STUDYING THE EFFECT OF CHITOSAN ON BACILLUS CEREUS PRODUCING CEREULIDE TOXIN IN MILK AND SOME DAIRY DESSERTS

A total of 150 samples of raw milk and two types of refrigerated processed dairy desserts dishes (cooked rice with milk and Mehalabia dishes) (50 for each) were collected from dairy shops and local restaurants in Assuit City, Egypt. ISO7932 method was used for enumeration of B. cereus, afterwards a PCR was performed to confirm the presence of cereulide toxin (ces) gene. In addition, the effect of different concentrations of chitosan and chitosan nanoparticles (CNPs) (0.05, 1%) on B. cereus was studied in pasteurized milk.The shelf life was evaluated by clot on boiling test and Ph value. Their pathological effect was detected by orally administration to experimental rats to investigate their adverse effect on rats liver and intestine using histopathological examination. The most prominent potent bactericidal effect was observed inCNPs 1%, B. cereus count reached to undetectable level at 5th day of refrigerated storage. The pasteurized milk inoculated with B. cereus, chitosan and CNPs showed some pathological lesions in rats treated with B. cereus,while the chitosan 0.5% had antibacterial activities without cytotoxic effect on rat tissues, alternatively to CNPs 1%, which exist edematous tips of intestinal villi and mild kupffer cells activation in liver.Therefore, our results contribute data that are primary to indicate the risk of food poisoning due to B. cereus and trials to control that in food by careful using of nanotechnology. However, the additional researches are needed to safe using of this technology even on natural nano-materials as it at nano-size gain new qualities.


INTRODUCTION
B. cereus is a spore forming opportunistic environmental wide spread pathogen, cause food-borne outbreaks in human (Yu et al., 2019). Zhao et al. (2020) had isolated B. cereus from different dairy products, and considered it as a potential risk pathogen need to contribute an effective prevention and control program. In addition, several publications reported an increasing number of foodborne intoxication cases caused by B.cereus.For instance, European Union about 600-700 annually reported confirmed cases of foodborne outbreaks linked to B. cereus toxins (Messelhäußer and Ehling-Schulz, 2018). Bacillus cereus strains have shown to produce seven different toxins (Papan et al., 2019),the most dangerous one is cereulide toxin, which performed in food. This toxin is a small cyclic dodecadepsipeptide encoded by the ces gene. The cereulide is heat and pH stable, highly resistant to protease activity and it remains active through the gastro-intestinal passage but, due to the lack of a suitable assay, it considered the least well known (Ceuppens et al., 2012;Zhang et al., 2015). The toxin produced in food during vegetative growth, and after the toxin produced, no treatment can destroy this stable molecule, including proteolysis and extreme pH (Chica et al., 2019). Generally, food matrices rich in carbohydrates, such as pasta and rice, as well as milk and dairy products have the highest risk of causing cereulide intoxications (Papan et al., 2019). Outbreaks of B. cereus foodborne illness associated with consumption of cooked rice have been reported worldwide (Kumari and Sarkar, 2014;Yang et al., 2017;Alvarenga et al., 2018). Chitosan is a modified natural carbohydrate polymer prepared by the partial N-deacetylation of chitin, a natural biopolymer derived from crustacean shells such as crabs, shrimps and lobsters. The antimicrobial activity of chitosan depends on the degree of deacetylation and a molecular weight (Tantala et al.,2012). Chitosan is safe, non-toxic and can interact with polyanions to form complexes and gels (Sukmark et al., 2011). It has many antibacterial mechanisms activity, as intercellular leakage hypothesis is widely accepted (Silalahi et al., 2016), blockage of nutrient flow by forming a polymer layer around bacterial cells (Fernandes et al., 2009). In addition, it may affect the structure of the phospholipid bilayer in the cell membrane resulting in the release of some of the cellular components (Yusman, 2006;Dutta and Dutta, 2011). Because chitosan has many advantages properties and mostly safe, so it used in nanotechnology as chitosan nanoparticles (CNPs) depend on ionic gelation method, which describes the crosslinking reaction of chitosan with sodium tripolyphosphate (TPP) (Nguyen et al., 2017;Sreekumar et al., 2018) Therefore, the objective of our study was to detect the incidence and count of B. cereus in raw milk and two dairy desserts then detect the cerulide producing toxin (ces gene) in isolates by using PCR. Another purpose was to investigate the potential of applying chitosan and its nanoparticles as a natural food preservative to control the growth of B. cereus in pasteurized milk during storage. In addition, to provide a histopathological evaluation for the antimicrobial activity of chitosan and CNPs in different concentrations against B. cereus on intestine and liver of experimental rats.

MATERIAL AND METHODS
The prevalence of emetic B. cereus strains in some food samples:

Collection of samples:
A total of 150 raw milk, cooked rice with milk and Mehalabia dishes samples (50 samples each) were collected from dairy shops of different localities in Assiut city, Egypt and brought to the laboratory by means of refrigerated transport within a timeframe of max 2 h. There, all samples were prepared according to APHA (1998).

Statistical analysis
The effect of different concentrations of the chitosan and its nanoparticles on B. cereus count in pasteurized milk was analyzed using oneway analysis of variance and repeated measures ANOVA and the differences among group means were analyzed using the Tukey's multiple comparisons test. A p-value of <0.01 was considered significant. The Graph Pad Prism software (GraphPad, Inc., San Diego, USA) (version 5) was employed for the statistical analysis.

The antibacterial efficacy results of chitosan and CNPs to inhibit the growth of B. cereus.
Regarding the effect of different concentrations of chitosan and CNPs on B. cereus count inoculated in pasteurized milk. Firstly, detect the size and shape of nanoparticles using TEM as shown in photo (4). Also, detection of freshness by clot on boiling test which revealed positive results by the 13 th day of the experiment in positive control sample and in pasteurized milk inoculated by 0.5% chitosan samples so, these samples were not tested (Table 2). In this study, the mean pH value of pasteurized milk control sample ranged from 6.8±0.04 to 3.4±0.2 by the end of the experiment. While by adding chitosan to pasteurized milk, it ranged from 6.8±0.05 to 4.2±0.09 and 5.8±0.04 in concentrations 0.5 and 1%, respectively. Nearly similar values obtained in pasteurized milk sample with CNPs. It reached 5.7±0.07 and 5.9±0.08 at day 15 th of the experiment for concentration 0.5 and 1% of CNPs, respectively (Table 2). It was found that B. cereus mean count decreased from 8 log10 CFU/ml at zero time to 3.8 log10 CFU/ml in positive control sample after 11 th days of storage at 4±1 ºC (Table 3). On the other hand, both concentrations of chitosan 0.5 and 1% had displayed antibacterial activity against B. cereus(1.10 and 1.2 log10 CFU/ml at the 7 th day of the experiment). CNPs still had good bactericidal activity at concentration 0.5% after 7 days from cooling storage comparing with positive control group at which the bacterial growth mean count reached 6.5 log10 CFU/ml as shown in Table (3). The most prominent potent bactericidal effect was observed inCNPs 1% in whichB. cereus count reached to undetectable level at the 5 th day of refrigerated storage.
Photo 4 TEM for chitosan nano-particles with average size 27.6 nm.     (2) which administrated milk with B. cereus (8 log10 CFU/ml), most rats showed mild congested and hyperemic intestine and enlarged pale liver.

Light microscopic examination.
The intestinein Group 1 (control negative) and Groups 3, 4 and 5 showed normal structure of intestinal villi with intact lamina epithelialis (Fig 2 A, B and C). While, The liver of same group's demonstrated normal hepatic parenchyma (Fig 3 A, B). In Group (6), rats take milk with 1% CNPs revealed mild edema appeared at the tip of some villi of the intestine (Fig 2D and E) and mild proliferation of kupffer cells in sinusoids of liver (Fig 3 C). Control positive milk with B. cereus only (Group 2) showed swollen villi, goblet cells hyperplasia, some villi appeared blunted associated with congestion of lamina propria and edema in submucosa (Fig 2 F, G & H). While, the liver of the same group showed characteristic vacuolar hepatocytes degeneration, perivascular mononuclear cells infiltration and kupffer cells proliferation in perivascular and hepatocyte plates (Fig 3 D & E). Group (7) which rats take milk with B. cereus and 0.5% chitosan, the intestine showed mild hyperplasia of goblet cells and slight aggregation of mononuclear inflammatory cells in villus core (Fig 2 I & J) and the liver showed mild vacuolar degeneration of hepatocytes (Fig 3 F). In Group (8), the intestine appeared almost normal mucosa except for slight increase in goblet cells population (Fig 2 K & L) and liver showed almost normal hepatocytes (Fig 3 G). On the other hand, Group (9) which rats take milk with B. cereus and 0.5% CNPs, the intestine showed increase population of goblet cells and some villi showed slight edematous tips with mild vacuolar degenerative changes in overlying epithelium (Fig 2 M and N). In addition, the liver showed mild necrobiotic changes of hepatocytes (Fig 3 H). In group (10) which rats take milk with B. cereus and 1% CNPs, intestinal villi showed nearly normal mucosa except for appearance of edema at the tips of some villi (Fig 2 O & P), liver showed mild sinusoidal dilatation and some engorged with RBCs accompanied by kupffer cells activation (Fig 3 I ).

DISCUSSION
The combined abilities of B. cereus spores and toxins, to survive in pasteurization and of certain strains to multiply at low temperatures make this organism a unique milk-borne pathogen. Since B. cereus grows best in a starch-rich environment (Samapundo et al., 2011), cooked rice with milk and Mehalabia are popular dessert prepared and sold all day by dairy shops or local restaurants in Egypt and many middle east countries. They produced by adding whole rice grains or rice flour or cornstarch to milk, cooked and stored at refrigeration temperature. In the present study, high incidence of B. cereus (52 and 62%) was reported in cooked rice with milk and mehalabia compared to raw milk samples. These investigated dairy products are all known to be subjected to different types of heat treatment during processing according to each product. Heat treatment is not completely efficient to eliminate spore-forming bacteria in foods, among Bacillus spp. This high incidence may be attributed also to the improper temperature control during storage of such products. Similar incidences were reported by  et al., 2018).The present article demonstrated that strains of B. cereuswhich isolated from milk and dairy desserts may contain genes encoding the toxic gene (ces), so the risk of food poisoning should not be neglected. For that reason, our present study was focused on the application of chitosan and CNPs in pasteurized milk contaminated with B. cereus as an example of spoilage and pathogenic microorganism. Significant decrease in B. cereus count was noticed in treated pasteurized milk with different concentrations of chitosan and the organism could not be detected by the 9 th day of the experiment. While, by adding CNPs (0.5 and 1%), the count could not be detected at 7 th and 5 th days of storage, respectively. On the other hand no significance differences (p >0.05) were observed between concentrations of chitosan and CNPs.  Benhabiles et al., 2012). Raafat et al. (2008) declare one of the suggested hypothesis for this effect, who stated that, this difference in sensitivity is largely ascribed to the different structure of Gram positive and Gram-negative bacterial cell envelopes. As a possible mechanism of action for chitosan, antimicrobial activity is due to binding to teichoic acids present in the cell wall of Gram-positive bacteria, coupled with membrane lipids extraction, which trigger a series of event that resulted in bacterial cell death. Tamara et al. (2018) showed that B. cereus was generally more resistant to the CNPs and suggested that B. cereus had more hydrophilic and negatively charged cellular structure. Another study (Hassan et al., 2016) investigated that the antibacterial activity of CNPs on bacillus spp. was recorded in percentage 100 % inhibition at different concentrations. Furthermore, CNPs can be used as bioactive ingredients carriers and in wide approach arrays, due to their favorable biological properties such as non-toxicity, biocompatibility, biodegradability and antibacterial ability (Zhao et al., 2011). Increasing the degree of de-acetylation has a major effect on the antimicrobial activity of chitosan and in dependency CNPs, that due to increase the free amino groups in the produced chitosan, which leads to higher antimicrobial activity, as the -NH2, -OH groups are the main reactive site in chitosan. As, it is determinant in the charge development and solubility of chitosan (Zaghloul and Ibrahim, 2019).
CNPs have been very effective in increasing the shelf life of different meats and their products (Quesada et al., 2016). In the present study, the shelf life of pasteurized milk with chitosan and CNPs was extended compared to control samples. Clot on boiling test was used to detect freshness of pasteurized milk. Positive control and pasteurized milk with 0.5% chitosan samples soured by the 13 th day of the experiment so, these samples were not tested. On the other hand, the shelf life of pasteurized milk extended more than 15 days by adding 1% chitosan, 0.5, and 1% CNPsas shown in table (2). Generally, treated milk either by chitosan or CNPs were considered the most preferable and strongly accepted as nearly no difference from control as shown in Figure (1). That may be the chitosan in nature white color and have no special taste or flavor so, haven't effect on flavor or appearance. Although chitosan produce potent bactericidal effect against most bacterial forms, neverthless it is important to examine the constituents of the food matrix (Awad and El Sohaimy, 2020). The change in pH value by adding chitosan and CNPs should be taken into consideration before apply chitosan as a natural preservative. Chitosan was reported to be positively charged and have high antimicrobial activity, mainly at pH values below its pKa of 6.5 (Chang et al., 2015). The molecular weight and viscosity development of chitosan in aqueous solution also play a significant role in the biochemical and pharmacological application of chitosan. Other major parameters are crystallinity, ash content, moisture content, heavy metal content and so on (Rinaudo, 2006). On the other hand, CNPs are natural materials with excellent physicochemical, antimicrobial and biological properties, which make them a superior environmentally friendly material and they possess bioactivity that does not harm humans ( With regarding to the effect of chitosan and CNPs on B. cereus in groups (7,8,9 and 10) at both concentrations 0.5% and 1% we found that both of CNPs and large molecule chitosan had antibacterial effect on B. cereus and markedly reduced its enterotoxins degenerative effects on intestine and liver. However, it was observed that the large molecule chitosan especially higher concentration 1% had much better antibacterial effect without cytotoxic effects on rat's intestinal villi in-compared to the same concentration 1% of CNPs, which exerted degenerative action on intestinal epithelium similar to group (6). In spite of antibacterial effect of 1% CNPs on B. cereus and its toxin, but it has mild side effect on biological tissue. While, this cytotoxic effect was less prominent with lesser concentration (0.5%) of CNPs. Our findings are inconsistent with those previously reported by Tokura et al., (1996) who mentioned that large chitosan molecules revealed greater antibacterial activates than smaller chitosan molecules. Additionally, the report of

CONCLUSION
This study demonstrated that the toxin gene ces is present in some strains of B. cereus isolated from some of raw milk and dairy desserts samples. Therefore, effective prevention and control measures of emetic B. cereus in food is demanded. Both chitosan and CNPs showed antibacterial activity in treated pasteurized milk against B. cereus. The shelf life of pasteurized milk with chitosan and CNPs solution was extended in compared to control samples. Furthermore, CNPs will be used increasingly in the fields of food storage, preservation and microbial inhibition with excellent physicochemical, antimicrobial and biological properties, which make them a superior environmentally friendly material and they possess bioactivity that does not harm humans. The results of experimental study revealed that chitosan 1% and CNPs 0.5% can be applied safely against B. cereus in dairy products without harmful effects on biological tissues.