The present investigation pertains to the starch adulteration of Burfi (a popular milk product consumed in Jammu and Kashmir), along with the study of prevalence and toxigenicity studies of Bacillus cereus emetic strain isolates of Burfi in Kashmir valley. The investigation was conducted during October 2009-June 2010. A total of 25 samples of Burfi were tested. Starch/rice flour was detected in 5 samples which only revealed Bacillus cereus emetic strains on isolation, making a prevalence of 20 percent contamination of Bacillus cereus emetic strain in Burfi available in and around the Srinagar city. Different areas of the Srinagar city differed with respect to starch adulteration as well as contamination of Bacillus cereus emetic strains. Central, north and east zones had the highest percentage of positive samples of starch as well as Bacillus cereus emetic strain (20% each). The field isolates and the standard strain of Bacillus cereus had similar cultural, morphological and biochemical characteristics.
Foods of animal and plant origin primarily being the main source of nutrients, also act as vehicles for many food borne infections and intoxications, as they provide an excellent media for growth and perpetuation of micro-organisms. Despite improved levels of hygiene and sanitation in handling and preparation of food items, the incidence of food-borne infections is increasing, owing to better reporting and diagnostic techniques, changes in eating habits and identification of new human pathogens (Crerar et al., 1996). Among the various food borne pathogens, Bacillus cereus, a gram positive, spore forming bacterium is wide spread in environment (soil, water and dust), and easily contaminates the foods of both plant and animal origin such as cereals, vegetables, milk and milk products, meat and meat products etc, there by causing food borne illnesses in humans (Larsen and Jorgeusen, 1996). Outbreaks of food poisoning due to Bacillus cereus have been described since the beginning of the last century with the first confirmed report in Norway in 1948 (Hauge, 1955). Since then many food-borne outbreaks were reported (Lund et al., 2000, Hussain et al., 2006). Milk, being ideal medium for growth of microorganisms, makes it suitable for multiplication of B. cereus as well as elucidation of its toxin in it. With the advent of increased number of psychrotolerant B. cereus strains, the dairy industry has witnessed increased reports of food poisoning outbreaks due to this organism (Granum et al., 1993).
Two distinct types of gastrointestinal disorders caused by B. cereus in humans viz; an early “emetic syndrome” and a late onset “diarrheal syndrome” involving two different types of enterotoxins, have been recognized (Kramer and Gilbert, 1989). The emetic syndrome, a food borne intoxication, caused by preformed B. cereus emetic enterotoxin (BCEET) in food has a rapid onset (1-5 hours) characterized predominantly by nausea and vomition, resembling closely to staphylococcal food poisoning (Adams and Moss, 2007). In contrast the diarrhoeal syndrome is caused due to production of B. cereus diarrhoeal enterotoxins (BCDET) during the vegetative growth of bacteria in the foods or in the intestines following ingestion, has a longer incubation period (12-24 hours) and is characterized by symptoms like diarrhoea, abdominal pain and rectal tenesmus, resembling closely Clostridium perfringens type A food poisoning (Kramer and Gilbert, 1989, Adams and Moss, 2007).
Apart from gasteroenteritis B. cereus is also involved in a variety of non-GIT infections like meningitis, endopthalmitis, endocarditis, periodonitis, osteomyelitis, wound infection and septicaemia in humans (Schoeni and Wong, 2005). It is also emerging as potential pathogen of serious concern in animals owing to increased reports of its role in diseases like, osteomyelitis, middle ear infections, abortions and mastitis (Schiefer et al. 1976). These reports unfold its explosive pathogenic role in various infections of animals.
The biological effects of B. cereus toxins have been studied extensively. The diarrhoeal enterotoxin produced during the exponential growth phase of the organism is destroyed at 56oC in 20 minutes, a temperature which is far less than the temperature attained in conventional cooking. However, emetic enterotoxin, commonly called as cerulide, produced in the stationary growth phase, is highly heat stable (126oC for 90 minutes), withstanding extremes of pH (2-11) and is unaffected by the temperature attained during conventional cooking process. As a result the episodes of food poisoning out breaks due to B. cereus emetic toxin outnumber the diarrhoeal ones.
The production of enterotoxins by B. cereus is dictated by the type of food rather than the strain involved. Studies on food ingredients have indicated rice and rice flour containing a high percentage of B. cereus emetic strains whereas diarrhoeal strains are found in almost all foods of animal origin. During growth, harvesting, milling, and other agricultural operations rice can variabily become contaminated with B. cereus spores from a wide variety of environmental sources including soil, dust, sediment, and water (Johnson et al., 1984). The spores survive normal cooking temperatures (Vijaylakshmi et al., 1981) and proliferate when the cooked rice is stored at room temperatures for long time leading to the episodes of intoxication (emetic type) or toxi-infection (diarrhoeal type) due to consumption of such temperature-abused rice. The emetic enterotoxin is selectively produced in rice and vegetable sprouts, milk and milk products, the later mostly being adulterated by the addition of rice powder/flour. The long transportation associated with continuous shaking of milk from rural areas to urban consumption sites provides an excellent shake culture media for the production of B.cereus emetic enterotoxin.
Materials and Methods
A total of twenty five samples of Burfi along with fluid (100 ml each) were collected in sterile urocols from local retail outlets of five arbitrary zones of Srinagar city viz. North (Hazratbal and surrounding areas), South (Sonawar and dalgate), East (Harwan, Shalimar and Nishat), West (Qamarwari aand aadjacent areas) and Central (Habbakadal, Khanyar and adjacent areas). The samples (Table 1) were brought to the laboratory on ice and processed within two hours of collection.
Table 1: Source and detail of Burfi collected from different zones of Srinagar city
Detection of starch/ rice flour
The samples of Burfi were subjected to test for the detection of starch/rice flour as an adulterant. For this 1% iodine solution was used as the standard. 3grams of test sample of Burfi was thoroughly mixed and boiled for 15min. After cooling to the room temperature 2 to 3 drops of 1 % iodine was added drop by drop. A positive test was indicated by change of colour to blue, which disappeared after boiling and reappeared on cooling.
Standard bacillus cereus emetic strain
The standard Bacillus cereus emetic strain (NCTC 11143) originally recovered from a natural emetic type food poisoning outbreak by Public health laboratory service Coollindale, London was obtained from Department of Veterinary Public Health, Ranchi Veterinary College, Ranchi, Bihar.
Isolation & Identification
The samples were subjected to the standard procedures for isolation of Bacillus cereus Emetic strains as described by Sakurai et al.(1994) with slight modification. The samples were heated to 70 0C for 10-15 minutes and then cooled down to 25-35 0C for inoculation into brain heart infusion broth (BHIB). After inoculation, the BHIB tubes were incubated at 370C for 24-48 hours and subjected to a temperature of 80-90 0C for 2 hours in a preset water bath. The tubes were allowed to cool and subsequently streaked on Mannitol Egg Yolk and polymixin B-sulphate agar plates (MYPA) and incubated at 37 0C for 24 hours. The discrete flat pink colonies with serrated borders, surrounded by a zone of lecithinase reaction and starch negative were taken as Bacillus cereus. The colonies were picked up on nutrient agar slants for biochemical and other tests (Table 2) as confirmatory to B. cereus as described by Cowan (1974), Gibson and Gordan (1974) and Norris et al. (1981). Starch hydrolysis negative colonies were designated as emetic strains of B. cereus.
Table 2: Biochemical and other characteristics of Bacillus cereus emetic strains
|8||Voges proskauer test|
|9||Fermentation of Ammonium salt sugars viz.
a) Glucose b) Arabinose c) Mannitol
Starch Adulteration and Bacillus cereus emetic strain contamination of Burfi
Out of total of twenty five samples screened, 5 samples were found adulterated with the starch (rice flour) showing an overall prevalence of 20%. All the starch positive samples revealed positive results for Bacillus cereus emetic strain contamination. However none of the starch negative samples revealed positive results for Bacillus cereus emetic strain contamination so making a prevalence of 20% only.
Among different representative zones of Srinagar city, central and north zones revealed the highest percentage of starch adulterated Burfi samples with 2 out of 5 samples (40%) from each zone showing adulteration. While as, west and south zones revealed 20 and 0 percent adulteration respectively, table (Table 3).
Table 3: Zone wise starch adulteration in barfi samples
|S.No||Zone||Samples tested||Samples positive||Percent positive|
Out of 25 samples tested only 20% of barfi samples were contaminated with Bacillus cereus emetic strains. Central and north zones showed maximum contamination (40.0%) followed by west zone (20.0% ). East and south zones did not reveal any contamination with B. cereus emetic strains, as shown in the table 4.
Table 4: Zone wise contamination of barfi samples with Bacillus cereus
|S. No.||Zone||Samples tested||Samples contaminated||Percent positive|
Cultural and Biochemical Properties of Field Isolates of Bacillus Cereus Emetic Strains Recovered From Milk and Milk Product
The morphological, cultural and biochemical characteristics of field isolates recovered from Burfi were compared with standard Bacillus cereus strain NCTC 11143. The isolate and the standard strains revealed the similar biochemical tests and morphological characteristics. The results are presented in Table 5.
Table 5: Biochemical and other characteristics of Bacillus cereus emetic strains recovered from raw milk and milk products.
NCTC 11143 Field strains
|9||Fermentation of sugars
Milk and milk products serve as a potential reservoirs for many bacterial pathogens. The role of staphylococci, Listeriae and Salmonellae in milk-borne infections and intoxications has been elucidated. These organisms are easily killed by heat treatment either in the form of pasteurization or boiling, the later being more acceptable method under local conditions. The combined abilities of the spores and toxins, to survive pasteurization and of certain strains to multiply at low temperatures make Bacillus cereus a unique organism as a milk-borne pathogen. There is hardly any organism with such diversified characteristics like aerobic and anaerobic, motile and/or non-motile, psychrotropic (40C) and thermophilic (500C) as Bacillus cereus. These characteristics make Bacillus cereus a unique and one of the most important food poisoning causing organisms. Bacillus cereus is ubiquitous and appears to have adjusted to the environmental changes in such a way that its presence is anticipated in almost all foods of domestic consumption, whether in the form of vegetative cells, spores or preformed toxins, thus posing a great public health threat. Raw market milk, being the source of all the three, acts as a good source of Bacillus cereus food poisoning, either directly or in the form of products utilizing milk as an ingredient. Milk and milk products are naturally contaminated with diarrhoeal strains of Bacillus cereus, which on conventional heating are easily killed as is true with other such food poisoning causing organisms. The presence of emetic strains of B. cereus in milk and of emetic enterotoxins is anticipated in situations where milk is adulterated with starchy foods like rice powder etc.
As milk is transported from the rural production areas to the urban consumer sites, for either direct consumption or as a basic ingredient of other milk products, it acts as an important shake culture medium for toxin production especially when the ambient temperature is coinciding with the growth requirements of the organism. Besides, absence of cold chain favours the emetic toxin production by the bacteria, even in low numbers. The toxin, because of its heat stability and the capacity to withstand a wider range of pH (2-11), leads to the onset of emetic syndrome in human beings when such contaminated milk is consumed. Milk being utilized as the basic ingredient in many milk products such as cheese, burfi, rasgola, rasmalai and ice-cream, if contaminated with the emetic strains of Bacillus cereus, poses the greatest public health challenge as toxins are not destroyed by any such process employed in the manufacture of these products.
In the present study all the samples of Burfi were subjected to starch test and both starch positive as well as starch negative samples were tested for detection of B. cereus emetic strains by standard procedure. The study revealed 20% adulteration of starch in the samples of the Burfi.
In the present study, all the starch positive samples of the Burfi revealed presence of Bacillus cereus emetic strains which otherwise form the part of the microflora of the rice and rice products, establishing a fact that rice powder was used as an adulterant for increasing the physical attributes, texture and the bulk without affecting the specific gravity of the milk. Twenty per cent of Burfi samples were contaminated with Bacillus cereus emetic strains. The results are in agreement with the findings of jayachandra et al. (1985) who reported distribution of Bacillus cereus as 12.30 percent in milk samples collected from street vendors, dairy plants, organized dairies and villages, while as Ahmed et al. (1983) reported a prevalence of 21% in raw milk in Egypt. Both these findings are a strong foundation of the fact that the adulteration of milk with rice flour (powder), to earn more from the less production of milk, is at the cost of human health. Among others Abdel khaliq et al. (1996) found 40.00 and 30.00 percent of raw and pasteurized milk samples, respectively contaminated with Bacillus cereus, while as Sharma et al .(2003) reported 66% contamination of milk with B. cereus.
Contamination of milk by Bacillus cereus has also been reported by many workers and established that entry of Bacillus cereus in the market milk and milk products could be due to many reasons including the adulteration of milk by water and rice flour (Kamat et al., 1989, Lin, 1998), use of contaminated utensils (Giffel et al., 1996) or even the mastitis in the cows (Adlan et al., 1980).
The presence of Bacillus cereus emetic strains in raw milk and milk products is alarming owing to the heat and pH resistance of the bacteria with subsequent production of toxins in milk in stationary phase. So such kinds of adulterations can prove detrimental since Bacillus cereus, besides causing GIT syndromes, is also involved in several non-GIT syndromes like meningitis, endocarditis etc. The present study, therefore unveils the faulty practice of adulteration of milk and milk products with rice powder, which needs a stronger monitoring and frequent testing, followed by subsequent control measures on the part of municipal authorities. Since all the starch positive samples were confirmed for Bacillus cereus emetic strain contamination so the presence of Bacillus cereus may act as an indicator bacteria for starch adulteration in milk and milk products and vice versa thus advocating total condemnation of starch contaminated milk and milk products, owing to the presence of its toxins.
Although there are only a few reports of food poisoning out breaks due to Bacillus cereus following consumption of milk and milk products, yet in most of the cases the organisms were capable of producing emetic enterotoxins (Giffel et al., 1996). The emetic enterotoxin producing strains of Bacillus cereus is of the serious public health significance, since rice powder harbouring Bacillus cereus emetic strains will get a suitable media in milk for toxin production, which is not destroyed even at a temperature of 1260C for 90 minutes, causing Bacillus cereus emetic syndrome particularly in children and the older people who are the major consumers of milk. Therefore the mere presence of Bacillus cereus emetic strains in milk and milk products needs to be reviewed for acceptibility of the later keeping in view the stability of the toxins that are formed. The inherent capacity of these organisms to adapt to a particular food or the presence, in the medium, of substances that promote/suppress enterotoxin production have been proposed (Singh, 1994). This study was primarily initiated to satisfy the need for data, which could be used as the background information in hazard analysis of milk and milk products in different representative zones of Srinagar city. Different zones revealed different percentage of starch and Bacillus cereus emetic strain contamination which may be correlated with higher population density, vehicular stress and poor living conditions.
Although, Bacillus cereus outbreaks were associated mostly with the ingestion of cateressd meals. Only several outbreaks were mentioned in which milk products could have been the cause of food poisoning. The poisoning, however, seems to be underestimated in the literature and statistics because of its short duration and resemblance to S. aureus food posoning. However all the isolates revealed similar morphological characteristics and biochemical tests as depicted by the standard strain (NCTC 11143).
The authors thanks to the Dean, FVSc and A.H, Shuhama, Alusteng, Srinagar, for providing necessary facilities at time of research.
Abdel- Khalak, A. and El- Sharbini, M. 1996. Prevalence of enterotoxigenic Bacillus cereus in raw and pasteurized milk. Vet. Med. J., Giza 44: 157- 161 (CAB Abstracts, 1996-98/ 07 record 96 of 153).
Adams, M. R. and Moss, M. O. (2007). Royal Society of Chemistry Great Britain, England. Food Microbiology. 3:60-64.
Adlan, A.M., Shommein, A. M., Amin, E. D. M-El. And El-Amin, E. D. M. 1980. A survey of bovine mastitis in four dairy farms in the Sudan. Sudan Journal of Veterinary Research 2: 27-39 (CAB international record 136 of 153).
Ahmad, A. A. H., Moustafa, M. K. and Marth, E. H. 1983. Incidence of Bacillus cereus in milk and milk products. Journal of Food Protection 46: 126-28.
Cowan, S. T. 1974. Characteristics of gram-positive bacteria. In cown and Steels Manual for the identification of medical bacteria. 2nd Edn. Cambridge press, London, Chap. 6, pp. 71.
Crerar, S. K., Dalton, C. B., Longbottom, H. M. and Kraa, E. 1996. Foodborne diseases: current trends and future surveillance needs in Australia. Medical Journal of Australia 165: 672-75.
Gibson, T. and Gordan, R. E. 1974. Genus 1 : Bacillus in Bergey’s Manual of Determinative Bacteriology. 8th Edn. Eds. Buchanan, N. E. pp.529-550. Baltimore, Williams and Wilkins.
Giffel-MC-te., Beumer, R. R., Leijendekkers, S., Rombouts, F. M. and Te Giffel, M. C. 1996. Incidence of Bacillus cereus and Bacillus subtilis in foods in Netherlands. Food Microbiology 13: 53-58 (FSTA Current 1990-9/96, Record 12 of 153).
Granum, P. E., Brynestad, S., O’Sullivan, K. and Nissen, H. 1993. Enterotoxin from Bacillus cereus: production and biochemical characterisation. Netherlands Milk Dairy Journal 47: 63-70.
Hauge, S. 1955. Food poisoning caused by aerobic spore forming Bacilli. Journal of Applied Bacteriology 18: 591-95.
Hussain, S. A., Munshi, Z. H. and Hussain, I. 2006. Food poisoning due to Bacillus cereus. A case report. Journal of Veterinary Public Health. 5: 57-59.
Jayachandra, T., Krishnamurthi, P. S. and Naterajan, N. 1985. Study on the incidence and distribution of Bacillus subtilis and other spore formers in various sources of milk supplies. Cherion, 14 : 98-100.
Johnson, K. M., Nelson, C. L. and Busta, F. F., 1984. Influence of temperature on germination and growth of spores of emetic and diarrheal strains of Bacillus cereus in a broth medium and in rice. J. Food Sci. 48: 286–287.
Kamat, P. R. 1989. Studies on some properties of emetic and diarrhoeal enterotoxin standard strains of Bacillus cereus. M.V.Sc thesis, submitted to B.A.U., Ranchi, Bihar.
Kramer, J. M. and Gilbert, R. J. 1989. Bacillus cereus and other Bacillus species. M.P Doyle (ed.) Food-borne Bacterial pathogens. pp 21-70 Marcel Dekker, New York.
Larsen, H. D.and Jorgeusen, K. 1996. The occurrence of Bacillus cereus in Danish Pasteurised milk, International journal of food microbiology 34: 179-186.
Lin, S., Scraft, H., Odumeru, J. A. and Griffths, M. W. 1998. Identification of contamination sources of Bacillus cereus in Pasteurized milk. International Journal of Food Microbiology 43: 159-71.
Lund, T., DeBuyser, M. L. and Granum, P. E. 2000. A new cytotoxin from Bacillus cereus that may cause necrotic enteritis. Molecular Microbiology. 38: 254-61.
Norris, J. R., Berkeley, R. C. W., Logan, N. A. and O’Donnell, A. G. 1981. The genera bacillus and sporolactobacillus. In “The Prokaryotes- A Handbook on Habitats, Isolation and Identification of Bacteria”. Vol. 11. Ed. Starr. M. P., Stolp. H., Truper, H. G., Balows, A. and Schlegal, H. G., pp. 1735.
Sakurai, N., Koike, K. A. Y. and Hayashi, H. 1994. The rice culture filtrate of Bacillus cereus isolated from emetic type and food poisoning causes mitochondrial swelling in a HEp-2 cell. Microbial. Immunol. 38 : 337-343.
Schiefer, B., Macdonald, K. R., Klavano, G. G. and Van-Dreumel, A. A. 1976. Pathology of Bacillus cereus mastitis in dairy cows. Canadian Veterinary Journal 17: 239-43.
Schoeni, J. L. and Wong, A. C. L. 2005. Bacillus cereus food poisoning and its toxins. Journal of Food Protection 68: 636-48.
Sharma, C. S., Sharma, D. K., Gill, J. P. S., Aulakh, R. S. and Sharma, J. K. 2003. Bacillus cereus from foods of animal origin in India and its public health significance. Actaveterinaria Scandinavica 44: 118.
Singh, D. K. and Narayan, K. G. 1994. Effect of hydrogen ion concentration and sucrose on enterotoxin production by Bacillus cereus. Journal of Food Science and Technology, 31: 338-40.
Vijaylakshmi, G., Dwarkanath, C. T. and Sreenivasa Murthy, V. 1981. Studies on Bacillus cereus contamination of rice and rice preparation. J. Food Sci. Technol. 18, 231-234.