(Months)

CC + EC

(a)

A A

B

B

C

D

AA

B

C

CDD

14

16

18

20

22

24

26

28

30

32

34

0 1 2 3 4 5

mM

MD

Am

g pr

otei

n

(Months)

CC + EC

(b)

Figure 2 Changes in HPOx content (a) and MDA content (b) values of common carp fillets stored at minus18∘C for 5 months The results arethe mean of three replications C fillet carp without coating C + EC fillet carp with edible coating The different letters indicate significantdifferences between treatment times for the same treatment (119901 lt 005)

CC + EC

ABB

A

B B

AB

ABD

C

ADE

B

0

10

20

30

40

50

60

70

0 1 2 3 4 5(Months)

120583m

-SH

g p

rote

in

(a)

CC + EC

A

BC

B

DD

A ABAB

CBC

AB

0010203040506070809

1

0 1 2 3 4 5

nM re

activ

e car

bony

lsm

g pr

otei

n

(Months)

(b)

Figure 3 Changes in sulfhydryl content (a) and protein carbonyl content (b) values of common carp fillets stored at minus18∘C for 5 monthsTheresults are the mean of three replications C fillet carp without coating C + EC fillet carp with edible coating The different letters indicatesignificant differences between treatment times for the same treatment (119901 lt 005)

34 Products of Protein Oxidation Protein oxidation in carpfillet during freezer storage is shown in Figure 3 therewas a significant increase (119901 lt 005) in the formation ofcarbonylated proteins during storage in the control sampleby the first month while the sample with coating presentedan increase until the third month and does not presentdifferences at the end of storage The increase was from 035to 089 nmmg of protein for the control sample and from045 to 059 nmmg of protein at maximum oxidation pointThe samples of processed fillet include red and white muscleand thus it is possible to find a high proportion of hemeproteins (hemoglobin and myoglobin) and consequently

a concentration of iron in addition to what can be foundchelated to proteins favoring protein and lipid oxidation Bythe fourth month both samples present a reduction in theconcentration of carbonyl groups which has been reportedby other authors during a storage at minus20∘C suggesting inter-actions between carbonyls and other cell components forwhich the formation of carbonyl groups should be consideredas a step in oxidation processes and not as a sole stablemarker of protein oxidation [40]The oxidation of proteins isassociated with the decrease in sulfhydryl groups which areconverted to disulfides During freezer storage a significantdecrease (119901 lt 005) was observed during the second month


in both samples Proteins are attacked by reactive oxygenspecies (ROS) where its creation in fish is due to diverseexternal factors such as noise manipulation slaughter orthe presence of metals such as iron The interaction canlead to the formation of carbonyl groups and the loss ofsulfhydryl groups [3 40] affecting structural functional andnutritional properties for which the use of EC with chitosancould reduce mentioned effects

35 Aminoacid Content The protein content in fresh carpis similar to that reported by FAO (21) in frozen speciesas well as frozen species but with coating (Table 1) Theprotein of fish is considered of the highest quality comparedto standard proteins reported by FAO and although theinformation concerning its nutritional value is widely knownfew are the studies that refer to its composition the scarcestbeing in common carp [41] It is known that the contentand bioavailability of aminoacids can be affected by differentoperations such as drying fermentation extrusion and evengermination [42] In this study it was observed that theaminoacid content in carp suffered a significant decrease(119901 lt 005) after 5 months of storage due to freezing whilethe species with the coating only presented a decrease inthe levels of Cys His Tyr Thr Met and Lys this could bedue to protein oxidation by intermediaries of lipid oxidationand environment factors such as pH temperature wateractivity and the presence of promoters and inhibitors likephenolic compounds [43]The sulfur aminoacids such asMetand Cys are highly susceptible to oxidation in the presenceof oxidized lipid products which lead to the formation ofa variety of compounds such as sulphone sulfoxides anddisulfide derivatives [44] In the present study a decreasein Met and Cys can be observed in the frozen fillet withregard to the control Once the edible coating is added aprotective activity can be observed for Cys (084 g100 g infrozen fillet and 091 g100 g in coated fillet) this can be dueto cross-linking which is usually attributed to the formationof Cys (disulfide bonds) and dityrosines (from two Cys andtwo Tyr residues) [45] The loss of Thr and Lys could bedue to the formation of intermediary adducts such as 120572-amino-3-keto butyric acid and 120572-amino adipic semialdehydeas a consequence of oxidation catalyzed by metals from themetalloproteins of the food matrix [43]

Common carp protein is characterized by a high con-tent of essential aminoacids compared to the standardprotein established by the FAO exceeding values in thecase of Met + Cys and Val (Table 2) in fresh carp havingLeu as the limiting aminoacid and presenting a ldquolimitingaminoacidrdquo index (or chemical index) of 8227 this indexis a basic parameter used for the evaluation of the nutri-tional value of a food which refers to the minor contentof an essential aminoacid with regard to a given standardprotein called the ldquolimiting aminoacidrdquo In this study theessential aminoacids in the sample in freezing without ECdecreased only maintaining Val as the excess aminoacid andhaving Lys as the limiting aminoacid This change in thelimiting aminoacid could be a consequence of enzymaticreactions andor bacterial growth during storage [46] On theother hand the frozen carp with EC continued to present

Table 1 Aminoacid composition of common carp fillet proteins 5-month frozen carp and 5-month frozen carp + edible coating storedat minus18∘C

Aminoacids Carp 5-month frozencarp

5-month frozencarp + edible

coatingAsparagine 628 plusmn 018a 503 plusmn 012b 598 plusmn 022a

Glutamic acid 1075 plusmn 022a 853 plusmn 018c 905 plusmn 015b

Alanine 498 plusmn 018a 376 plusmn 015c 428 plusmn 023b

Arginine 374 plusmn 010a 311 plusmn 012b 345 plusmn 018a

Cysteine 098 plusmn 007a 084 plusmn 008b 091 plusmn 006a

Phenylalanine 351 plusmn 024a 271 plusmn 028b 329 plusmn 019a

Glycine 334 plusmn 012a 276 plusmn 019b 311 + 016a

Histidine 248 plusmn 008a 15 plusmn 005c 201 + 012b

Isoleucine 389 plusmn 024a 231 plusmn 022b 328 + 017a

Leucine 543 plusmn 032a 476 plusmn 038b 521 plusmn 034a

Lysine 553 plusmn 028a 412 plusmn 022b 53 plusmn 019c

Methionine 273 plusmn 008a 108 plusmn 007b 206 plusmn 009c

Serine 272 plusmn 007a 197 plusmn 008c 225 plusmn 011b

Tyrosine 267 plusmn 015a 186 plusmn 021b 222 plusmn 013c

Threonine 316 plusmn 022a 242 plusmn 012b 308 plusmn 015a

Valine 428 plusmn 019a 376 plusmn 032b 412 plusmn 023a

Protein () 2239 plusmn 17a 2265 plusmn 12a 2287 plusmn 14aabc119901 lt 005

a higher concentration in the aforementioned aminoacids(Iso Met + Cys and Val) thereby presenting a protectiveeffect in Lys which results in great benefit since fish couldbe maintained as an important source of this aminoacidafter 5 months of storage in the freezer [42 47] Thechemical index diminishes 11 units in the fillet withoutEC and 3 units for the fillet with the EC after freezingwhich suggests that EC has a protective effect over essentialaminoacids

36 SDS-PAGE The molecular weight profiles obtainedthrough SDS-PAGE of the MPs extracted from the differenttreatments during storage are shown in Figure 4 in whichthe composition of the MPs of common carp was myosinheavy chains (MHC) actin (A) and troponin (T) observingthat the sample without coating presents after 5 months ofstorage molecular weight bands lower than those of myosinwith an interval of 150 100 and 75 kDa approximately this isprobably due to carbonylation of MHC coinciding with thatreported by Kjaeligrsgard et al [48] in rainbow trout LikewisePassi et al (2005) reported an increase of oxidized proteins indifferent species of Mediterranean fish after lipid oxidationpossibly due to the presence of the heme group in myoglobin[49] For the samples with coating no notable changes wereobserved in the SDS-PAGE profile which could suggest thatthe coating is retarding the oxidation mechanism showingthat actin was the least oxidized during freezing coincidingwith that reported by Eymard et al [40] In spite of the highsusceptibility of myofibrillary proteins to oxidation ediblecoatings could help in the preservation of protein integrity


150100

5037

2520

15

10

C C C CM1 M2 M4 M5

MHC

A

T

75

C + EC C + EC C + EC C + EC250

(KD)

Figure 4 SDS-PAGE of common carp fillets stored at minus18∘C control batch (C) fillet without coating and filleted carp with edible coating (C+ EC) M1 M2 M4 and M5 from first to fifth month during storage

Table 2 Values of the limiting aminoacid index ()

Aminoacids Standard FAOWHO (1991)c Carp 5-month frozen carp 5-month frozen carp + edible coatingg100 g protein g100 g protein g100 g protein

Phe + Tyra 630 618 9810 457 7254 551 8746Isoleucine 280 389 13893 231 8250 328 11714Leucine 660 543 8227 476 7212 521 7894Lysine 580 553 9534 412 7103 530 9138Met + Cysb 250 371 14840 192 7680 297 11880Threonine 340 316 9294 242 7118 308 9059Valine 350 428 12229 376 10743 412 11771Amino acid index 8227 7103 7894aPhenylalanine + tyrosine bmethionine + cysteine caccording to Usydus et al [47]

of aquaticmarine species stored during freezing keepingfunctional and nutritional properties for more time

4 Conclusions

The use of EC added with chitosan allows retention ofthe physicochemical and nutritional characteristics of thecommon carp for more time during storage diminishingthe loss of moisture bacterial growth nutritional value andspeed of lipid and protein oxidation in oxidation products(hydroperoxides lipoperoxidation and carbonyl proteins)as well as an indication of the aminoacids present in thecommon carp fillet with EC being an excellent alternative asan adjuvant in the conservation through freezing of aquaticspecies of economic importance on a global scale

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

Ana Gabriela Morachis Valdez thanks the National Councilof Science and Technology (Consejo Nacional de Ciencia yTecnologıa Mexico) for a graduate scholarship

References

[1] T Li J Li W Hu and X Li ldquoQuality enhancement in refrig-erated red drum (Sciaenops ocellatus) fillets using chitosancoatings containing natural preservativesrdquo Food Chemistry vol138 no 2-3 pp 821ndash826 2013

[2] A B Falowo P O Fayemi and V Muchenje ldquoNatural antioxi-dants against lipid-protein oxidative deterioration in meat andmeat products a reviewrdquo Food Research International vol 64pp 171ndash181 2014

[3] C O Mohan C N Ravishankar K V Lalitha and T KSrinivasaGopal ldquoEffect of chitosan edible coating on the qualityof double filleted Indian oil sardine (Sardinella longiceps) duringchilled storagerdquo Food Hydrocolloids vol 26 no 1 pp 167ndash1742012

[4] E Indergard I Tolstorebrov H Larsen and T M EikevikldquoThe influence of long-term storage temperature and type ofpackaging materials on the quality characteristics of frozenfarmed Atlantic Salmon (Salmo Salar)rdquo International Journal ofRefrigeration vol 41 pp 27ndash36 2014

[5] R Pamplona ldquoAdvanced lipoxidation end-productsrdquo Chemico-Biological Interactions vol 192 no 1-2 pp 14ndash20 2011

[6] E Latou S F Mexis A V Badeka S Kontakos and MG Kontominas ldquoCombined effect of chitosan and modifiedatmosphere packaging for shelf life extension of chicken breastfilletsrdquo LWTmdashFood Science and Technology vol 55 no 1 pp263ndash268 2014


[7] M Ahmad S Benjakul P Sumpavapol and N P NirmalldquoQuality changes of sea bass slices wrapped with gelatin filmincorporated with lemongrass essential oilrdquo International Jour-nal of Food Microbiology vol 155 no 3 pp 171ndash178 2012

[8] P K Dutta S Tripathi G K Mehrotra and J Dutta ldquoPer-spectives for chitosan based antimicrobial films in food appli-cationsrdquo Food Chemistry vol 114 no 4 pp 1173ndash1182 2009

[9] S M Ojagh M Rezaei S H Razavi and S M H HosseinildquoEffect of chitosan coatings enriched with cinnamon oil on thequality of refrigerated rainbow troutrdquo Food Chemistry vol 120no 1 pp 193ndash198 2010

[10] F Nowzari B Shabanpour and S M Ojagh ldquoComparison ofchitosan-gelatin composite and bilayer coating and film effecton the quality of refrigerated rainbow troutrdquo Food Chemistryvol 141 no 3 pp 1667ndash1672 2013

[11] J Huang Q Chen M Qiu and S Li ldquoChitosan-based EdibleCoatings for Quality Preservation of Postharvest WhitelegShrimp (Litopenaeus vannamei)rdquo Journal of Food Science vol77 no 4 pp C491ndashC496 2012

[12] I Garcia-Argueta O Dublan-Garcia B Quintero-Salazar ADominguez-Lopez LM Gomez-Olivan andA F ZM SalemldquoEffect of lactic acid bacteria on the textural properties of anedible film based on whey inulin and gelatinrdquo African Journalof Biotechnology vol 12 pp 2659ndash2669 2013

[13] AOACOfficial Methods of Analysis of the Association of Analyt-ical Chemistry Association of Official Analytical Chemists 4thedition 1984

[14] J E Owen F A Nunez M T Arias et alManual De Practicasde Cursos de Tecnologıas de la Carne Facultad de ZoontecniaUniversidad de Chihuahua Chihuahua Mexico 1982

[15] T Ngapo B Wilkinson R Chong et al ldquoGelation of bovinemyofibrillar protein induced by 15 Gluconolaconerdquo in Pro-ceedings of the 38th International Congress of Meat Science andTechnology pp 1095ndash1098 Clermont Ferrand France 1992

[16] A G Gornall C J Bardawill andMMDavid ldquoDeterminationof serum proteins by means of the biuret reactionrdquoThe Journalof Biological Chemistry vol 177 no 2 pp 751ndash766 1949

[17] A M Pilosof ldquoSolubilidadrdquo in Caracterizacion funcional yestructural de proteınas A M R and G B Barholomai Eds pp60ndash75CeudebaCYTED (Programa IberoamericanodeCienciay Tecnologıa para el Desarrollo) 2000

[18] G L Ellman ldquoTissue sulfhydryl groupsrdquo Archives of Biochem-istry and Biophysics vol 82 no 1 pp 70ndash77 1959

[19] E J Conway and A Byrne ldquoAn absorption apparatus forthe micro-determination of certain volatile substances I Themicro-determination of ammoniardquo Journal of Biochemistry vol27 pp 419ndash429 1936

[20] Z Y Jiang J V Hunt and S P Wolff ldquoFerrous ion oxidation inthe presence of xylenol orange for detection of lipid hydroper-oxide in low density lipoproteinrdquo Analytical Biochemistry vol202 no 2 pp 384ndash389 1992

[21] R L Levine J A Williams E R Stadtman and E ShacterldquoCarbonyl assays for determination of oxidatively modifiedproteinsrdquoMethods in Enzymology vol 233 pp 346ndash357 1994

[22] S Parvez and S Raisuddin ldquoProtein carbonyls Novel biomark-ers of exposure to oxidative stress-inducing pesticides in fresh-water fish Channa punctata (Bloch)rdquo Environmental Toxicologyand Pharmacology vol 20 no 1 pp 112ndash117 2005

[23] P C Burcham ldquoModified protein carbonyl assay detects oxi-dised membrane proteins A new tool for assessing drug- andchemically-induced oxidative cell injuryrdquo Journal of Pharmaco-logical and Toxicological Methods vol 56 no 1 pp 18ndash22 2007

[24] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[25] K Ibrahim Sallam ldquoAntimicrobial and antioxidant effects ofsodium acetate sodium lactate and sodium citrate in refriger-ated sliced salmonrdquo Food Control vol 18 no 5 pp 566ndash5752007

[26] S Fakhreddin Hosseini M Rezaei M Zandi and F F GhavildquoPreparation and functional properties of fish gelatin-chitosanblend edible filmsrdquo Food Chemistry vol 136 no 3-4 pp 1490ndash1495 2013

[27] N M Soares M S Oliveira and A A Vicente ldquoEffectsof glazing and chitosan-based coating application on frozensalmon preservation during six-month storage in industrialfreezing chambersrdquo LWTmdashFood Science and Technology vol 61no 2 pp 524ndash531 2015

[28] W Fan J Sun Y Chen J Qiu Y Zhang and Y Chi ldquoEffects ofchitosan coating on quality and shelf life of silver carp duringfrozen storagerdquo Food Chemistry vol 115 no 1 pp 66ndash70 2009

[29] N M Soares T S Mendes and A A Vicente ldquoEffect ofchitosan-based solutions applied as edible coatings and waterglazing on frozen salmon preservationmdasha pilot-scale studyrdquoJournal of Food Engineering vol 119 no 2 pp 316ndash323 2013

[30] D Liu L Liang W Xia J M Regenstein and P ZhouldquoBiochemical and physical changes of grass carp (Ctenopharyn-godon idella) fillets stored at -3 and 0 ∘crdquo Food Chemistry vol140 no 1-2 pp 105ndash114 2013

[31] J J Connell ldquoMethods of assessing and selecting for qualityrdquo inControl of Fish Quality Springer Berlin Germany 3rd edition1990

[32] B Gimenez P Roncales and J A Beltran ldquoModified atmo-sphere packaging of filleted rainbow troutrdquo Journal of theScience of Food and Agriculture vol 82 no 10 pp 1154ndash11592002

[33] G Olafsdottir E Martinsdottir J Oehlenschager et al ldquoMeth-ods to evaluate fish freshness in research and industryrdquo Trendsin Food Science and Technology vol 8 pp 258ndash265 1997

[34] ICMSFMicroorganisms in Foods 2 Sampling forMicrobiologicalAnalysis Principles and Specific Applications University ofToronto Press New York NY USA 2nd edition 1986

[35] P Fernandez-Saiz G Sanchez C Soler J M Lagaron and MJ Ocio ldquoChitosan films for the microbiological preservation ofrefrigerated sole and hake filletsrdquo Food Control vol 34 no 1 pp61ndash68 2013

[36] Y-J Jeon J Y V A Kamil and F Shahidi ldquoChitosan as an edibleinvisible film for quality preservation of herring and Atlanticcodrdquo Journal of Agricultural and Food Chemistry vol 50 no 18pp 5167ndash5178 2002

[37] M E Lopez-Caballero M C Gomez-Guillen M Perez-Mateos and P Montero ldquoA chitosan-gelatin blend as a coatingfor fish pattiesrdquo Food Hydrocolloids vol 19 no 2 pp 303ndash3112005

[38] T LiW Hu J Li X Zhang J Zhu and X Li ldquoCoating effects oftea polyphenol and rosemary extract combined with chitosanon the storage quality of large yellow croaker (Pseudosciaenacrocea)rdquo Food Control vol 25 no 1 pp 101ndash106 2012

[39] B W S Souza M A Cerqueira H A Ruiz et al ldquoEffect ofChitosan-based coatings on the shelf life of Salmon (Salmosalar)rdquo Journal of Agricultural and Food Chemistry vol 58 no21 pp 11456ndash11462 2010


[40] S Eymard C P Baron and C Jacobsen ldquoOxidation of lipidand protein in horsemackerel (Trachurus trachurus)mince andwashedminces during processing and storagerdquo Food Chemistryvol 114 no 1 pp 57ndash65 2009

[41] K A Skibniewska J Zakrzewski J Kłobukowski et al ldquoNutri-tional value of the protein of consumer carp cyprinus carpio LrdquoCzech Journal of Food Sciences vol 31 no 4 pp 313ndash317 2013

[42] J Boye R Wijesinha-Bettoni and B Burlingame ldquoProteinquality evaluation twenty years after the introduction of theprotein digestibility corrected amino acid scoremethodrdquoBritishJournal of Nutrition vol 108 no 2 pp S183ndashS211 2012

[43] M Estevez ldquoProtein carbonyls in meat systems a reviewrdquoMeatScience vol 89 no 3 pp 259ndash279 2011

[44] R L Levine N Wehr J A Williams et al ldquoDeterminationof carbonyl groups in oxidized proteinsrdquo Methods MolecularBiology vol 99 pp 15ndash24 2000

[45] MN LundMHeinonen C P Baron andM Estevez ldquoProteinoxidation in muscle foods a reviewrdquo Molecular Nutrition andFood Research vol 55 no 1 pp 83ndash95 2011

[46] A Ciampa G Picone L Laghi H Nikzad and F CapozzildquoChanges in the amino acid composition of Bogue (Boopsboops) fish during storage at different temperatures by 1H-NMRspectroscopyrdquo Nutrients vol 4 no 6 pp 542ndash553 2012

[47] Z Usydus J Szlinder-Richert and M Adamczyk ldquoProteinquality and amino acid profiles of fish products available inPolandrdquo Food Chemistry vol 112 no 1 pp 139ndash145 2009

[48] I V H Kjaeligrsgard M R Noslashrrelykke C P Baron and F JessenldquoIdentification of carbonylated protein in frozen rainbow trout(Oncorhynchus mykiss) fillets and development of protein oxi-dation during frozen storagerdquo Journal of Agricultural and FoodChemistry vol 54 no 25 pp 9437ndash9446 2006

[49] S Passi S Cataudella L Tiano andG P Littarru ldquoDynamics oflipid oxidation and antioxidant depletion inMediterranean fishstored at different temperaturesrdquo BioFactors vol 25 no 1-4 pp241ndash254 2005

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201


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


for which the use of edible coatings (EC) as adjuvants ofpreservation have demonstrated to provide an increase inshelf-life due to their function as a barrier to oxygen besidesbeing employed as a vehicle of diverse components suchas essential oils bacteriocins organic acids and chitosanwhich help in the control of oxidation and diminish thedeterioration by microorganisms [6ndash8] Chitosan (poly-b-(1ndash4)-D-glucosamine) is a versatile biopolymer having abroad range of applications in the food industry It has beenreported to have a number of functional properties thatmake chitosan useful in food preservation these include itsantimicrobial activity [2] and antioxidant activity [9] and itsability to form protective films or coatings [10] Althoughstudies have been carried out concerning the use of chitosanas an antioxidant andor an antimicrobial agent in EC [3 9ndash11] none have thoroughly discussed its effect on nutritionalproperties The purpose of this study was the use of an ECcontaining 15 chitosan in order to reduce the speed ofdeterioration caused by oxidation andor microbial growthevaluating physicochemical textural and nutritional prop-erties during storage at commercial freezing temperatures(minus18∘C) in common carp

2 Materials and Methods

21 Preparation and Treatment of Fish Samples

211 Chemicals Chitosan medium molecular weight dea-cetylation value of 75ndash85 and viscosity of 200ndash800 cP waspurchased from Aldrich Chemical Co

Bovine serum albumin acrylamide NN1015840-methylene-bisacrylamide trichloroacetic acid (TCA) FeSO4 sulfu-ric acid cumene hydroperoxide (CHP) butylhydroxy-toluene methanol xylenol orange di-nitrophenylhydrazine(DNPH) guanidine ethanol ethyl acetate hydrochloric acidCoomassie Brilliant Blue R-250 thioglycolic acid NBD-Cland o-phthalaldehyde (OPA) were purchased from Sigma-Aldrich (St Louis Missouri USA)

Thiobarbituric acid was purchased (TBA) from Fluka(Sigma-Aldrich Toluca MX) sodium chloride EDTA dis-odium salt NN1015840N1015840- tetramethylethylenediamine (TEMED)Tris (base) urea 120573-mercaptoethanol glycine acetic acidglacial sodium phosphate monobasic sodium phosphatedibasic and copper sulfate pentahydrate were purchasedfrom JT Baker (Pennsylvania USA) sodium carbonate andlactic acid were purchased from Fermont (Monterrey MX)sodium dodecyl sulfate (SDS) and bromophenol blue wereobtained from Hycel (Mexico MX) and plate count agarwas purchased from Bioxon Becton and Dickinson (MexicoMX) All reagents used were of analytical grade

212 Fish Sample Preparation A total of 40 freshwater carps(Cyprinus carpio) with an average weight of 550ndash650 g werepurchased at Tiacaque Aquacultural Center in Toluca Stateof Mexico Mexico and were transferred to the Food ScienceLaboratory in the School of Chemistry at the UniversidadAutonoma del Estado de Mexico and were filleted by handusing knives and cutting boards sanitized in chlorine solution

and rinsed in sterile distilled water The fish were harvestedduring May 2016 Two fillets were obtained from each fishafter removing the head and bone and were then immersedin the coating solution

213 Preparation of Coating Solution and Treated FilletsChitosan solution was prepared with 15 (wv) chitosanin 1 vv lactic acid To achieve complete dispersion ofchitosan the solution was stirred at 40∘C for 15 h on ahotplatemagnetic stirrer the final coating forming solutionconsisted of 13 whey 6 gelatin 13 glycerol and 4inulin according to Garcia-Argueta et al [12] with a finalpH of 35 Fillet samples were randomly assigned to twotreatment batches consisting of one control batch (uncoated)and one batch treated with the coating solution For eachcoated batch approximately 20 carp fillets (12ndash15 cm) wereimmersed for 15 s in the coating solution and then allowedto stand for 1min Then the fish fillets were drained on asterile metal net and air-dried for 20min in order to formthe edible coatings placed on polyethylene containers andthen stored at minus18 plusmn 1∘C for subsequent quality assessmentin a commercial freezer (Torrey Mexico MX) Chemicaland microbiological analyses were performed at monthlyintervals to determine the overall quality of fish for fivemonths

22 Chemical Analyses

221 Moisture and Total Protein Analysis Moisture contentwas determined by difference in weight between the freshsample of minced fillet and the dried sample after drying inan oven at 105∘C until reaching constant weight The result isexpressed as a percentage of moisture The content of totalprotein was determined through the Kjeldahl method andresults are expressed as g of protein100 g of fish as describedin AOAC [13]

222 pH 10 g of fillet muscle was weighed and homoge-nized at high speed in mixerblender (Osterizer 450-20) for1min with 90mL of distilled water Connective tissue waseliminated by filtering with cloth in accordance with thatdescribed by Owen et al [14] pH was determined with adigital pH meter (Conductronic pH 120 New York USA)

223 Myofibrillar Protein Extraction Myofibrillar protein(MP) was obtained in accordance with the methodologydescribed byNgapo et al [15] with slightmodifications 100 gof common carp muscle was homogenized with a blenderfor 10min with a mixture of ice-cold water 1 1 1 (wwv)and was then placed in an ice bath with a magnetic stirrerThemyofibrillar suspensionwas filtered through two layers ofcloth in order to remove the connective tissue this procedurewas carried out twice The homogenized muscle was thencentrifuged at 3000timesg at 4∘C for 25min and the supernatantwas discarded The protein concentration of the myofibrillarprecipitate was determined using the Biuret method [16]25mgmL of MP was stored in a glass container with a lid forthe formation of the gel Gel forming was developed in two-step heating first incubation at 40∘C for 30min followed by





times 100 (1)























A B C D D E

A A A AB CD

CC + EC

0 1 2 3 4 5(Months)

30

40

50

60

70

80

90

m

oistu

re

(a)

A AAB BC

C C

A B C DE F

CC + EC

0

5

10

15

20

25

0 1 2 3 4 5(Months)

mg

N100

g

(b)

A

B CD

E D

A

BC D D

E

CC + EC

552545658

662646668

7

0 1 2 3 4 5

pH

(Months)

(c)

A A

B CD E

A A

B CD D

CC + EC

log 1

0U

FCg

sam

ple

0 1 2 3 4 5(Months)

107

106

105

104

103

(d)







A

B

BC BC BC

D

A A AA A

B

03

04

05

06

07

08

09

0 1 2 3 4 5

nM H

POx

mg

prot

ein

(Months)

CC + EC

(a)

A A

B

B

C

D

AA

B

C

CDD

14

16

18

20

22

24

26

28

30

32

34

0 1 2 3 4 5

mM

MD

Am

g pr

otei

n

(Months)

CC + EC

(b)


CC + EC

ABB

A

B B

AB

ABD

C

ADE

B

0

10

20

30

40

50

60

70

0 1 2 3 4 5(Months)

120583m

-SH

g p

rote

in

(a)

CC + EC

A

BC

B

DD

A ABAB

CBC

AB

0010203040506070809

1

0 1 2 3 4 5

nM re

activ

e car

bony

lsm

g pr

otei

n

(Months)

(b)































150100

5037

2520

15

10

C C C CM1 M2 M4 M5

MHC

A

T

75


(KD)






4 Conclusions




Acknowledgments


References



























































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





times 100 (1)























A B C D D E

A A A AB CD

CC + EC

0 1 2 3 4 5(Months)

30

40

50

60

70

80

90

m

oistu

re

(a)

A AAB BC

C C

A B C DE F

CC + EC

0

5

10

15

20

25

0 1 2 3 4 5(Months)

mg

N100

g

(b)

A

B CD

E D

A

BC D D

E

CC + EC

552545658

662646668

7

0 1 2 3 4 5

pH

(Months)

(c)

A A

B CD E

A A

B CD D

CC + EC

log 1

0U

FCg

sam

ple

0 1 2 3 4 5(Months)

107

106

105

104

103

(d)







A

B

BC BC BC

D

A A AA A

B

03

04

05

06

07

08

09

0 1 2 3 4 5

nM H

POx

mg

prot

ein

(Months)

CC + EC

(a)

A A

B

B

C

D

AA

B

C

CDD

14

16

18

20

22

24

26

28

30

32

34

0 1 2 3 4 5

mM

MD

Am

g pr

otei

n

(Months)

CC + EC

(b)


CC + EC

ABB

A

B B

AB

ABD

C

ADE

B

0

10

20

30

40

50

60

70

0 1 2 3 4 5(Months)

120583m

-SH

g p

rote

in

(a)

CC + EC

A

BC

B

DD

A ABAB

CBC

AB

0010203040506070809

1

0 1 2 3 4 5

nM re

activ

e car

bony

lsm

g pr

otei

n

(Months)

(b)































150100

5037

2520

15

10

C C C CM1 M2 M4 M5

MHC

A

T

75


(KD)






4 Conclusions




Acknowledgments


References



























































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














A B C D D E

A A A AB CD

CC + EC

0 1 2 3 4 5(Months)

30

40

50

60

70

80

90

m

oistu

re

(a)

A AAB BC

C C

A B C DE F

CC + EC

0

5

10

15

20

25

0 1 2 3 4 5(Months)

mg

N100

g

(b)

A

B CD

E D

A

BC D D

E

CC + EC

552545658

662646668

7

0 1 2 3 4 5

pH

(Months)

(c)

A A

B CD E

A A

B CD D

CC + EC

log 1

0U

FCg

sam

ple

0 1 2 3 4 5(Months)

107

106

105

104

103

(d)







A

B

BC BC BC

D

A A AA A

B

03

04

05

06

07

08

09

0 1 2 3 4 5

nM H

POx

mg

prot

ein

(Months)

CC + EC

(a)

A A

B

B

C

D

AA

B

C

CDD

14

16

18

20

22

24

26

28

30

32

34

0 1 2 3 4 5

mM

MD

Am

g pr

otei

n

(Months)

CC + EC

(b)


CC + EC

ABB

A

B B

AB

ABD

C

ADE

B

0

10

20

30

40

50

60

70

0 1 2 3 4 5(Months)

120583m

-SH

g p

rote

in

(a)

CC + EC

A

BC

B

DD

A ABAB

CBC

AB

0010203040506070809

1

0 1 2 3 4 5

nM re

activ

e car

bony

lsm

g pr

otei

n

(Months)

(b)































150100

5037

2520

15

10

C C C CM1 M2 M4 M5

MHC

A

T

75


(KD)






4 Conclusions




Acknowledgments


References



























































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


A

B

BC BC BC

D

A A AA A

B

03

04

05

06

07

08

09

0 1 2 3 4 5

nM H

POx

mg

prot

ein

(Months)

CC + EC

(a)

A A

B

B

C

D

AA

B

C

CDD

14

16

18

20

22

24

26

28

30

32

34

0 1 2 3 4 5

mM

MD

Am

g pr

otei

n

(Months)

CC + EC

(b)


CC + EC

ABB

A

B B

AB

ABD

C

ADE

B

0

10

20

30

40

50

60

70

0 1 2 3 4 5(Months)

120583m

-SH

g p

rote

in

(a)

CC + EC

A

BC

B

DD

A ABAB

CBC

AB

0010203040506070809

1

0 1 2 3 4 5

nM re

activ

e car

bony

lsm

g pr

otei

n

(Months)

(b)































150100

5037

2520

15

10

C C C CM1 M2 M4 M5

MHC

A

T

75


(KD)






4 Conclusions




Acknowledgments


References



























































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




























150100

5037

2520

15

10

C C C CM1 M2 M4 M5

MHC

A

T

75


(KD)






4 Conclusions




Acknowledgments


References



























































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





















































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

effect of chitosan edible coating on the biochemical and ... · researcharticle effect of chitosan...

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