at

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Keywords:

Chitosan

ttaCwbilin ale obain

reamyey proe eventh. Reposed

food contact material in Europe is pending (Fernandez, Cava, Ocio,& Lagaron, 2008). Chitosan exhibits its antimicrobial and otherdesirable properties, such as decreased transpiration losses (ElGhaouth, Arul, & Pannampalam, 1991; Jiang & Li, 2001) and

changes throughout a 30-days storage period were monitored todetermine if the lms could lead to an extension of shelf-life.

2. Materials and methods

2.1. Materials

Chitosan, prepared as described by Baxter, Dillon, Taylor, andRoberts (1992) with a degree of N-acetylation 9.0%, was a gift

Abbreviations: CWP, chitosan/whey protein; MAP, modied atmosphere pack-aging; WP, whey protein.* Corresponding author. Tel./fax: 39 0812539473.

Contents lists availab

LWT - Food Science

w.e

LWT - Food Science and Technology 44 (2011) 2324e2327E-mail address: raffaele.porta@unina.it (R. Porta).(Del Nobile, Conte, Incoronato, & Panza, 2009). The current studyinvestigated if an edible chitosan/whey protein (CWP) lm coatingwould be able to delay the appearance of an acid taste andmicrobial growth of Ricotta cheese packaged under MAP.

Chitosan [poly-a-(1-4)-2-amino-2-deoxy-D-glucose] is a hydro-philic biopolymer obtained by alkaline N-deacetylation of chitin(Kurita, 2001) that possesses broad-spectrum antimicrobial prop-erties against fungi, bacteria and viruses (Coma et al., 2002; Liu,Guan, Yang, Li, & Yao, 2001; Rabea, Badawy, Stevens, Smagghe, &Steurbaut, 2003; Rhoades & Roller, 2000). Approval of chitosan as

Ko1odziejska, 2008). Recently, we reported the preparation ofboth chitosan/WP (CWP) (Di Pierro, Chico, Villalonga, Mariniello,Damiao, Masi et al., 2006) and chitosan/ovalbumin lms (DiPierro et al., 2007). These lms showed good visual characteris-tics, and they are likely edible since they are easily degraded byserine proteases.

In the present study, fresh Ricotta cheese was coated withCWP lm and stored under 40% CO2/60% N2 MAP conditions toreduce microbial growth (Dermiki, Ntzimani, Badeka, Savvaidis, &Kontominas, 2008). Cheese microbiological and physicochemicalWhey protein

1. Introduction

Ricotta cheese is an unripened, cby heat-induced coagulation of whperishable and has a limited shelf-lifto textural changes and mold growsphere packaging (MAP) has been pro0023-6438/$ e see front matter 2010 Elsevier Ltd.doi:10.1016/j.lwt.2010.11.031in the chitosan/whey protein coated cheese, compared to the control, at each storage time. Our ndingssuggest a potential utility of chitosan/whey protein coatings to extend fresh dairy product shelf-life.

2010 Elsevier Ltd. All rights reserved.

dairy product obtainedtein (WP). It is highlyunder refrigeration duecently, modied atmo-for packaging of Ricotta

delayed ripening of fruits and vegetables (El Ghaouth,Pannampalam, Castaigne, & Arul, 1992; Jiang & Li, 2001), in chito-san-only lms (Butler, Vergano, Testin, Bunn, &Wiles, 1996; Chen &Hwa, 1996; Kittur, Kumar, & Tharanathan, 1998; Kumar, Bristow,Smith, & Payne, 2000) and in combination with other polymerssuch as pectin (Hoagland & Parris, 1996), methylcellulose (Chen,Yeh, & Chiang, 1996) or proteins (Jia, Fang, & Yao, 2009; Sztuka &Shelf-lifeEdible lm coating(0.34 0.02milliequivalent/100 g of analyzed sample) of the control only on day 30. The viable numbers oflactic acid bacteria andmesophilic and psychrotrophic microorganisms were signicantly lower (p< 0.05)Ricotta cheesecoated Ricotta cheese did not change signicantly during the rst 21 days and reached the acidity levelChitosan/whey protein lm as active co

Prospero Di Pierro a, Angela Sorrentino a, LoredanaRaffaele Porta a,*aDipartimento di Scienza degli Alimenti, Universit di Napoli Federico II, Parco Gussob Institute of Food Research, Norwich Research Park, Colney, NR4 7UA Norwich, UK

a r t i c l e i n f o

Article history:Received 4 August 2010Received in revised form16 November 2010Accepted 23 November 2010

a b s t r a c t

Shelf-life extension of Ricomodied atmosphere at 4

and carbon dioxide permealm prepared with chitosacoated Ricotta cheeses werthe rst two weeks and rem

journal homepage: wwAll rights reserved.ing to extend Ricotta cheese shelf-life

ariniello a, Concetta Valeria L. Giosafatto a,b,

0055 Portici, Napoli, Italy

cheese coated with a chitosan/whey protein edible lm and stored underas evaluated. The chitosan/whey proteinlm had 35% and 21% lower oxygenty, respectively, and about three times higherwater vapor permeability thanone. Over a 30-day storage period, no differences in the pH of control andserved. While the titratable acidity of the control increased linearly duringed constant for the rest of the storage period, the corresponding values for

le at ScienceDirect

and Technology

lsevier .com/locate/ lwt

for 10min and the obtained supernatantswere ltered through bothcotton lint and paper lter.

Measurement of pH was carried out using a digital pH meter,model 211 (Hanna instruments, PBI International), according toAOAC procedures (AOAC, 1990).

2.6. Titratable acidity

Samples of 10 g of Ricotta cheese were added to 50 mL of

e and Technology 44 (2011) 2324e2327 2325from Professor R. A. A. Muzzarelli (University of Ancona, Italy).Spray-dried whey from bovine milk (product no. W1500, lot no.81K0279), containing 11% w/w proteins (biuret) and 65% w/wlactose, was purchased from Sigma Chemical Co. (St. Louis, MO).Buffered Peptone Water (BPW), Plate Count Agar (PCA) and deMan Rogosa Sharpe Agar (MRS)media formicrobiological analyseswere from Merck (Darmstadt, Germany). All other chemicals wereanalytical grade.

2.2. Preparation of CWP lm forming solution

A CWP lm forming solution, containing 0.8% w/v chitosan and2.4% w/v lyophilized milk whey (11% WP), was prepared by mixingof chitosan dissolved in 0.1 N HCl and lyophilized milk whey dis-solved in distilled water under continuous stirring. The pH of thechitosan-whey solution was adjusted to pH 5.0 with 0.1 N HCl, andthe solution was lter-sterilized through a Sterilcup ltrationsystem (0.45 mm cut off, Millipore, Bedford, MA, USA).

2.3. Film formation and characterization

Films were prepared by casting 32.5 mL of either chitosan (0.8%w/v) or CWP (0.8% w/v chitosan and 2.4% w/v lyophilized milkwhey) solutions into polystyrene Petri dishes (60 15 mm). Thesolutions were previously de-aerated under vacuum in order toprevent small bubbles which could form pinholes in the nishedlms, and then transferred into Petri dishes to be dried at 50 Covernight under air circulation. The lms were peeled from thePetri dishes and stored at 20 C in a desiccator (50% RH). Filmthickness was measured using a micrometer model HO62 withsensitivity of 2 mm (Metrocontrol Srl, Casoria (NA), Italy). Filmwater vapor permeability (WVP) was evaluated by gravimetric test(Di Pierro et al., 2010) according to ASTM E96 (ASTM,1993). Oxygenand carbon dioxide barrier properties were examined at 25 C and50% relative humidity by using a Multiperm Oxygen and CarbonDioxide Analyzer (ExtraSolution s.r.l., Pisa, Italy).

2.4. Ricotta cheese packaging

Ricotta cheese was obtained from a local dairy manufacturer(Casa Madaio s.r.l., Castelcivita, Salerno, Italy) both in June and inSeptember 2009 and three different lots of production were sub-jected to two different shelf-life experiments. One set of Ricottacheese samples, supplied within 4 h from their production ina traditional polypropylene thermoformed colander (net weightapproximately 100 g), was dipped 30 s into the sterile CWP lmforming solution. Excess solution was allowed to drain from thesurface of the samples for 30 s before they were placed into plastictrays and packaged under modied atmosphere. Both dipped andundipped (control) cheeses were placed into plastic trays andpackaged under modied atmosphere (40% CO2/60% N2 mixture),as described by Dermiki et al. (Dermiki, Ntzirnani, Badeka,Savvaidis & Kontominas, 2008), by using an OS 600 INOX VGP(Ormad macchine s.n.c., Terlizzi, Bari, Italy) and nally stored at4 0.5 C. After 7, 14, 21 and 30 days of storage, duplicate samples(5e10 g) from both CWP-coated and uncoated (control) Ricottacheeses were taken with pre-sterilized cork-borers.

2.5. pH measurement

Samples of Ricotta cheese (10 g each) were homogenized, at themaximumspeed for 5min, in 100mLof distilledwaterwith anUltra-Turrax T8 homogenizer (IKA-WerkeGmbH, Staufen, Germany). After

P. Di Pierro et al. / LWT - Food Sciencstirring for 15 min, the homogenates were centrifuged at 3000 gdistilled water and homogenized for 5 min at the maximum speedwith an Ultra-Turrax T8 homogenizer. The homogenates were rstheated, under stirring, until a temperature of 40 C was reachedand then diluted to the nal volume of 150 mL with distilledwater. After centrifugation at 3000 g for 10 min, the superna-tants were ltered through both cotton lint and paper lter. Then,5 drops of phenolphthalein (1% in ethanol) were added to 25 mLof ltered supernatant and the titratable acidity (TA) was deter-mined by addition of 0.1 N NaOH until the solution became pink.The TA, expressed as milliequivalent/100 g, was calculated asfollow:

TA a b 100=cwhere a and b correspond to the concentration and the volume oftitrant solution, respectively, and c refers to the grams of analyzedsample.

2.7. Microbiological analysis

Samples of Ricotta cheese (5 g each) were aseptically transferredinto sterile plastic falcon tubes and homogenized in 45 mL of 0.1%(w/v) sterile BPW for 5 min, at room temperature and at themaximum speed, by an Ultra-Turrax T8 homogenizer. The volumewas adjusted to 50 mL and then serial decimal dilutions, preparedwith 0.1% (w/v) sterile BPW,were inoculated in duplicate on growthmedia for the estimation of aerobic plate counts (APC). The micro-biological analyses were carried out according to the pour platemethod (APHA, 1992).

Both mesophilic and psychrotrophic microorganisms wereenumerated on PCA plates which were separately incubated underdifferent experimental conditions (at 30 C for 48 h for the meso-philic microora count; at 12 C for 4e5 days for the psychrotrophicdetermination). Lactic acid bacteria were determined on MRSplates according to the pour plate method, with overlay, afterincubation at 30 C for 2e3 days.

2.8. Sensory evaluation

A simple, unstructured sensory evaluation was performed byseven trained individuals every time the samples were submittedto microbiological analyses. Colour, avour, odour and texture werethe selected parameters. The tasters evaluated the differentparameters by using a hedonistic scale from 0 to 4 (0 unaccept-able; 1 poor; 2 fair; 3 good; 4 very good).

Table 1Barrier properties of lms obtained by casting chitosan or chitosan/whey proteinsolution.

Film Thickness(mm)

Permeability(g mm m2 day2 kPa 2)

O2 CO2 Water vapor

Chitosan 55.3 0.4 32.8 0.7 22.5 0.9 0.4 0.3Chitosan/whey protein 61.5 0.3 21.3 0.9 17.7 1.2 1.3 0.2The results are expressed as means of ten samples SD. Further experimentaldetails are given in the text.

also could have provided some buffering effect (Salaun, Mietton, &Gaucheron, 2005). The pH values remained relatively constant untilday 30 (Fig. 1).

The TA showed a different trend in CWP-coated than inuncoated cheese (Fig. 2). In the control sample, it increased linearlyduring the rst twoweeks and then remained stable, whereas it didnot change signicantly in CWP-coated cheese until day 21 andthen reached the same level as measured for the control sample onday 30. The increase in TA was likely caused by organic acidproduction primarily by lactic acid producing bacteria whichincreased during storage (Fig. 3-C). The CWP-coated cheesesshowed only a slight increase in viable lactic acid bacteria duringthe rst three weeks which could explain the constant TA values

6.2

6.3

6.4

6.5

6.6

6.7

6.8

0 5 10 15 20 25 30Storage time (days)

pH

Fig. 1. Effect of CWP coating on the evolution of pH during Ricotta cheese storage at4 C under modied atmosphere (40% CO2/60% N2). C, uncoated samples (control);B, CWP-coated samples. Data are the means SD of two different experiments run on

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P. Di Pierro et al. / LWT - Food Science and Technology 44 (2011) 2324e232723262.9. Statistical analysis

JMP software 5.0 (SAS Campus Drive, Building S, Cary, NC, USA)was used for all statistical evaluations. Data were subjected toanalysis of variance and statistical differences at a given samplingtime were determined using the TukeyeKramer HSD test. Differ-ences were considered to be signicant at p < 0.05.

3. Results and discussion

We hypothesized that CWP lm would be useful as coatingmaterial for Ricotta cheese since it could combine the antimicrobialactivity of chitosan with desirable oxygen, carbon dioxide andwater vapor permeability (Table 1). The coating would allow for themovement of water vapor across the lm, thus preventing watercondensation and, as a consequence, microbial spoilage. The pHvalues of control and CWP-coated Ricotta cheeses during storageare shown in Fig. 1. The pH values decreased, after 7 days of storagelikely because of lactic acid synthesis by Lactobacillus spp. (Dermikiet al., 2008; Whitley, Muir, & Waites, 2000) and acidic amino acidsand free fatty acids formation due to proteolysis and lipolysis. Inaddition, dissolution of some of the CO2 in the modied atmo-sphere (approximately 15% during the rst 5 days of storage) mighthave contributed to the pH decrease (Daniels, Krishnamurthi, &

different occasions (June and September, see Materials and methods).Rizvi, 1985; Farber, 1991; Gonzalez-Fandos, Sanz, & Olarte, 2000;Olarte, Gonzalez-Fandos, Gimenez, Sanz, & Portu, 2002), but it

0.20

0.25

0.30

0.35

0.40

0 5 10 15 20 25 30Storage time (days)

TA

(m

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Fig. 2. Effect of CWP coating on titratable acidity (TA) during Ricotta cheese storage at4 C under modied atmosphere (40% CO2/60% N2). C, uncoated samples (control);B, CWP-coated samples. Data are the means SD of two different experiments run ondifferent occasions (June and September, see Materials and methods).

5.05.56.06.5Storage time (days)

2.02.53.03.54.04.5

0 5 10 15 20 25 30

Fig. 3. Effect of CWP coating on the development of mesophilic (A), psychrotrophic (B)and lactic acid (C) bacteria during Ricotta cheese storage at 4 C under modiedatmosphere (40% CO2/60% N2). C, uncoated samples (control); B, CWP-coated

samples. Data are the means SD of two different experiments run on differentoccasions (June and September, see Materials and methods).

during the rst 21 days (Fig. 2). As the number of lactic acid bacteriaincreased (Fig. 3-C), the TA also increased (Fig. 2). In both samplesmaximum TA was observed when the lactic acid bacteria were inthe range of 5e6 log cfu/g and this occurred at day 14 for thecontrol and at day 30 for the CWP-coated cheese.

The viable numbers of mesophilic and psychrotrophic micro-organisms were signicantly lower (p < 0.05) in CWP-coated than

Coma, V., Martial-Gros, A., Garreau, S., Copinet, A., Salin, F., & Deschamps, A. (2002).Edible antimicrobial lms based on chitosan matrix. Journal of Food Science, 67,1162e1169.

Daniels, J. A., Krishnamurthi, R., & Rizvi, S. S. H. (1985). A review of the effects ofcarbon dioxide on microbial growth and food quality. Journal of Food Protection,48, 532e537.

Del Nobile, M. A., Conte, A., Incoronato, A. L., & Panza, O. (2009). Modied atmo-sphere packaging to improve the microbial stability of Ricotta. African Journal ofMicrobiology Research, 3(4), 137e142.

Dermiki, M., Ntzimani, A., Badeka, A., Savvaidis, I. N., & Kontominas, M. G. (2008).

P. Di Pierro et al. / LWT - Food Science and Technology 44 (2011) 2324e2327 2327in control samples (Fig. 3-A and -B). In fact, the microbiologicallimit of acceptability, 7 log cfu/g (ICMSF, 1986), was reached incontrol samples within 7 days for mesophilic bacteria (Fig. 3-A),between days 7 and 14 for psychrotrophs (Fig. 3-B), and betweendays 14 and 21 for lactic acid bacteria (Fig. 3-C). In contrast, the limitwas never reached in CWP-coated Ricotta cheeses. Thus, CWP lmmight be an effective coating to extend Ricotta cheese shelf-life.

No differences in visual appearance, texture, avor and odorbetween uncoated and CWP lm-coated Ricotta cheese sampleswere detected during sensory evaluation (data not shown).Furthermore, CWP-coated Ricotta cheese maintained its texturebetter than the control cheese.

4. Conclusions

Coating of Ricotta surface with a CWP edible lm reducedgrowth of microbial contaminants and extended the shelf-life ofthe product packed under modied atmosphere. The coatingdelayed the development of undesirable acidity, better maintainedthe texture and did not seem to modify sensory characteristics. It ispossible that the benets derived from the biopolymers can also berealized with other dairy products.

Acknowledgements

The authors would like to thank Mrs. Maria Fenderico for herskilful technical assistance. This research was supported by a grantto R.P. from POR 2000e2006 Regione Campania Asse prioritario diriferimento 3 e Risorse Umane- Misura 3.16 Promozione dellaRicerca e del Trasferimento Tecnologico nei settori connessi allacrescita ed allo sviluppo sostenibile del sistema Campania.

References

AOAC (1990). Ofcial methods of analysis (15th ed.). Washington, DC, USA:Association of Ofcial Analytical Chemists.

APHA (1992). Compendium of methods for the microbiological examination of foods.Washington: American Public Health Association.

ASTM (1993). Standard test methods for water vapor Transmission of Materials.E96-93. In Annual book of ASTM standards (pp 701e708). Philadelphia, PA:American Society for testing and materials.

Baxter, A., Dillon, M., Taylor, K. D. A., & Roberts, G. A. F. (1992). Improved method forIR determination of the degree of N-acetylation of chitosan. InternationalJournal of Biological Macromolecules, 14, 166e169.

Butler, B. L., Vergano, P. J., Testin, R. F., Bunn, J. N., & Wiles, J. N. (1996). Mechanicaland barrier properties of edible chitosan lms as affected by composition andstorage. Journal of Food Science, 61, 953e955.

Chen, M., Yeh, G. H., & Chiang, B. J. (1996). Antimicrobial and physicochemicalproperties of methylcellulose and chitosan lms containing a preservative.Journal of Food Processing and Preservation, 20, 379e390.

Chen, R. H., & Hwa, H. (1996). Effect of molecular weight of chitosan with the samedegree of deacetylation on the thermal, mechanical, and permeability proper-ties of the prepared membrane. Carbohydrate Polymers, 29, 353e358.Shelf-life extension and quality attributes of the whey cheese Myzithra Kala-thaki using modied atmosphere packaging. LWT-Food Science and Technology,41, 284e294.

Di Pierro, P., Chico, B., Villalonga, R., Mariniello, L., Damiao, A. E., Masi, P., et al.(2006). Chitosan-whey protein edible lms produced in the absence orpresence of transglutaminase: analysis of their mechanical and barrier prop-erties. Biomacromolecules, 7, 744e749.

Di Pierro, P., Chico, B., Villalonga, R., Mariniello, L., Masi, P., & Porta, R. (2007).Transglutaminase-catalyzed preparation of chitosaneovalbumin lms. Enzymeand Microbial Technology, 40, 437e441.

Di Pierro, P., Mariniello, L., Sorrentino, A., Villalonga, R., Chico, B., & Porta, R.(2010). Putrescine-polysaccharide conjugates as transglutaminase substratesand their possible use in producing crosslinked lms. Amino Acids, 38,669e675.

El Ghaouth, A., Arul, J., & Pannampalam, R. (1991). Use of chitosan coating to reducewater loss and maintain quality of cucumbers and bell pepper fruits. Journal ofFood Processing and Preservation, 15, 359e368.

El Ghaouth, A., Pannampalam, R., Castaigne, F., & Arul, J. (1992). Chitosan coating toextend the storage life of tomatoes. Hortscience, 27, 1016e1018.

Farber, J. M. (1991). Microbiological aspects of modied atmosphere packagingtechnology: a review. Journal of Food Protection, 54, 58e70.

Fernandez, A., Cava, D., Ocio, M. J., & Lagaron, J. M. (2008). Perspectives for bio-catalysts in food packaging. Trends in Food Science and Technology, 19,198e206.

Gonzalez-Fandos, E., Sanz, S., & Olarte, C. (2000). Microbiological, physicochemicaland sensory characteristics of Cameros cheese packaged under modiedatmospheres. Food Microbiology, 17, 407e414.

Hoagland, P. D., & Parris, N. J. (1996). Chitosan/pectin laminated lms. Journal ofAgriculture and Food Chemistry, 44, 1915e1919.

ICMSF (1986). Sampling for microbiological analysis: Principles and scientic appli-cations (2nd ed.). Toronto, Canada: International Commission on Microbiolog-ical Specications for Foods.

Jia, D., Fang, Y., & Yao, K. (2009). Water vapor barrier and mechanical properties ofkonjac glucomannanechitosanesoy protein isolate edible lms. Food and Bio-products Processing, 87, 7e10.

Jiang, Y., & Li, Y. (2001). Effects of chitosan coating on postharvest life and quality oflongan fruit. Food Chemistry, 73, 139e143, (5).

Kittur, F. S., Kumar, K. R., & Tharanathan, R. N. (1998). Functional packaging prop-erties of chitosan lms. Z Lebensm Unters Forsch A, 206, 44e47.

Kumar, G., Bristow, J. F., Smith, P. J., & Payne, G. F. (2000). Enzymatic gelation of thenatural polymer chitosan. Polymer, 41, 2157e2168.

Kurita, K. (2001). Controlled functionalization of the polysaccharide chitin. Progressin Polymer Science, 26, 1921e1971.

Liu, X. F., Guan, Y. L., Yang, D. Z., Li, Z., & Yao, K. D. (2001). Antibacterial action ofchitosan and carboxymethylated chitosan. Journal of Applied Polymer Science,79, 1324e1335.

Olarte, C., Gonzalez-Fandos, E., Gimenez, M., Sanz, S., & Portu, J. (2002). The growthof Listeria monocytogenes in fresh goat cheese (Cameros cheese) packagedunder modied atmospheres. Food Microbiology, 19, 75e82.

Rabea, E. I., Badawy, M. E. I., Stevens, C. V., Smagghe, G., & Steurbaut, W. (2003).Chitosan as antimicrobial agent: applications and mode of action. Bio-macromolecules, 4, 1457e1465.

Rhoades, J., & Roller, S. (2000). Antimicrobial actions of degraded and nativechitosan against spoilage organisms in laboratory media and foods. AppliedEnvironmental Microbiology, 66, 80e86.

Salaun, F., Mietton, B., & Gaucheron, F. (2005). Buffering capacity of dairy products.International Dairy Journal, 15, 95e109.

Sztuka, K., & Ko1odziejska, E. I. (2008). Effect of transglutaminase and EDC onbiodegradation of sh gelatin and gelatin-chitosan lms. European FoodResearch and Technology, 226, 1127e1133.

Whitley, E., Muir, D., & Waites, W. M. (2000). The growth of Listeria monocytogenesin cheese packed under modied atmosphere. Journal of Applied Microbiology,88, 52e57.

Chitosan/whey protein film as active coating to extend Ricotta cheese shelf-life1 Introduction2 Materials and methods2.1 Materials2.2 Preparation of CWP film forming solution2.3 Film formation and characterization2.4 Ricotta cheese packaging2.5 pH measurement2.6 Titratable acidity2.7 Microbiological analysis2.8 Sensory evaluation2.9 Statistical analysis

3 Results and discussion4 Conclusions Acknowledgements References