nisin-loaded chitosan biopreservative for cheese

NISIN-LOADED CHITOSAN/ALGINATE NANOPARTICLES:A HOPEFUL HYBRID BIOPRESERVATIVEMARYAM ZOHRI1,8, MOHAMMAD SHAFIEE ALAVIDJEH2, SEYED SAEED MIRDAMADI3, HOMA BEHMADI4,SEYED MEHDI HOSSAINI NASR2, SIMA ESHGHI GONBAKI5, MEHDI SHAFIEE ARDESTANI6 andALI JABBARI ARABZADEH7

1Department of Food Science and Technology, Science and Research Branch, Islamic Azad University, Tehran, Iran2Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Science, Tehran, Iran3Iranian Research Organization Science & Technology, Biotechnology Department, Tehran, Iran4Department of Food Engineering and Post-Harvest Technology Research, Agricultural Engineering Institute, Karaj, Iran5Department of Engineering, Science and Research Branch, Islamic Azad University, Tehran, Iran6Research and Development and Hepatitis B Department, Research and Production Complex, Pasteur Institute of Iran, Tehran, Iran7Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran

8Corresponding author.TEL: +989364593253;FAX: +982612704532;EMAIL: [email protected]

Received for Publication August 29, 2012Accepted for Publication December 18, 2012

doi: 10.1111/jfs.12021

ABSTRACT

To improve the deficiencies concerning the physicochemical instability of nisin, ahybrid of nisin at concentration of 450 IU/mL with chitosan/alginate nanopar-ticles was prepared. Antibacterial strength of the hybrid was compared with freenisin against Listeria monocytogenes ATCC 25923 and Staphylococcus aureus ATCC19117 in ultra filtered (UF) Feta cheese. The effects of nisin and the nisin-loadednanoparticles on the chemical composition, rheological parameters, color indicesand sensory attributes of UF Feta cheese were studied. Antibacterial experimentsindicated that the nisin-loaded nanoparticles were able to decrease the popula-tions of S. aureus and L. monocytogenes up to five- and sevenfold on a logarithmicscale in comparison with free nisin, respectively. Sensory acceptance and physico-chemical features of UF Feta cheese were also significantly improved using thenisin-loaded nanoparticles as compared with those of free nisin. Overall, greaterantibacterial strengths and less undesirable influences of this hybrid than those offree nisin on the original quality of UF Feta cheese would make this hybrid apromising biopreservative in dairy products.

PRACTICAL APPLICATIONS

This study investigates the use of chitosan/alginate nanoparticles as an auxiliaryadjuvant in food preservation process. Considering our former promising anti-bacterial strength observed for this hybrid against S. aureus in the milk samplesand also our new findings, it can be concluded that this hybrid would actually bean effectual potential biopreservative against common foodborne pathogenswithout any harmful side effects on the original qualities of the assessed dairyproducts. These outstanding features would be an incentive for further futureinvestigation and probable industrialization of this hybrid as a highly productivebiopreservative in food preservation technology.

INTRODUCTION

Today, nanotechnology is incorporating into all fields ofscience; it can not be an exception for biosciences-relatedrealms. One of these domains affected by this hopeful tech-nology is food science. Extension of the shelf life of food

products is one of the pivotal purposes in food technology. Itis usually achieved by use of efficient and optimum amountof chemical and/or biological antimicrobial agents during themanufacturing process. Nowadays, making use of biopreser-vatives in food industry realm is gradually becoming currentand established (Mauriello et al. 2005). One class of these

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Journal of Food Safety ISSN 1745-4565

40 Journal of Food Safety 33 (2013) 40–49 © 2013 Wiley Periodicals, Inc.

biological preservatives which has just become widespread isthe group of antimicrobial proteins. An example of this classis nisin which is considered an effective antimicrobial agentagainst a wide range of gram-positive and some gram-negative bacterial strains (Stevens et al. 1991; Kramer et al.2004). Nevertheless, nisin and the other antimicrobialproteins/peptides are readily denatured by the proteaseenzymes acting commonly in the acidic rich environment ofthe food materials. In addition, they also easily react withother ingredients inside of the food matrices like lipids and soforth. These interactions can possibly give rise to diminutionof intrinsic antimicrobial strength and bioavailability ofthese entities and ultimately their imperfect actions (Liu andHansen 1990). Regarding these obstacles, finding a way toreduce these disadvantages would be beneficial. One of theways which has been proposed recently as a possible strategyto overcome these deficiencies is the encapsulation approachfor this nature-originated peptides and proteins by usingsuitable polymeric micro and nanoparticle systems (Wanet al. 1997). Nanoparticle systems are displaying variouspotential applications in food technology realms such as con-trolled release of the active components, particle coating,flavor stabilization, taste masking and improvement of theshelf life through the physical/chemical stabilization of theactive constituents involved in this regard. Among the micro-bial pathogens which decrease the shelf life of food productslies Listeria species. This pathogen is regarded as a publichealth concern due to the severity of its pathogenesisstrength, its prevalence among the immunosuppressed andits high case-fatality rate (Schmidt et al. 2009). For instance,Listeriosis disease is a case caused by a gram-positive nons-porulating bacillus i.e., Listeria monocytogenes. In addition,L. monocytogenes is psychotropic and can even grow at refrig-eration temperatures; therefore, subsequent contaminationby this organism could be regarded as a high risk for the con-sumers (Benedito et al. 2000). Considering the problemsrelating to this microbial pathogen, many researchers havefocused and performed experiments against this pathogenicbacterium until now with the aim of considerable reductionof its harmful effects in dairy products. In a study, noticeablesensitivity of L. monocytogenes to nisin was observed (Harriset al. 1998). Another study also proved antimicrobial pro-perty of nisin when added to cottage cheese (Benkerroumand Sandine 1998). Growth inhibition of L. innocua was alsoobserved in Cheddar cheese by either adding nisin Z to aliposome or by its in situ production in mixed culture(Benech et al. 2002). It was found that encapsulation of nisinZ in liposomes can provide a new strategy for improvementof the physicochemical stability of nisin by protection ofnisin from the detrimental action of the proteases being pro-duced from the starter during the process of cheese making,which causes the partial inactivation of the function of nisin.A research concerning the use of calcium alginate micro-

particles containing nisin showed the same activity as freenisin against an indicator culture of Lactobacillus curvatusboth in De Man, Rogosa and Sharpe medium broth andreconstituted skim milk (Wan et al. 1997). Suppression ofL. monocytogenes colonization following the adsorption ofnisin onto silica surfaces also revealed the antimicrobialefficiency of nisin against this bacterium (Bower et al.1995). Staphylococcus aureus is regarded as the other mostepidemic bacteria being one of the reasons for respiratoryinfections or enteritis. Moreover, it can give rise to post-contamination of dairy food preparations during thestorage time; consequently, it is necessary to inhibit itsgrowth in dairy products for more safety and better quality.Growth inhibitory effect of nisin-containing modified algi-nate films/beads was evaluated on S. aureus by Millette et al.(2007). Based on the results, it was suggested that sterile,hydrophobic and biodegradable films or beads containingdifferent concentration of nisin could conceivably be usedto control the growth of pathogens or microorganismsresponsible for spoilage at the surface of round beef orother meat products. A comparative study between the anti-bacterial effect of nisin and nisin-loaded chitosan/alginatenanoparticles was recently performed on the growth ofS. aureus in raw and pasteurized milk samples (Zohri et al.2010). Twofold increase in the antibacterial strength wasobserved for the nisin-loaded nanoparticles as against freenisin at concentration of 450 IU/mL. These outcomesresults from the more penetrability and slow liberation ofnisin from chitosan/alginate nanoparticles during storageand consequently its gradual action over more time periodscompared with the samples lacking the nanoparticles.Grounded on the promising results reported from ourformer study and also different research studies concerningthe use of biocompatible polymeric nanoparticles, it wasintended to compare the antimicrobial strength of theoptimized nisin-loaded chitosan/alginate nanoparticleswith free nisin in the ultra filtered (UF) Feta cheese as well.For this purpose, the growth inhibitions of S. aureus andL. monocytogenes were investigated in the UF Feta cheeseas the two prevalent microbes usually being responsiblefor post-contamination in such products. Microstructurefeatures, sensory specifications and rheological propertiesof the UF Feta cheese containing nisin or nisin-loadedchitosan/alginate nanoparticles were also examined. Theseinvestigations are also crucial and should be examinedbecause they undoubtedly have great impact on the qualityand marketing of the food products. It is hoped that thesefindings can help establish a reliable new way to improvethe antimicrobial strength of nisin with the contributionand synergisms from these biocompatible nanoparticles.These new hybrid biopreservatives may be employed forextending the shelf life of food products showing high riskof post-contamination over the period of storage.

M. ZOHRI ET AL. NISIN-LOADED CHITOSAN/ALGINATE NANOPARTICLES

41Journal of Food Safety 33 (2013) 40–49 © 2013 Wiley Periodicals, Inc.

MATERIALS AND METHODS

Materials and Instruments

Acetic acid (Merck Co., Darmstadt, Germany), sodium algi-nate (BDH Co., London, U.K.), low molecular weight chito-san (CAS No. 9012-76-4; Sigma-Aldrich Co., St. Louis,MO), Coomassie Brilliant Blue G-250 (Sigma Chemical Co.,Perth, WA), phosphoric acid 85% (Sigma-Aldrich Co.),nisin (Nisaplin containing 2.5% w/w of nisin, Sigma-Aldrich Co.), starter culture (Choozit MR804 FRO 500DCU; Danisco Co., Denmark) and Listeria selective agarbase (Oxoid Co., Basingstoke, U.K.) were purchased andused as received. S. aureus ATCC 19117 and L. monocytoge-nes ATCC 25923 were prepared from Persian Type Collec-tion of Culture (PTCC) in Iranian Research Organizationfor Science and Technology. Baird–Parker agar was boughtfrom Difco Co. (Detroit, MI). The Universal TestingMachine (S-Series Bench U.T.M. Model H5KS, HounsfieldTest Equipment Ltd., Redhill, U.K.), scanning electronmicroscope (SEM; LEO440I, 10 kV, U.K.), Hunter lab Colo-rimeter Model D25-DP9000 (Hunter lab Associates Inc.,Reston, VA), Digital pH-meter (pH 537; Wissenschaftlich-Technische Werkstätten GmbH, Weilheim in Oberbayern,Germany), and Pelco-CPD-2 critical point drier (Ted PellaCo., Redding, CA) were also applied for executing theexperiments.

Preparation and Characterization ofNisin-Loaded Chitosan/AlginateNanoparticles

Nisin-loaded chitosan/alginate nanoparticles were preparedand physicochemically characterized according to our pre-vious study (Zohri et al. 2011). The nisin-loaded nanopar-ticles were prepared from nisin with optimum activity atfinal concentration of 450 IU/mL and with a mean size of205 nm by a two-step procedure adapted from Rajaonar-ivony’s method of preparing poly-L-lysine nanoparticles(Rajaonarivony et al. 1993). Briefly, for preparing the nano-particles, 540 mL of the nisin solution (10 mg/mL) wasadded dropwise to 8 mL aqueous solution of sodium algi-nate (250 mg/mL) and stirred for 30 min. Then, 4 mL of thechitosan stock solution (250 mg/mL in 1% v/v acetic acidsolution) was added to the resulting alginate solution andstirred for another 1 h. The nanoparticles were obtained bycentrifugation at 2,500 g for 10 min at 4C to separate thefree polymers from the nanoparticles (Zohri et al. 2010).

Culture Maintenance and Revival Procedures

L. monocytogenes ATCC 25923 and S. aureus ATCC 19117strains were obtained from PTCC. Revival process was

accomplished by three sequential subcultures. The bacterialstrains were cultivated in 20% w/v glycerol and skim milk15%. After 48 h of incubation, the population of the bacte-ria was measured with a spectrophotometer. The populationof 106 cfu/mL was used for the inoculation of the cheesesamples (Schmidt et al. 2009).

Cheese-Making Procedure

The UF Feta cheese samples were prepared according to theinstruction of Tetra Pak Company (Bylund 1995). Accord-ingly, the milk samples were initially bactofugated, past-eurized (72C for 15 s), ultra filtrated, homogenized andpasteurized (80C for 20 s) for the second time. The pH ofmilk reached the level of 6.2 by adding the starter (1.0 g for10 kg of retentate). Then, inside the cheese blocks, rennet(�2,200 international milk-clotting units/g) was mixedwith water (2 g for 100 kg of retentate) and used for coagu-lation process. Before coagulation, 106 cfu/mL of L. monocy-togenes and S. aureus and 2 mL (450 IU/mL) of nisin-loadedchitosan/alginate nanoparticles were added to the block ofUF milk including starter. They were then incubated at 37Cfor 24 h and then stored at 4C for another 24 h. The pre-pared samples were the cheese plus L. monocytogenes andor/S. aureus as the controls, the cheese + L. monocytogenesand or/S. aureus + nisin, and the cheese + L. monocytogenesand or/S. aureus + nisin-loaded nanoparticles in three repli-cates. Then, the tests were performed at the determinedtime intervals.

Cheese Sampling Method forMicrobial Counts

The cheese samples (1 g) were vortexed with 9 mL of sterilesodium citrate solution (2% w/w) in triplicate as describedby Sipahioglu et al. (1999). Samples (three types at differenttimes) were again serially diluted by sodium citrate to 0.1 oftheir initial amounts. Then, 1 mL of the final dilution wasplated in triplicate on Listeria Selective agar (tryptose,10.0 g; yeast extract, 5.0 g; beef extract, 5.0 g; sodium chlo-ride, 20.0 g; disodium hydrogen phosphate, 1.35 g; esculin,1.00 g; nalidixic acid, 0.02 g; acriflavin hydrochloride).Plates were then incubated at 37C for 48 h and after thatwere ready for counting L. monocytogenes population.Necessary dilutions were done on Baird–Parker agar to enu-merate S. aureus population inside the cheese samples(Romeih et al. 2002).

Microbial Assay

The populations of L. monocytogenes and S. aureus inside ofthe cheese samples were measured according to the methodsof Sipahioglu et al. (1999) Romeih et al. (2002), respectively.

NISIN-LOADED CHITOSAN/ALGINATE NANOPARTICLES M. ZOHRI ET AL.


Color Indices Measurement

For color indices assessments, Hunter lab ColorimeterModel D25-DP9000 was calibrated with black and whitereference standard sheets and used for running the experi-ments. The samples with a height of 1.5 cm were placedonto the standard sheets with the following specification:Y = 83.32, X = 81.26 and Z = 98.03. The scale used wasbased on CIELAB system in which L* was a representativefor luminance ranging from 0 (black) to 100 (white); a*(green to red); b* (blue to yellow) spanning from -120 to+120. Hue angle (h°) was calculated based on the followingformula i.e., h° = arc tan (b*/a*). Chroma (C*) was deter-mined by this formula i.e., C* = [(a*)2 + (b*)2]1/2 and thedifference i.e., DE is a single number that measures thedistance between the two colors (DE) was calculatedby the next formula i.e., DE = [(L* - L*0)2 + (a* - a*0)2 +(b* - b*0)2]1/2 with L*0, a*0 and b*0 are the color indices forthe control cheese samples at time zero (Lawless andHeymann 1998). Mean values and standard deviation werecalculated for nine runs.

Chemical Analysis

Chemical composition including fat content, pH, proteincontent, ash content, moisture and total nitrogen/protein ofthe cheese samples were analyzed in untreated or treatedsamples with nisin or nisin-loaded chitosan/alginate nano-particles at days 0 and 14. The pH of the cheese sampleswas measured using a digital pH-meter (microprocessorpH meter, model pH 537; Wissenschaftlich-TechnischeWerkstätten GmbH). The cheese samples were analyzed formoisture content by vacuum-oven method (Association ofOfficial Analytical Chemists; AOAC 2000). For measuringthe ash contents of the samples, 10 g of the samples wereaccurately weighed in a crucible and dried by hot air oven at105C. The samples were then ignited on a flame and finallyturned into ash in a furnace at 550C to reach a constantweight while white ash was left (AOAC 2000). The fat contentof the cheese samples was determined by the Gerber method(James 1995). Total nitrogen amounts were determined byKjeldahl method (AOAC 1997). The total nitrogen calcu-lated was then used for determination of protein contentby multiplying the total nitrogen by 6.38. All chemical mea-surements were done in triplicate (Romeih et al. 2002).

Uniaxial Constant Speed Compression Test

Uniaxial compression is a standard technique for evaluationof mechanical and fracture properties and one of the severalways for the primary assessment of cheese texture quality(Wium and Qvist 1997). The stress at yield point andmaximum force at fracture point as the index for hardness

evaluation were measured form the compression curves andconducted by means of a HTE Universal Testing Machine(S-Series Bench U.T.M. Model H5K-S; Hounsfield TestEquipment Ltd.) with a 500-N load cell. The cheese sampleswere prepared in cubic shape with 20 mm3 volume each.Samples used were taken from approximately a depth of2 cm in each of the cheese blocks and kept at 12C prior toanalysis. The samples under study were uniaxially com-pressed up to 50% of their initial height at compression rateof 20 mm/min (Sipahioglu et al. 1999). Each sample wasanalyzed in six repetitions. Force-displacement data wereemployed for calculation of stress and Hencky strain(eHencky) and stress at yield point was obtained for the stress-Hencky strain curve. H0 and Ht are regarded as sampleheights before and after deformation at times zero and taccording to the following formula

εHencky = ( )LnH

H

t

0(1)

Sensory Assessment

Sensory attributes examinations were conducted on thecontrol UF Feta cheese and the treated UF Feta cheesesamples containing nisin and/or nisin + chitosan/alginatenanoparticles. Properties such as flavor, aroma, oral textureand overall acceptability were measured and compared witheach other. These experiments were carried out by 14 expe-rienced panelists through a 5-point scale hedonic test with1 being bad and 5 being excellent and the rest were inbetween (Foegeding et al. 2003). The samples of about 30 gin duplicate in three batches were prepared and examinedconcurrently for each of the sensory experiments at20 � 3C. Panelist employed water and unsalted crackers toclean their palates between the runs. In order to keep awayfrom any misjudgment on final decision, the experimentswere conducted under red lighting.

SEM Study of the Morphologyof Nanoparticles

The cheese samples were prepared and the microstructurewas investigated microscopically by SEM grounded on theSipahioglu et al. (1999) method. The cheeses were slicedinto 4–5 mm3 cubes and fixed overnight in 2.8% w/v glut-araldehyde solution. The cheese samples were then rinsedwith distilled water for 1 min three times. Graded ethanolseries (20, 40, 60, 80, 95 and 100%) was employed for dehy-dration process of the cheese samples for 30 min each.Chloroform was used for defatting of the cheese samplesfor 15 min. Defatted samples were refrigerated until freeze-fractured in liquid nitrogen. Drying of the freeze-fracturedsamples was carried out using a Pelco-CPD-2 critical point



drier (Ted Pella Inc., Redding, CA). The samples were thencoated with 30 nm gold-palladium and the micrographswere taken by JEOL SEM (SLEO 440 I, 10 kV, U.K.).

Statistical Analysis

The data were analyzed through full factorial design withJMP software SAS Inc. The probabilities of P < 0.05 andP < 0.01 were regarded as being significant and highlysignificant, respectively.

RESULTS AND DISCUSSION

Microbial Evaluation

Population counts of S. aureus and L. monocytogenes werecarried out at time intervals of 0, 7, 14, 28 and 35 (in days)as depicted in Figs. 1 and 2, respectively. As is shown inFig. 1, twofold inhibition of S. aureus population is seen forfree nisin compared with the nisin-loaded nanoparticles atday 7. The initial reduction in the antibacterial strength ofthe nisin-loaded nanoparticles follows from the fact that thetighter and protective binding of the nanoparticles withnisin may delay the antibacterial action of nisin inside incomparison with free nisin. Nevertheless, at the later timeintervals, a progressive and increasing antibacterial strengthfor the nisin-loaded nanoparticles is observed in compari-son with free nisin due to gradual liberation of nisin fromthe nanoparticles and also intrinsic antibacterial effectsof chitosan which can be assignable to two reasons. First, formation of a polymeric membrane resulted from the posi-

tioning of chitosan nanoparticles onto the surface of the cellinhibits nutrients from entering the cell (Zheng and Zhu2003). Second, the harmful interaction between the poly-cationic chitosan and the negatively charged surface ofbacteria interferes with the permeability of the bacterialcell wall and leads to the outflow of intracellular electrolytesand proteins and subsequent death of microorganism(Dutta et al. 2003). The difference between antimicrobialstrength of free nisin and the nisin-loaded nanoparticlesstarts at day 14 (~ twofold decrease in the microbial popula-tion in the case of nisin-loaded ones compared with freenisin samples). This decrease continues until day 35, atwhich point a fivefold logarithmic reduction in the micro-bial population is observed for the nisin-loaded nanopar-ticles as against free nisin. This outcome was in harmonywith our previous report on more antibacterial strength ofthe nisin-loaded nanoparticles than free nisin on S. aureuspopulation count in the milk samples (Zohri et al. 2010). Inthe samples containing L. monocytogenes, as shown in Fig. 2,a similar and noticeable decrease in the bacterial populationis observed for free nisin and the nisin-loaded nanoparticlesat day 7. Afterwards, i.e., at day 14, growth inhibition isalmost the same for the cheese samples containing free nisin

FIG. 1. MICROBIAL COUNT FOR STAPHYLOCOCCUS AUREUS INULTRA FILTERED FETA CHEESE SAMPLES AT DIFFERENT DAYS(•) i.e., cheese + S. aureus; (�) cheese + S. aureus + nisin; (�) thecheese + S. aureus + nisin-loaded chitosan/alginate nanoparticles.

FIG. 2. MICROBIAL COUNT FOR LISTERIA MONOCYTOGENES INULTRA FILTERED FETA CHEESE SAMPLES AT DIFFERENT DAYS(�) i.e., cheese + L. monocytogenes (�) i.e., cheese + L. mono-cytogenes + nisin; (�) i.e., cheese + L. monocytogenes + nisin-loadedchitosan/alginate nanoparticles.



as day 7 while much greater growth inhibition about fourtimes on a logarithmic scale is observed for the nisin-loadednanoparticles than free nisin at this time. From days 28through 35, the antibacterial power of free nisin continuesto be decreasing dramatically as against the previous days.However, the antibacterial strength for the nisin-loadednanoparticles is increasing at these times. Approximately, alogarithmic reduction of about seven was observed for thenisin-loaded nanoparticles compared with free nisin atday 35. The more antibacterial strength of the nisin-loadednanoparticles for L. monocytogenes than S. aureus issomehow related to the competitive effect of the starter withL. monocytogenes in acquiring its energy sources (Maisnier-Patin et al. 1992). In addition, the more strength of thenisin-loaded nanoparticles than free nisin can be attributedto protection potentiality of these nanoparticles. Thisattribute can prevent nisin from unfavorable interactionwith lipid and proteins which naturally decreases the anti-microbial power of nisin. The other reason is concernedwith chitosan itself as an innate antimicrobial agent whichdisplays synergistic antimicrobial effects with free nisin(Lisbeth 1998; Kyoon No et al. 2002; Fernandez-Saiz et al.2009). Considering the favorable and hopeful resultsobtained, it is reasonable to conclude that the biocompat-ible chitosan/alginate nanoparticles are able to promote andoptimize the antibacterial strength of nisin and preparethe way for more potential applications of this efficientbiopreservative in various fields of food industry.

Color Indices Analysis

In agreement with the data presented in Table 1, it can beconcluded that the two variables (factors) of cheese typeand storage time and their interactions show no statisticallysignificant effect on luminescence index. On the whole, thefactor cheese type displays more significant effect (P < 0.01)than the storage time (P < 0.05) on the other remaining

parameters (indices) except for the index a*. The changes insome color indices according to Table 2 can be attributed tothe differences in the physical structure of the three types ofcheese over the course of storage times. One reason is due tohigh fat content of the UF Feta cheese. Actually, the fat glob-ules can possibly be coalesced and agglomerated owing toincrease in contact surface over the storage times. This stageinduces a reduction in plasticizing effect of the fat globules.The lessening in the plasticizing impact of fat with waterconfines the junctions in-between casein chains (Madadlouet al. 2007). This process results in denaturation of caseinnetworks due to exodus of whey protein molecules. As aresult, void spaces on the surface of cheese matrix areformed, and which give rise to the meaningful variations inthe color indices. The other reason is that the employmentof the chitosan/alginate nanoparticles can in nature causevariations in color indices due to the inherent color of thenanoparticles spreading onto the surface of the cheesesample. In comparison, cheese + nisin samples exhibit moredifference in some color indices than the cheese + nisin-loaded nanoparticles and cheese samples as the controls.The results can arise from the more interaction of unpro-tected nisin with fats and proteins inside of the cheesesamples and probable formation of new mixtures withunknown color features which is least in the case of thenisin-loaded nanoparticles (Laloy et al. 1998). In summary,no significant changes lowering the color quality areobserved for the two types of the treated cheese samplesby comparison with the control cheese samples over thestorage times.

Chemical Investigation

As Table 3 shows, there is no difference in the moisturecontent among the three types of cheeses in each time inter-vals. But reduction in the moisture content is observed forthe different types of cheeses at day 14 in contrast with time

TABLE 1. F-VALUE SUMMARY OF THE MAIN VARIABLES AND THEIR INTERACTIONS FOR COLOR INDICES MEASUREMENTS, CHEMICALCOMPOSITIONS, RHEOLOGICAL PARAMETERS AND SENSORY FEATURES IN ULTRA FILTERED FETA CHEESE SAMPLES

Variablescorrelatedwith sensoryproperties A B A ¥ B

Variablescorrelatedwith colorevaluations A B A ¥ B

Variablescorrelatedwith chemicalcompositions A B A ¥ B

Flavor 70.08** 154.14** 16.05** L* 0.1 0.55 1.97 Moisture (%) 4.00* 100.00** 1.00Aroma 613.00** 904.00** 121.00** a* 38.15** 4.95** 1.59 Fat (%) 1.06 8.25* 1.69Oral texture 99.90** 61.18** 7.18** b* 37.74** 3.19* 6.86** pH 39.25** 148.56** 5.73*Color 218.15** 279.38** 2.00** c* 39.19** 3.17* 7.67** Ash (%) 33.63** 30.83** 12.16**Overall acceptability 1,027.00** 505.00** 199.00** h° 45.60** 1.77 6.10** Protein (%) 19.46** 1.28 5.82*Yield point 6.93** 3.06* 6.05** DE 22.41** 2.44 2.40 Nitrogen (%) 2.20 129.44** 2.22Maximum force 1.25 1.55 2.08 – – – – – – – –

Letter A is representative for main variable cheese type; letter B is representative for main variable storage time; the multiplied letters A ¥ B is repre-sentative for the interaction of the two variables. The symbols * and ** are indicative of P < 0.05 and P < 0.01, respectively.



zero. This trend is regarded as normal with respect to thecourse of storage time. In addition, the storage time variableshows a higher contribution to statistical significance(P < 0.01) than the cheese type variable (P < 0.05) whileno interactions between the main variables is observed(Table 1). The data related to the fat amount in Table 3 dis-plays no change between the three cheese types and storagetimes at all. This observation is expected because the pro-teolysis process in the UF Feta cheese often begins at the laststage of cheese ripening which is about 45 days after theinitial stage of the cheese-making process. Therefore, thereis no enough time (2 weeks) for the cheese samples to beinvolved in this reaction. In addition, only the storage timeshows statistical significance (P < 0.05) in this experiment asalso supported by Karami et al.’s (2008) study. The changesseen in the pH measurements revealed a decrease over timein all cheese types. The more decrease in the pH over thetime intervals observed for the cheese + nisin sample than

that of the nisin-loaded nanoparticles is due possibly to theintrinsic buffering effect of the chitosan/alginate nanopar-ticles which is least in the case of free nisin to prevent thepH changes. Both of the variables (P < 0.01) and their inter-actions is pertinent to the fluctuations in the pH measure-ments. The ash contents were higher at day 14 for the twotypes of the treated cheeses than those of the ash at timezero. Besides, the main variables of cheese type and storagetime and their interactions are involved in the observed dif-ferences (P < 0.01). Table 3 exhibits nearly the same totalprotein content for all the three types of cheese samples atthe two time intervals. The variable cheese type (P < 0.01)and the interactions between the two main variables(P < 0.05) shows principal contribution to this very littledifferences. Total nitrogen reveals some increment at day14 for all the cheese types as compared with time zero.This can be followed from the reaction of cheese ingredientswith their environments over the course of storage and

TABLE 2. IMPACT OF CHEESE TYPE AND STORAGE TIME ON COLOR MEASUREMENT PROPERTIES OF ULTRA FILTERED FETA CHEESE SAMPLES

Cheese samplesStoragetimes (day)

Measured variables

L* a* b* C* h° DE

Cheese 0 99.09 � 0.02a -0.07 � 0.01abcd 7.92 � 0.28c 7.92 � 0.28c -1.56 � 0.00b 1.57 � 0.26abc

2 99.73 � 0.25a -0.76 � 0.12d 8.98 � 0.36ab 9.01 � 0.36ab -1.48 � 0.01b 0.56 � 0.26bc

4 99.14 � 0.06a -0.41 � 0.04cd 9.00 � 0.27ab 9.00 � 0.27ab -1.52 � 0.00b 0.56 � 0.14bc

8 99.53 � 0.41a -0.48 � 0.08cd 9.36 � 0.04a 9.37 � 0.04a -1.51 � 0.00b 0.33 � 0.13c

Cheese + free nisin 0 99.29 � 0.43a 0.69 � 0.73a 8.23 � 0.77bc 8.28 � 0.72bc 0.43 � 1.7ab 1.71 � 0.99ab

2 99.33 � 0.26a 0.45 � 0.04ab 7.47 � 0.33c 7.48 � 0.33c 1.51 � 0.02a 2.13 � 0.28a

4 99.33 � 0.41a 0.72 � 0.25a 7.52 � 0.14bc 7.55 � 0.11c 1.47 � 0.03a 2.23 � 0.23a

8 99.61 � 0.21a 0.31 � 0.03abc 7.85 � 0.08bc 7.86 � 0.08c 1.53 � 0.00a 1.73 � 0.04ab

Cheese + nisin-loadedchitosan/alginatenanoparticles

0 100.07 � 0.35a 0.26 � 0.07abc 7.92 � 0.09c 7.92 � 0.10c 1.53 � 0.00a 1.73 � 0.05ab

2 98.61 � 1.61a -0.36 � 0.33bcd 8.02 � 0.14c 8.03 � 0.12c -0.92 � 1.1ab 1.95 � 1.02a

4 99.43 � 0.19a -0.45 � 0.40cd 8.15 � 0.23bc 8.17 � 0.25bc -1.51 � 0.04b 1.26 � 0.18abc

8 99.61 � 0.52a -0.05 � 0.03abcd 8.15 � 0.00bc 8.15 � 0.00bc -1.56 � 0.00b 1.35 � 0.04abc

Means � standard errors of the measured parameters are shown. The common superscripts in each column is indicative of statistical insignificancei.e., P > 0.05.a*, green to red; b*, blue to yellow; C*, chroma; h°, hue angle; L*, luminance; DE, color difference.

TABLE 3. CHEMICAL COMPOSITION OF THE ULTRA FILTERED FETA CHEESE SAMPLES UNTREATED AND TREATED WITH NISIN AND/ORNISIN-LOADED CHITOSAN/ALGINATE NANOPARTICLES AT VARIOUS TIME INTERVALS

Factors

Measured parameters

Moisture (%) Fat (%) pH Ash (%) Total protein (%) Total nitrogen (%)

Cheese at zero time 61.33 � 0.57a 6.57 � 0.28a 4.50 � 0.00a 1.04 � 0.04c 18.08 � 0.00bc 19.49 � 0.08b

Cheese + free nisin at zero time 60.33 � 0.57a 6.32 � 0.10a 4.25 � 0.06b 1.14 � 0.03bc 18.18 � 0.03ab 19.49 � 0.08b

Cheese + nisin-loaded chitosan/alginatenanoparticles at zero time

61.00 � 0.00a 6.58 � 0.26a 4.49 � 0.05a 1.06 � 0.04c 18.15 � 0.03ab 19.49 � 0.08b

Cheese at 14th day 58.66 � 0.57b 6.33 � 0.11a 4.00 � 0.00c 1.02 � 0.01c 18.00 � 0.00c 19.85 � 0.06a

Cheese + free nisin at 14th day 58.33 � 0.57b 6.26 � 0.16a 3.69 � 0.12d 1.43 � 0.10a 18.21 � 0.08ab 20.03 � 0.04a

Cheese + nisin-loaded chitosan/alginatenanoparticles at 14th day

59.00 � 0.00b 6.11 � 0.11a 4.22 � 0.11b 1.21 � 0.03b 18.28 � 0.09a 19.88 � 0.10a

Means � standard errors of the measured parameters are presented. The common superscripts in each column is indicative of statistical insignifi-cance i.e., P > 0.05.



production of new nitrogen compounds which consequentlyincreases the mass of nitrogen. Also, only the storage time isa significant factor in this respect (P < 0.01). Totally, varia-tions in the two treated cheese types are not regarded asimportant as compared with the untreated cheese samples.

Analysis of Rheological Parameters

Regarding the results of the experiments in Tables 1 and 4,there can generally be seen no difference between threetypes of the cheeses during the specified time intervals forthe maximum forces at fracture and stress at yield points.Table 1 reveals higher statistical significance for the variablecheese type (P < 0.01) than the storage time variable(P < 0.05) on the whole. Trivial change is observed for thecheese + nisin at day 4 with a reduction in the stress at yieldpoint (P < 0.05). In contrast, no statistical changes in thestress at yield points and the maximum forces are viewedfor the cheese samples containing the nanoparticles due tothe low amounts of these polymers and consequently lack offormation of gel-like structures (Yanes et al. 2002). To sumup, incorporation of the nanoparticles displays rheologicalfeatures almost comparable to the control cheese samples.

Sensory Evaluations

Variations in the flavor scoring scale as observed in Tables 1and 4 show a sharp reduction in the flavor quality of thecheese + nisin samples as against the other two types ofcheese samples mainly at days 4 and 8. This fall can beattributed to the faster and more facile reaction of free nisinwith the natural protease inside of the cheese environment.This reaction generally produces nitrogen-based com-

pounds over time which gives unpleasant flavor to thecheese sample. This is least in the case of the control cheesesamples and the protected nisin with the chitosan/alginatenanoparticles. A related study on liposome also revealed thepotentiality of taste masking for such nanosystem by aremarkable decrease in the bittering impact of the bacterialresidues producing this biopreservative (Benech et al. 2003).In addition, the cheese type and storage time variables andtheir interactions are statistically significant (P < 0.01) andresponsible for these changes. Investigation of aroma inTable 4 reveals no difference between the control cheesesamples and the cheese + nisin-loaded nanoparticles untilday 2. However, a marked lessening in the quality of aromais viewed for the cheese + nisin samples especially at day 4and onwards in comparison to the control and nisin-loadednanoparticles cheese samples. One of the reasons can beassigned to this is the direct reaction between the free nisinand proteins and high load of fat present inside of the UFFeta cheese as against the nisin-loaded nanoparticles cheesesamples. This interaction can destroy the structure of nisinand brings about unpleasant aroma. This issue had previ-ously been confirmed in a study by Benech et al. (2003).Both variables and their interactions (P < 0.01) are involvedin this outcome (Table 1). Analysis of oral texture for thecheese + nisin samples shows a noticeable reduction in itsoral acceptance from time zero onwards. The more reducedquality of the cheese + nisin samples than those of the othercheese types is due to both loss of water and easy reaction offree nisin with the cheese matrix. The cheese samples con-taining the nisin-loaded nanoparticles display almost thesame behavior as the control cheese samples. This is becauseof the low amount of the nanoparticles, avoidance of directand instant reaction of nisin with the cheese matrix and

TABLE 4. CHEESE TYPE AND STORAGE TIME INFLUENCE ON THE RHEOLOGICAL AND SENSORY PROPERTIES OF ULTRA FILTERED FETA CHEESESAMPLES

Cheese samplesStoragetimes (day)

Measured variables

Flavor Aroma Oral texture ColorOverallacceptability

Yield point(in kPa)

Maximum force(in Newton)

Cheese 0 5.00 � 0.00a 5.00 � 0.00a 4.88 � 0.33a 5.00 � 0.00a 5.00 � 0.00a 155 � 12ab 1.98 � 0.13a

2 5.00 � 0.00a 5.00 � 0.00a 4.88 � 0.33a 5.00 � 0.00a 5.00 � 0.00a 173 � 13a 2.04 � 0.01a

4 4.00 � 0.00c 4.00 � 0.00b 3.88 � 0.33b 5.00 � 0.00a 4.00 � 0.00b 172 � 14a 2.04 � 0.01a

8 4.00 � 0.00c 4.00 � 0.00b 3.88 � 0.33b 4.00 � 0.00c 4.00 � 0.00b 170 � 9a 2.04 � 0.0a

Cheese + free nisin 0 4.88 � 0.33ab 4.88 � 0.33a 3.88 � 0.33b 4.88 � 0.33ab 4.88 � 0.33a 173 � 6a 2.04 � 0.01a

2 4.00 � 0.00c 4.00 � 0.00b 3.88 � 0.33b 4.00 � 0.00c 4.00 � 0.00b 147 � 35ab 2.05 � 0.01a

4 4.00 � 0.00c 4.00 � 0.00b 3.88 � 0.33b 4.00 � 0.00c 4.00 � 0.00b 128 � 35b 2.04 � 0.01a

8 3.00 � 0.00d 3.00 � 0.00c 2.88 � 0.00c 3.00 � 0.00d 3.00 � 0.00c 172 � 14a 1.88 � 0.34a

Cheese + nisin-loadedchitosan/alginatenanoparticles

0 5.00 � 0.00a 5.00 � 0.00a 4.88 � 0.33a 5.00 � 0.00a 5.00 � 0.00a 166 � 11a 2.04 � 0.01a

2 4.66 � 0.50ab 5.00 � 0.00a 5.00 � 0.00a 4.66 � 0.50b 5.00 � 0.00a 171 � 12a 2.04 � 0.01a

4 4.55 � 0.52b 5.00 � 0.00a 4.88 � 0.33a 5.00 � 0.00a 5.00 � 0.00a 169 � 7a 2.05 � 0.01a

8 4.00 � 0.00c 4.00 � 0.00b 3.88 � 0.33b 4.00 � 0.00c 5.00 � 0.00a 172 � 9a 2.04 � 0.01a

In each column mean � standard error is shown. Different superscripts in each column show statistical significance i.e., P < 0.05. kPa (N/m2) meanskilopascal.



their small size making them not being recognizablewith taste buddies. Both variables and their interactions(P < 0.01) are assigned to these variations (Table 1). Thechanges observed in the color scores according to Table 4can be owing to the natural diffusion of the original con-stituent materials outwards from the serum phase of beta-lactoglobulin networks. Both of the main variables and theirinteractions (P < 0.01) are involved in these differences(Table 1). An overall acceptability is expected due to theimproving effects of the nanoparticles on the sensory char-acteristics of the UF Feta cheese. Main variables and theirinteractions (P < 0.01) are contributed to these differences(Table 1). In all, sensory evaluations exhibit an equivalentand parallel trend for the nisin-loaded nanoparticles ascompared with the control cheese samples while showingunfavorable impacts for the cheese + nisin samples.

Microscopic Analysis

Figure 3 shows a clear representation of the chitosan/alginate nanoparticles with smooth surface, regular shapeand almost similar sizes inside of the casein structure ofthe UF Feta cheese sample after 72 h. These properties willcause the nanoparticles to easily penetrate and interactstronger with the microorganisms entering inside of cheesematrix during post-contamination stage.

CONCLUSIONS

In connection with the whole results, it was indicated thatnisin-loaded chitosan/alginate nanoparticles shows moreantibacterial potency than free nisin without any unwantedeffects on the quality of the original UF Feta cheese. There-fore, the use of these biocompatible nanoparticles can be

regarded as a new strategy for constructing composites ofnisin with more antibacterial efficacies in the near future.These features make this composite an excellent biopreser-vative candidate in food science. These promising outcomesalso provide further impetus for more investigation on thishybrid biopreservative and prepare the way for the practicaluse of these biocompatible nanoparticles in food industry.

ACKNOWLEDGMENT

Authors are thankful to Dr. Parviz Aberumand for construc-tive guidance in some process of performing the research.

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