Impact of Probiotics on Some Food Poising Microorganisms In Milk Based Deserts

Hanan R. Ghanayem⁽¹⁾,  Naglaa  AA⁽¹⁾, Asmaa T. Talayea⁽²⁾ and Tamer MH El. Sharawey ⁽¹⁾

• Food Hygiene Department, Animal Health Research Institute, Tanta Branch, ARC.
• Microbiology, Animal Health Research Institute, Tanta Branch, ARC.

ABSTRACT

          Currently, the increased interest in the field of food safety are shifted toward using natural food preservatives instead of chemicals which may have some harmful effect either on human health or food constituents. A total of 60 milk-based desserts samples, rice pudding, Mahallabia, and custard (20of each) were collected from different dairies and pastry shops in Tanta city, Egypt. The collected samples were examined for isolation and identification of Staphylococcus aureus (Staph. aureus) and Bacillus cereus (B. cereus), detection of virulence genes for both organisms using PCR and finally, studying the effect of two probiotics strains of Lactobacillus acidophilus L. acidophilus) and Bividobacterium lactis ( B.lactis) on the survival of Staph. aureus and B. cereus in rice pudding during refrigerator storage for 14 days. The incidence of Staph. aureus was 40%, 20% and 10% in the examined rice pudding, Mahallabia, and custard samples, respectively. While the incidence of B. cereus was 60%, 45% and 30% in the same examined samples, respectively. The result of PCR for virulence genes revealed presence of spa and coa gens in all Staph. aureus isolates(100%) and revealed presence of nhe and cytk gens in all B. cereus isolates (100%). Staph. aureus and B. cereus counts were decreased in rice pudding samples significantly (p<0.05) at 3^rd, 7^thand10^th day of storage in samples with added both probiotics strains. So, Rice pudding was considered suitable food for the delivery of probiotic micro-organisms, with sufficient viability and acceptable sensory characteristics.

Key words: milk-based desserts, B. cereus, Staph. aureus, PCR, probiotic, L. acidophilus and B. lactis

INTRODUCTION:

Recently, the development of food products has been directed toward producing healthier foods that have characteristics similar to those observed in fresh products, with increased nutritional and functional properties, and no chemical additives (Normanno et al. 2012)

Dairy desserts in Egypt are locally produced by traditional retail shops and the production takes place manually in stores. They are made from highly nutritive raw materials; milk and sugar and are easily spoiled by the multiplication of specific microbial contaminants (Arakawa et al. 2008).

Staphylococcus is facultative anaerobic round-shaped and Gram-positive cocci; which produces various virulence genes, toxins and aggressive enzymes. It can be found in numerous food products as well as raw milk and its products, it was responsible for a variety of symptoms and diseases, this micro-organism is known as one of the world’s most important causes of disease epidemics associated with food consumption (Zhao et al. 2020). Staph. aureus is one of the most important pathogen in food poisoning, due to its wide spread and ability of many strains to synthesize enterotoxin. It causes gastroenteritis symptoms like nausea, vomiting, abdominal cramps and diarrhea (Scherrer et al. 2004).

Bacillus cereus is a Gram-positive, aerobic or facultative anaerobic spore-forming bacilli, commonly present in various natural environments and food ingredients (Fiedoruk et al. 2017). Bacillus cereus associated with two forms of gastrointestinal diseases. The emetic form of illness manifests in vomiting and nausea while, the diarrheal form mainly appears to manifest only in gastrointestinal symptoms such as watery diarrhea and abdominal cramps (Chai et al. 2019).The pathogenicity of B. cereus is caused by different toxins produced by this bacterium. The diarrheal type of food poisoning B. cerus is caused by enterotoxins such as non-hemolytic enterotoxin (NHE) and cyto-toxin K (CytK) produced during vegetative growth in the small intestine (Sergeev et al. 2005)

PCR is a powerful molecular biological technique that was introduced to facilitate the detection of virulence factors by using DNA probes that detect specific virulence factors and also, a  powerful diagnostic tool for the detection of pathogenic micro-organisms (Malorny et al. 2003)

Food preservation is a continuous effort that aims to eliminate and reduce the out-growth of spoilage and pathogenic micro-organisms in foods. Probiotics are known as live micro-organisms that have alternative effect to antibiotics in the treatment of food borne diseases as it provide a health benefit to the host when administered in adequate amounts.(Sanders et al. 2007).

Probiotics are defined as live micro-organisms which when administered in adequate amounts (10⁶-10⁷cfu/g) confer a health benefit on the host" (FAO/WHO 2010).Mainly, they are used as natural food preservatives and important factors for preventing diseases, controlling bacterial and fungal contamination, and improving human and animal health (Lucatto et al. 2020).

The viability of probiotic micro-organisms during processing and storage besides the acceptance by the consumers is the major criterion to determine the potency and the market prosperity of the probiotic product. Lactic acid bacteria are widely used in food preservation at refrigerator temperatures due to their ability to produce high amount of hydrogen peroxide and/or other antibacterial substances at refrigerator temperatures which inhibit food borne pathogens and psychrophilic spoilage micro-organisms.(Alireza et al. 2016).

There are numerous health benefits reported for Bifidobacterium spp. and Lactobacillus spp. that they faster recolonization of the intestinal microbiota after administration of antibiotics, treatment and prevention of diarrhea, alleviation of constipation, possible treatment of inflammatory bowel disease, reduction in lactose intolerance in some individuals, reduction in serum cholesterol level, increased resistance to microbial infections, impact on immune function, and potential role in cancer prevention(Zavisic et al. 2012).

Lactobacillus acidophilus is a probiotic micro-organism available in conventional food (milk, yoghurt and toddler formula) and dietary supplements. Antagonistic activity of L. acidophilus against food borne microorganisms such as E. coli, Staph. aureus, L. monocytogenes and Cl. perfringens has been previously reported.(Kasimoglu and Akgun 2004).

Therefore, the objective of this study is isolation and identification of Staph. aureus and B. cereus from some milk based dessert samples, detection of virulence genes of these pathogens, followed by evaluation to the effect of  probiotic L. acidophilus and B. lactis on  sensory attributes and growth of Staph. aureus and B. cereus in rice pudding during storage for 14 days at 4^oC.

MATERIAL and METHODS:

1- Collection and handling of the samples:

A total of 60 milk-based desserts samples, rice pudding, Mahallabia, and custard (20 of each) collected from different dairies and pastry shops in Tanta city, Egypt. Each sample was obtained as sold to the public and transferred directly to the laboratory in ice box under complete aseptic condition without delay for further examination.

2. isolation and identification of Staph. aureus and B. cereus:

The isolation and identification of  Staph. aureus was performed according to (APHA, 1992;Quinn et al. 2002) using brain heart infusion broth and Baird Parker agar, Mannitol salt agar (Oxoid). Meanwhile , B. cereus was isolated and identified according to (Procopet al. 2017; Sandra and Tallen 2012) using brain heart infusion broth and polymyxin– pyruvate- egg yolk–mannitol-bromothymol blue agar (PEMBA, Oxoid) .

3. 3. Detection of virulence genes of aureus and B. cereus:-

DNA extraction: DNA was extracted from the isolated Staph. aureus and B. cereus using QIAamp DNA mini kit. It was applied on 3 random isolates. PCR Master Mix and cycling conditions of the primers during PCR was prepared according to Emerald Amp GT PCR mastermix (Takara) kit. Oligonucleotide primers used in PCR have specific sequence and amplify a specific product (Table A)

Table (A): Oligonucleotide primers sequences of virulence genes of Staph. aureus  and  B. cereus.

4.Impact of probiotic bacteria on the growth and survival of   Staph. aureus and B. cereus in vitro:

4.1. Preparation of bacterial strains:

Staph. aureus and B. cereus strains which isolated and identified from  the proceeded samples(rice pudding, Mahallabia, and custard)  then confirmed by PCR at Animal Health Research Institute (AHRI), Dokki, Egypt. The strains were adjusted at concentration of 10⁷cfu/ml (Kantachote and Charernjiratrakul 2008).

Probiotic strains ( L. acidophilus and B. lactis) were obtained from faculty of Agriculture-Ain Shams University, and adjusted to obtain desired inoculum level of 10⁸cfu/ml (Maha et al. 2015).

4.2. Preparation of rice pudding:

The rice pudding was produced in amounts to obtain 3 kg of the final product. Using mixed ingredients (Buffalo's milk, rice, sucrose sugar, starch) by weight, heated 80-85 °C until the ingredients dissolved completely, and cooled to 40 °C in an water bath with continuous stirring.

4.3. Experimental trials:

As soon as the mixture reached the desired cooling temperature, the samples were divided into 12 equal groups (200g of each) and placed in aseptic polypropylene trays designed for disposable food packaging. The suspension of both probiotics (2ml for each group except C -ve) approximately 8 log cfu/ml in rice pudding was added at the beginning of the storage (1^st) day. Also the suspension of both Staph. aureus and B. cereus (2ml for each group except C-ve) approximately 7 log cfu/ml were added. The inoculums of probiotic Staph. aureus and B. cereus strains were evenly distributed in the rice pudding each group separately by mixing with a sterile spatula for 50 second. Then cold and stored at 4±1°C for up to 14 days.

The samples divided into 12 equal groups (200gm each) arranged as follows:

• C–ve: Control non inoculated either with aureus, B. cereus or probiotic strains.
• Ca +ve: Inoculated with 10⁷cfu/ml aurus strain.
• Cb +ve: Inoculated with 10⁷cfu/ml cereus strain.
• GI : Inoculated with10⁷cfu/ml aureus and 10⁸cfu/ml L. acidophilus.
• GII: Inoculated with both10⁷cfu/ml aureus and10⁸cfu/ml B. lactis.
• GIII: Inoculated with 10⁷cfu/ml aureus strain and mix of 10⁸cfu/ml L. acidophilus and B. lactis.
• GIV: Inoculated with 10⁷cfu/ml cereus and10⁸cfu/ml L. acidophilus.
• GV: Inoculated with both10⁷cfu/ml cereus and10⁸cfu/ml B. lactis.
• GVI: Inoculated with 10⁷cfu/ml cereus and mix of10⁸ cfu/ml L. acidophilus and B. lactis.
• GVII: Inoculated with 10⁸cfu/ml acidophilus only.
• G VIII: Inoculated with 10⁸cfu/ml lactis strain only.
• GVIIII: Inoculated with 10⁸cfu/ml of both acidophilus and B. lactis strains.

4.4-Determination of pH value:

The pH values of rice pudding was measured according to the method no.981.12 of AOAC (2003) using a digital pH meter.

4.5-Microbiological Analysis:

• acidophilus La-5 and B. lactis were counted by pour-plating 1 mL of each dilution in De-Man Regosa and Sharp medium (MRS) broth and agar, to which lithium chloride (0.2 g 100 mL^–1) (Merck, Darmstadt, Germany) and sodium propionate (0.3 g 100 mL^–1) (Sigma–Aldrich, St. Louis, MO, USA) solutions were added (LP-MRS agar).L. acidophilus after 2 days of aerobic incubation at 37°C for 48hours.B. lactis after 72 h of anaerobic incubation (Anaerobic System Anaerogen, Oxoid) at 37 °C (Vinderola and Reinheimer 1999) .
• aureus count was determined for all trials throughout storage period according to (ISO 2003).
• Enumeration of cereus was done according to (Merzougui et al. 2014).

4.6- Sensory evaluation:

Nine panelists, selected depending on their availability and willingness to participate in the study, from staff of Department of Food Hygiene Animal Health Research Institute, Tanta Branch. Evaluated the organoleptic characteristics of the rice pudding  samples. The judges were asked to evaluate the coded samples (C -ve, GVII, GVIII and GVIIII, all of them from the same replicate), using a 9-point balanced hedonic scale (1: dislike extremely - 9: like extremely) based on color, texture, odor, and taste. The samples were presented at random digits and containing 20g of the product at 4 ± 1 °C. Water was provided to judges to clean their palates between samples (Cardarelli et al. 2008).

4.7. Statistical analysis:-

The data was statistically analyzed using one-way ANOVA of SPSS program for windows SPSS Inc. Chicago, IL and USA (Version 20)and Duncan’s post hoc test with p<0.05 considered to be statistically significant.

RESULTS:-

Table(1) : Incidence % of  Staph. aureus and B. cereus isolated from examined milk based dessert  samples (n=20)

1. B : % was calculated according to the total number of examined samples

Figure(1):Gel electrophoresis pattern for detection of virulence genes of Staph. aureus.

Agarose gel electrophoresis showed amplification of spa gene of Staph. aureus at 226bpandcoa gene of Staph. aureus at570bp,lane: 1,2and3 show positive amplification of spa and coa genes at 226bpand 570 respectively L:ladder (100-1000bp) Pos: positive control; Neg: negative control.

Figure (2):Gel electrophoresis pattern for detection of virulence genes of B. cerus figure .

Agarose gel electrophoresis showed amplification of cytk gene of B. cereus at 421bpandnhe gene of B. cereus at 766bp,lane: 1,2and3 show positive amplification of cytk and nhe genes at 421bpand 766, respectively. L:ladder (100-1000bp) Pos: positive control; Neg: negative control.

Table (2):  Impact of Probiotic on survival of Staph. aureus as log cfu/g in Rice pudding during refrigeration storage.

Ca  +ve – mean control +ve Staph. aurus

The values represent Mean ± SE of three experiments.

Means within a column followed by different letters are significantly different (P < 0.05).

Table(3): Impact of Probiotic on survival of B. cereus in Rice pudding during  refrigeration storage.

Cb +ve mean control +ve  for B. cereus

The values represent Mean ± SE of three experiments.

Means within a column followed by different letters are significantly different (P < 0.05).

Table (4): Viability of probiotic in rice pudding during refrigeration storage.

The values represent Mean ± SE of three experiments.

Mean with capital letters in a column and small letters in a row differ significantly (P<0.

Table (5): Changes in sensory scores of rice pudding samples artificially inoculated with probiotics L. acidophilus and B. lactis strains during  refrigeration storage at 4°cin comparison with control (c-ve)..

Mean ±SE with different capital letters in a column and small letters in a row differ significantly (P<0.05).

Table (6) :  pH of controls and inoculated groups with  probiotics and bacterial strains in rice pudding during refrigerator storage at 4±1°C

Means within a column followed by different letters are significantly different (P < 0.05).

Figure ( 3):Change in pH of controls and inoculated groups with  probiotics and bacterial strains in rice pudding during refrigeration  storage at 4±1°C.

Figure (4): Change in pH of groups inoculated with  probiotics  strains only in rice pudding during refrigeration storage at 4±1°C.

DISCUSSION

Dairy-based desserts are considered a good media for the growth of many micro-organisms due to its high nutritive value and they are important means for transmission of different pathogens mainly in places where the hygienic measures are low (Meyer-Broseta et al. 2003).

The results showed in Table (1) indicated that the incidence of Staph. aureus was 40%, 20% and 10% in the examined rice pudding, Mahallabia, and custard samples, respectively. These results nearly similar to that obtained by Hassan and Afifi ( 2016) for the rice pudding and Mahallabia. While, Eman and Saad (2016) recorded higher incidence80%in examined rice pudding for CPS samples, but Sotohy et al. (2022) recorded lower incidence for rice pudding and Mahallabia .

The high contamination rate of dairy products with Staph. aureus is mainly due to the use of poor quality milk, environmental contamination as unclean hands of worker who either suffering from diseased or apparent healthy carriers, unsanitary production and marketing practices (Araujo et al. 2002).

While B. cereus incidence was 60%, 45% and 30% in the examined rice pudding, Mahallabia, and custard samples, respectively. These results nearly similar to that of  Wallaa (2017) who found that B. cereus incidence was 62%, 44% and 32% in the examined rice pudding, Mahallabia and custard samples. While, Hassan and Afifi (2016) recorded  lower incidence for the rice pudding. Also, Hussein et al. (2015) recorded higher incidence for Mahallabia. Lower incidence for custard were recorded by Van Netten et al. (1990) .

Bacillus cereus has emerged as a major food borne pathogen during the last few decades and causes two types of illness through the elaboration of enterotoxins (Jay 2005). These different results were due to regional, seasonal, sampling and the degree of contamination that depends on the precautions observed during processing.

Staphylococcal infections are usually associated with the presence of virulence genes. The spa gene is mostly used for typing of Staph. aureus, encodes for protein A and the coagulase (coa) gene is another example of Staph. aureus virulence gene that is regarded as important, because it plays an essential role in the alliance with other genes to survive inside host cells and to invade its immune system  (Balaban and Rasooly, 2000).

As showed in figure (1) PCR of virulence genes revealed presence of spa and coa gens in all Staph. aureus tested samples(100%). These results agreed with Abdel-Tawab et al. (2016) who established the presence of spa gene in nearly all of the isolates. Meanwhile, the results disagreed with that recorded by Momtaz et al. (2013) who reported that spa and coa were detected with 26.82% and 63.41%, respectively.

The pathogenecity of B. cereus is attributed to the production of two main toxin types (diarrheal and emetic toxins) that are responsible for emesis and diarrhea (Schoeni and Wong 2005).

The obtained results in Figure (2) regarded that all the examined B. cereus isolates were positively by PCR technique for virulence genes and revealed presence of nhe and cytk gens in all tested samples (100%), similar study conducted by Cui et al. (2016) who detected nhe by 100%, other findings reported by Glasset et al. (2016) who added the presence of nhe and cytk with 96% and 42%, respectively.

Milk and dairy products are one of the most important human needs because they rich in organic matter. Further, they are a suitable environment for the growth of various food borne pathogens and this indicates the necessity for milk hygiene and elimination of pathogenic micro-organisms and their products, including toxins (Amelia et al. 2021).

Probiotic bacteria affect many of micro-organisms through changing the pH and glucose levels secreting enzymes and antimicrobial toxins, changing the expression of pathogenic genes and competition on food intake (Kerry et al. 2018).Since Staph. aureus, which is salt and nitrite tolerant, it also able to grow under anaerobic conditions, there is an increased risk that it will grow and produce toxins (Kaban and Kaya  2006)

Table (2) explained the effect of the two different probiotics on the growth pattern of Staph. aureus in experimentally inoculated rice pudding samples. At zero day, there were no significance difference between all
examined groups (Ca+ve, GI, GII and GIII),from 2^nd day till end of storage period, there were significantly difference (p< 0.05).Staph. aureus counts in rice pudding samples declined from (6.73±0.1 to 3.8±0.05) ,(6.46±0.2 to 3.6±0.08) and (6.64±0.1 to 3.9±0.05)log cfu/gin treated  probiotics groupsG1,G II and G III respectively, when compared to (Ca +ve) during the storage period. So it was observed that Staph. aureus count was declined, when increasing probiotics concentrations to10⁸cfu/g. These results agreed with previously reported by Nassif et al. (2015) who stated that staph. aureus count was decreased from6.48 at zero day to 3.52 log₁₀cfu/g at the 9^thday of storage. While Hemmat et al. (2018) found that L. acidophilus mitigate staph. aureus count from 4.26 at zero day of storage  to 1.72 cfu/g at 6^thday of storage , and B. lactis also mitigate staph. aureus count from 4.26  at zero day to 1.49 log10cfu/g for at 6^th day of storage, respectively. Also, Eman and Saad. (2016) reported that Staph. aureus count decreased significantly (p<0.05) at 3^rd, 7^thday of storage in comparison to zero day. The results indicated that Staph. aureus was reduced in all treatments especially group GII followed by group GI and then group G III, this agreed with Kebary et al. (2005) who found that all studied Bifidobacteria strains strongly inhibited the growth of Staph. aureus.

Probiotic are LAB which produces antimicrobial substances, such as organic acids, fatty free acids, ammonia, hydrogen-peroxide and bio surfactant. It also produces low-molecular-weight antibacterial peptide-bacteriocins that inhibit both gram positive and gram-negative enteric pathogens (Gomes et al. 2012).

Bacillus cereus is an important pathogenic organism for estimating the risk for food borne diseases. The actual disease symptoms, however, are caused by toxins, either produced during growth in the gut; enterotoxins or during growth in food emetic toxin (Adams and Moss 2008).

Table (3) showed that both probiotics strains had inhibitory effect against B. cereus growth in rice pudding as lowering its count from (6.13±0.08to3.73±0.1), (6.03±0.8 to3.46±0.2) and (6.32±0.1 to 4.03±0.08) log cfu/g through 14 day of refrigeration storage in probiotics inoculated groups G IV, GV and GV1, respectively when  compared to Cb +ve group. Moreover, B. lactis alone has better effect than combination of L. acidophilus and B. lactis. These results agreed with Servin (2004) and Parada et al. (2007) who revealed that the use of Bifidobacterium  lactis BB-12 is profitable on hygienic quality of rice pudding due to the cumulative effects of antimicrobial agents such as hydrogen peroxide, bacteriocins and organic acids. To exert health benefits, probiotic bacteria must be viable and available in high concentrations, above 10⁶ CFU/g product (Shah. 2007). Generally, the recorded data mentioned in table 2 and 3 agreed with  Berenice Arias et al. (2013) who reported that LAB induce its antagonistic effects against  pathogenic bacteria through its ability to produce bacteriocins and bacteriocins like substances which are narrow-spectrum proteinaceous toxins that serve to kill closely related bacteria.

Table (4) revealed the change of viability of Probiotic in rice pudding during refrigeration storage for 14 day. The viable cell numbers of GVII, GVIII and GVIIII (7.6±0.05,7.66±0.08 and 7.5±0.08 log cfu/g, at the 1^stday), respectively. Significantly decreased during 14 days of storage period (P<0.05).No significant differences observed between GVII, GVIII and GVIIII during zero, 3^thand 7^thdays of storage period (P>0.05).Satisfactory probiotic viability above 7 log cfu g⁻¹ was found in the present study for L. acidophilus and B.lactis, which observed throughout the storage period. This observation is similar to that reported by Abghari et al. (2011) and nearly similar to Ranadheera et al. (2013) who found L. acidophlus-5count was ranged from 7.70 to 7.38 logcfu/g. While lower results recorded by Cardarelli et al. (2008) for the L. acidophilus strain, which were below 7 log cfu /g. So, results indicated that probiotics in rice puddings pioneered in the present study could have a potentiality to be used as carriers of L. acidophilus (LA-5) and B. lactis in food systems, as similar to the literature depending on viability and activity of probiotic bacteria incorporated into dairy desserts (Aragon-Alegro et al. 2007).

As observed in this study, that B. lactis populations was higher than those of L. acidophilus, and this agreed with Pereira et al. (2010), nevertheless, the viability of  L. acidophilus (97.1 %) was less affected than B. lactis (94.7%) at 14 day of storage due to the sensitivity of this strain to air during storage. Moreover, there seems to be a substantial influence of the food matrix on the viability of such micro-organisms in products.

Table (5) discussed the sensory properties, however, there were no significant differences in (color, odor, texture and taste) for GVII, GVIII and GVIIII (P>0.05) compared with Control –ve at zero and 3^rdday of storage, but at 7^th day, showed significant difference (P<0.05) between c-ve and other probiotic treated groups. While there was no significant difference between probiotic treated groups from 7^th until 14^th of storage period. Control –ve was still acceptable until the 7^th day. Use of probiotic bacteria showed the best score through the days (0, 3 and 7) of storage (taste, texture and odor). GVIII was favored for its taste, color and odor. However, GVII was preferred for its texture at 10 and 14 day of storage period, when compared to GVIII and GVIIII due to occurrence of heterogenic texture, while GVIII most preferred for its taste and odor, and this agreed with MC Breaty et al. (2001) who observed more extensive proteolysis and improved flavor in B. lactis BB-12 of samples during the storage. No abnormal odor in the examined probiotic rice pudding was detected and this agreed with  Tulay Ozcan et al. (2010) and Eman and Saad (2016).

The presence of probiotic strains in the product improved its sensory characteristics. These micro-organisms might also be able to produce metabolites with antimicrobial activity, such as organic acids, alcohols, bacteriocins and reducing deterioration of product (Buriti et al. 2012).  

PH is an important factor which can dramatically affect bacterial growth, probiotics spp. that able to tolerate a wide range of pH (1-9) and grow well at acidic pH 1-5 (Chowdhury et al. 2012).

Table (6) and Fig. (3) Illustrated the pH values of the rice pudding samples on day one were very similar and no significant differences (P>0.05) were observed among different groups for pH. However, by the end of the storage period, pH reduced significantly (P<0.05) for both Staph. aureus and B. cereus bacteria during storage period as a result of acid production by probiotic bacteria, in compared to those of controls one(C-ve, Ca +ve  and Cb +ve).The pH levels in probiotic rice pudding depended on the strain used where the pH values obtained in all L. acidophilus inoculated samples were significantly lower than those of B. lactis and controls samples, since L. acidophilus has higher acid formation ability. In general, the three trials studied showed increasing values of acidity and decreasing values of pH. This agreed with Tulay Ozcan et al. (2010).

Figure (4) Showed that during the storage period there was a small decrease but not significant in pH values was observed in G VII,G VIII and GVIIII groups(p < 0.05). However, the changes of pH values did not affect the viability of the probiotic micro-organism strains Since, the pH values of the food samples remained close to neutrality, which is considered optimal for survival of probiotics in  rice pudding and this agreed with Homayouni et al. (2012).

CONCLUSION:

Proper sanitation and hygiene during handling of rice pudding are important factors to protect the consumer health and prevent spoilage of the product. The results suggested that high levels of viable L. acidophilus and B. lactis in rice pudding is a good source for probiotic bacteria delivery with appreciated organoleptic quality and microbiological safety leading to good kinds for its future commercial production. L. acidophilus and B. lactis had antagonistic effect on Staph. aureus and B. cereus in rice pudding at refrigeration storage temperature. Moreover, B. lactis alone has better effect than combination of L. acidophilus and B. lactis.