Antimicrobial Effects of Selenium and Chitosan Nanoparticles on Raw Milk and Kareish Cheese Текст научной статьи по специальности «Биологические науки»

2022, Scienceline Publication

Worlds Veterinary Journal

World Vet J, 12(3): 330-338, September 25, 2022

DOI: https://dx.doi.org/10.54203/scil.2022.wvj42

Antimicrobial Effects of Selenium and Chitosan Nanoparticles on Raw Milk and Kareish Cheese

Shimaa N. Mohamed 1* , Hamdy A. Mohamed2 , Hend A. Elbarbary 2(S , and Nahla Abo EL-Roos 1 B

'Food Hygiene Animal Health Research Institute, Egypt

2Food Hygiene and Control Department, Faculty of Veterinary Medicine, Benha University, Egypt *Corresponding author's Email: vet_shimaanabil @yahoo.com

ABSTRACT

The contamination of milk and its dairy products with different microorganisms could cause public health hazards. Antibacterial nanoparticles (NPs) are a novel way to ensure that milk and milk products are safe. The present study investigated the effect of chitosan NPs (CS-NPs) and selenium NPs (Se-NPs) on some microorganisms, which consequently affect raw milk and Kareish cheese. Small-sized nanomaterials of Se-NPs and CS-NPs at the size of approximately 20 nm were used in this study. The samples were 700 ml raw milk and 700g Kareish cheese manufactured from 3000 mg milk. The concentrations of used nanoparticles were 0.5%, 1%, and 1.5% for Se-NPs and 2.5%, 5%, and 10% for CS-NPs. They were used to improve the microbial properties of milk and Kareish cheese samples during storage at the refrigerated temperature of 4°C. The aerobic plate count, Enterobacteriaceae count, Staphylococcus count, and mold count were significantly reduced in milk and Kareish cheese samples treated with CS-NPs and Se-NPs. The study has confirmed that CS-NPs and Se-NPs indicated high antimicrobial activity against the studied microorganisms at all concentrations although CS-NPs were more effective than Se-NPs. It can be concluded that these NPs can be used as preservatives in milk and milk products, such as Kareish cheese. In addition, increasing the concentrations of these NPs by 10% for CS-NPS and 1.5% for Se-NPS boosted their effects.

Keywords: Chitosan, Enterobacteriaceae, Kareish cheese, Nanoparticle, Selenium, Staphylococcus aureus INTRODUCTION

In many parts of the world, milk and dairy products are perfect media for microorganism growth due to their high nutritional content (Ledenbach and Marshall, 2009). It is impossible to eliminate microbe contamination of milk during the preparation of various dairy products; consequently, the microbiological content of milk is an important factor in its quality from a safety standpoint (Singh et al., 2011). Many zoonotic bacteria, such as Escherichia coli (E. coli), Salmonella, and Staphylococcus aureus (S. aureus), can be found in dairy products and can cause serious diseases, especially in immunocompromised consumers (Pal, 2007).

Nanotechnology has made its way into improving the quality of food as well as unique food supplements, additives, and nutrients (Huang et al., 2017). It is a new technology that could mark the beginning of the second technological generation. It focuses on the characterization, fabrication, and manipulation of structures or materials smaller than 100 nm (Ozimek et al., 2010). It aims to improve the tastes, textures, and bioavailability of minerals and supplements, as well as extending the shelf life of the products (Chaudhry and Castle, 2011). As a result, nanotechnological advantages have lately been used to tackle food and environmental challenges (Jaiswal et al., 2019) by enhancing the quality of micronutrients during processing, storage, and distribution (Chen et al., 2006). Nanomaterials are now used in the food industry for various purposes, such as food ingredients or additives, or as part of packaging materials (Rhim et al., 2013). Selenium nanoparticles (Se-NPs) can be used instead of antibiotics, such as ampicillin, to prevent and treat a variety of bacterial diseases and infections in people. Nano selenium is 60 times more effective than traditional treatments in treating infections caused by S. aureus, E. coli, and P. aeruginosa.

It can improve absorption into plants, animals, people, and microbes and act as an antioxidant with a lower risk of selenium toxicity. Furthermore, one of the most important uses of Se-NPs is chemoprevention via immune activation (Majeed et al., 2018). Chitin is a polysaccharide of animal origin that has a fibrous structure and is abundant in nature. Chitosan (CS) can be made by removing the acetyl groups from the chitin structure that is the major component of the exterior skeleton of insects and crustaceans, such as shrimp, crabs, and lobster (Kumar et al., 2005). Because of its nontoxicity, biodegradability, and antibacterial characteristics (Widnyana et al., 2021), CS is used in biomedical research, agriculture, genetic engineering, as well as the food industry, and water treatment (El-Dahma et al., 2017). Chitosan has a stronger effect on Gram-positive bacteria (S. aureus, Lactobacillus plantarum, Lactobacillus brevis, and

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Lactobacillus bulgaris) than Gram-negative bacteria (E. coli, Salmonella typhy (Coma et al., 2003). In contrast, Chung et al. (2004) found that Gram-negative bacteria are more sensitive to CS than Gram-positive bacteria as they have substantially more hydrophilicity. Chitosan's antifungal effect is thought to be fungistatic rather than fungicidal, with the potential to transmit regulatory changes in both the host and the fungus (Raafat and sahl, 2009).

This study aimed to investigate the way CS-NPS and Se-NPs effectively reduce pathogens in milk and Kareish cheese during cold storage.

MATERIAL AND METHODS

Collection of samples

Fresh raw milk (700ml) used in this study was purchased from dairy shops in El Monofiya Governorate, Egypt. All samples were kept in an ice box, transferred to the laboratory with minimum delay under completely hygienic conditions, and examined as rapidly as possible. The total sample was divided into two parts, one for raw milk examination and the other for manufacturing Kareish cheese for cheese examination. The experiment was repeated three times on different batches of milk dairy shops.

Preparation of milk sample

The milk samples (700 ml of raw milk) were divided into seven groups, 100 ml per group. The first three groups were treated with Se-NPs at concentrations of 0.5%, 1%, and 1.5%. The second three groups were treated with 2.5%, 5%, and 10% CS-NPs. The seventh group served as control. All samples were kept at 4°C. The analysis of the samples was performed on at days 1, 3, 6, 9, 12, and 15 of storage.

Kareish cheese manufacturing

Cheese manufacture essentially involves the coagulation of casein. Raw milk was heated at 74°C for 15 seconds and then cooled rapidly to 40°C AQt this point, 1.5% yogurt starter culture was added for coagulation. When coagulation had been completed, the curd was transferred into gauze to get rid of whey in 24 hours. In the next step, cut and stored in its pasteurized salted whey (7% salt) for 24 hours. Cheese samples were stored at 4°C (Phelan et al., 1993). In Kareish cheese, the fat level in dry matter and the moisture content should not exceed 10% and 75%, respectively (Egyptian Standard 2000/4-1008). To manufacture 700g of Kareish cheese, 3000 ml of raw milk was used. After that, the cheese was divided into seven groups followed by the addition of Se-NPs (0.5%, 1%, and 1.5%) and CS-NPs (2.5%, 0.5%, and 1%).

Nanomaterials

The Se-NPs and CS-NPs were prepared at the Naqaa Foundation for Scientific Research, Technology, and Development in Giza, Egypt. The Se-NPs were prepared according to the modified method of Qian Li et al. (2010). The Se solution was obtained by adding 100 mM of Sodium selenite to 50 mM ascorbic acid. Varied sodium selenite to ascorbic acid ratios (1:1, 1:2, 1:3, 1:4, 1:5, 1:6) had been reacted from the stock solution. The ascorbic acid was added drop by drop to the sodium selenite under magnetic stirring at various rpm (200, 600, 1000 rpm) at room temperature for 30 minutes. Combos had been allowed to react with each other's in the targeted shape until the shade alternate was observed from colorless to mild orange. Soon after the shad alternate was once determined, the combination used to be diluted to 25 ml with double distilled water.

Chitosan NPs were prepared according to Calvo et al. (1998). Chitosan deacetylation was 75%, with a molecular weight of 200 KDa. The CS solution was made using the ionotropic gelation process, which involved dissolving 100 mg of CS in a 1 percent v/v acetic acid solution and stirring it at room temperature until it turned transparent. A 0.1 molar sodium hydroxide solution was added to the mixture with a pH of 6.5. In a Pyrex glass flask, 10 ml of 0.80 mg/ml tripolyphosphate aqueous solution was added dropwise at room temperature under a magnetic stirrer at 750 rpm. The solution was then sonicated at room temperature for 10 minutes at 80 percent amplitude using the SB-5200 DTD Ultrasonic Cleaner, China (Vaezifar, 2013). The CS-NP solution was filtered by nylon syringe 0.22^m mesh and then freeze-dried for subsequent analysis.

Microbiological assay

Serial dilution was prepared according to ISO 1999; the surface plate method determined aerobic plate count at 35°C (Petran et al., 2015). After that, Baird Parker Agar was used to isolate and differentiate coagulase-positive staphylococci in food (FDA, 2001). Colonies appeared in gray-black, and a clear halo was developed around colonies from coagulase-positive S. aureus. Enterobacteriaceae counts, E coli, and Salmonella spp., were determined according to ISO 21528: 2017. Neutral red colonies resulted in pink colonies due to glucose fermentation resulting from produced acid and decreased PH. Finally, Enumeration and isolation of fungi were done according to International Commission on

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Microbiological Specifications for Foods (ICMSF, 1996) using Sabouraud dextrose agar with chloramphenicol (0.05 mg/ml), which was then incubated at 28-30°C for 2-21 days.

Statistical analysis

Microbiological data were converted into logarithms of the colony number of forming units (CFU/gm). The analysis of variance (ANOVA) was performed in SPSS software (Version 22, SPSS Inc. Chicago, IL, and USA). Means and standard deviations were calculated. By applying Duncan's Multiple Range test, multiple mean comparisons were made to measure the specific differences between pairs of means. Values were statistically significant at the p < 0.05 level

RESULTS AND DISCUSSION

Selenium has an antibacterial effect at extremely high concentrations (1.5 to 3 mg/kg body weight), which are fatal to living organisms. Therefore, its use is limited to medicinal purposes (Khiralla and El-Deeb 2015). According to some researchers, Se-NPs are better than elemental selenium because they have antibacterial activity at low doses of 20 ^g/mL (Huang et al., 2017).

Because of its nontoxicity, biodegradability, and antibacterial characteristics, CS is used for various purposes (Widnyana et al., 2021), including food processing (Cheba, 2011). In vitro tests have shown that Gram-negative bacteria are more sensitive to CS than Gram-positive bacteria, with higher morphological alterations after treatment (Chen et al., 2002; Simunek et al., 2006; and Eaton et al., 2008). The amount of adsorbed CS is determined by the charge density on the cell surface. Adsorbed CS at higher levels would cause more cell membrane structure and permeability alterations. This indicates that the host-microbe influences the antibacterial method of action (Masson et al., 2008). In the current study, the microbiological changes as aerobic plate count (APC), Staphylococcus spp. count, Enterobacteriaceae count, and mold count of milk and cheese samples were estimated throughout the cooling storage at 4°C for 15 days

Aerobic bacterial count

Aerobic bacterial count in milk samples

The initial total bacterial load was reduced over time when CS-NPs and Se-NPs were added to milk samples. During chilling storage of milk samples treated with CS-NPs, APC decreased from 5.71 to 4.2 ~1 log10CFU/ml at a concentration of 2.5%. The microbial effect of CS-NPs against total bacterial count increased by increasing the concentration of CS-NPs, so when the concentration reached 10%, the count of total bacterial count significantly decreased from 5.71 to 3.86 (~2 log10) CFU/ml (Table 1, p < 0.05). In milk samples treated with 0.5% Se-NPs, APC decreased from 5.71 to 4.2 ~1 log10 CFU/ml, but when the concentration reached 1.5%, APC decreased from 5.71 to 3.8 ~2 log10 CFU/ml (p < 0.05, Table 1). The Egyptian standards for raw milk (ES:154-1/2005) mentioned that the acceptable count of total bacterial count should be less than 200 count /ml (EOS, 2005).

Aerobic bacterial count in Kareish cheese samples

In Kareish cheese samples treated with 10% CS-NPs, the APC count decreased from 4.56 to 2.63 ~2 log10 CFU/ml (Table 2) and the APC count significantly decreased from 4.56 to 3.4 ~1 log10 CFU/ml in cheese samples treated with 1.5% Se-NPS (Table 2, p < 0.05). According to the obtained results, 10% CS-NPs was the most effective antimicrobial agent (against total bacterial count followed by 1.5% Se-NPs. As Hariharan et al. (2012) stated, the antibacterial activity was related to the concentration of nanoparticles. Gram-positive and Gram-negative bacteria are both inhibited by the antibacterial mechanism of Se-NPs, which is unknown yet. Currently, it is thought that Se-NPs break the bacterial cell wall by interacting with the peptidoglycan layer and damaging the double-stranded DNA structure (Sonkusre et al., 2014). Chitosan NPs have antimicrobial activity due to electrostatic interaction between their positive charge and the negative charge of bacterial membranes leading to cell membrane lysis of bacterial cells (Rabea et al., 2003; Tripathi et al., 2008). Chitosan NPs can also interact with essential microbial nutrients, causing microbial growth disruption and eventually death (Jia et al., 2001; Rabea et al., 2003).

Staphylococci bacterial count

As shown in figures 1 and 2, the antibacterial effect of nanoparticles against staphylococci in milk and Kareish cheese, respectively, was confirmed.

Staphylococci count in milk samples

Staphylococci count decreased in milk samples from 4.66 to 3.36 ~1 log10 CFU/ml at the concentration of 2.5% CS-NPs. Moreover, the microbial effect of CS-NPs against staphylococci increased by increasing the concentration of CS-NPs, so the count of staphylococci decreased from 4.66 to 2.66 (~2 log10 ) CFU/ml when the concentration reached 10% (Figure 1). In milk samples treated with 0.5% Se-NPs, staphylococci decreased from 4.66 to 3.3 ~1 log10 CFU/ml, and when the concentration reached 1.5%, staphylococci decreased from 4.66 to 2.7 ~2 log10 CFU/ml (Figure 1).

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Table 1. Effects of different concentrations of chitosan and selenium nanoparticles on the aerobic plate count of the examined milk samples during storage at 4°C

Groups First day Third day Sixth day Ninth day Twelfth day Fifteenth day

Control 5.71 ± 0.05a 5.84 ± 0.089b 6.48 ± 0.094 d 6.87 ± 0.061d 7.51 ± 0.047d 7.88 ± 0.04d

2.5% chitosan 5.71 ± 0.05a 5.57 ± 0.02ab 5.25 ± 0.031ab 4.84 ± 0.01e 4.61 ± 0.02e 4.2 ± 0.03f

5% chitosan 5.71 ± 0.05a 5.43 ± 0.03ab 5.19 ± 0.02ab 4.72 ± 0.01e 4.27 ± 0.01f 3.86 ± 0.04fg

10% chitosan 5.71 ± 0.05a 5.11 ± 0.035ab 4.86 ± 0.03c 4.5 ± 0.01f 3.74 ± 0.04fg 3.17 ± 0.05g

0.5% selenium 5.71 ± 0.05a 5.67 ± 0.01ab 5.46 ± 0.06ab 4.91 ± 0.03e 4.80 ± 0.02e 4.62 ± 0.02f

1% selenium 5.71 ± 0.05a 5.58 ± 0.01ab 5.43 ± 0.01ab 4.79 ± 0.02e 4.53 ± 0.01f 4.39 ± 0.01fg

1.5% selenium 5.71 ± 0.05a 5.38 ± 0.03ab 5.17 ± 0.02ab 4.67 ± 0.01e 4.38 ± 0.01f 3.8 ± 0.07fg

The values represented as mean ± standard deviation of three experiments. a- b-c- d-e- f- g means superscript letters within a column are significantly

different (p < 0.05).

Table 2. Effect of different concentrations of chitosan and selenium nanoparticles on the aerobic plate count of the examined cheese samples during storage at 4°C

Groups First day Third day Sixth day Ninth day Twelfth day Fifteenth day

Control 4.56 ± 0.3a 4.76 ± 0.3b 4.98 ± 0.3e 5.39 ± 0.13e 5.56 ± 0.13g 6.3 ± 0.4g

2.5% chitosan 4.56 ± 0.3 a 4.37 ± 0.2ab 4.22 ± 0.15c 3.85 ± .02d 3.45 ± 0.1d 3.33 ± 0.11d

5% chitosan 4.56 ± 0.3a 4.31 ± 0.11ab 4.19 ± 0.11cd 3.63 ± 0.1d 3.4 ± 0.17d 3.32 ± 0.16d

10% chitosan 4.56 ± 0.3 a 4.24 ± 0.2ab 4.15 ± 0.21d 3.42 ± 0.15d 3.25 ± 0.14f 2.63 ± 0.2h

0.5% selenium 4.56 ± 0.3 a 4.45 ± 0.15ab 4.37 ± 0.2c 4.25 ± 0.2c 4.13 ± 0.1c 3.82 ± 0.13d

1% selenium 4.56 ± 0.3 a 4.4 ± 0.1ab 4.28 ± 0.1cd 4.13 ± 0.3c 3.85 ± 0.3d 3.6 ± 0.11d

1.5% selenium 4.56 ± 0.3 a 4.32 ± 0.2ab 4.22 ± 0.1cd 3.91 ± 0.2cd 3.61 ± 0.1d 3.41 ± 0.20d

The values represented as mean ± standard deviation of three experiments. a different (p < 0.05).

; means superscript letters within a column are significantly

Staphylococcal

count (log10 CFU/ml)

- Control 2.5% Chitosan 5% Chitosan 10% Chitosan 0.5% selinium 1% selinium 1.5% selinium

1 st day 3rd day 6th day 9th day

Days of storage

12th day

15th day

Figure 1. Effect of different concentrations of chitosan and selenium nanoparticles on Staphylococci count of the examined milk samples during storage at 4°C

Staphylococci count in Kareish cheese samples

Staphylococci count decreased in cheese samples from 4.43 to 3.24 ~1 log10CFU/ml using 2.5% CS-NPs. The microbial effect of CS-NPs against staphylococci increased by increasing the concentration of CS-NPs, so when the concentration reached 10%, the count of staphylococci decreased from 4.43 to 2.58 ~2 log10 CFU/ml (Figure 2). In cheese samples treated with Se-Nps at the concentration of 0.5%, staphylococci decreased from 4.43 to 3.63~1 log10 CFU/ml, and when the concentration reached 1.5%, staphylococci decreased from 4.43 to 3.2 ~1 log10 CFU/ml as seen in Figure 2. Similar to the findings of Qi et al. (2004), Ro-drigus-Nunez et al. (2012), Salmabi and Seema (2013), Van Toan et al. (2013), Younes et al. (2014), and Widnyana et al. (2021), the S. aureus was inhibited by CS. Moreover, the antimicrobial effect of Se-NPs recorded by Khiralla and El-Deeb (2015) indicated that the inhibition zone increased with an increase in the concentration of Se-NPs. The Se-NPs were reported to be a potent antimicrobial agent against S. aureus (Chudobova et al., 2014). According to Phong et al. (2011), the proportion of live S. aureus decreased in the presence of Se-NPs at 7.8, 15.5, and 31 g/mL after 3, 4, and 5 hours. The Egyptian standards for Kareish cheese (No.1008/2000) mentioned that S. aureus (coagulate-positive) was absent in 1 g (EOS, 2000).

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Mohamed ShN, Mohamed HA, Elbarbary HA, and EL-Roos NA (2022). Antimicrobial Effects of Selenium and Chitosan Nanoparticles on Raw Milk and

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2

Staphylococcal

count (log 10 cfu/gm)

^^^ Control

2.5% Chitozan 5% Chitozan )l( 10% Chitozan 0.5% selinium 1% selinium 1.5 % selinium

Figure 2. Effect of different concentrations of chitosan and selenium nanoparticles on Staphylococci count of the examined cheese samples during storage at 4°C

Enterobacteriaceae count

Tables 3 and 4 showed that results obtained from treated samples significantly differed from the control result (p < 0.05). The antibacterial and antibacterial assays of CS-NPs against Gram-negative and Gram-positive bacteria were applied at different concentrations. The inhibition increased by increasing the concentration of CS-NPs. Gram-negative bacteria were more sensitive to CS-NPs than gram-positive bacteria (Coma et al., 2003).

Enterobacteriaceae count in milk samples

Enterobacteriaceae count has been decreased in milk samples from 4.57 to 3.27 ~1 log10CFU/ml at a concentration of 2.5%, and the microbial effect of CS-NPs against Enterobacteriaceae increased by increasing the concentration of CS-NPs. Enterobacteriaceae were not detected at the highest concentration of chitosan, 10% (Table 3). In milk samples treated with 0.5% Se-Nps, Enterobacteriaceae decreased from 4.57 to 3.39 ~1 log10 CFU/ml, but when the concentration reached 1.5%, staphylococci decreased from 4.57 to 2.65 ~1 log10 CFU/ml (Table 3). The Egyptian standards for raw milk (ES:154-1/2005) declared that the total coliforms are less than 10 count/ml E. coli and Salmonella and other pathogens absent in 1 ml (EOS, 2005).

Enterobacteriaceae count in Kareish cheese samples

Enterobacteriaceae count significantly decreased in cheese samples from 3.44 to 2.63 ~1 log10CFU/ml in different concentrations of CS-NPs and Se-Nps (p < 0.05, Table 4). The results agreed with Balicka-Ramisz et al. (2005), Liu et al. (2006), and Chung and Chen (2008), who reported that CS had antibacterial activity against E. coli. Balicka-Ramisz et al. (2005) and Benhabiles et al. (2012) recorded the antibacterial activities of CS against Salmonella sp. Results agreed with Hassanien and Shaker (2020), who used CS-NPs at a 30 ^g/mL concentration. Chitosan NPs exerted a high bactericidal effect on isolates, such as E. coli O157:H7 recovered from Kareish cheese samples, which significantly increased with an increase in concentration. Khiralla and El-Deeb (2015) evaluated the effect of Se-NPs against foodborne pathogens, such as E. coli and S. aureus. They found that the inhibition zone increases with increasing Se-Nps concentration. According to Shrestha et al. (2010) and Khurana et al. (2019), CS-NPs and Se-NPs have antibacterial effects against Enterococcus faecalis. Selenium NPs were highly effective against E. faecalis biofilm at the concentration of 1mg/ml (Sanjay et al., 2021). The Egyptian standards for Kareish cheese (No.1008/2000) mentioned that the total coliforms should be less than 10 CFU/g, E. coli should be absent in 1g Kareish cheese, Salmonella and other pathogens absent in 25 g (EOS, 2000).

Molds count

The antibacterial action of nanoparticles against molds was demonstrated by the fact that the counts of treated and control samples were significantly different (p < 0.05). Molds were not detected at high concentrations of CS-NPs (10%) and Se-NPs (1.5%) in milk samples (Table 5). However, the mold count significantly decreased by ~1 log10 CFU/ml in cheese samples at high concentrations CS-NPs (10%) and Se-NPs (1.5%), respectively (p < 0.05, Table 6). Antifungal activity in the current study agreed with that of Yien et al. (2012), indicating that the CS-NPs were observed to be natural antifungal agents when used in concentrations of 1-3 mg/ml against Candida albicans, Aspergillus niger, and Fusarium solani pathogenic strain isolated from clinical specimens. Moreover, a study by Shakibaie et al. (2015) indicated the anti-biofilm activity of biologically generated (Se-NPs) in concentrations ranging from 10 to 200 mg/mL against the biofilm

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days of storage

produced by clinically isolated fungus strains, such as Aspergillus fumigatus and Candida albicans. The obtained results of the current study agreed with those of Rasha et al. (2019), indicating that the use of CS-NPS in concentrations of 0.25% and 0.5% before or after manufacturing Kareish cheese could prolong safe preservation as the nano-chitosan have the antimicrobial potential against several bacteria and fungi, such as Aspergillus flavus. Furthermore, Elsharawy et al. (2019) revealed that the mold counts in Kareish cheese treated with 1% CS were lower than untreated cheese samples during the storage period. Since direct contact with CS causes hyphae to weaken and swell, the fungistatic characteristics of CS are linked to its ability to induce morphological changes in the cell wall (Rabea et al., 2003). The Egyptian standards for Kareish cheese (No.1008/2000) mentioned that yeasts and molds should be less than 10 CFU/g (EOS, 2000).

Table 3. Effect of different concentrations of chitosan and selenium nanoparticles on Enterobacteriaceae count of the examined milk samples during storage at 4°C

Groups First day Third day Sixth day Ninth day Twelfth day Fifteenth day

Control 4.57 ± 0.0 2a 4.76 ± 0.09a 4.97 ± 0.04a 5.53 ± 0.06a 5.89 ± 0.05e 6.2 ± 0.04g

2.5% chitosan 4.57 ± 0. 02a 4.17 ± 0.02a 3.92 ± 0.031b 3.79 ± 0.01c 3.65 ± 0.02c 3.27 ± 0.03bc

5% chitosan 4.57 ± 0. 02a 4.34 ± 0.03a 3.73 ± 0.02c 3.11 ± 0.01bc 3.14 ± 0.01dc 2.75 ± 0.04f

10% chitosan 4.57 ± 0. 02a 4.19 ± 0.04b 3.36 ± 0.03 bc 2.82 ± 0.01d 2.74 ± 0.04f *ND

0.5% selenium 4.57 ± 0.0 2a 4.49 ± 0.01a 4.17 ± 0.06b 3.9 ± 0.03c 3.87 ± 0.02c 3.39 ± 0.02c

1 % selenium 4.57 ± 0. 02a 4.36 ± 0.01a 3.76 ± 0.01c 3.44 ± 0.02c 3.57 ± 0.01dc 2.95 ± 0.01f

1.5 % selenium 4.57 ± 0.02a 4.23 ± 0.03b 3.57 ± 0.02c 3.35 ± 0.01d 2.9 ± 0.01f 2.65 ± 0.07f

The values represented as mean ± standard deviation of three experiments. a ^ c 4 e f g means superscript letters within a column are significantly

different (p < 0.05). *ND: Not detected

Table 4. Effect of different concentrations of chitosan and selenium nanoparticles on Enterobacteriaceae count of the examined cheese samples during storage at 4°C.

Groups First day Third day Sixth day Ninth day Twelfth day Fifteenth day

Control 3.44 ± 0.1a 3.56 ± 0.3a 3.98 ± 0.1e 4.26 ± 0.13e 4.56 ± 0.13f 4.73 ± 0.2f

2.5% chitosan 3.44 ± 0.1 a 3.33 ± 0.2ab 3.27 ± 0.1b 3.12 ± .02c 2.91 ± 0.1cd 2.63 ± 0.11d

5% chitosan 3.44 ± 0.1 a 3.28 ± 0.1ab 3.21 ± 0.1b 2.85 ± 0.2cd 2.66 ± 0.2d 2.42 ± 0.14d

10% chitosan 3.44 ± 0.1a 3.2 ± 0.1ab 3.15 ± 0.1c 2.69 ± 0.15d 2.52 ± 0.14d 2.32 ± 0.2g

0.5% selenium 3.44 ± 0.1a 3.4 ± 0.2ab 3.33 ± 0.2b 3.22 ± 0.2c 3.12 ± 0.1c 2.83 ± 0.15cd

1% selenium 3.44 ± 0.1a 3.35 ± 0.1ab 3.29 ± 0.1b 3.13 ± 0.3c 2.84 ± 0.1cd 2.64 ± 0.21cd

1.5% selenium 3.44 ± 0.1a 3.3 ± 0.2ab 3.25 ± 0.1b 2.98 ± 0.2cd 2.72 ± 0.1cd 2.58 ± 0.23d

The values represented as mean ± standard deviation of three experiments means superscript letters within a column are significantly

different (p < 0.05).

Table 5. Effect of different concentrations of chitosan and selenium nanoparticles on molds count of the examined milk samples during storage at 4°C._

Groups First day Third day Sixth day Ninth day Twelfth day Fifteenth day

Control 3.8 ± 0.04a 3.96 ± 0.01a 4.73 ± 0.09e 4.95 ± 0.061e 5.67 ± 0.04g 5.92 ± 0.04g

2.5% chitosan 3.8 ± 0.04a 3.51 ± 0.02a 3.35 ± 0.031ab 3.22 ± 0.01c 2.81 ± 0.02d 2.3 ± 0.03d

5% chitosan 3.8 ± 0.04a 3.36 ± 0.03ab 3.21 ± 0.02ab 2.7 ± 0.01cd 2.63 ± 0.01d *ND

10% chitosan 3.8 ± 0.04a 3.11 ± 0.03b 2.9 ± 0.03c 2.59 ± 0.01d 2.35 ± 0.04f *ND

0.5% selenium 3.8 ± 0.04a 3.75 ± 0.01a 3.67 ± 0.06ab 3.41 ± 0.03c 3.11 ± 0.02cd 2.7± 0.02d

1% selenium 3.8 ± 0.04a 3.68 ± 0.01ab 3.56 ± 0.01ab 3.28 ± 0.02cd 2.94 ± 0.01d *ND

1.5% selenium 3.8 ± 0.04a 3.59 ± 0.03ab 3.34 ± 0.02ab 2.91 ± 0.01cd 2.55 ± 0.01d *ND

The values represented as mean ± standard deviation of three experiments. ^ c, d e, f g means superscript letters within a column are significantly

different (p < 0.05). *ND: Not detected.

Table 6. Effect of different concentrations of chitosan and selenium nanoparticles on molds count of the examined

cheese samples during storage at 4°C

Groups First day Third day Sixth day Ninth day Twelfth day Fifteenth day

Control 3.54 ± 0.3a 3.73 ± 0.2a 3.92 ± 0.21e 4.34 ± 0.13e 4.57 ± 0.2f 5.35 ± 0.3f

2.5% chitosan 3.54 ± 0.3a 3.43 ± 0.14ab 3.33 ± 0.15b 3.22 ± 0.2c 2.83 ± 0.12cd 2.63 ± 0.12d

5% chitosan 3.54 ± 0.3a 3.36 ± 0.12ab 3.26 ± 0.11b 3.13 ± 0.11cd 2.72 ± 0.15d 2.53 ± 0.1d

10% chitosan 3.54 ± 0.3a 3.24 ± 0.2ab 3.16 ± 0.14c 2.77 ± 0.12d 2.53 ± 0.13d 2.42 ± 0.14g

0.5% selenium 3.54 ± 0.3a 3.48 ± 0.13ab 3.4 ± 0.21b 3.32 ± 0.14c 3.13 ± 0.1c 2.76 ± 0.11cd

1% selenium 3.54 ± 0.3a 3.39 ± 0.11ab 3.31 ± 0.11b 3.15 ± 0.20c 2.92 ± 0.14cd 2.68 ± 0.2cd

1.5% selenium 3.54 ± 0.3a 3.3 ± 0.21ab 3.25 ± 0.2b 2.91 ± 0.21d 2.65 ± 0.1cd 2.52 ± 0.13d

The values represented as mean ± standard deviation of three experiments. b, c d e, f g means superscript letters within a column are significantly

different (p < 0.05).

6

CONCLUSION

Biocompatible Se-NPs and CS-NPs had high antimicrobial activity against pathogenic and spoilage Gram-positive and Gram-negative bacteria, as well as molds that affect raw milk and Kareish cheese. According to this study, nanoparticles can be employed as a preservative in milk and Kareish cheese to extend their shelf life. Further studies should be conducted on the effectiveness of nanotechnology and nanoparticles on dairy products, their prevention of microbial contamination, and the limitation of mold excretions like aflatoxins.

DECLARATIONS

Acknowledgments

The authors are grateful to the Animal Health Research Institute in Egypt for all of their assistance in providing materials, media, instruments, and devices used in this research. Naqaa Foundation for Scientific Research, Technology, and Development in Giza, Egypt, provided the nanoparticles. This article did not receive any other financial support.

Authors' contribution

Hend Ahmed Elbarbary and Hamdy Abd El Samea Mohamed created the study plan and revised the research article. Shimaa Nabil Mohamed and Nahla Abo EL-Roos examined the data and conducted laboratory experiments. Shimaa Nabil did the statistical analysis and wrote the paper. Nahla Abo EL-Roos, who also revised the research manuscript, provided the experimental instruments. All authors read and approved the final version of the manuscript for publishing in the present journal

Competing interests

There are no conflicts of interest declared by the authors. Ethical consideration

The author checked the manuscript for ethical issues, such as plagiarism, consent to publish, misconduct, data fabrication and/or falsification, double publishing and/or submission, and redundancy.

REFERENCES

Assis OBG, Goy R, and Britto D (2008). A review of the antimicrobial activity of chitosan. Polimeros: Ciencia e Tecnologia, 19(3): 241-247. DOI: https://www.doi .org/10.1590/S0104-14282009000300013

Balicka-Ramisz A, Wojtasz-Pajak A, Pilarczyk B, Ramisz A, and Laurans L (2005). Antibacterial and antifungal activity of chitosan. International Society for Animal Hygiene, 2: 406-408. Available at: https://www.isah-soc.org/userfiles/downloads/proceedings/2005/sections/95 vol 2.pdf

Benhabiles MS, Tazdai't S, Lounici H, Drouiche N, Goosend MFA, and Mamerib N (2012). Antibacterial activity of chitin, chitosan and its oligomers prepared from shrimp shell waste. Food Hydrocolloids, 29(1): 48-56. DOI: https://www.doi.org/10.1016/j.foodhyd.2012.02.013

Calvo P, Remunan-Lopez C, Vila-Jata JL, and Alonso MJ (1998). Chitosan and chitosan: Ethylene oxide-propylene oxide blocks copolymer nanoparticles as novel carriers for protein and vaccines. Pharmaceutical Research, 14: 1431-1436. DOI: http://www.doi .org/10.1023/A:1012128907225

Chaudhry Q and Castle L (2011). Food applications of nanotechnologies: An overview of opportunities and challenges for developing countries. Trends in Food Science & Technology, 22 (11): 595-603. DOI: https://www.doi.org/10.1016/j.tifs.2011.01.001

Cheba BA (2011). Chitin and chitosan: Marine biopoly-mers with unique properties and versatile applications. Global Journal Biotechnology & Biochemistry 6(3): 149-153. Available at: http://idosi.org/gjbb/gjbb6(3)11/7.pdf

Chen H, Weiss J, and Shahidi F (2006). Nanotechnology in nutraceuticals and functional foods. Food technology, 60(3): 30-36. Available at: https://agris.fao.org/agrissearch/search.do?recordID=US201301069370

Chen YM, Chung YC, Woan Wang L, Chen KT, and Li SY (2002). Antibacterial properties of chitosan in waterborne pathogen. Journal of Environmental Science and Health, Part A, 37(7): 1379-1390. DOI: https://www.doi.org/10.1081/ESE-120005993

Chudobova D, Cihalova k, Dostalova S, Ruttkay B, Nedecky MA, Merlos Rodrigo K, Tmejova K, Kopel P, Nejd lL, Kudr J et al. (2014). Comparison of the effects of silver phosphate and selenium nanoparticles on Staphylococcus aureus growth reveals potential for selenium particles to prevent infection. FEMS Microbiology Letters, 351(2): 195-201. DOI: https://www.doi.org/10.1111/1574-6968.12353

Chung YC, Su YP, Chen CC, Jia G, Wang HL, Wu JCG, and Lin JG (2004). Relationship between antibacterial activity of chitosan and surface characteristics of cell wall. Acta Pharmacologica Sinica, 25(7): 932-936. Available at:

https://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.1056.2469&rep=rep 1 &type=pdf

Chung YC and Chen CY (2008). Antibacterial characteristics and activity of acid-soluble chitosan. Bioresource Technology, 99(8): 2806-2814. DOI: https://www.doi.org/10.1016/j.biortech.2007.06.044

Coma V, Deschamps A, and Martial-Gros A (2003). Bioactive packaging materials from edible chitosan polymer—antimicrobial activity assessment on dairy-related contaminants. Journal of Food Science, 68(9): 2788-2792. DOI: https://www.doi.org/10.1111/i.1365-2621.2003.tb05806.x

Eaton P, Fernandes JC, Pereira E, Pintado ME, and Malcata FX (2008). Atomic force microscopy study of the antibacterial effects of chitosan on Escherichia coli and Staphylococcus aureus, Ultramicroscopy, 8(10): 1128-1134. DOI: https://wwwdoi.org/10.1016/j.ultramic.2008.04.015

Egyptian organization for standardization and quality (EOS) (2000). Egyptian standards for Kareish cheese, part 4. Available at: https://archive.org/details/es.1008.4.2005/page/n11/mode/2up

7

Egyptian organization for standardization and quality (EOS) (2005). Egyptian standards for Kareish cheese, part 1. Available at: https://archive.org/details/es.154.1.2005

El-Dahma MM, Khattab AA, Gouda E, El-Saadany KM, and Ragab WA (2017). The antimicrobial activity of chitosan and its application on Kareish cheese shelf life. Alexandria Science Exchange Journal, 38(4): 733-745. Available at: https://aseiaiqjsae.iournals.ekb.eg/article 4183 afad3386e619bf5ce3ca00a51d35bd1e.pdf

El Sharawy TMH, Hamdi AM, Ramadan MS, Hend AE, and EL-Diasty EM (2019). Incidence of fungi in soft skimmed milk cheese from pasteurized milk and trials to control them. Benha Veterinary Medical Journal, 37: 91-96. DOI: https://www.doi.org/10.21608/bvmi.2019.16980.1092

Food and Drug Administration (FDA) (2001). Laboratory methods (food). Bacteriological analytical manual (BAM). Available at: http://www.fda.gov/Food/FoodScienceResearch/Laboratory Methods/ucm2006949. htm>

Hariharan H, Al-Dhabi NA, Karuppiah P, and Rajaram SK (2012). Microbial synthesis of selenium nanocomposite using Saccharomyces cerevisiae and its antimicrobial activity against pathogens causing nosocomial infection. Chalcogenide Letters, 9(12): 509-515. Available at: https://chalcogen.ro/509_Hariharan.pdf

Hassanien AA and Shaker EM (2020). Investigation of the effect of chitosan and silver nanoparticles on the antibiotic resistance of Escherichia coli O157:H7 isolated from some milk products and diarrheal patients in Sohag city, Egypt. Veterinary World, 13(8): 1647-1653. DOI: https://www.doi.org/10.14202/vetworld.2020.1647-1653

Helander IM, Nurmiaho-Lassila EL, Ahvenainen R, Rhoades J, and Roller S (2001). Chitosan disrupts the barrier properties of the outer membrane of gram-negative bacteria. International Journal of Food Microbiology, 71(2-3): 235-244. DOI: https://www.doi.org/10.1016/s0168-1605(01)00609-2

Huang X, Dai Y, Cai J, Zhong N, Xiao H, McClements DJ, and Hu K (2017). Resveratrol encapsulation in core-shell biopolymer nanoparticles: Impact on antioxidant and anticancer activities. Food Hydrocolloids, 64: 157-165. DOI: https://www.doi.org/10.1016/i.foodhyd.2016.10.029

International Commission on Microbiological Specifications for Foods: Update (ICMSF) (1996). Food Control, 7(2): 99-101. DOI: https://www.doi.org/10.1016/S0956-7135(96)90007-9

ISO (2017). Microbiology of the food chain, horizontal method for the detection and enumeration of Enterobacteriaceae, Part 2: Colony-count technique. Available at: https://webstore.ansi.org/Standards/SIS/SSENISO215282017-1433308

ISO (1999). Microbiology of food and animal feeding stuffs. Preparation of test samples, initial suspension and decimal dilutions for microbiological examination, Part 1: General rules for the preparation of the initial suspension and decimal dilutions. Available at: https://products.ihs.com/Ohsis-SEOZ345152.html

Jaiswal L, Shankar S, and Rhim JW (2019). Applications of nanotechnology in food microbiology. Methods in Microbiology, Chapter 3, 46: 43-60. DOI: https://www.doi.org/10.1016/BS.MIM.2019.03.002

Jia Z, Shen D, and Xu W (2001). Synthesis and antibacterial activities of quaternary ammonium salt of chitosan. Carbohydrate Research, 333(1): 1-6. DOI: https://www.doi.org/10.1016/s0008-6215(01)00112-4

Khiralla GM and El-Deeb BA (2015). Antimicrobial and antibiofilm effects of selenium nanoparticles on some foodborne pathogens. LWT-Food Science and Technology, 63(2): 1001-1007. DOI: https://www.doi.org/10.1016/Uwt.2015.03.086

Khurana A, Tekula S, Saifi MA, Venkatesh P, and Godugu C (2019). Therapeutic applications of selenium nanoparticles. Biomedicine & Pharmacotherapy, 111: 802-812. DOI: https://www.doi.org/10.1016/i.biopha.2018.12.146

Kumar ABV, Varadaraj MC, Gowda LR, and Tharanathan RN (2005). Characterization of chito-oligosaccharides prepared by chitosanolysis with the aid of papain and Pronase, and their bactericidal action against Bacillus cereus and Escherichia coli. Biochemical Journal, 391(2): 167-175. DOI: https://www.doi .org/10.1042/BJ20050093

Ledenbach LH and Marshall RT (2009). Microbiological spoilage of dairy products. In: W. Sperber and M. Doyle (Editors) Compendium of the Microbiological Spoilage of Foods and Beverages. Food Microbiology and Food Safety. Springer, New York, pp. 1-28. DOI: https://www.doi .org/10.1007/978-1 -4419-0826-1 2

Liu XF, Guan YL, Yang DZ, Li Z, and Yao KD (2006). Antibacterial action of chitosan and carboxy- methylated chitosan. Journal of Applied Polymer Science, 79(7): 1324-1335. DOI: https://www.doi.org/10.1002/1097-4628(20010214)79:7%3C1324::AID-APP210%3E3.0.CO;2-L

Majeed W, Zafar M, Bhatti A, and John P (2018). Therapeutic potential of selenium nanoparticles. Journal of Nanomedicine & Nanotechnology, 9(1): 1000487. Available at: https://www.walshmedicalmedia.com/open-access/therapeutic-potential-of-selenium-nanoparticles-2157-7439-1000487.pdf

Masson M, Holappa J, Hjalmarsdöttir M, Rünarsson ÖV, Nevalainen T, and Järvinen T (2008). Antimicrobial activity of piperazine derivatives of chitosan. Carbohydrate Polymers, 74(3): 566-571. DOI: https://www.doi.org/10.1016/i.carbpol.2008.04.010

Muehlhoff E, Bennett A, and McMahon D (2013). Milk and dairy products in human nutrition. Food and agriculture organisation of the united nations (FAO), Rome, Italy. International Journal of Diary Technology, 67(2): 303-304. DOI: https://www.doi.org/10.1111/1471-0307.12124

Ozimek L, Pospiech E, and Narine S (2010). Nanotechnologies in food and meat processing. ACTA Scientiarum Polonorum Technologia Alimentaria, 9(4): 401-412. Available at: https://www.food.actapol.net/pub/1 4 2010.pdf

Pal M (2007). Zoonoses, 2nd Edition. Satyam Publishers, Jaipur, India. Available at: www. sciepub .com/reference/310463

Petran RL, Grieme LE, and Foong-Cunningham S (2015). Culture methods for enumeration of microorganisms. Compendium of Methods for the Microbiological Examination of Foods. DOI: https://www.doi .org/10.2105/MBEF.0222.011

Phelan JA, Renaud J, and Fox PF (1993). Cheese: Chemistry, Physics, and Microbiology. In: P. F. Fox (Editor), Vol. I. Chapman & Hall., London. Available at: https://link.springer.com/book/10.1007/978-1-4615-2650-6

Phong AT and Thomas JW (2011). Selenium nanoparticles inhibit Staphylococcus aureus growth. International Journal of Nanomedicine, 6: 15531558. DOI: https://www.doi. org/ 10.2147/IJN.S21729

Qian Li, Chen T, Yang F, and zhing JW (2010). Facile and controllable one-step fabrication of selenium nanoparticles assisted by L-cysteine. Materials Letters, 64(5): 614-617. DOI: https://www.doi.org/10.1016/i.matlet.2009.12.019

Qi L, Zirong X, Jiang X, Hu C, and Zou X (2004). Preparation and antibacterial activity of chitosan nanoparticles. Journal of Carbohydrate Research, 339(16): 2693-2700. DOI: https://www.doi.org/10.1016/i.carres.2004.09.007

Raafat D and Sahl HG (2009). Chitosan and its antimicrobial potential - a critical literature survey. Microbial Biotechnology, 2(2): 186-201. DOI: https://www.doi .org/10.1111/i.1751-7915.2008.00080.x

Rabea EI, Badawy ME, Stevens CV, Smagghe G, and Steurbaut W (2003). Chitosan as antimicrobial agent: Applications and mode of action. Biomacromolecules, 4(6): 1457-1465. DOI: http://www.doi .org/10.1021/bm034130m

Rasha MH, Noha M, and Khaled N (2019). A trial for improvement of Kareish cheese quality by using chitosan nanoparticle. Egyptian Journal Of Vetrinary Sciences, 50: 69-80. Available at: https://eivs.iournals.ekb.eg/article_70825_c5a6650ce16f34b71ec2f2ab129c926f.pdf

8

Rhim JW, Park HM, and Ha CS (2013). Bio-nanocomposites for food packaging applications. Progress in Polymer Science, 38(10-11): 1629-1652. DOI: https://www.doi.org/10.1016li.progpolymsci.2013.05.008

Ro-drigus-Nunez JR, López-Cervantes J, Sánchez-Machado DI, Ramírez-Wong B, Torres-Chavez P, and Cortez-Rocha MO (2012). Antimicrobial activity of chitosan-based films against Salmonella typhimurium and Staphylococcus aureus. International Journal of Food Science Technology, 47(10): 2127-2133. DOI: https://www.doi.org/10.1111/i.1365-2621.2012.03079.x

Salmabi KA and Seema PN (2013). Action of Chitosan and its derivatives on clinical pathogens. International Journal of Current Microbiology and Applied Sciences, 3(10): 748-759. Available at: https://www.iicmas.com/vol-3-

10/Salmabi.%20K.%20Assainar%20and%20Seema%20P%20Nair.pdf

Saniay M and Nobuyuki T (2021). Biosynthesized selenium nanoparticles: Characterization, antimicrobial, and antibiofilm activity against Enterococcusfaecalis, Peer Journals, 9: e11653. DOI: https://www.doi.org/10.7717/peeri.11653

Shakibaie M, Salari Mohazab N, and Ayatollahi Mousavi SA (2015). Antifungal activity of selenium nanoparticles synthesized by Bacillus species Msh-1 against Aspergillus fumigatus and Candida albicans. Jundishapur Journal of Microbiology, 8(9): e26381. DOI: https://www.doi.org/10.5812/iim.26381

Shrestha A, Shi Z, Neoh KG, and Kishen A (2010). Nanoparticulates for antibiofilm treatment and effect of aging on its antibacterial activity. Basic Research, 36(6): 1030-1035. DOI: https://www.doi.org/10.1016/i.ioen.2010.02.008

Simunek J, Tishchenko G, Hodrová B, and Bartonová H (2006). Effect of chitosan on the growth of human colonic bacteria. Folia Mocrobiologica, 51: 306-308. DOI: https://www.doi.org/10.1007/BF02931820

Singh P, Wani AB, Saengerlaub S, and Langowski H (2011). Understanding critical factors for the quality and shelf-life of MAP fresh meat: A review. Critical Reviews in Food Science and Nutrition, 51(2): 146-177. DOI: https://www.doi.org/10.1080/10408390903531384

Sonkusre P, Nanduri R, Gupta P, and Cameotra SS (2014). Improved extraction of intracellular biogenic selenium nanoparticles and their specificity for cancer chemoprevention. Journal of Nanomedicine & Nanotechnology, 5(2): 1000194. DOI: https://www.doi.org/10.4172/2157-7439.1000194

Tripathi S, Mehrotra GK, and Dutta PK (2008). Chitosan based antimicrobial films for food packaging applications. e-Polymers, 8(1): 093. DOI: https://www.doi.org/10.1515/epoly.2008.8.1.1082

Vaezifar S, Razavi S, Golozar MA, Karbasi S, Morshed M, and Kamali M (2013). Effects of some parameters on particle size distribution of Chitosan nanoparticles prepared by ionic gelation method. Journal of Cluster Science, 24: 891-903. DOI: https://link. springer.com/article/10.1007/s10876-013-0583-2

Van Toan N, Hanh TT, and Thien PVM (2013). Antibacterial activity of chitosan on some common food contaminating microbes. Journal of Biotechnology and Biomaterials, 4(1): 1-5. DOI: https://www.doi.org/10.2174/1876502501304010001

Widnyana IMS, Subekti S, and Kismiyati (2021). Potential of chitosan from shrimp waste for treating Staphylococcus aureus bacteria in skin wound. World's Veterinary Journal, 11(4): 705-708. DOI: https://www.doi.org/10.54203/scil.2021.wvi88

Yien L, Zin NM, Sarwar A, and Katas H (2012). Antifungal activity of chitosan nanoparticles and correlation with their physical properties. International Journal of Biomaterials, 2012: 632698. DOI: https://www.doi.org/10.1155/2012/632698

Younes I, Haiii S, Frachet V, Rinaudo M, Jellouli K, and Nasria M (2014). Chitin extraction from shrimp shell using enzymatic treatment. Antitumor, antioxidant and antimicrobial activities of chitosan. International Journal of Biological Macromolecules, 69: 489-498. DOI: https://www.doi.org/10.1016/i.iibiomac.2014.06.013

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