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Journal of Food Safety ISSN 1745-4565

NISIN-LOADED CHITOSAN/ALGINATE NANOPARTICLES: A HOPEFUL HYBRID BIOPRESERVATIVE

MARYAM ZOHRI1,8, MOHAMMAD SHAFIEE ALAVIDJEH2, SEYED SAEED MIRDAMADI3, HOMA BEHMADI4, SEYED MEHDI HOSSAINI NASR2, SIMA ESHGHI GONBAKI5, MEHDI SHAFIEE ARDESTANI6 and ALI JABBARI ARABZADEH7
1 2

Department of Food Science and Technology, Science and Research Branch, Islamic Azad University, Tehran, Iran Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Science, Tehran, Iran 3 Iranian Research Organization Science & Technology, Biotechnology Department, Tehran, Iran 4 Department of Food Engineering and Post-Harvest Technology Research, Agricultural Engineering Institute, Karaj, Iran 5 Department of Engineering, Science and Research Branch, Islamic Azad University, Tehran, Iran 6 Research and Development and Hepatitis B Department, Research and Production Complex, Pasteur Institute of Iran, Tehran, Iran 7 Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran

Corresponding author. TEL: +989364593253; FAX: +982612704532; EMAIL: mzohri@srbiau.ac.ir Received for Publication August 29, 2012 Accepted for Publication December 18, 2012 doi: 10.1111/jfs.12021

ABSTRACT
To improve the deciencies concerning the physicochemical instability of nisin, a hybrid of nisin at concentration of 450 IU/mL with chitosan/alginate nanoparticles was prepared. Antibacterial strength of the hybrid was compared with free nisin against Listeria monocytogenes ATCC 25923 and Staphylococcus aureus ATCC 19117 in ultra ltered (UF) Feta cheese. The effects of nisin and the nisin-loaded nanoparticles on the chemical composition, rheological parameters, color indices and sensory attributes of UF Feta cheese were studied. Antibacterial experiments indicated that the nisin-loaded nanoparticles were able to decrease the populations of S. aureus and L. monocytogenes up to ve- and sevenfold on a logarithmic scale in comparison with free nisin, respectively. Sensory acceptance and physicochemical features of UF Feta cheese were also signicantly improved using the nisin-loaded nanoparticles as compared with those of free nisin. Overall, greater antibacterial strengths and less undesirable inuences of this hybrid than those of free nisin on the original quality of UF Feta cheese would make this hybrid a promising biopreservative in dairy products.

PRACTICAL APPLICATIONS
This study investigates the use of chitosan/alginate nanoparticles as an auxiliary adjuvant in food preservation process. Considering our former promising antibacterial strength observed for this hybrid against S. aureus in the milk samples and also our new ndings, it can be concluded that this hybrid would actually be an effectual potential biopreservative against common foodborne pathogens without any harmful side effects on the original qualities of the assessed dairy products. These outstanding features would be an incentive for further future investigation and probable industrialization of this hybrid as a highly productive biopreservative in food preservation technology. products is one of the pivotal purposes in food technology. It is usually achieved by use of efcient and optimum amount of chemical and/or biological antimicrobial agents during the manufacturing process. Nowadays, making use of biopreservatives in food industry realm is gradually becoming current and established (Mauriello et al. 2005). One class of these
Journal of Food Safety 33 (2013) 4049 2013 Wiley Periodicals, Inc.

INTRODUCTION
Today, nanotechnology is incorporating into all elds of science; it can not be an exception for biosciences-related realms. One of these domains affected by this hopeful technology is food science. Extension of the shelf life of food
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biological preservatives which has just become widespread is the group of antimicrobial proteins. An example of this class is nisin which is considered an effective antimicrobial agent against a wide range of gram-positive and some gramnegative bacterial strains (Stevens et al. 1991; Kramer et al. 2004). Nevertheless, nisin and the other antimicrobial proteins/peptides are readily denatured by the protease enzymes acting commonly in the acidic rich environment of the food materials. In addition, they also easily react with other ingredients inside of the food matrices like lipids and so forth. These interactions can possibly give rise to diminution of intrinsic antimicrobial strength and bioavailability of these entities and ultimately their imperfect actions (Liu and Hansen 1990). Regarding these obstacles, nding a way to reduce these disadvantages would be benecial. One of the ways which has been proposed recently as a possible strategy to overcome these deciencies is the encapsulation approach for this nature-originated peptides and proteins by using suitable polymeric micro and nanoparticle systems (Wan et al. 1997). Nanoparticle systems are displaying various potential applications in food technology realms such as controlled release of the active components, particle coating, avor stabilization, taste masking and improvement of the shelf life through the physical/chemical stabilization of the active constituents involved in this regard. Among the microbial pathogens which decrease the shelf life of food products lies Listeria species. This pathogen is regarded as a public health concern due to the severity of its pathogenesis strength, its prevalence among the immunosuppressed and its high case-fatality rate (Schmidt et al. 2009). For instance, Listeriosis disease is a case caused by a gram-positive nonsporulating bacillus i.e., Listeria monocytogenes. In addition, L. monocytogenes is psychotropic and can even grow at refrigeration temperatures; therefore, subsequent contamination by this organism could be regarded as a high risk for the consumers (Benedito et al. 2000). Considering the problems relating to this microbial pathogen, many researchers have focused and performed experiments against this pathogenic bacterium until now with the aim of considerable reduction of its harmful effects in dairy products. In a study, noticeable sensitivity of L. monocytogenes to nisin was observed (Harris et al. 1998). Another study also proved antimicrobial property of nisin when added to cottage cheese (Benkerroum and Sandine 1998). Growth inhibition of L. innocua was also observed in Cheddar cheese by either adding nisin Z to a liposome or by its in situ production in mixed culture (Benech et al. 2002). It was found that encapsulation of nisin Z in liposomes can provide a new strategy for improvement of the physicochemical stability of nisin by protection of nisin from the detrimental action of the proteases being produced 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 microJournal of Food Safety 33 (2013) 4049 2013 Wiley Periodicals, Inc.

particles containing nisin showed the same activity as free nisin against an indicator culture of Lactobacillus curvatus both in De Man, Rogosa and Sharpe medium broth and reconstituted skim milk (Wan et al. 1997). Suppression of L. monocytogenes colonization following the adsorption of nisin onto silica surfaces also revealed the antimicrobial efciency of nisin against this bacterium (Bower et al. 1995). Staphylococcus aureus is regarded as the other most epidemic bacteria being one of the reasons for respiratory infections or enteritis. Moreover, it can give rise to postcontamination of dairy food preparations during the storage time; consequently, it is necessary to inhibit its growth in dairy products for more safety and better quality. Growth inhibitory effect of nisin-containing modied alginate lms/beads was evaluated on S. aureus by Millette et al. (2007). Based on the results, it was suggested that sterile, hydrophobic and biodegradable lms or beads containing different concentration of nisin could conceivably be used to control the growth of pathogens or microorganisms responsible for spoilage at the surface of round beef or other meat products. A comparative study between the antibacterial effect of nisin and nisin-loaded chitosan/alginate nanoparticles was recently performed on the growth of S. aureus in raw and pasteurized milk samples (Zohri et al. 2010). Twofold increase in the antibacterial strength was observed for the nisin-loaded nanoparticles as against free nisin at concentration of 450 IU/mL. These outcomes results from the more penetrability and slow liberation of nisin from chitosan/alginate nanoparticles during storage and consequently its gradual action over more time periods compared with the samples lacking the nanoparticles. Grounded on the promising results reported from our former study and also different research studies concerning the use of biocompatible polymeric nanoparticles, it was intended to compare the antimicrobial strength of the optimized nisin-loaded chitosan/alginate nanoparticles with free nisin in the ultra ltered (UF) Feta cheese as well. For this purpose, the growth inhibitions of S. aureus and L. monocytogenes were investigated in the UF Feta cheese as the two prevalent microbes usually being responsible for post-contamination in such products. Microstructure features, sensory specications and rheological properties of the UF Feta cheese containing nisin or nisin-loaded chitosan/alginate nanoparticles were also examined. These investigations are also crucial and should be examined because they undoubtedly have great impact on the quality and marketing of the food products. It is hoped that these ndings can help establish a reliable new way to improve the antimicrobial strength of nisin with the contribution and synergisms from these biocompatible nanoparticles. These new hybrid biopreservatives may be employed for extending the shelf life of food products showing high risk of post-contamination over the period of storage.
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MATERIALS AND METHODS

Materials and Instruments
Acetic acid (Merck Co., Darmstadt, Germany), sodium alginate (BDH Co., London, U.K.), low molecular weight chitosan (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, SigmaAldrich Co.), starter culture (Choozit MR804 FRO 500 DCU; Danisco Co., Denmark) and Listeria selective agar base (Oxoid Co., Basingstoke, U.K.) were purchased and used as received. S. aureus ATCC 19117 and L. monocytogenes ATCC 25923 were prepared from Persian Type Collection of Culture (PTCC) in Iranian Research Organization for Science and Technology. BairdParker agar was bought from Difco Co. (Detroit, MI). The Universal Testing Machine (S-Series Bench U.T.M. Model H5KS, Hounseld Test Equipment Ltd., Redhill, U.K.), scanning electron microscope (SEM; LEO440I, 10 kV, U.K.), Hunter lab Colorimeter Model D25-DP9000 (Hunter lab Associates Inc., Reston, VA), Digital pH-meter (pH 537; WissenschaftlichTechnische Werksttten GmbH, Weilheim in Oberbayern, Germany), and Pelco-CPD-2 critical point drier (Ted Pella Co., Redding, CA) were also applied for executing the experiments.

accomplished by three sequential subcultures. The bacterial strains were cultivated in 20% w/v glycerol and skim milk 15%. After 48 h of incubation, the population of the bacteria was measured with a spectrophotometer. The population of 106 cfu/mL was used for the inoculation of the cheese samples (Schmidt et al. 2009).

Cheese-Making Procedure
The UF Feta cheese samples were prepared according to the instruction of Tetra Pak Company (Bylund 1995). Accordingly, the milk samples were initially bactofugated, pasteurized (72C for 15 s), ultra ltrated, homogenized and pasteurized (80C for 20 s) for the second time. The pH of milk reached the level of 6.2 by adding the starter (1.0 g for 10 kg of retentate). Then, inside the cheese blocks, rennet (2,200 international milk-clotting units/g) was mixed with water (2 g for 100 kg of retentate) and used for coagulation process. Before coagulation, 106 cfu/mL of L. monocytogenes and S. aureus and 2 mL (450 IU/mL) of nisin-loaded chitosan/alginate nanoparticles were added to the block of UF milk including starter. They were then incubated at 37C for 24 h and then stored at 4C for another 24 h. The prepared samples were the cheese plus L. monocytogenes and or/S. aureus as the controls, the cheese + L. monocytogenes and or/S. aureus + nisin, and the cheese + L. monocytogenes and or/S. aureus + nisin-loaded nanoparticles in three replicates. Then, the tests were performed at the determined time intervals.

Preparation and Characterization of Nisin-Loaded Chitosan/Alginate Nanoparticles

Nisin-loaded chitosan/alginate nanoparticles were prepared and physicochemically characterized according to our previous study (Zohri et al. 2011). The nisin-loaded nanoparticles were prepared from nisin with optimum activity at nal concentration of 450 IU/mL and with a mean size of 205 nm by a two-step procedure adapted from Rajaonarivonys method of preparing poly-L-lysine nanoparticles (Rajaonarivony et al. 1993). Briey, for preparing the nanoparticles, 540 mL of the nisin solution (10 mg/mL) was added dropwise to 8 mL aqueous solution of sodium alginate (250 mg/mL) and stirred for 30 min. Then, 4 mL of the chitosan stock solution (250 mg/mL in 1% v/v acetic acid solution) was added to the resulting alginate solution and stirred for another 1 h. The nanoparticles were obtained by centrifugation at 2,500 g for 10 min at 4C to separate the free polymers from the nanoparticles (Zohri et al. 2010).

Cheese Sampling Method for Microbial Counts

The cheese samples (1 g) were vortexed with 9 mL of sterile sodium citrate solution (2% w/w) in triplicate as described by Sipahioglu et al. (1999). Samples (three types at different times) were again serially diluted by sodium citrate to 0.1 of their initial amounts. Then, 1 mL of the nal dilution was plated in triplicate on Listeria Selective agar (tryptose, 10.0 g; yeast extract, 5.0 g; beef extract, 5.0 g; sodium chloride, 20.0 g; disodium hydrogen phosphate, 1.35 g; esculin, 1.00 g; nalidixic acid, 0.02 g; acriavin hydrochloride). Plates were then incubated at 37C for 48 h and after that were ready for counting L. monocytogenes population. Necessary dilutions were done on BairdParker agar to enumerate S. aureus population inside the cheese samples (Romeih et al. 2002).

Microbial Assay Culture Maintenance and Revival Procedures

L. monocytogenes ATCC 25923 and S. aureus ATCC 19117 strains were obtained from PTCC. Revival process was
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The populations of L. monocytogenes and S. aureus inside of the cheese samples were measured according to the methods of Sipahioglu et al. (1999) Romeih et al. (2002), respectively.
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Color Indices Measurement

For color indices assessments, Hunter lab Colorimeter Model D25-DP9000 was calibrated with black and white reference standard sheets and used for running the experiments. The samples with a height of 1.5 cm were placed onto the standard sheets with the following specication: Y = 83.32, X = 81.26 and Z = 98.03. The scale used was based on CIELAB system in which L* was a representative for 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 following formula i.e., h = arc tan (b*/a*). Chroma (C*) was determined by this formula i.e., C* = [(a*)2 + (b*)2]1/2 and the difference i.e., DE is a single number that measures the distance between the two colors (DE) was calculated by 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 for the control cheese samples at time zero (Lawless and Heymann 1998). Mean values and standard deviation were calculated for nine runs.

evaluation were measured form the compression curves and conducted by means of a HTE Universal Testing Machine (S-Series Bench U.T.M. Model H5K-S; Hounseld Test Equipment Ltd.) with a 500-N load cell. The cheese samples were prepared in cubic shape with 20 mm3 volume each. Samples used were taken from approximately a depth of 2 cm in each of the cheese blocks and kept at 12C prior to analysis. The samples under study were uniaxially compressed up to 50% of their initial height at compression rate of 20 mm/min (Sipahioglu et al. 1999). Each sample was analyzed in six repetitions. Force-displacement data were employed for calculation of stress and Hencky strain (eHencky) and stress at yield point was obtained for the stressHencky strain curve. H0 and Ht are regarded as sample heights before and after deformation at times zero and t according to the following formula

Hencky =

Ln

( )
Ht H0

(1)

Sensory Assessment
Sensory attributes examinations were conducted on the control UF Feta cheese and the treated UF Feta cheese samples containing nisin and/or nisin + chitosan/alginate nanoparticles. Properties such as avor, aroma, oral texture and overall acceptability were measured and compared with each other. These experiments were carried out by 14 experienced panelists through a 5-point scale hedonic test with 1 being bad and 5 being excellent and the rest were in between (Foegeding et al. 2003). The samples of about 30 g in duplicate in three batches were prepared and examined concurrently for each of the sensory experiments at 20 3C. Panelist employed water and unsalted crackers to clean their palates between the runs. In order to keep away from any misjudgment on nal decision, the experiments were conducted under red lighting.

Chemical Analysis
Chemical composition including fat content, pH, protein content, ash content, moisture and total nitrogen/protein of the cheese samples were analyzed in untreated or treated samples with nisin or nisin-loaded chitosan/alginate nanoparticles at days 0 and 14. The pH of the cheese samples was measured using a digital pH-meter (microprocessor pH meter, model pH 537; Wissenschaftlich-Technische Werksttten GmbH). The cheese samples were analyzed for moisture content by vacuum-oven method (Association of Ofcial Analytical Chemists; AOAC 2000). For measuring the ash contents of the samples, 10 g of the samples were accurately weighed in a crucible and dried by hot air oven at 105C. The samples were then ignited on a ame and nally turned into ash in a furnace at 550C to reach a constant weight while white ash was left (AOAC 2000). The fat content of the cheese samples was determined by the Gerber method (James 1995). Total nitrogen amounts were determined by Kjeldahl method (AOAC 1997). The total nitrogen calculated was then used for determination of protein content by multiplying the total nitrogen by 6.38. All chemical measurements were done in triplicate (Romeih et al. 2002).

SEM Study of the Morphology of Nanoparticles

The cheese samples were prepared and the microstructure was investigated microscopically by SEM grounded on the Sipahioglu et al. (1999) method. The cheeses were sliced into 45 mm3 cubes and xed overnight in 2.8% w/v glutaraldehyde solution. The cheese samples were then rinsed with distilled water for 1 min three times. Graded ethanol series (20, 40, 60, 80, 95 and 100%) was employed for dehydration process of the cheese samples for 30 min each. Chloroform was used for defatting of the cheese samples for 15 min. Defatted samples were refrigerated until freezefractured in liquid nitrogen. Drying of the freeze-fractured samples was carried out using a Pelco-CPD-2 critical point
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Uniaxial Constant Speed Compression Test

Uniaxial compression is a standard technique for evaluation of mechanical and fracture properties and one of the several ways for the primary assessment of cheese texture quality (Wium and Qvist 1997). The stress at yield point and maximum force at fracture point as the index for hardness
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drier (Ted Pella Inc., Redding, CA). The samples were then coated with 30 nm gold-palladium and the micrographs were taken by JEOL SEM (SLEO 440 I, 10 kV, U.K.).

Statistical Analysis
The data were analyzed through full factorial design with JMP software SAS Inc. The probabilities of P < 0.05 and P < 0.01 were regarded as being signicant and highly signicant, respectively.

RESULTS AND DISCUSSION

Microbial Evaluation
Population counts of S. aureus and L. monocytogenes were carried out at time intervals of 0, 7, 14, 28 and 35 (in days) as depicted in Figs. 1 and 2, respectively. As is shown in Fig. 1, twofold inhibition of S. aureus population is seen for free nisin compared with the nisin-loaded nanoparticles at day 7. The initial reduction in the antibacterial strength of the nisin-loaded nanoparticles follows from the fact that the tighter and protective binding of the nanoparticles with nisin may delay the antibacterial action of nisin inside in comparison with free nisin. Nevertheless, at the later time intervals, a progressive and increasing antibacterial strength for the nisin-loaded nanoparticles is observed in comparison with free nisin due to gradual liberation of nisin from the nanoparticles and also intrinsic antibacterial effects of chitosan which can be assignable to two reasons. First,

FIG. 2. MICROBIAL COUNT FOR LISTERIA MONOCYTOGENES IN ULTRA FILTERED FETA CHEESE SAMPLES AT DIFFERENT DAYS () i.e., cheese + L. monocytogenes () i.e., cheese + L. monocytogenes + nisin; () i.e., cheese + L. monocytogenes + nisin-loaded chitosan/alginate nanoparticles.

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

formation of a polymeric membrane resulted from the positioning of chitosan nanoparticles onto the surface of the cell inhibits nutrients from entering the cell (Zheng and Zhu 2003). Second, the harmful interaction between the polycationic chitosan and the negatively charged surface of bacteria interferes with the permeability of the bacterial cell wall and leads to the outow of intracellular electrolytes and proteins and subsequent death of microorganism (Dutta et al. 2003). The difference between antimicrobial strength of free nisin and the nisin-loaded nanoparticles starts at day 14 (~ twofold decrease in the microbial population in the case of nisin-loaded ones compared with free nisin samples). This decrease continues until day 35, at which point a vefold logarithmic reduction in the microbial population is observed for the nisin-loaded nanoparticles as against free nisin. This outcome was in harmony with our previous report on more antibacterial strength of the nisin-loaded nanoparticles than free nisin on S. aureus population count in the milk samples (Zohri et al. 2010). In the samples containing L. monocytogenes, as shown in Fig. 2, a similar and noticeable decrease in the bacterial population is observed for free nisin and the nisin-loaded nanoparticles at day 7. Afterwards, i.e., at day 14, growth inhibition is almost the same for the cheese samples containing free nisin
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as day 7 while much greater growth inhibition about four times on a logarithmic scale is observed for the nisin-loaded nanoparticles than free nisin at this time. From days 28 through 35, the antibacterial power of free nisin continues to be decreasing dramatically as against the previous days. However, the antibacterial strength for the nisin-loaded nanoparticles is increasing at these times. Approximately, a logarithmic reduction of about seven was observed for the nisin-loaded nanoparticles compared with free nisin at day 35. The more antibacterial strength of the nisin-loaded nanoparticles for L. monocytogenes than S. aureus is somehow related to the competitive effect of the starter with L. monocytogenes in acquiring its energy sources (MaisnierPatin et al. 1992). In addition, the more strength of the nisin-loaded nanoparticles than free nisin can be attributed to protection potentiality of these nanoparticles. This attribute can prevent nisin from unfavorable interaction with lipid and proteins which naturally decreases the antimicrobial power of nisin. The other reason is concerned with chitosan itself as an innate antimicrobial agent which displays synergistic antimicrobial effects with free nisin (Lisbeth 1998; Kyoon No et al. 2002; Fernandez-Saiz et al. 2009). Considering the favorable and hopeful results obtained, it is reasonable to conclude that the biocompatible chitosan/alginate nanoparticles are able to promote and optimize the antibacterial strength of nisin and prepare the way for more potential applications of this efcient biopreservative in various elds of food industry.

Color Indices Analysis

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

parameters (indices) except for the index a*. The changes in some color indices according to Table 2 can be attributed to the differences in the physical structure of the three types of cheese over the course of storage times. One reason is due to high fat content of the UF Feta cheese. Actually, the fat globules can possibly be coalesced and agglomerated owing to increase in contact surface over the storage times. This stage induces a reduction in plasticizing effect of the fat globules. The lessening in the plasticizing impact of fat with water connes the junctions in-between casein chains (Madadlou et al. 2007). This process results in denaturation of casein networks due to exodus of whey protein molecules. As a result, void spaces on the surface of cheese matrix are formed, and which give rise to the meaningful variations in the color indices. The other reason is that the employment of the chitosan/alginate nanoparticles can in nature cause variations in color indices due to the inherent color of the nanoparticles spreading onto the surface of the cheese sample. In comparison, cheese + nisin samples exhibit more difference in some color indices than the cheese + nisinloaded nanoparticles and cheese samples as the controls. The results can arise from the more interaction of unprotected nisin with fats and proteins inside of the cheese samples and probable formation of new mixtures with unknown color features which is least in the case of the nisin-loaded nanoparticles (Laloy et al. 1998). In summary, no signicant changes lowering the color quality are observed for the two types of the treated cheese samples by comparison with the control cheese samples over the storage times.

Chemical Investigation
As Table 3 shows, there is no difference in the moisture content among the three types of cheeses in each time intervals. But reduction in the moisture content is observed for the 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, CHEMICAL COMPOSITIONS, RHEOLOGICAL PARAMETERS AND SENSORY FEATURES IN ULTRA FILTERED FETA CHEESE SAMPLES Variables correlated with sensory properties Variables correlated with color evaluations A 0.1 38.15** 37.74** 39.19** 45.60** 22.41** Variables correlated with chemical compositions A Moisture (%) Fat (%) pH Ash (%) Protein (%) Nitrogen (%) 4.00* 1.06 39.25** 33.63** 19.46** 2.20

AB

B 0.55 4.95** 3.19* 3.17* 1.77 2.44

AB 1.97 1.59 6.86** 7.67** 6.10** 2.40

AB

Flavor 70.08** Aroma 613.00** Oral texture 99.90** Color 218.15** Overall acceptability 1,027.00** Yield point 6.93** Maximum force 1.25

154.14** 16.05** L* 904.00** 121.00** a* 61.18** 7.18** b* 279.38** 2.00** c* 505.00** 199.00** h 3.06* 6.05** DE 1.55 2.08

100.00** 1.00 8.25* 1.69 148.56** 5.73* 30.83** 12.16** 1.28 5.82* 129.44** 2.22

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

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TABLE 2. IMPACT OF CHEESE TYPE AND STORAGE TIME ON COLOR MEASUREMENT PROPERTIES OF ULTRA FILTERED FETA CHEESE SAMPLES Storage times (day) 0 2 4 8 0 2 4 8 0 2 4 8 Measured variables L* 99.09 0.02 99.73 0.25a 99.14 0.06a 99.53 0.41a 99.29 0.43a 99.33 0.26a 99.33 0.41a 99.61 0.21a 100.07 0.35a 98.61 1.61a 99.43 0.19a 99.61 0.52a
a

Cheese samples Cheese

a* -0.07 0.01 -0.76 0.12d -0.41 0.04cd -0.48 0.08cd 0.69 0.73a 0.45 0.04ab 0.72 0.25a 0.31 0.03abc 0.26 0.07abc -0.36 0.33bcd -0.45 0.40cd -0.05 0.03abcd
abcd

b* 7.92 0.28 8.98 0.36ab 9.00 0.27ab 9.36 0.04a 8.23 0.77bc 7.47 0.33c 7.52 0.14bc 7.85 0.08bc 7.92 0.09c 8.02 0.14c 8.15 0.23bc 8.15 0.00bc
c

C* 7.92 0.28 9.01 0.36ab 9.00 0.27ab 9.37 0.04a 8.28 0.72bc 7.48 0.33c 7.55 0.11c 7.86 0.08c 7.92 0.10c 8.03 0.12c 8.17 0.25bc 8.15 0.00bc
c

h -1.56 0.00 -1.48 0.01b -1.52 0.00b -1.51 0.00b 0.43 1.7ab 1.51 0.02a 1.47 0.03a 1.53 0.00a 1.53 0.00a -0.92 1.1ab -1.51 0.04b -1.56 0.00b
b

DE 1.57 0.26abc 0.56 0.26bc 0.56 0.14bc 0.33 0.13c 1.71 0.99ab 2.13 0.28a 2.23 0.23a 1.73 0.04ab 1.73 0.05ab 1.95 1.02a 1.26 0.18abc 1.35 0.04abc

Cheese + free nisin

Cheese + nisin-loaded chitosan/alginate nanoparticles

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

zero. This trend is regarded as normal with respect to the course of storage time. In addition, the storage time variable shows a higher contribution to statistical signicance (P < 0.01) than the cheese type variable (P < 0.05) while no interactions between the main variables is observed (Table 1). The data related to the fat amount in Table 3 displays no change between the three cheese types and storage times at all. This observation is expected because the proteolysis process in the UF Feta cheese often begins at the last stage of cheese ripening which is about 45 days after the initial stage of the cheese-making process. Therefore, there is no enough time (2 weeks) for the cheese samples to be involved in this reaction. In addition, only the storage time shows statistical signicance (P < 0.05) in this experiment as also supported by Karami et al.s (2008) study. The changes seen in the pH measurements revealed a decrease over time in all cheese types. The more decrease in the pH over the time intervals observed for the cheese + nisin sample than

that of the nisin-loaded nanoparticles is due possibly to the intrinsic buffering effect of the chitosan/alginate nanoparticles which is least in the case of free nisin to prevent the pH changes. Both of the variables (P < 0.01) and their interactions is pertinent to the uctuations in the pH measurements. The ash contents were higher at day 14 for the two types of the treated cheeses than those of the ash at time zero. Besides, the main variables of cheese type and storage time and their interactions are involved in the observed differences (P < 0.01). Table 3 exhibits nearly the same total protein content for all the three types of cheese samples at the 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 little differences. Total nitrogen reveals some increment at day 14 for all the cheese types as compared with time zero. This can be followed from the reaction of cheese ingredients with their environments over the course of storage and

TABLE 3. CHEMICAL COMPOSITION OF THE ULTRA FILTERED FETA CHEESE SAMPLES UNTREATED AND TREATED WITH NISIN AND/OR NISIN-LOADED CHITOSAN/ALGINATE NANOPARTICLES AT VARIOUS TIME INTERVALS Measured parameters Factors Cheese at zero time Cheese + free nisin at zero time Cheese + nisin-loaded chitosan/alginate nanoparticles at zero time Cheese at 14th day Cheese + free nisin at 14th day Cheese + nisin-loaded chitosan/alginate nanoparticles at 14th day Moisture (%) 61.33 0.57 60.33 0.57a 61.00 0.00a
a

Fat (%) 6.57 0.28 6.32 0.10a 6.58 0.26a

a

pH 4.50 0.00 4.25 0.06b 4.49 0.05a

a

Ash (%) 1.04 0.04 1.14 0.03bc 1.06 0.04c

c

Total protein (%) 18.08 0.00 18.18 0.03ab 18.15 0.03ab

bc

Total nitrogen (%) 19.49 0.08b 19.49 0.08b 19.49 0.08b 19.85 0.06a 20.03 0.04a 19.88 0.10a

58.66 0.57b 58.33 0.57b 59.00 0.00b

6.33 0.11a 6.26 0.16a 6.11 0.11a

4.00 0.00c 3.69 0.12d 4.22 0.11b

1.02 0.01c 1.43 0.10a 1.21 0.03b

18.00 0.00c 18.21 0.08ab 18.28 0.09a

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

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production of new nitrogen compounds which consequently increases the mass of nitrogen. Also, only the storage time is a signicant factor in this respect (P < 0.01). Totally, variations in the two treated cheese types are not regarded as important 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 three types of the cheeses during the specied time intervals for the maximum forces at fracture and stress at yield points. Table 1 reveals higher statistical signicance for the variable cheese type (P < 0.01) than the storage time variable (P < 0.05) on the whole. Trivial change is observed for the cheese + nisin at day 4 with a reduction in the stress at yield point (P < 0.05). In contrast, no statistical changes in the stress at yield points and the maximum forces are viewed for the cheese samples containing the nanoparticles due to the low amounts of these polymers and consequently lack of formation of gel-like structures (Yanes et al. 2002). To sum up, incorporation of the nanoparticles displays rheological features almost comparable to the control cheese samples.

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

pounds over time which gives unpleasant avor to the cheese sample. This is least in the case of the control cheese samples and the protected nisin with the chitosan/alginate nanoparticles. A related study on liposome also revealed the potentiality of taste masking for such nanosystem by a remarkable decrease in the bittering impact of the bacterial residues producing this biopreservative (Benech et al. 2003). In addition, the cheese type and storage time variables and their interactions are statistically signicant (P < 0.01) and responsible for these changes. Investigation of aroma in Table 4 reveals no difference between the control cheese samples and the cheese + nisin-loaded nanoparticles until day 2. However, a marked lessening in the quality of aroma is viewed for the cheese + nisin samples especially at day 4 and onwards in comparison to the control and nisin-loaded nanoparticles cheese samples. One of the reasons can be assigned to this is the direct reaction between the free nisin and proteins and high load of fat present inside of the UF Feta cheese as against the nisin-loaded nanoparticles cheese samples. This interaction can destroy the structure of nisin and brings about unpleasant aroma. This issue had previously been conrmed in a study by Benech et al. (2003). Both variables and their interactions (P < 0.01) are involved in this outcome (Table 1). Analysis of oral texture for the cheese + nisin samples shows a noticeable reduction in its oral acceptance from time zero onwards. The more reduced quality of the cheese + nisin samples than those of the other cheese types is due to both loss of water and easy reaction of free nisin with the cheese matrix. The cheese samples containing the nisin-loaded nanoparticles display almost the same behavior as the control cheese samples. This is because of the low amount of the nanoparticles, avoidance of direct and 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 CHEESE SAMPLES Measured variables Cheese samples Cheese Storage times (day) 0 2 4 8 0 2 4 8 0 2 4 8 Flavor 5.00 0.00a 5.00 0.00a 4.00 0.00c 4.00 0.00c 4.88 0.33ab 4.00 0.00c 4.00 0.00c 3.00 0.00d 5.00 0.00a 4.66 0.50ab 4.55 0.52b 4.00 0.00c Aroma 5.00 0.00a 5.00 0.00a 4.00 0.00b 4.00 0.00b 4.88 0.33a 4.00 0.00b 4.00 0.00b 3.00 0.00c 5.00 0.00a 5.00 0.00a 5.00 0.00a 4.00 0.00b Oral texture 4.88 0.33a 4.88 0.33a 3.88 0.33b 3.88 0.33b 3.88 0.33b 3.88 0.33b 3.88 0.33b 2.88 0.00c 4.88 0.33a 5.00 0.00a 4.88 0.33a 3.88 0.33b Color 5.00 0.00a 5.00 0.00a 5.00 0.00a 4.00 0.00c 4.88 0.33ab 4.00 0.00c 4.00 0.00c 3.00 0.00d 5.00 0.00a 4.66 0.50b 5.00 0.00a 4.00 0.00c Overall acceptability 5.00 0.00a 5.00 0.00a 4.00 0.00b 4.00 0.00b 4.88 0.33a 4.00 0.00b 4.00 0.00b 3.00 0.00c 5.00 0.00a 5.00 0.00a 5.00 0.00a 5.00 0.00a Yield point (in kPa) 155 12ab 173 13a 172 14a 170 9a 173 6a 147 35ab 128 35b 172 14a 166 11a 171 12a 169 7a 172 9a Maximum force (in Newton) 1.98 0.13a 2.04 0.01a 2.04 0.01a 2.04 0.0a 2.04 0.01a 2.05 0.01a 2.04 0.01a 1.88 0.34a 2.04 0.01a 2.04 0.01a 2.05 0.01a 2.04 0.01a

Cheese + free nisin

Cheese + nisin-loaded chitosan/alginate nanoparticles

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

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regarded as a new strategy for constructing composites of nisin with more antibacterial efcacies in the near future. These features make this composite an excellent biopreservative candidate in food science. These promising outcomes also provide further impetus for more investigation on this hybrid biopreservative and prepare the way for the practical use of these biocompatible nanoparticles in food industry.

ACKNOWLEDGMENT
Authors are thankful to Dr. Parviz Aberumand for constructive guidance in some process of performing the research.

FIG. 3. SCANNING ELECTRON MICROGRAPH OF ULTRA FILTERED FETA CHEESE BLOCKS CONTAINING NISIN-LOADED CHITOSAN/ALGINATE NANOPARTICLES AFTER 72 H

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CONCLUSIONS
In connection with the whole results, it was indicated that nisin-loaded chitosan/alginate nanoparticles shows more antibacterial potency than free nisin without any unwanted effects on the quality of the original UF Feta cheese. Therefore, the use of these biocompatible nanoparticles can be
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