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Growth and enterotoxin production by sporeforming bacterial pathogens from spices
Food Control 15 (2004) 491–496 www.elsevier.com/locate/foodcont
Growth and enterotoxin production by sporeforming bacterial pathogens from spices Mousumi Banerjee, Prabir K. Sarkar
*
Microbiology Laboratory, Department of Botany, University of North Bengal, Siliguri 734 430, India Received 2 March 2003; received in revised form 27 July 2003; accepted 28 July 2003
Abstract Spices often act as important vectors for various micro-organisms, specially sporeformers implicating possible health problems for consumers. Bacillus cereus enterotoxin (BCET) and Clostridium perfringens enterotoxin (PET) were estimated using the latex agglutination method. Seventy-four percent of the tested 23 strains of B. cereus were able to produce 8 to >256 ng BCET ml 1 brain heart infusion broth supplemented with glucose. Of the selected 16 strains of Cl. perfringens, 19% produced 2–32 ng PET ml 1 modified Duncan Strong medium. Some market spices, like cumin powder contained a high BCET titre (64 ng g 1 ). After intentional inoculation of black pepper powder with a toxigenic strain (120-B1) of B. cereus and 14 d-storage at room temperature, there was no significant (P < 0:05) change in the cell count and BCET production. To assess safety of spicy foods, aloo dam (a potato-based food) and goat meat gravy were taken as subjects for the respective growths of B. cereus and Cl. perfringens. Freshly prepared aloo dam did not contain B. cereus, however immediate to seasoning with small cardamom the count of B. cereus cells was 533 g 1 and the BCET content was 8 ng g 1 . After keeping the food at 30 C for 21 h, the cell count increased to 106 g 1 and the BCET content increased to 128 ng g 1 . A similar situation happened when aloo dam was intentionally inoculated with B. cereus 120-B1. A toxigenic strain of Cl. perfringens multiplied rapidly in the gravy; after 19 h at 37 C the cell count increased from 103 to 107 g 1 , however the PET content (2 ng g 1 ) remained unchanged. After boiling the 19 h-long incubated gravy for 15 min in a water bath, the cell count fell to 103 g 1 and the PET content went below the limit of detection. The results confirmed that these foods are capable of supporting outgrowth of bacterial pathogens introduced in contaminated spices and the production of enterotoxins. 2003 Elsevier Ltd. All rights reserved. Keywords: Bacillus cereus; Clostridium perfringens; Enterotoxins; Spices; Challenge study
1. Introduction Spices can transform an ordinary meal into an adventure as well as they are classed as important vehicle for various micro-organisms implicating possible health problems for consumers and shelf life problems for foods. Foodborne disease is perhaps the most widespread health problem in the contemporary world and an important cause of reduced economic productivity (WHO, 1992). In most of the cases of foodborne illness, the pathogenic effect occurs in the alimentary tract giving rise to symptoms of diarrhoea and vomiting. Toxins
*
Corresponding author. Tel.: +91-353-2582106; fax: +91-3532581546. E-mail address: [email protected] (P.K. Sarkar). 0956-7135/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2003.07.004
frequently play a pivotal role in this process. Contaminated spices may cause a microbiological problem, depending on the end use. Cuisines that incorporate spices may pose a risk to public health because they are often added to foods that undergo no further processing or are eaten raw. Spices are the principal source of sporeforming bacteria in large volumes of foods, such as soups, stews and gravies produced by catering establishments. Under favourable conditions, they germinate and multiply to infective and toxic levels (Pafumi, 1986). Previous studies on the microbiology of spices have demonstrated that Bacillus cereus and Clostridium perfringens are the sporeformers commonly found in spices (Antai, 1988; Banerjee & Sarkar, 2003; Kneifel & Berger, 1994; Powers, Latt, & Brown, 1976). Numerous reports of B. cereus foodborne illness have been cited by Goepfert, Spira, and Kim (1972). For these outbreaks the levels of B. cereus in the food rem-
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nants were found to be 106 –109 cfu g 1 or ml 1 (Gilbert, 1979). There are two distinct syndromes caused by separate toxins produced by B. cereus. While the diarrhoeal toxin is produced during the late exponential phase of growth, emetic toxin is produced during late exponential to stationary phase of growth (Kramer & Gilbert, 1989). Cl. perfringens occurs in a variety of foods and causes food poisoning (Labbe, 1991). Spices and herbs containing Cl. perfringens, even in low numbers (<102 cfu g 1 ), may play an important role in contributing to the microbial load of prepared foods (De Boer, Spiegelenberg, & Janssen, 1985). Although foods related to food poisoning outbreaks caused by Cl. perfringens usually contain a large number (105 –106 cfu g 1 ) of cells (Labbe, 1991), spores, even at low levels, could germinate and grow to reach a significant number (Nakamura & Kelly, 1968). Symptoms associated with the disease are caused by an enterotoxin that is expressed during sporulation of enterotoxigenic strains (Czeczulin, Hanna, & McClane, 1993). Ingested vegetative cells that survive the stomach’s acidity pass to the small intestine where they grow, sporulate and release enterotoxin. Many surveys have shown that Cl. perfringens is found in raw and processed food, most notably, raw meat products and spices (Labbe, 1989, 2000). The present study was undertaken to understand the potential health hazard caused by these bacterial pathogens from spices.
broth culture or blended sample (1.5 ml) was taken in an Eppendorf tube and centrifuged (Remi CM12) (900 g; 20 min for broth culture, 30 min for spice/food sample) at 4 C. For sample, the supernatant was filtered through a 0.2 lm-cellulose acetate membrane (Sartorius AG, G€ ottingen, Germany). The supernatants/filtrates were used immediately for the assay of enterotoxin. Similarly, for the extraction of Cl. perfringens enterotoxin (PET), Cl. perfringens strains were grown at 37 C for 20 h in cooked meat medium. The culture was heated at 75 C for 20 min, and a 0.8 ml of the aliquot (taken from the base of the tube) was inoculated into 16 ml modified Duncan Strong (DS) medium (HiMedia M1237) (Duncan & Strong, 1968). The tubes were incubated at 37 C for 24 h. The culture was centrifuged (900 g, 20 min) at 4 C and the supernatant was used for the assay. Extraction of enterotoxin from food was done following the method as described above for the extraction of BCET.
2. Materials and methods
2.3. Challenge testing in black pepper powder
2.1. Organisms
In a sterile screw-capped glass bottle a 100 g-sample of black pepper powder was mixed well with spores of B. cereus 120-B1 which was prepared by growing it at 37 C for 72 h on sterile cellophane (British cellophane grade 325P, Avonmouth, UK) overlaid on nutrient agar (HiMedia M561) plate. The bottle was left at room temperature (31–33 C). B. cereus count and enterotoxin titre were monitored at 0 h, and after 7 and 14 d of storage using B. cereus selective agar (HiMedia M833, FD003, FD045) (BCSA) and BCET-RPLA kit, respectively.
The bacterial strains were isolated from different spices collected from retailed outlets in India (Banerjee & Sarkar, 2003). While Cl. perfringens strains were maintained at 4 C in cooked meat medium (Cat. No. M149; HiMedia Laboratories Pvt Ltd, Mumbai, India), B. cereus strains were maintained at 4 C on tryptone soya agar (HiMedia M290) slants, with sub-culturing after every six months.
2.2.2. Assay Qualitative as well as semi-quantitative tests for the enterotoxins were performed using BCET-RPLA (reversed passive latex agglutination) toxin detection kit (Oxoid TD950; Unipath Limited, Basingstoke, Hampshire, UK) and PET-RPLA toxin detection kit (Oxoid TD930) for BCET (diarrhoeal type) and PET (type A), respectively, as directed in the manual.
2.2. Enterotoxins 2.4. Safety of foods on storage 2.2.1. Extraction For the extraction of B. cereus enterotoxin (BCET), B. cereus strains were inoculated into 10 ml brain heart infusion broth (HiMedia M210) supplemented with 10 g glucose l 1 (BHIG) and incubated at 33 C for 18 h on a shaker (200 rpm). For the isolation of enterotoxin from spice samples, a 10 g-sample was blended with 10 ml of 8.5 g sodium chloride l 1 (extra dilution done when needed) using a Stomacher lab-blender 400 (Seward Medical, London, UK) for 5 min at Ônormal’ speed. The
2.4.1. Safety of aloo dam (a potato-based ethnic food) against B. cereus 2.4.1.1. Preparation of aloo dam. Ingredients: Potato (2–4 cm dia), 1 kg; cumin, 1/4 tsp.; tejpat, 2 pcs, cumin powder, 2 tbsp.; turmeric powder, 1/2 tsp., grated fresh ginger, 2 tsp; oil, 4 tbsp.; water, 4 cups (600 ml); salt to taste. Recipe: Whole deskinned potatoes were fried in hot oil until golden brown and kept aside. Tejpat and cumin
M. Banerjee, P.K. Sarkar / Food Control 15 (2004) 491–496
were sauted followed by addition of turmeric powder, cumin powder and grated ginger. The mixture was fried till spices separated from oil. Previously-fried potatoes along with an appropriate amount of water were added to the mixture. Salt was sprinkled to taste and mixed well. The pan was covered and allowed to cook on low heat for 15 min until the potatoes were tendered and a typical aroma developed. 2.4.1.2. Challenge study on aloo dam. Fifty grams of freshly prepared aloo dam were taken in each of three sterile glass bottles. Aloo dam of one bottle was seasoned with ground small cardamom (where B. cereus was detected in 100% of the samples analysed; Banerjee & Sarkar, 2003), while another bottle was intentionally inoculated with a 24 h-old culture of B. cereus 120-B1 (an enterotoxigenic strain) in BHIG. An uninoculated bottle was kept as control. All the three bottles were plugged with sterile cotton wool and kept at 30 C. Sampling from each bottle was done at 0 h and after 21 h for B. cereus count and BCET production using BCSA and BCET-RPLA kit, respectively. 2.4.2. Safety of meat (goat) gravy against Cl. perfringens 2.4.2.1. Preparation of meat curry. Ingredients: Minced goat meat, 1 kg; small cardamom, 1/2 tsp.; clove, 1/2 tsp.; cinnamon, 1/2 tbsp.; tejpat, 4 pcs; turmeric powder, 2 1/2 tbsp.; red chilli powder, 1 tsp.; grated fresh ginger, 3 tsp.; grated garlic, 3 tsp.; grated onion, 8 tbsp.; oil, 5 tbsp.; water, 5 cups (750 ml); salt to taste. Recipe: Minced goat meat, marinaded with grated onion, garlic and ginger, was refrigerated at 4–6 C for 1 h. Tejpat, clove, cinnamon and small cardamom were sauted followed by addition of turmeric powder and red chilli powder. Marinaded minced meat was added in it and fried till the mass separated from oil. The fried meat along with water and salt was cooked for 15 min in a pressure cooker (1.1 kg cm 2 ).
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2.5. Statistical analysis Data were analysed statistically by determining analysis of variance of triplicate sets (Snedecor & Cochran, 1989).
3. Results 3.1. Bacillus cereus and BCET Qualitative tests on BCET production of selected 23 strains (one from each kind of spices which showed maximum bacterial count; Banerjee & Sarkar, 2003) showed that 74% of the strains were able to produce it in BHIG (Table 1). Of these enterotoxigenic strains, 88% showed a highly positive (+++) reaction and were selected for quantitative estimation. The BCET content in BHIG varied from 8 to >256 ng ml 1 . Sixty percent of the tested strains produced 32 ng BCET ml 1 , two strains produced 128 ng BCET ml 1 and one strain produced 256 ng ml 1 . The profiles of load of B. cereus cells and BCET content in selected spices are shown in Table 2. Cumin powder had the highest BCET content, while cardamom had the lowest. In ajmud the cell count of B. cereus was maximum, however the BCET titre was in between the values of the other two spices. Intentional inoculation of black pepper powder with 108 cfu g 1 of an enterotoxigenic strain (120-B1) of B. cereus showed no significant (P < 0:05) changes in the cell count as well as enterotoxin titre (48 ng g 1 ) on storage for 14 d. Freshly prepared aloo dam did not contain any B. cereus and its toxin at detectable level. But when the same was seasoned with small cardamom, the aloo dam contained both cells and toxin. After 21 h-storage at 30 C both the levels increased significantly (P < 0:05). A similar situation was observed when aloo dam was intentionally inoculated with B. cereus (Table 3). 3.2. Clostridium perfringens and PET
2.4.2.2. Challenge study on meat gravy. Fifty grams of freshly prepared goat meat gravy were taken in a sterile screw-capped glass bottle and mixed well with spores of Cl. perfringens 16-C2 (an enterotoxigenic strain). An uninoculated bottle was taken as a control. Both the bottles were capped tightly and incubated at 37 C for 19 h. Cell counts and enterotoxin titres were measured at 0 h and after 19 h of storage using perfringens agar (HiMedia M579, FD011, FD012) in an anaerobic jar filled with carbon dioxide (HiMedia LE002A) and PETRPLA kit, respectively. The same parameters were again measured after boiling the cultured gravy for 15 min in a water bath.
Qualitative detection of PET of selected 16 strains (one from each kind of sample which had the highest cell count) showed that 19% of the strains were able to produce PET in modified DS medium. The PET content ranged from 2 to 32 ng ml 1 (Table 4). Table 5 shows that the Cl. perfringens count as well as its toxin content were not at detectable levels in freshly prepared meat gravy (pH 6.21). It was inoculated with an enterotoxigenic strain (16-C2) of Cl. perfringens. After 19 h of storage at 37 C, although the cell count increased from 103 to 107 g 1 , the toxin content remained unchanged. When boiled for 15 min, the cell
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Table 1 Production of BCET in glucose supplemented brain heart infusion broth by B. cereus isolates from spices Strain no.
BCET
Source
+/) 3-B2 10-B3 14-B1 16-B2 17-B1 18-B2 20-B1 32-B1 33-B2 36-B1 37-B2 40-B1 56-B2 57-B1 68-B1 85-B1 95-B1 96-B2 98-B2 107-B3 120-B1 128-B1 135-B1
Turmeric (Curcuma longa) powder Coriander (Coriandrum sativum) powder Black pepper (Piper nigrum) Mustard (Brassica juncea) Fenugreek (Trigonella foenum-graecum) Aniseed (Pimpinella anisum) Cinnamon (Cinnamomum zeylanicum) Turmeric Clove (Syzygium aromaticum) Black cumin (Nigella sativa) Coriander Red chilli (Capsicum fruitescens) powder Bishop’s weed (Trachyspermum ammi) Poppy seed (Papaver somniferum) Ginger (Zingiber officinale) Asafoetida (Ferula asafoetida) Large cardamom (Amomum subulatum) Small cardamom (Elettaria cardamomum) Cumin (Cuminum cyminum) Ajmud (Trachyspermum roxburghianum) Cumin powder Allspice (Pimenta dioica) Tejpat (Cinnamomum tamala)
Table 2 Load of B. cereus cells and their enterotoxin (BCET) in selected spicesa Spice Ajmud Cumin powder Small cardamom
Cfu g
1
BCET
4000 367 (200–500) 833 (700–1000)
+/)
ng g
+++ +++ +++
36 64 32
1
a
Values are means of determinations on triplicate sets. Where individual determinations differed by more than 5% from the means, ranges of values are shown in parentheses.
Table 3 Growth of B. cereus and production of its enterotoxin (BCET) in aloo dam (a potato-based food) on storage at 30 C till 21 ha Sampling time (h)
Growth (cfu g 1 )b 0 21 BCET (ng g 1 )c 0 21
Aloo dam
Aloo dam + small cardamom
Aloo dam + B. cereus 120-B1
533 (400–700)
1 · 106
3233 (3000–3500) 4 · 107
8 128
16 256
a Values are means of determinations on triplicate sets. Where individual determinations differed by more than 5% from the means, ranges of values are shown in parentheses. b DL (detection limit), 100 cfu g 1 . c DL (detection limit), 2 ng g 1 .
ng ml
+++ +++ ) +++ +++ ++ +++ ) +++ +++ +++ +++ ) +++ +++ +++ ) ) +++ ) +++ + +++
1
32 32 32 >256 NDa 128 64 32 32 32 128 32 32
8 256 ND 32
Table 4 Production of PET in modified DS medium by Cl. perfringens isolates from spices Strain no.
Source
PET +/)
7-C2 11-C3 14-C1 16-C2 17-C1 18-C1 19-C1 20-C2 36-C3 45-C1 47-C2 68-C2 91-C1 130-C3 131-C1 135-C3
Coriander Cumin powder Black pepper Mustard Fenugreek Aniseed Clove Cinnamon Black cumin Cumin Coriander powder Ginger Bishop’s weed Black pepper powder Caraway (Carum carvi) Tejpat
) ) ) +++ ) ) ) ++ ) ) )
ng ml
1
32
2
) ) ) ) +++
8
count in the 19 h-old cultured gravy decreased to 103 g and the toxin content went below the detection limit.
1
4. Discussion B. cereus was found as one of the most frequent foodborne bacterial pathogens in spices (Banerjee &
M. Banerjee, P.K. Sarkar / Food Control 15 (2004) 491–496 Table 5 Growth of Cl. perfringens and production of its enterotoxin (PET) in goat meat gravy on storage at 37 C till 19 h, and effect of boilinga Sampling time (h)
Gravy
Gravy + Cl. perfringens 16-C2
Growth (cfu g 1 )b 0 19 Followed by boiling (100 C, 15 min)
2517 (2350–2700) 5 · 107 1206 (1120–1300)
PET (ng g 1 )c 0 19 Followed by boiling (100 C, 15 min)
2 2
a
Values are means of determinations on triplicate sets. Where individual determinations differed by more than 5% from the means, ranges of values are shown in parentheses. b DL (detection limit), 10 cfu g 1 . c DL (detection limit), 2 ng g 1 .
Sarkar, 2003). To define the extent of its dreadfulness, an analysis of BCET was undertaken. Since the stimulatory effect of glucose on growth and enterotoxin production by B. cereus is well documented (Garcia-Arribas & Kramer, 1990; Kramer, 1984), BHIG was the medium of choice for this purpose. Our results suggest that, in principle, B. cereus isolates from spices are able to produce enterotoxins. It should, however, be borne in mind that the BCET-RPLA test does not specifically react with the diarrhoeal toxin, but with an indicator protein. In addition, this test cannot detect the B. cereus emetic toxin. Consequently, in vivo toxicity tests should be carried out in case of doubt of toxicity of samples having high BCET levels (Nout, Bakshi, & Sarkar, 1998). While selecting for the BCET level in spices, the spice kinds where all the samples of that kind contained B. cereus (Banerjee & Sarkar, 2003) were chosen. Cumin powder had the highest content of BCET however with the lowest cell count which indicates the production of a considerably high level of BCET by the strain 120-B1 (a cumin powder isolate). The results suggest a potential health hazard of foods containing spices with this bacterium. To understand how much the environment of a spice is congenial for growth of B. cereus and its enterotoxin production, black pepper powder was taken as a support spice since none of the five samples of black pepper powder tested contained B. cereus (Banerjee & Sarkar, 2003). After intentional inoculation of the spice with B. cereus 120-B1, there was no significant (P < 0:05) changes in the cell count as well as BCET production even after 14 d incubation at room temperature (as spices are conventionally stored). This indicates that a dry spice, like black pepper, can support survival of pathogenic organisms and act as a vector for contaminating foods. One is usually fond of spicy foods, but the issue of their safety needs to be assessed. Aloo dam, a potato-
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based ethnic and popular side dish, was taken as a model food for the growth of B. cereus. The rationale behind selecting this starchy food was the findings of GarciaArribas and Kramer (1990) that the starch-supplemented BHI supported near-optimum growth of B. cereus and production of enterotoxin at levels comparable to those produced in BHIG. Freshly prepared aloo dam did not contain B. cereus, but small cardamom acted as a carrier of B. cereus when the food was seasoned with it (as is practiced routinely). The inoculum, whether through the spice vector or as a pure culture intruder, multiplied rapidly in this starch-based food at room temperature and produced enterotoxin. The only commercially available serological assay for PET is RPLA test (Labbe, 1989). Out of 16 strains of Cl. perfringens tested, only three strains showed a positive response towards this test. Hence, those three isolates were subjected to quantitative assay. However, this culture method is less reliable due to problems in encouraging Cl. perfringens to produce sufficient toxin in artificial media. The direct detection of PET in faeces should be carried out parallelly as much larger amount of toxin is formed in vivo (Labbe, 1989). In a study similar to that carried out on aloo dam with B. cereus, goat meat gravy was taken as a test food for the growth of a meat-loving organism, Cl. perfringens and its enterotoxin production. This anaerobic sporeforming bacterial pathogen occurs in 30–80% of raw and frozen meat and also poultry (Labbe, 1989). In this study, it was found that the freshly prepared meat gravy was free from this bacterium. When the gravy was inoculated with Cl. perfringens 16-C2, it multiplied rapidly, however the enterotoxin level remained unchanged. Although low levels of production have been observed in vegetative cell cultures, the PET is synthesized by the sporulating cells (Adams & Moss, 1995; Brynestad & Granum, 2002). For this, it has been recommended by Juneja and Marmer (1998) that control measures for Cl. perfringens food-poisoning must ensure that large numbers of vegetative cells are not consumed. Possibly the gravy and the duration of incubation were not conducive for sporulation. After boiling the 19 h-long incubated gravy the cell count decreased significantly (P < 0:05) due to the destruction of vegetative cells and the enterotoxin level went below the detection limit. The enterotoxin is heat labile, and heating in saline at 60 C for 5 min destroys 80% of its serological activity (Labbe, 1989). It is likely the reason for non-detection of PET using RPLA method after boiling for 15 min. It is concluded that contaminated spices, when added to foods, are able to introduce the pathogens which may subsequently outgrow and produce enterotoxins. The spice microflora can shorten market life of the products through spoilage and/or conceivably contribute to consumer illness. The application of knowledge of the factors inhibiting the growth of different pathogenic
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bacteria can contribute to minimize the risk of food manufacturing operations.
References Adams, M. R., & Moss, M. O. (1995). Food microbiology. Cambridge: Royal Society of Chemistry. Antai, S. P. (1988). Study of the Bacillus flora of Nigerian spices. International Journal of Food Microbiology, 6, 259–261. Banerjee, M., & Sarkar, P. K. (2003). Microbiological quality of some retail spices in India. Food Research International, 36, 469–474. Brynestad, S., & Granum, P. E. (2002). Clostridium perfringens and foodborne infections. International Journal of Food Microbiology, 74, 195–202. Czeczulin, J. R., Hanna, P. C., & McClane, B. C. (1993). Cloning, nucleotide sequencing and expression of the Clostridium perfringens enterotoxin gene in Escherichia coli. Infection and Immunity, 61, 3429–3439. De Boer, E., Spiegelenberg, W. M., & Janssen, F. W. (1985). Microbiology of spices and herbs. Antonie van Leeuwenhoek, 51, 435–438. Duncan, C. L., & Strong, D. H. (1968). Improved medium for sporulation of Clostridium perfringens. Applied Microbiology, 16, 82–89. Garcia-Arribas, M. L., & Kramer, J. M. (1990). The effect of glucose, starch and pH on growth, enterotoxin and haemolysin production by strains of Bacillus cereus associated with food poisoning and non-gastrointestinal infection. International Journal of Food Microbiology, 11, 21–34. Gilbert, R. J. (1979). Bacillus cereus gastroenteritis. In H. Riemann, & F. L. Bryan (Eds.), Foodborne infections and intoxications (2nd ed., pp. 495–518). New York: Academic Press. Goepfert, J. M., Spira, W. M., & Kim, H. U. (1972). Bacillus cereus: food poisoning organisms. A review. Journal of Milk and Food Technology, 35, 213–227. Juneja, V. K., & Marmer, B. S. (1998). Thermal inactivation of Clostridium perfringens vegetative cells in ground beef and turkey as
affected by sodium pyrophosphate. Food Microbiology, 15, 281– 287. Kneifel, W., & Berger, E. (1994). Microbiological criteria of random samples of spices and herbs retailed on the Austrian market. Journal of Food Protection, 57, 893–901. Kramer, J. M. (1984). Bacillus cereus enterotoxins: production, isolation, detection and properties. In J. F. Alouf, J. H. Freer, F. J. Ferhenbach, & J. Jeljaszewicz (Eds.), Bacterial protein toxins (pp. 385–386). London: Academic Press. Kramer, J. M., & Gilbert, R. J. (1989). Bacillus cereus and other Bacillus species. In M. P. Doyle (Ed.), Foodborne bacterial pathogens (pp. 21–70). New York: Marcel Dekker. Labbe, R. G. (1989). Clostridium perfringens. In M. P. Doyle (Ed.), Foodborne bacterial pathogens (pp. 191–234). New York: Marcel Dekker. Labbe, R. G. (1991). Clostridium perfringens. Journal of the Association of Official Analytical Chemists, 74, 711–714. Labbe, R. G. (2000). Clostridium perfringens. In B. Lund, T. BairdParker, & G. Gould (Eds.), The microbiological safety and quality of food (pp. 1110–1135). Gaithersburg, Maryland: Aspen Publishers. Nakamura, M., & Kelly, K. D. (1968). Clostridium perfringens in dehydrated soups and sauces. Journal of Food Science, 33, 424– 426. Nout, M. J. R., Bakshi, D., & Sarkar, P. K. (1998). Microbiological safety of kinema, a fermented soya bean food. Food Control, 9, 357–362. Pafumi, J. (1986). Assessment of the microbiological quality of spices and herbs. Journal of Food Protection, 49, 958–963. Powers, E. M., Latt, T. G., & Brown, T. (1976). Incidence and levels of Bacillus cereus in processed spices. Journal of Milk and Food Technology, 39, 668–670. Snedecor, G. W., & Cochran, W. G. (1989). Statistical methods (8th ed.). Ames, Iowa: Iowa State University Press. WHO (World Health Organization) (1992). WHO Commission on Health and Environment. Report of the Panel on Food and Agriculture, WHO/EHE/92.2.
Growth and enterotoxin production by sporeforming bacterial pathogens from spices Mousumi Banerjee, Prabir K. Sarkar
*
Microbiology Laboratory, Department of Botany, University of North Bengal, Siliguri 734 430, India Received 2 March 2003; received in revised form 27 July 2003; accepted 28 July 2003
Abstract Spices often act as important vectors for various micro-organisms, specially sporeformers implicating possible health problems for consumers. Bacillus cereus enterotoxin (BCET) and Clostridium perfringens enterotoxin (PET) were estimated using the latex agglutination method. Seventy-four percent of the tested 23 strains of B. cereus were able to produce 8 to >256 ng BCET ml 1 brain heart infusion broth supplemented with glucose. Of the selected 16 strains of Cl. perfringens, 19% produced 2–32 ng PET ml 1 modified Duncan Strong medium. Some market spices, like cumin powder contained a high BCET titre (64 ng g 1 ). After intentional inoculation of black pepper powder with a toxigenic strain (120-B1) of B. cereus and 14 d-storage at room temperature, there was no significant (P < 0:05) change in the cell count and BCET production. To assess safety of spicy foods, aloo dam (a potato-based food) and goat meat gravy were taken as subjects for the respective growths of B. cereus and Cl. perfringens. Freshly prepared aloo dam did not contain B. cereus, however immediate to seasoning with small cardamom the count of B. cereus cells was 533 g 1 and the BCET content was 8 ng g 1 . After keeping the food at 30 C for 21 h, the cell count increased to 106 g 1 and the BCET content increased to 128 ng g 1 . A similar situation happened when aloo dam was intentionally inoculated with B. cereus 120-B1. A toxigenic strain of Cl. perfringens multiplied rapidly in the gravy; after 19 h at 37 C the cell count increased from 103 to 107 g 1 , however the PET content (2 ng g 1 ) remained unchanged. After boiling the 19 h-long incubated gravy for 15 min in a water bath, the cell count fell to 103 g 1 and the PET content went below the limit of detection. The results confirmed that these foods are capable of supporting outgrowth of bacterial pathogens introduced in contaminated spices and the production of enterotoxins. 2003 Elsevier Ltd. All rights reserved. Keywords: Bacillus cereus; Clostridium perfringens; Enterotoxins; Spices; Challenge study
1. Introduction Spices can transform an ordinary meal into an adventure as well as they are classed as important vehicle for various micro-organisms implicating possible health problems for consumers and shelf life problems for foods. Foodborne disease is perhaps the most widespread health problem in the contemporary world and an important cause of reduced economic productivity (WHO, 1992). In most of the cases of foodborne illness, the pathogenic effect occurs in the alimentary tract giving rise to symptoms of diarrhoea and vomiting. Toxins
*
Corresponding author. Tel.: +91-353-2582106; fax: +91-3532581546. E-mail address: [email protected] (P.K. Sarkar). 0956-7135/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2003.07.004
frequently play a pivotal role in this process. Contaminated spices may cause a microbiological problem, depending on the end use. Cuisines that incorporate spices may pose a risk to public health because they are often added to foods that undergo no further processing or are eaten raw. Spices are the principal source of sporeforming bacteria in large volumes of foods, such as soups, stews and gravies produced by catering establishments. Under favourable conditions, they germinate and multiply to infective and toxic levels (Pafumi, 1986). Previous studies on the microbiology of spices have demonstrated that Bacillus cereus and Clostridium perfringens are the sporeformers commonly found in spices (Antai, 1988; Banerjee & Sarkar, 2003; Kneifel & Berger, 1994; Powers, Latt, & Brown, 1976). Numerous reports of B. cereus foodborne illness have been cited by Goepfert, Spira, and Kim (1972). For these outbreaks the levels of B. cereus in the food rem-
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nants were found to be 106 –109 cfu g 1 or ml 1 (Gilbert, 1979). There are two distinct syndromes caused by separate toxins produced by B. cereus. While the diarrhoeal toxin is produced during the late exponential phase of growth, emetic toxin is produced during late exponential to stationary phase of growth (Kramer & Gilbert, 1989). Cl. perfringens occurs in a variety of foods and causes food poisoning (Labbe, 1991). Spices and herbs containing Cl. perfringens, even in low numbers (<102 cfu g 1 ), may play an important role in contributing to the microbial load of prepared foods (De Boer, Spiegelenberg, & Janssen, 1985). Although foods related to food poisoning outbreaks caused by Cl. perfringens usually contain a large number (105 –106 cfu g 1 ) of cells (Labbe, 1991), spores, even at low levels, could germinate and grow to reach a significant number (Nakamura & Kelly, 1968). Symptoms associated with the disease are caused by an enterotoxin that is expressed during sporulation of enterotoxigenic strains (Czeczulin, Hanna, & McClane, 1993). Ingested vegetative cells that survive the stomach’s acidity pass to the small intestine where they grow, sporulate and release enterotoxin. Many surveys have shown that Cl. perfringens is found in raw and processed food, most notably, raw meat products and spices (Labbe, 1989, 2000). The present study was undertaken to understand the potential health hazard caused by these bacterial pathogens from spices.
broth culture or blended sample (1.5 ml) was taken in an Eppendorf tube and centrifuged (Remi CM12) (900 g; 20 min for broth culture, 30 min for spice/food sample) at 4 C. For sample, the supernatant was filtered through a 0.2 lm-cellulose acetate membrane (Sartorius AG, G€ ottingen, Germany). The supernatants/filtrates were used immediately for the assay of enterotoxin. Similarly, for the extraction of Cl. perfringens enterotoxin (PET), Cl. perfringens strains were grown at 37 C for 20 h in cooked meat medium. The culture was heated at 75 C for 20 min, and a 0.8 ml of the aliquot (taken from the base of the tube) was inoculated into 16 ml modified Duncan Strong (DS) medium (HiMedia M1237) (Duncan & Strong, 1968). The tubes were incubated at 37 C for 24 h. The culture was centrifuged (900 g, 20 min) at 4 C and the supernatant was used for the assay. Extraction of enterotoxin from food was done following the method as described above for the extraction of BCET.
2. Materials and methods
2.3. Challenge testing in black pepper powder
2.1. Organisms
In a sterile screw-capped glass bottle a 100 g-sample of black pepper powder was mixed well with spores of B. cereus 120-B1 which was prepared by growing it at 37 C for 72 h on sterile cellophane (British cellophane grade 325P, Avonmouth, UK) overlaid on nutrient agar (HiMedia M561) plate. The bottle was left at room temperature (31–33 C). B. cereus count and enterotoxin titre were monitored at 0 h, and after 7 and 14 d of storage using B. cereus selective agar (HiMedia M833, FD003, FD045) (BCSA) and BCET-RPLA kit, respectively.
The bacterial strains were isolated from different spices collected from retailed outlets in India (Banerjee & Sarkar, 2003). While Cl. perfringens strains were maintained at 4 C in cooked meat medium (Cat. No. M149; HiMedia Laboratories Pvt Ltd, Mumbai, India), B. cereus strains were maintained at 4 C on tryptone soya agar (HiMedia M290) slants, with sub-culturing after every six months.
2.2.2. Assay Qualitative as well as semi-quantitative tests for the enterotoxins were performed using BCET-RPLA (reversed passive latex agglutination) toxin detection kit (Oxoid TD950; Unipath Limited, Basingstoke, Hampshire, UK) and PET-RPLA toxin detection kit (Oxoid TD930) for BCET (diarrhoeal type) and PET (type A), respectively, as directed in the manual.
2.2. Enterotoxins 2.4. Safety of foods on storage 2.2.1. Extraction For the extraction of B. cereus enterotoxin (BCET), B. cereus strains were inoculated into 10 ml brain heart infusion broth (HiMedia M210) supplemented with 10 g glucose l 1 (BHIG) and incubated at 33 C for 18 h on a shaker (200 rpm). For the isolation of enterotoxin from spice samples, a 10 g-sample was blended with 10 ml of 8.5 g sodium chloride l 1 (extra dilution done when needed) using a Stomacher lab-blender 400 (Seward Medical, London, UK) for 5 min at Ônormal’ speed. The
2.4.1. Safety of aloo dam (a potato-based ethnic food) against B. cereus 2.4.1.1. Preparation of aloo dam. Ingredients: Potato (2–4 cm dia), 1 kg; cumin, 1/4 tsp.; tejpat, 2 pcs, cumin powder, 2 tbsp.; turmeric powder, 1/2 tsp., grated fresh ginger, 2 tsp; oil, 4 tbsp.; water, 4 cups (600 ml); salt to taste. Recipe: Whole deskinned potatoes were fried in hot oil until golden brown and kept aside. Tejpat and cumin
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were sauted followed by addition of turmeric powder, cumin powder and grated ginger. The mixture was fried till spices separated from oil. Previously-fried potatoes along with an appropriate amount of water were added to the mixture. Salt was sprinkled to taste and mixed well. The pan was covered and allowed to cook on low heat for 15 min until the potatoes were tendered and a typical aroma developed. 2.4.1.2. Challenge study on aloo dam. Fifty grams of freshly prepared aloo dam were taken in each of three sterile glass bottles. Aloo dam of one bottle was seasoned with ground small cardamom (where B. cereus was detected in 100% of the samples analysed; Banerjee & Sarkar, 2003), while another bottle was intentionally inoculated with a 24 h-old culture of B. cereus 120-B1 (an enterotoxigenic strain) in BHIG. An uninoculated bottle was kept as control. All the three bottles were plugged with sterile cotton wool and kept at 30 C. Sampling from each bottle was done at 0 h and after 21 h for B. cereus count and BCET production using BCSA and BCET-RPLA kit, respectively. 2.4.2. Safety of meat (goat) gravy against Cl. perfringens 2.4.2.1. Preparation of meat curry. Ingredients: Minced goat meat, 1 kg; small cardamom, 1/2 tsp.; clove, 1/2 tsp.; cinnamon, 1/2 tbsp.; tejpat, 4 pcs; turmeric powder, 2 1/2 tbsp.; red chilli powder, 1 tsp.; grated fresh ginger, 3 tsp.; grated garlic, 3 tsp.; grated onion, 8 tbsp.; oil, 5 tbsp.; water, 5 cups (750 ml); salt to taste. Recipe: Minced goat meat, marinaded with grated onion, garlic and ginger, was refrigerated at 4–6 C for 1 h. Tejpat, clove, cinnamon and small cardamom were sauted followed by addition of turmeric powder and red chilli powder. Marinaded minced meat was added in it and fried till the mass separated from oil. The fried meat along with water and salt was cooked for 15 min in a pressure cooker (1.1 kg cm 2 ).
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2.5. Statistical analysis Data were analysed statistically by determining analysis of variance of triplicate sets (Snedecor & Cochran, 1989).
3. Results 3.1. Bacillus cereus and BCET Qualitative tests on BCET production of selected 23 strains (one from each kind of spices which showed maximum bacterial count; Banerjee & Sarkar, 2003) showed that 74% of the strains were able to produce it in BHIG (Table 1). Of these enterotoxigenic strains, 88% showed a highly positive (+++) reaction and were selected for quantitative estimation. The BCET content in BHIG varied from 8 to >256 ng ml 1 . Sixty percent of the tested strains produced 32 ng BCET ml 1 , two strains produced 128 ng BCET ml 1 and one strain produced 256 ng ml 1 . The profiles of load of B. cereus cells and BCET content in selected spices are shown in Table 2. Cumin powder had the highest BCET content, while cardamom had the lowest. In ajmud the cell count of B. cereus was maximum, however the BCET titre was in between the values of the other two spices. Intentional inoculation of black pepper powder with 108 cfu g 1 of an enterotoxigenic strain (120-B1) of B. cereus showed no significant (P < 0:05) changes in the cell count as well as enterotoxin titre (48 ng g 1 ) on storage for 14 d. Freshly prepared aloo dam did not contain any B. cereus and its toxin at detectable level. But when the same was seasoned with small cardamom, the aloo dam contained both cells and toxin. After 21 h-storage at 30 C both the levels increased significantly (P < 0:05). A similar situation was observed when aloo dam was intentionally inoculated with B. cereus (Table 3). 3.2. Clostridium perfringens and PET
2.4.2.2. Challenge study on meat gravy. Fifty grams of freshly prepared goat meat gravy were taken in a sterile screw-capped glass bottle and mixed well with spores of Cl. perfringens 16-C2 (an enterotoxigenic strain). An uninoculated bottle was taken as a control. Both the bottles were capped tightly and incubated at 37 C for 19 h. Cell counts and enterotoxin titres were measured at 0 h and after 19 h of storage using perfringens agar (HiMedia M579, FD011, FD012) in an anaerobic jar filled with carbon dioxide (HiMedia LE002A) and PETRPLA kit, respectively. The same parameters were again measured after boiling the cultured gravy for 15 min in a water bath.
Qualitative detection of PET of selected 16 strains (one from each kind of sample which had the highest cell count) showed that 19% of the strains were able to produce PET in modified DS medium. The PET content ranged from 2 to 32 ng ml 1 (Table 4). Table 5 shows that the Cl. perfringens count as well as its toxin content were not at detectable levels in freshly prepared meat gravy (pH 6.21). It was inoculated with an enterotoxigenic strain (16-C2) of Cl. perfringens. After 19 h of storage at 37 C, although the cell count increased from 103 to 107 g 1 , the toxin content remained unchanged. When boiled for 15 min, the cell
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Table 1 Production of BCET in glucose supplemented brain heart infusion broth by B. cereus isolates from spices Strain no.
BCET
Source
+/) 3-B2 10-B3 14-B1 16-B2 17-B1 18-B2 20-B1 32-B1 33-B2 36-B1 37-B2 40-B1 56-B2 57-B1 68-B1 85-B1 95-B1 96-B2 98-B2 107-B3 120-B1 128-B1 135-B1
Turmeric (Curcuma longa) powder Coriander (Coriandrum sativum) powder Black pepper (Piper nigrum) Mustard (Brassica juncea) Fenugreek (Trigonella foenum-graecum) Aniseed (Pimpinella anisum) Cinnamon (Cinnamomum zeylanicum) Turmeric Clove (Syzygium aromaticum) Black cumin (Nigella sativa) Coriander Red chilli (Capsicum fruitescens) powder Bishop’s weed (Trachyspermum ammi) Poppy seed (Papaver somniferum) Ginger (Zingiber officinale) Asafoetida (Ferula asafoetida) Large cardamom (Amomum subulatum) Small cardamom (Elettaria cardamomum) Cumin (Cuminum cyminum) Ajmud (Trachyspermum roxburghianum) Cumin powder Allspice (Pimenta dioica) Tejpat (Cinnamomum tamala)
Table 2 Load of B. cereus cells and their enterotoxin (BCET) in selected spicesa Spice Ajmud Cumin powder Small cardamom
Cfu g
1
BCET
4000 367 (200–500) 833 (700–1000)
+/)
ng g
+++ +++ +++
36 64 32
1
a
Values are means of determinations on triplicate sets. Where individual determinations differed by more than 5% from the means, ranges of values are shown in parentheses.
Table 3 Growth of B. cereus and production of its enterotoxin (BCET) in aloo dam (a potato-based food) on storage at 30 C till 21 ha Sampling time (h)
Growth (cfu g 1 )b 0 21 BCET (ng g 1 )c 0 21
Aloo dam
Aloo dam + small cardamom
Aloo dam + B. cereus 120-B1
533 (400–700)
1 · 106
3233 (3000–3500) 4 · 107
8 128
16 256
a Values are means of determinations on triplicate sets. Where individual determinations differed by more than 5% from the means, ranges of values are shown in parentheses. b DL (detection limit), 100 cfu g 1 . c DL (detection limit), 2 ng g 1 .
ng ml
+++ +++ ) +++ +++ ++ +++ ) +++ +++ +++ +++ ) +++ +++ +++ ) ) +++ ) +++ + +++
1
32 32 32 >256 NDa 128 64 32 32 32 128 32 32
8 256 ND 32
Table 4 Production of PET in modified DS medium by Cl. perfringens isolates from spices Strain no.
Source
PET +/)
7-C2 11-C3 14-C1 16-C2 17-C1 18-C1 19-C1 20-C2 36-C3 45-C1 47-C2 68-C2 91-C1 130-C3 131-C1 135-C3
Coriander Cumin powder Black pepper Mustard Fenugreek Aniseed Clove Cinnamon Black cumin Cumin Coriander powder Ginger Bishop’s weed Black pepper powder Caraway (Carum carvi) Tejpat
) ) ) +++ ) ) ) ++ ) ) )
ng ml
1
32
2
) ) ) ) +++
8
count in the 19 h-old cultured gravy decreased to 103 g and the toxin content went below the detection limit.
1
4. Discussion B. cereus was found as one of the most frequent foodborne bacterial pathogens in spices (Banerjee &
M. Banerjee, P.K. Sarkar / Food Control 15 (2004) 491–496 Table 5 Growth of Cl. perfringens and production of its enterotoxin (PET) in goat meat gravy on storage at 37 C till 19 h, and effect of boilinga Sampling time (h)
Gravy
Gravy + Cl. perfringens 16-C2
Growth (cfu g 1 )b 0 19 Followed by boiling (100 C, 15 min)
2517 (2350–2700) 5 · 107 1206 (1120–1300)
PET (ng g 1 )c 0 19 Followed by boiling (100 C, 15 min)
2 2
a
Values are means of determinations on triplicate sets. Where individual determinations differed by more than 5% from the means, ranges of values are shown in parentheses. b DL (detection limit), 10 cfu g 1 . c DL (detection limit), 2 ng g 1 .
Sarkar, 2003). To define the extent of its dreadfulness, an analysis of BCET was undertaken. Since the stimulatory effect of glucose on growth and enterotoxin production by B. cereus is well documented (Garcia-Arribas & Kramer, 1990; Kramer, 1984), BHIG was the medium of choice for this purpose. Our results suggest that, in principle, B. cereus isolates from spices are able to produce enterotoxins. It should, however, be borne in mind that the BCET-RPLA test does not specifically react with the diarrhoeal toxin, but with an indicator protein. In addition, this test cannot detect the B. cereus emetic toxin. Consequently, in vivo toxicity tests should be carried out in case of doubt of toxicity of samples having high BCET levels (Nout, Bakshi, & Sarkar, 1998). While selecting for the BCET level in spices, the spice kinds where all the samples of that kind contained B. cereus (Banerjee & Sarkar, 2003) were chosen. Cumin powder had the highest content of BCET however with the lowest cell count which indicates the production of a considerably high level of BCET by the strain 120-B1 (a cumin powder isolate). The results suggest a potential health hazard of foods containing spices with this bacterium. To understand how much the environment of a spice is congenial for growth of B. cereus and its enterotoxin production, black pepper powder was taken as a support spice since none of the five samples of black pepper powder tested contained B. cereus (Banerjee & Sarkar, 2003). After intentional inoculation of the spice with B. cereus 120-B1, there was no significant (P < 0:05) changes in the cell count as well as BCET production even after 14 d incubation at room temperature (as spices are conventionally stored). This indicates that a dry spice, like black pepper, can support survival of pathogenic organisms and act as a vector for contaminating foods. One is usually fond of spicy foods, but the issue of their safety needs to be assessed. Aloo dam, a potato-
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based ethnic and popular side dish, was taken as a model food for the growth of B. cereus. The rationale behind selecting this starchy food was the findings of GarciaArribas and Kramer (1990) that the starch-supplemented BHI supported near-optimum growth of B. cereus and production of enterotoxin at levels comparable to those produced in BHIG. Freshly prepared aloo dam did not contain B. cereus, but small cardamom acted as a carrier of B. cereus when the food was seasoned with it (as is practiced routinely). The inoculum, whether through the spice vector or as a pure culture intruder, multiplied rapidly in this starch-based food at room temperature and produced enterotoxin. The only commercially available serological assay for PET is RPLA test (Labbe, 1989). Out of 16 strains of Cl. perfringens tested, only three strains showed a positive response towards this test. Hence, those three isolates were subjected to quantitative assay. However, this culture method is less reliable due to problems in encouraging Cl. perfringens to produce sufficient toxin in artificial media. The direct detection of PET in faeces should be carried out parallelly as much larger amount of toxin is formed in vivo (Labbe, 1989). In a study similar to that carried out on aloo dam with B. cereus, goat meat gravy was taken as a test food for the growth of a meat-loving organism, Cl. perfringens and its enterotoxin production. This anaerobic sporeforming bacterial pathogen occurs in 30–80% of raw and frozen meat and also poultry (Labbe, 1989). In this study, it was found that the freshly prepared meat gravy was free from this bacterium. When the gravy was inoculated with Cl. perfringens 16-C2, it multiplied rapidly, however the enterotoxin level remained unchanged. Although low levels of production have been observed in vegetative cell cultures, the PET is synthesized by the sporulating cells (Adams & Moss, 1995; Brynestad & Granum, 2002). For this, it has been recommended by Juneja and Marmer (1998) that control measures for Cl. perfringens food-poisoning must ensure that large numbers of vegetative cells are not consumed. Possibly the gravy and the duration of incubation were not conducive for sporulation. After boiling the 19 h-long incubated gravy the cell count decreased significantly (P < 0:05) due to the destruction of vegetative cells and the enterotoxin level went below the detection limit. The enterotoxin is heat labile, and heating in saline at 60 C for 5 min destroys 80% of its serological activity (Labbe, 1989). It is likely the reason for non-detection of PET using RPLA method after boiling for 15 min. It is concluded that contaminated spices, when added to foods, are able to introduce the pathogens which may subsequently outgrow and produce enterotoxins. The spice microflora can shorten market life of the products through spoilage and/or conceivably contribute to consumer illness. The application of knowledge of the factors inhibiting the growth of different pathogenic
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bacteria can contribute to minimize the risk of food manufacturing operations.
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