PREVALENCE OF AEROBIC SPORE FORMING BACILLI WITH SPECIAL REFERENCE TO BACILLUS CEREUS IN YOGHURT AND RICE BUDDING

Dept. of Food Control,

Faculty of Veterinary Medicine, ZagazigUniversity

Prevalence of aerobic spore forming Bacilli with special reference

to Bacillus cereus in yoghurt

and rice budding

(With 4 Tables)

By

E.I. Awad

(Received at 4/9/2008)

تواجد العصويات الهوائية المتحوصلة مع إشارة خاصة الى ميکروب الباسيلس سيريس فى الزبادى والارز باللبن

عصمت ابراهيم السعيد

أجريت الدراسة علي ثلاثون عينة من کل من الزبادي والأرز باللبن المجمعة من أماکن مختلفة بمدينة الزقازيق وذلک بهدف استبيان مدي تواجد البکتيريا الهوائية المتجرثمة خاصة الباسيلس سيرس وقد أسفرت النتائج علي أن متوسط العدد الکلي  للبکتيريا الهوائية المتجرثمة 5.3x10^4و 4.6x10⁵ في الزبادي والأرز باللبن علي التوالي بينما کان نسبة تواجد الباسيلس سيرس في الزبادي والأرز باللبن علي التوالي40% و 73% وعند تصنيف 60 عتره من البکتيريا الهوائية المتجرثمة المعزولة من الزبادي کان الباسيلس ليشينيفورميز هو أکثر المعزولات ويمثل38% من جملة المعزولات ولکن عند تصنيف عتره معزولة من الأرز باللبن تبين أن الباسيلس سيرس هو الأکثر تواجدا ويمثل42% وعند اختبار قدرة ميکروب الباسيلس سيرس علي تحليل البروتين تبين الأتي80% من معزولات ميکروب الباسيلس سيرس المعزولة من الأرز باللبن لهل القدرة علي تحليل البروتين بينما55.5% من الميکروبات المعزولة من الزبادي لها القدرة علي إفراز الإنزيم المحلل للبروتين وقد دلت النتائج علي أن عدد و نسبة تواجد ميکروب الباسيلس سيرس في الأرز باللبن أکثر من الزبادي مما يشير إلي أن مصدر تلوث اللبن الخام بهذا الميکروب هو الأرز هذا وقد تمت مناقشة النتائج لبيان أهميتها الصحية والاقتصادية.

ٍSUMMARY

A total of 60 samples including (30 plain yoghurt and 30 rice budding) were collected from dairy shops, supermarkets and groceries in Zagazig city, Egypt and analyzed for enumeration of their Aerobic spore-formers and Bacillus cereus counts. The proteolytic activity of isolated Bacillus cereus was also investigated. The results obtained showed that mean counts of Aerobic spore-formers in the examined yoghurt and rice budding samples respectively were 5.3x10⁴ and4.6x10⁵, while Bacillus cereus could be isolated from 12 (40%) and 22 (73%) with mean counts of 1.3x10³ and 1.6x10⁵of the examined samples, respectively. Out of 60 Bacillus spp. isolated from examined yoghurt samples, Bacillus licheniformis was predominating and comprising 23(38%) while 9 (15%) isolates proved to be Bacillus cereus and on the other hand Bacillus cereus was predominating other Bacillus spp. isolated from rice budding samples 25(42%). 20 (80%) out of 25 Bacillus cereus obtained from rice budding samples possessed proteolytic activity compared to    5 (55.5%) isolates from yoghurt samples. Results were discussed in the context of present microbiological specifications and the need for its implementation by regulatory agencies to ward off possible health hazards arising from these microorganisms.

Key words: B. cereus, yoghurt, rice budding

Introduction

Fermented milk is the most popular fermented dairy products produced in Egypt and world wide. The value of fermented milk in human nutrition is based not only on the nutritive value of milk from which it is made and increased digestibility due to changes of milk constituents during fermentation period  but also on the beneficial effect of intestinal micro flora , prophylactic and healing effect (buttress, 1997; Hussein and Kebary, 1999 and Zedan et al., 2001).

Fermented milk has a positive effect on human defense system and cardiovascular diseases (Piaia, 2001) and high intake of fermented products was associated with decrease risk of ulcer (Elmstahl et al., 1998).

Yoghurt is highly nutritious food of useful therapeutic value as it prevents the intestinal putrefaction resulting from anaerobic decomposition, prevents the gastrointestinal disorder, prevents coronary heart disease, reduces the risk of colon cancer, exerts a hypocholesterolaemic effect and produces antibiotics as acidophilin, lactocidin, nicin and lactoline that inhibit the growth of many pathogens  (Robinson, 1991).

The rice budding and custard are the most popular varieties of Egyptian dairy desserts used not only in villages but also in cities .Rice budding ingredients are milk sugar, rice and other additives such as vanillin, raisins and shaved coconut to enhance its nutritive value and improve its flavor (Ray, 1996).

Heat resistant Psychrotrophs predominating Bacillus spp. can be introduced into milk supplies from water, udder, teat surfaces, soil or from milk stone deposits on farm bulk tanks, pumps, pipelines, gaskets and processing equipment and these organisms produce proteinases which result in the development of objectionable flavor and quality defects in dairy products (Meer et al., 1991).

Bacillus cereus had been reviewed in food poisoning cases linked with milk and dairy products  and there are two clinical forms of Bacillus cereus food poisoning , one is called diarrheal  form that resemble to Clostridium perfringens food poisoning while the second form is called emetic syndrome food poisoning (Mossel, 1982). Infection of human by Bacillus cereus may be complicated by bronchopneumonia and otitis (Eley, 1996).

The unique combination of both thermoduric and Psychrotrophic properties of Bacillus cereus represents recurring problems for dairy industries such as spoilage of pasteurized milk (sweet curdling) and cream (bitty cream) under the effect of proteolytic and lipolytic actions  (Griffiths, 1990).

In recognition of the public health and economic significance of Aerobic spore-formers especially Bacillus cereus, therefore the present study was undertaken to investigate its presence in milk and dairy products.

Materials and methods

A total of 60 samples including (30 plain yoghurt and 30 rice budding) were collected from dairy shops, supermarkets and groceries in Zagazig city, Egypt.

Preparation of samples (A.P.H.A., 1992):

The sample containers were mixed thoroughly using sterile spatula before examination. Eleven gram of the prepared samples were added to 99 ml of peptone water in a sterile bottles and thoroughly mixed to make a dilution of 1/10, from which decimal serial dilutions were prepared.

 Methodology (A.P.H.A., 1992):

1- Enumeration and identification of Aerobic spore-formers and Bacillus cereus.

Dextrose tryptone agar medium (Oxoid, 1999) and Bacillus cereus medium (Biolife, 1993) were used for enumeration of Aerobic spore-formers and Bacillus cereus respectively. Isolated colonies were purified and identified according to Cowan and Steel (1974)

2-Determination of proteolytic activity of isolated Bacillus cereus (Harrigan and McCance, 1976)

Overnight cultures were spot inoculated onto pre-poured plates of standard plate count agar with 10% added skim milk. The inoculated plates were incubated at 25Ĉ for 48 hours and subsequently flooded with 10% W/V acetic acid, appearance of clear zone around the colonies after one minute exposure were regarded as positive. 

Results

 Table 1: Aerobic spore-formers count/gram of examined samples      (n=30).

*-ve samples >100

Table 2: Bacillus cereus count/gram of examined samples (n=30)

*-ve samples >100

Table 3: Frequency distribution of Bacillus spp. isolated from examined  samples. 

 Table 4: Proteolytic activity of isolated Bacillus cereus

Discussion

The results given in Table 1 showed that out of the examined 30 yoghurt samples 26 (87%) were positive for Aerobic spore-formers. The high level of Aerobic spore-formers contamination was 4.2x10⁶, the low level was 3.0x10³ and the mean value was 5.3x10⁴ while Aerobic spore-formers could be isolated from 28 (93%) of the examined rice budding samples and the count ranged from 2.8x10⁴ to 2.6x10⁷ with a mean value of 4.6x10⁵ .Nearly similar finding was reported by Rangasamy et al. (1993) and Said and Fahmy (1991)

Information derived from the results reported in Table 2 revealed that Bacillus cereus could be isolated from 12 (40%) and 22 (73%) with a mean counts of 1.3x10³ and 1.6x10⁵of the examined yoghurt and rice budding samples respectively. These findings were in agreement with those reported by Sallam et al. (1991), Rangasamy et al. (1993), Notermans et al. (1997) and Warke et al. (2000) while Giffel et al., (1996) recorded lower values.

The results presented in Table 2 declared that the incidence and count of Bacillus cereus in examined rice budding samples are higher than yoghurt. The Bacillus cereus spores are often found in cereals especially rice and some spores are surviving cooking and subsequently germinated into Bacilli which, under warm storage condition in cooked food grow and produce toxins (Hobbs and Roberts, 1993).Bacillus cereus can be introduced into milk via sources other than raw milk and the equipment is probably an important source of contamination. Bacillus cereus spores were predominantly transmitted from feed, via feces to farm bulk tank milk (Vissers et al., 2007). In fermented milk and cheese vegetative Bacillus cereus are rapidly eliminated during production process (Schoder et al., 2007)

Out of 60 Bacillus spp. isolated from examined yoghurt samples, Bacillus licheniformis was predominating and comprising 23(38%) while 13 (22.0%) isolates proved to be Bacillus cereus and on the other hand Bacillus cereus was predominating other Bacillus spp. isolated from rice budding samples 25(42%) (Table, 3). These findings were in agreement with those reported by Meer et al. (1993) and Zhou et al. (2008). Bacillus cereus is a common cause of two types of food-borne illnesses, the diarrheal type is characterized by abdominal cramps and diarrhea 8-16 hours following ingestion while emetic type is characterized by vomiting 1-5 hours after ingestion of incriminated food (Schoder et al., 2007 and Svensson et al., 2007). Other spore-forming organisms such as Bacillus licheniformis have also been reported as agents of food poisoning with similar characteristics to those of Bacillus cereus (Hobbs and Roberts, 1993).

The results given in Table 4 showed that out of 25 Bacillus cereus obtained from rice budding samples, 20 (80%) were possessed proteolytic activity while only 5 (38.5%) out of 13 Bacillus cereus isolates isolated from yoghurt samples. Nearly similar finding was reported by Awad (1996). The thermoduric bacteria do not influence on the keeping quality of either raw and heat treated milk except those produced spores. The later are responsible for spoilage of pasteurized (sweet curdling) and occasionally raw milk which has been kept at low temperature as cream (bitty cream) under the effect of proteolytic and lipolytic action (Schoder et al., 2007)

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