ASSAY OF BACILLUS CEREUS EMETIC TOXIN PRODUCED IN ORANGE SQUASH

The contamination of squash by B. cereus, an enterotoxin producer, was found to range between 7.5×104 and 1.8×104 CFU/g in orange squash (during storage), that is hazardous. Orange squash is widely produced and consumed in India, but has a low rating of 3 on the scale of 10 (on feedback), mostly due to high sugars, not preferred these days. It can be preserved for >9 months due to added sugars and preservatives. During processing squash, if juice is not quickly cooled and/or squash is kept for long at temperatures <48 °C after processing, it can be a source of food poisoning. Reason, a large number of toxins can be produced by B. cereus. B. cereus strains, isolated from squash, produce heat stable toxin. Vacuolar assay confirmed them as emetic toxins, produced in squash. The toxin behaved like an ionophore in assay using mitochondria, extracted from liver cells of chicken with potassium ions in buffer. The toxicity of toxin by assay was 3200 IU/ng (BC IV strain) and 800 IU/ng (BC X strain). By the vacuolar expansions of mitochondria in assay, toxins of B. cereus demonstrated a toxic effect, in the range of 20.93 to 60.94 % by BC IV toxin and 43.28 to 45.02 % by BC X toxin, on the 3rd day growth of B. cereus in squash and toxin extraction for assay. It was also possible to produce antibodies against the B. cereus whole cell and toxin of BC IV, as an attempt to detect B. cereus contaminations in foods, by Ouchterlony’s immune-diffusion test.


Introduction
Globally the total burden due to food borne diseases is not known [1]. Bacillus cereus is a ubiquitous contaminant of foods that cannot be completely eliminated and is also known to survive at temperatures as low as 4 °C to 6 °C [2]. It is an etiological agent of two distinct forms of food poisonings, caused by its toxins, showing syndromes of the emetic and diarrheal toxins [3] with >90 % of the poisonings by B. cereus being due to its emetic toxin [4], produced by growth of B. cereus in foods [5]. The rod shaped, aerobic gram-positive bacterium produces endospores in adverse conditions. The toxin, produced by emetic strains of B. cereus, has immune-modulating property in the human body [6]. This toxin is a low molecular weight-heat and acid-stable depsipeptide and can withstand intestinal proteolytic enzymes [7]. The organism is widely reported in foods like rice [8,9] pasta, noodles [10], milk and milk products [11,12], poultry products [13], cook chill meals [2], ready-to-serve meals [14], and various fruit products [15]. The toxin can maintain its activity after exposure to 126 °C for 90 min, and considered as one of the most stable enterotoxins [16]. It is thus advantageous to assay emetic toxins of B. cereus in contaminated foods.
The infective dose of B. cereus in foods is 10 5 -10 8 /g [17] that can lead to emetic poisoning if ingested [18,19]. The toxin dose of ≤8 μg toxin kg -1 body weight, is a toxic dose in humans and none of the enzymes in the human body are known to detoxify this toxin [20]. If present in a dose as low as 0.01 to 1.28 μg emetic toxin /g food, it results in severe and acute poisoning symptoms [21].
To assay this emetic toxin, a positive vacuolar expansion (of mitochondria) confirms the toxin of B. cereus, related to emetic-syndrome (9) and is a good screening assay for toxin in foods. The vacuole activity is pH stable (2 to 11) and resistant to proteolytic enzymes [22]. On the other Food Science and Technology hand, the toxin, a cereulide protein-a cyclic dodeca-depsipeptide [21,23,24] is tolerant and resistant to heat [10,25] and highly stable with a low molecular size ~1.2 kDa [17]. Considering the ionophoretic property of emetic toxin [26], PIPES buffer containing K+, was used in this study. The vacuolar assays confirmed toxic effects of emetic toxins, by two strains BC IV and BC X of B. cereus, isolated from squash. The effectiveness of the toxins ranged between 2 % to 44 % for BC IV and 4 % to 9 % for BC X toxins, in the squash medium.
The antigenic potential of emetic toxin of B. cereus has been reported to be very poor in rhesus monkey, compared to Staphylococcal enterotoxin [16]. However still, an attempt, made to raise antibodies against B. cereus strain BC IV and its emetic toxin in rabbits (New Zealand White males), yielded positive results, presented herein.

Materials and Methods
Preparation of Squash: Orange squash, assessed for specific organism Bacillus cereus counts, was prepared ( Fig. 1) with fruit juice using the standard protocol [27].

Enumeration of B. cereus counts
Spread Plate method was used to enumerate the microbial load (for required fruit products), on PEMB agar medium. If the quantity of food to be examined was large, uniformly distributed representative samples of the fruit product (50 g) were used. The samples of 10 2 to 10 6 dilutions were plated for enumeration. The enumeration of B. cereus in squash on PEMBA: Aliquots from the experimental flasks were diluted into 0.1 % Butterfield's phosphate buffered dilution water (10 2 -10 7 dilutions), surface was plated (0.2 mL) on PEMBA medium in 90 mm dia petri plates and spread evenly, and incubated (30 °C) for 5 days growth and counted (CFU/mL) at 1, 3 and 5 days after inoculation. Arithmetic counts were converted to log 10 CFU/mL values.

3. Isolation and cultural characteristics of the strains
Standard procedures [29,30] were followed to isolate B. cereus from squash. Preserved (60 days) squash sample(s) were serially diluted (10 -2 to 10 -5 ) in autoclaved Butterfield's phosphate buffered dilution water. The dilutions were plated in replicates (0.2 mL), on PEMBA and incubated 24 h at 30 °C±2 °C. Colonies, presenting a peacock blue colour with precipitation zone/halo, due to egg yolk hydrolysis (lecithinase test) were considered positive and were enumerated [31]. The lecithinase positive and mannitol utilization negative colonies of B. cereus [32], were picked from plates, purified and transferred to nutrient agar slants. Care was taken not to pick colonies turning yellow (mannitol utilizing colonies) [28].
The Emetic toxin Production, Extraction, Effective toxin assay, its Toxicity test and exploring it as an immuno diagnostic tool using the isolate strains BC IV and BC X, in the order of experimentation are mentioned (Fig. 2).

Antibodies Production Protocol
• Antigens :Whole cell and toxin of BC IV used as Antigens • Immunization protocol developed to raise polyclonal antibodies in sera • Ouchterlony's ID test confirmed antigenicity of the Whole cell and toxin .

5. Extraction of Mitochondria
A fresh, liver (from a broiler chicken ~1 kg weight, cut a BC IV and BC X fresh) was used for Extraction of Mitochondria [within an hour of procuring it] and a standard protocol [35] was followed for extraction ( Fig. 3).  Production of Emetic Toxin: a) in Orange squash: A 48 h culture growth of Bacillus cereus BC IV, was scrapped from two slants with 20 mL distilled water. The suspension was diluted to adjust OD 600 of ~1.0. The inoculum of 100 μL was added into sterilized squash (150 mL). The culture growth Food Science and Technology in squash was centrifuged (on day 1, 3&5) ~2000 ×g for 20 minutes to extract toxin (supernatant). The toxin was heated at 100 °C (7 min) to denature heat-labile diarrheal toxin of B. cereus before use for assay; b) in Nutrient broth [Titer determination & Toxicity test of toxin] Cultures (BC IV&X), grown in agar slants were diluted in NB. A suspension (150 μL having ~1×10 3 CFU/mL, OD ~1.0) of B. cereus culture inoculum was added to make a total volume of 150 mL nutrient broth (in 500 mL Erlenmeyer flasks), incubated at 30 °C±2 °C (100 rpm in shaker) for 48 h. Cultures were diluted in NB to give final concentrations of approximately 1×10 3 CFU/ mL in triplicate [36,37]. The growth in medium was centrifuged at ~2000 ×g for 30 minutes (4 °C). The molecular size of toxin (cereulide) is <1.2 kda. The cell pellet was separated and the supernatant (toxin extract) collected, filtered through 0.4 μm membrane filter, to produce toxin extract of B. cereus [23]. The toxin supernatant was heat-treated (100 °C for 5-7 minutes) to denature heat-labile diarrheal toxin of B. cereus, prior to assay for use as emetic toxin; c) the maximum population density of B. cereus (MPD): The Maximum population density (MPD) in a 5 days growth of B. cereus strains (BCIV & BCX), during toxin production in squash, was recorded and expressed as log 10 CFU/mL; d) biochemical changes in squash, used for toxin production: After inoculation of B. cereus strains, to produce toxin in squash, Reducing sugars and total Titratable acidity were assessed [27].
Vacuolar assay for effect of emetic toxin. The toxin was assayed on mitochondria expansions (Fig. 2). The toxin, produced either in squash or in Nutrient broth, was used for spectrophotometric and microscopic observations, respectively. Since emetic toxin has the potassium ionophore property, it may then selectively help in the uptake of K+ by mitochondria in such a way so as to be a cause of swelling, or vacuoles [24,38,39]. Other changes with inter-membrane spaces of mitochondria are a result of toxicity [40], leading to expansions, recognized as emetic activity of toxin. The K+ containing PIPES buffer was thus used to suspend CLM. [26,36]. The main criterion to assay B. cereus emetic toxin [5,41,42], was to assess if it was effective in a buffer suspension chosen, in this study.

Spectrophotometric assay & quantification of Mitochondrial Expansion by Emetic toxin
Mitochondria were extracted and suspended in sucrose solution along with heat treated toxin (in presence of K + cations in nitrate containing PIPES buffer solution pH 7.2) (Fig. 3). The vacuolar swellings in mitochondria in presence of toxin [26] were observed spectroscopically and reported as the effectiveness of toxin. The absorbance was observed with UV-VIS spectrophotometer (Model DIGSPEC 200 GL) using glass cuvettes. The swelling of mitochondria was observed as lowering of absorbance (λ 520nm ) due to toxin, and compared to control (without toxin). The various combination-mixes, of mitochondrial fractions (0.05, 0.04, 0.03, 0.02, 0.01&0.1 mL) in KNO 3 buffer solution with emetic toxin (BC IV/BC X) supernatants (0.1 mL or 0.2 mL), and in a total volume of 4.0 mL were made up to 4.4 mL with distilled water. These expansions were assayed due to B. cereus strains BC IV and BC X on 1 st , 3 rd & 5 th days of growth of organism using a mix of CLM with toxin volumes (0.1 mL or 0.2 mL), observed between 0 to 20 minutes exposure. The exposure to the toxin remained constant for experimental period of at least 4 hours (9, 21), in which the observations were conducted (at 30 °C). The Abs 520nm values were recorded in a 20 min period (with 5 min intervals, against blank without toxin). These trials, ascertained the toxic effects on mitochondria. All assays were run in duplicates.
Based on the results, it was observed, that the toxin was effective hence its titer (toxicity) also was determined as follows.
Toxicity test of Emetic toxin extracts for titer determination [9,21,36]. The toxin supernatants, filter sterilized and heated at 100 °C for 10 min, were used in the assay: a) toxin extracts, 25 μL aliquots of BC IV&BC X, were diluted (two-fold) in buffered potassium nitrate ( Food Science and Technology (25 µL) of emetic toxin. After 10 minutes reaction, smears were prepared on clean glass slide from each dilution, air-dried, stained with indigo carmine solution (0.1 %) and then dried in air; c) the morphological effect of toxin (mitochondrial expansion) was observed under binocular microscope (Leitz) (at 40 X and 100 X (Oil immersion lens) and also compared with the control (without toxin) slide; d) the highest dilution that showed the Positive morphological effect (vacuolar expansion) was observed as a positive toxin effect on mitochondria. The inverse of the highest dilution of toxin that showed vacuoles was recorded as the titer of toxin. An average of 3 observed fields of the dilution, with a minimum 10 vacuoles/field was recorded. A positive vacuolar expansion in the assay, confirmed the emetic toxin activity [21] of the strains BC IV & BC X. The minimum dose of titer of 1 U was measured as 5 ng/mL [24,43]. Based on 1 U value, the titer of B. cereus toxins of BC IV and BC X were calculated.
Production of polyclonal antibodies of the whole cell and emetic toxin of B. cereus strain BC IV.
The possibility to raise polyclonal antibodies (against the BC IV-whole cell antigen (WC-Ag) and emetic toxin Ag (Tx-Ag), a possible tool to detect B. cereus or its toxin in foods, was explored. The antigens (Ag) of B. cereus strain BC IV&BC X were prepared ( Table 1). Table 1 Preparation of Antigens -Whole cell (Ag) and Emetic toxn (Ag) for use in Immunizations [15] Object New Zealand White male rabbits as animal for experimentations The schedule ( Table 2) of intravenous and intramuscular immunization schedule was followed ( Fig. 4) for raising antisera against: BC IV whole cell (WC-Ag)&BC IV toxin (Tx-Ag).
New Zealand White male rabbits: 1) 2 kg by weight was used for WC-Ag; 2) 2.4 kg by weight was used for Tx-Ag, to raise polyclonal antibodies. Both rabbits were ~12 weeks old.

Antibodies Production Protocol in Rabbits.
After procuring the rabbits, they were initially fed and let to get use to the new environment for 10 days as well as to have a check on their proper health before immunizations. Antisera for B. cereus strain BC IV cellular and toxin fractions were raised in two separate rabbits as per schedule ( Table 2). On day 1, pre-immunization blood was collected, (15 mL) for control rabbit serum. The schedule of intravenous (Iv) and intramuscular (I M ) immunizations was followed until booster doses. The immunization booster dose (I M ) for whole cell (Ab) at 90 days and for toxin (Ab) at 83 days was completed. On 7 days after completion, a high volume of blood was collected for sera (Abs).
For final bleeding, the rabbit was restrained and outer surface ear was cleaned with alcohol. The blood sample was taken from the auricular artery of the ear. After blood collection, direct pressure was applied to the ear with sterile cotton to prevent bruising and unnecessary bleeding. The ears were cleaned with alcohol and rabbit was allowed to relax.
An extra booster dose, after 15 days of blood collection, did not help to increase the titer of serum.
The rabbits were observed daily, and any lesions if present were taken care of. Blood collections were stored in smaller volumes (-20 °C).

Food Science and Technology
The Ouchterlony's double diffusion test was used to study the banding patterns due to toxin sera interaction with different cellular antigens of different Bacillus cereus strains.

Results and Discussion
Orange squash (Kissan) is one of the summer drinks in India. The high content of sugar and preservatives gives it a rating of 3 on a scale of 10 [44]. In spite of high sugar content, the drink can be hazardous, when a pathogen like Bacillus cereus, can inhabit/adapt to the medium, which can emerge as a specific ecotype [45] to B. cereus. Along the food chain, it is also very unclear as to how pathogens originate or are transmitted, that many pathogenic organisms show their presence in newer/processed-foods. In scanning microbial loads of processed products, Bacillus cereus was present in orange squash, sweet mango and papaya bars and tomato pulp ( Table 3). Other foods like Soymilk (overnight refrigerated), soy tofu, blanched soybeans, tomato pulp powder from blanched tomatoes (preserved >9 months), maize and soy blended extruded products also contain B. cereus [15] in appreciable numbers. A high population of B. cereus in orange squash resulted in a decision to examine it as a medium to produce toxin by inoculation of this organism and assay presence of emetic toxin, produced by two isolates of B. cereus.

2. Orange squash as a medium for toxin production by B. cereus strains:
The orange squash as a medium for B. cereus (BC IV & BC X) inoculation showed the increase in acidity by 28-30 % and total reducing sugars by 16-21 %, in 5 days ( Table 5).

Production and Extraction of Bacillus cereus Emetic Toxin
The presence of >10 3 CFU/g in squash can cause foodborne illness and hence it was important to understand the toxicity and effectiveness of a toxin, produced by B. cereus. In the vacuolar assay, a threshold concentration of the cereulide, can lead to the loss of mitochondrial membrane potential in human cells, which is similar to that, observed in boar sperm mitochondria during expansion, due to the cereulide (emetic toxin). Human killer cell mitochondria were reported as equivalent to boar semen [20] and that boar sperms were equally sensitive to cereulide [36]. This vacuolar assay thus establishes the toxin effect, as if it were to be present in a human body. However since boar semen was difficult to obtain, we assayed mitochondrial expansions using CLM in the assay, instead. The vacuolar assay of B. cereus emetic toxins showed, that the extracted heat treated toxins (BC IV and BC X) were effective (toxic) on CLM, reported as the extent of expansions, were also related with the extent of growth of the organism ( Table 6). The results showed that: 1) the growth of B. cereus in squash reached the maximum population density (MPD) of log10 CFU/mL of 8.00 to 10.2, on 5 th day; 2) that B. cereus produces toxins in the end of the logarithmic growth phase of the organism and is known to be independent of sporulation [46,47]. The toxin effectiveness from 3 rd day's growth (in late logarithmic growth stage it was high as compared to other days (1 st or 5 th day). The specific microbial load of B. cereus also reached the peak value of ~ log 10 CFU/mL of 7.38-10; 3) the effectiveness of the toxin assay declined on 5 th day. By neutralizing toxin or using un-neutralized toxin, of both strains, a decrease in effectiveness of 5 th day's toxin extract as compared to 3 rd day's extract was observed. However so BC X toxin (un-neutralized) was as effective (43-45 %) as the neutralized toxin (45 %) of 3 rd day. On the other hand, the effect of un-neutralized BC IV toxin showed (~61 % lowering of the toxic effect) compared to its neutralized counterpart in assay conditions. The assay thus showed that the toxins of both strains did not have the same efficacy, even if produced in the same medium or are extracted at the same stage of growth of B. cereus. The production of toxin was definitely related to cell concentration of the organism in growth [21]; 4) B. cereus strains could be differentiated on their toxicity levels based on extent of mitochondrial expansions due to toxin (neutralized or otherwise); 5) this assay may be extended to check toxicity of B. cereus emetic toxin directly from other foods.
The heat stable emetic toxin was effective and active (as un-neutralized toxin) at low pH also [16]. Thus toxin is active in stored foods, unlike the diahorreal toxin, where activity of the toxin can be eliminated or reduced under variable conditions. Thus, potential hazard of emetic toxin as shown by their toxicity when extracted cannot be ignored. Table 6 Toxic effects (mitochondria swelling) of B. cereus toxins of BC X and BC IV from squash after inoculation with 10 6 cfu mL -1 of cultures BC X and BC IV in orange squash

4. Toxicity Test and Microscopic Examination of Titer of B. cereus Toxin:
The assay of emetic toxins (Fig. 2) showed high toxicity [21], with toxic concentration of BC IV>BC X. In the vacuolar assay with respect to B. cereus toxins BC IV and BC X were observed at highest toxin dilutions of 1/2048 and 1/512, respectively (Fig 6, Table 9). The concentration of toxin was calculated from the titer value in the total assay volume (125 µL) ( Table 7). B. cereus BC IV and BC X toxins were toxic at 3200 IU/ng and 800 IU/ng respectively.

5. Immunization Protocol and Immunodiffusion test
The sera booster dose on 83 rd day (for toxin Ag) and 90 th day (for cell Ag), of immunization schedule (Tables 1, 2, Fig. 7) was followed by blood collection and sera preparation for antibodies. The precipitin test on serum raised showed titers of 2048 and 1024, for WC-Ab and toxin-Ab respectively. The Ouchterlony's Immuno-diffusion test with the serum antibodies against Whole cells (WC-Ag) and Emetic toxin (Tx-Ag), of B. cereus BC IV strain showed clear banding patterns, confirming that the same single strain serum can be used to detect other B. cereus strains that may be present in contaminated foods (Table 8, Fig. 8). It is thus possible to use serum (with polyclonal antibodies) as an alternate tool to detect B. cereus in foods Table 8 Immuno-diffusion test, showing Banding pattern in Ag-Ab reaction. Toxin, used in a brown rabbit, Cellular Ag, used in a white rabbit (Fig. 8).  Fig. 8. Immunodiffusion test of B cereus antigens: a -cellular Ag in side wells banding with its antisera (in center well); b -emetic toxin Ag in side well banding with its antisera (in center well)

Conclusions
B. cereus toxin is known to be present in foods and fruit products [48][49][50]. In foods their viable counts ranged between 10 CFU mL -1 / g -1 to 10 6 CFU mL -1 /g -1 [15,48]. Under low pH (as in squash) there is a possibility of increase in the lag phase and generation times of this organism [51], to let the organism multiply later. Thus, the inoculum was added at higher active cell concentration for toxin production. Bacillus cereus emetic toxins in squash were effective and toxic. Its presence in squashes (acidic product) among other products [15] is one of the first reports and is a signal on quality defects that can arise in acidic fruit products, if preserved at room temperature (≥30 °C). In preparing a fruit squash, the juice is not heat-treated, and thus it is possible for entry of B. cereus from a raw material. B. cereus strain(s) can thus survive in storage. Orange Pineapple squash usually has a long shelf life (9-12 months). Contamination levels of this organism can reach between 10 2 -10 8 g -1 , in various foods [15,52]. Due to heat and acid resistance of B. cereus and its toxin, the organism can survive and propagate in foods [52][53][54]. A multiplication of the organism to threshold levels can lead to emetic toxin production [47]. Thus, if not detected early in storage may cause serious hazard in foods. A prolonged storage of such products may contribute to sporulation until favorable conditions Food Science and Technology return for growth and toxin production by this organism. Hence storage periods should be carefully recommended and good manufacturing practices in all Food Processing units are needed. The contamination level should not exceed 100 CFU•g -1 or mL -1 of food, to avoid the heat resistant toxin [52], which is adverse to hygiene [55,56].
This study used CLM and B. cereus toxin in assay of vacuolar expansions as a first report, to the best of our knowledge, on the use of chicken liver tissue for vacuolar assay. There is a possibility of using polyclonal antibodies (Cellular WC-Ab, Toxin Tx-Ab of B. cereus), as a tool, to detect B. cereus or occurrence of emetic toxin, where molecular diagnostic facilities may not be accessible in Labs. Improvements in immunization schedule can help to obtain higher titer of the sera. To mention, the sera, obtained for these two Ags, have shown the possibility to detect other strains of emetic strains of B. cereus (15 & not reported in this study). Their antigenicity can be used to further our knowledge and gain insights into B. cereus emetic toxin(s) and its antibodies. A large volume of high titer sera, required to detect large number of strains, is the only limitation.
The growth of B. cereus at temperatures <48 °C can cause food poisoning. To avoid this organism entering foods, a cook of 70 °C for 12s can help to achieve a 6 Log reduction of B. cereus [57] as shown by a study on D-values [(1 min (60 °C) to 33.2 min (50 °C)] of B. cereus vegetative cells]. In phosphate buffer the D-value was reported to be 10 min at 49-55 °C. A temperature of only 30 °C can form spores of B. cereus [46]. The spores of B. cereus BC IV germinated in presence of alanine (1 mM-100 mM), at 30 °C [15] (Fig. 9). Thus BC IV can propagate by spores at room temperature, in squash. The spores can allow the organism to escape pasteurization or sanitation procedures [58], due to its heat and acid resistance and can be problematic in convenience foods and mass catering. It is therefore important, to control B. cereus population [59] to avoid toxin production in foods. Hence the emetic toxin of B. cereus is certainly one of the most dangerous enterotoxins [60]. The PCR method has shown promise to detect a cereulide (emetic toxin) in Germany [61]. It is one of the most recent ways to detect the toxin. However series of at least 18 cereulide variants of emetic toxin have been reported to exist [62]. In India the detection or assay of this organism is not at a good scale. Its diversity in various Indian foods also needs to be studied. Among the serious damages, caused by emetic toxins, fulminant liver failure can be fatal, wherein the toxin can inhibit hepatic mitochondria fatty acid oxidation [63,64]. The loss of retinal structure and function [65,66] is also a severe disorder due to emetic toxin of B. cereus. There is a need to create larger awareness on contaminated food(s) and food poisoning symptoms. Good manufacturing practices in food-processing units are again emphasized.
Bacillus cereus can be present on surfaces, sporulate and survive heat treatments (especially in dairy industry), where hygiene methods often are not able to control B. cereus. This is because the spores have high hydrophobicity and high adhesion ability for surfaces. An easy, fast and cheap detection method is also a limitation. To detect it by PCR method or by the use of antibodies requires skilled hands and may not always be available in the food industry.

Food Science and Technology
The awareness of consumer preference for food safety, easy possibilities of detection shown, may not remain a limitation in future for its inclusion in critical control points in HACCP programs, where B. cereus is a problem.