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ASSESSMENT OF THE ABILITY OF SOME PROBIOTIC BACTERIA TO BIND AND REMOVE AFLATOXIN FROM CONTAMINATED WHEAT DURING BALADI BREAD MAKING
ELSANHOTY, R. M.1 AND AZEKE, M. A.2 1 Institute of Genetic Engineering and Biotechnology, Department of Industrial Biotechnology, Branch of Food and Dairy Biotechnology, Menoufia University, Egypt. Present address: Department of Food Science and Human Nutrition, Faculty of Agriculture and Veterinary Medicine, Al-Qassum University, Kingdom of Saudi Arabia. 2 Department of Biochemistry, Ambrose Alli University, P. M. B. 14, Ekpoma, Edo State, Nigeria

ABSTRACT This study was conducted to assess the potential of four strains of lactic acid and bifidobacteria (Lactobacillus acidophilus ATCC 20552, Lactobacillus sanfrasiscensis DSM20451, Lactobacillus rhamnosus TISTR 541 and Bifidobacterium angulatum DSMZ 20098) to bind aflatoxins in vivo, and the stability of the aflatoxins complexes formed. It was found that Lactobacillus rhamnosus TISTR 541 had the highest aflatoxin binding ability. The complexes formed by this strain were also most stable both in viable and non-viable (heat treated) forms. This strain was therefore used to study the reduction of aflatoxins from contaminated wheat flour during baladi bread making process. It was found that fermenting aflatoxin contaminated flour with backers yeast + Lactobacillus rhamnosus TISTR 541 was most effective in aflatoxin reduction than either yeast alone or the lactic acid bacteria alone. Keywords: Aflatoxin binding, Lactic acid bacteria, detoxification, fate, baladi bread, HPLC. Corresponding author. Tel. 0020102248605. E-mail address: rafaatmhamed@yahoo.com INTRODUCTION Aflatoxins, AFs, are a group of potent mycotoxins with mutagenic, carcinogenic, teratogenic, hepatotoxic and immunosuppressive properties. They are of particular importance because of their adverse effects on animal and human health (Lewis et al., 2005). AFs are produced as secondary metabolites of fungal strains (Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius) that grow on a variety of food and feed commodities during growth, harvest, storage, and transportation of these products (Peltonen et al., 2001; Jiang et al., 2005). Aflatoxin B1 (AFB1), the most toxic AF, is of particular interest because it is a frequent contaminant of many food products and one of the most potent naturally occurring mutagens and carcinogens known (Teniola et al., 2005). The primary classes of mycotoxins are aflatoxins, zearalenone, trichothecenes, fumonisins, ochratoxin A and the ergot alkaloids (Var and kabak 2009). Aflatoxins and fumonisins are known to be hazardous to the health of humans, in some cases directly causing illness

and even death. Aflatoxins have been implicated in liver cancer (JECFA, 1998; Wild and Hall, 2000). Chao et al. (1991) reported an incident where aflatoxins present in a foodstuff consumed by people in Malaysia in 1988 were strongly implicated as the cause of death of 13 children. Aflatoxins have been reported to impair growth in children from Benin and Togo (Gong et al., 2002). Fumonisins were reported to be associated with oesophageal cancer in rural areas in South Africa (Rheeder et al., 1992) and China (Chu and Li, 1994) and liver cancer in China (Ueno et al., 1997). Consumption of mouldy sorghum and maize containing fumonisin B1 has been associated with an outbreak of abdominal pain and diarrhoea in India (Bhat et al., 1997). Aflatoxins and fumonisins occur worldwide in maize, either alone or together (Kpodo et al., 2000). Effects of processing on mycotoxin contamination in food products are increasingly being investigated throughout the world, and this strategy is showing great promise for mycotoxin reduction. The use of physical

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methods, including cleaning, separation, screenings, washing, aqueous extraction, dehulling and milling, have been shown to be effective, to a certain extent, in reducing mycotoxins in cereals (Charmley and Prelusky, 1995; Shetty and Bhat, 1999; Voss et al., 2001). Aflatoxin and fumonisin levels in tortillas were found to be significantly reduced as a result of alkaline cooking (Voss et al., 2001). Due to the increasing number of reports on the toxic nature of aflatoxins, there is a need to control the aflatoxin levels in food and feed. Methods of control can be classified in two categories: (1) prevention of mould contamination and growth and (2) detoxification of contaminated products, a method not widely accepted (Mishra and Das, 2003). Fermentation has been employed as a method of food preservation for centuries, and lactic acid bacteria are reported to reduce mould growth and aflatoxin production (Mokoena et al., 2006). Lactic acid bacteria (LAB) and bifidobacteria, due in large part to their GRAS status and use as probiotics, are of particular interest for reducing the bioavailability of AFs. A number of studies have screened these microorganisms for the ability to bind AFs and have reported a wide range of genus, species and strains with specific binding capacities (ElNezami et al. 1998 Peltonen et al., 2001, Haskard et al., 2001, Gratz et al., 2005, Zinedine et al., 2005, Shahin 2007). This work was planned to study the following points: 1) to screen four lactic acid bacteria and

bifidobacteria strains for their ability to remove aflatoxin and to find other lactic acid bacteria and bifidobacteria strains that efficiently bind aflatoxins. 2) The best performing strains were further investigated for their ability to bind aflatoxin AFB and AFG from contaminated wheat flour during balady bread making. MATERIALS AND METHODS Bacterial strains and preparation of cultured bacteria and bacterial cell. Four strains of probiotic lactic acid and bifidobacteria were obtained from different international culture collections. Table 1 shows the list of bacteria according to their aeration level and their original suppliers. These strains were selected based either on their common use by food industry or on available information regarding their effects on food mutagens. Strains were cultured on de Mann, Rogosa and Sharpe (MRS, Difico Laboratories) agar plates. Anaerobic strains were kept in an anaerobic jar (Anaerogen, Oxoid). Fully grown colonies were stored on plates at 4C until needed with subculturing on a monthly basis. For long-term conservation of strains, spore or cell suspensions were kept in cryovials at 80 C with 90% glycerol as cryoprotectant. Lactobacilli and bifidobacteria strains were cultivated in MRS broth, but the broth for bifidobacteria was supplementated with cysteine. Both were incubated for 24 h at the appropriate growth temperature (Table 1).

Table 1. Bacterial strains used to investigate aflatoxin binding ability. Strains Source* Oxygen requirement Lactobacillus acidophilus ATCC 20552 Lactobacillus rhamnosus TISTR 541 Lactobacillus sanfrasiscensis DSM20451 Bifidobacterium angulatum DSMZ 20098 *(1) 1 2 3 3 Anaerobic Aerobic Aerobic Anaerobic

Egyptian Microbial Culture Collection (EMCC) at Cairo Microbiological Resources Centre (Cairo MIRCEN), Faculty of Agriculture, Ain Shams, University. (2) Thailand Institute of Scientific and Technological Research, Bangkok, Thailand. (3) Germany Collection of Microorganisms and Cell Cultures (DSMZ).

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A portion of overnight culture was used to inoculate 150 ml broth and incubated for 24 h at 37C in an anaerobic jar (Anaerogen, Oxoid). Cultured bacteria were collected by centrifuging at 6000g, 10 C, for 15 min under sterile conditions. The bacterial cells were then washed five times with 5 ml sterile dionoized water. The pelleted bacteria was resuspended in phosphate-buffered saline [PBS; pH 7.3, 0.01 M]) prior to use for aflatoxin binding ability (El-Nezami et al., 1998). Determination of aflatoxin binding properties Solid AFB1, AFB2, AFG1 and AFG2 (Sigma, St. Louis, Mo.) were dissolved in acetonitrilebenzene (3:97 v/v) to obtain an aflatoxin concentration of 4mg/ml. This was stored as stock solution. The concentration was determined spectrophotometrically at 348 nm. To prepare an AF solution, 50l of methanol was added and then made to volume with PBS, or PBS was added directly and the acetonitrilebenzene was evaporated by heating in a water bath at 80C for 10 min. The bacterial pellet was suspended in PBS (1.5 ml) containing 15 microgram of AFB1 per ml, incubated at 37C for 24 h. The bacteria were pelleted by centrifugation at 3000g, 10C for 15 min. 200 l supernatant containing unbound AFB 1 was collected and stored at -20C. All assays were performed in triplicates, and both positive controls (bacteria suspended in PBS for each strain) and AFB1 controls (5 ug/ml of AFB1 in PBS) were included prior to analysis by high performance liquid chromatography (HPLC). Determination of complex stability To the study surface binding of aflatoxin and the complex stability, cultured bacterial S=

samples (108 bacteria/ml) were used. Bacteria were either incubated as viable (in 4 ml of PBS for 1 h) or heat treated (boiled in 4 ml of PBS for 1 h) cultures. All bacterial samples were centrifuged, and the supernatant removed prior to aflatoxins binding assays. All incubations were carried out at 37C, and all centrifugations were at 2,500 g for 10 min at 10C. Preparation of grain samples Wheat grain samples were scratched by shaking with sand for 1 min disinfested by immersing in 5% sodium hypochlorite for 2 min, washed thoroughly with sterilized water and dried in hot-air oven at 44C for 42 hrs (Osman, 1982). Inoculation of spore suspension production of aflatoxin in wheat and

Spore suspension was prepared from pure cultures of Aspergillus flavus (21 days old) grown on PDA plates (9 cm). These plates were flooded with 15 ml of sterilized distilled water and brushed thoroughly for 12 min. The suspension was filtered through three layers of cheesecloth to remove the mycelia residues. Number of spores/ml was counted in the collected spore suspension by using a Spencer haemacytometer to about 10 6 spores/ml. Spore suspension was used to inoculate test grains, to give a final density of approximately 30003500 spore/g of wheat grains as described by (Eisa et al., 1996). Moisture content of wheat grains was adjusted appropriately. The required volume of water (S) needed for each moisture content was calculated according to the equation of Anon. (1962):

Required moisturecontent - initial moisture content 100 100 - requiredmoisture content


Balady bread making process Baladi bread was manufactured according to the traditional method Khorshied et al, (1989). The flour samples obtained from the inoculated wheat was divided into three portions of 300 g each. The first portion (A) was used in making balady bread using the traditional bakers yeast for fermentation. The second portion (B) was used for the same procedure except that fermentation was done by mixture 1 % yeast and 2 % Lactobacillus rhamnosus (TISTR 541). The third portion (C) was fermented with 5 %

After moisture adjustment inoculated grains were stored at room temperature for 20 days after which aflatoxins production was determined in 100g wheat grain samples. The grains were first of all dried, milled and then sieved through a 40 mesh sieve. This procedure gave a flour yield of 82 %. The flour samples thus produced from inoculated wheat were partly used for the assay of aflatoxins AFB1, AFB2, AFG1 and AFG2 and partly used for making balady bread.

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Lactobacillus rhamnosus (TISTR 541) during the bread making process. 1 % kitchen salt (NaCl) and 210 mL of tap water was added to all samples, properly mixed and fermented for 1 - 2 h. The dough thus obtained was divided into smaller portions, spread on wooden boards previously dusted with wheat bran. This was flattened manually and proofing was done at 28-30 C for 60 min under Relative Humidity of 75-85 %. The final flattening was done with removal of excess bran before baking at 450 500 C for 1 1.5 h. Finally the bread was allowed to cool at room temp for 30 min. Samples were collected at each stage of treatment to fermentation for 1h and 2 h, and after baking and cooling. These were kept at -2 C pending aflatoxin analysis by HPLC. Determination of aflatoxins The HPLC method of Roos et al., (1997) was used for analyses. The mobile phase consisted of water: methanol: acetonitrile (54:29:17, v/v/v) at flow rate of 1ml/min. The extinction was measured at the wavelength of 362 nm. Quantification was done on the basis of peak height using internal standards. The level of aflatoxins was measured as part per billion (ppb). Statistical analysis All assays were carried out in triplicate. Results were subjected to analyses of variance (ANOVA) and Turkeys test (Gomez and Gomez, 1984). Differences were considered to be statistically different at p < 0.05. Results and discussion Aflatoxin binding of (AFB1, AFB2, AFG1 and AFG2) and complex stability properties

The aflatoxin binding properties of four lactic acid and bifidobacterial strains are given in Tables 2 and 3. Results showed that strains under investigation were able to bind AFB1, AFB2, AFG1 and AFG2 efficiently. Lactobacillus rhamnosus (TISTR 541) had the highest percentage binding ability for AFB1, AFB2, AFG1 and AFG2 (P< 0.05) when compared to other microorganisms (Lactobacillus acidophilus ATCC 20552, Lactobacillus sanfrasiscensis DSM20451, Bifidobacterium angulatum DSMZ 20098 ) both in viable and heat treated stage. Results obtained by HPLC of aflatoxins from cultured and heat treatments indicated that the majority of bound AFB1, AFB2, AFG1 and AFG2 are attached to the bacterial surface. Aflatoxins were detected on the cultured pellets before and after the washing of the strains. There were significant differences (P < 0.05) between the amounts of AFB1, AFB2, AFG1 and AFG2 detected in each bacterial strain. There were also significant differences between the strains in their ability to bind aflatoxins in their viable and heat treated stage. There were considerable variations in the percentage of AFB1, AFB2, AFG1 and AFG2 bound both initially and after washing. Lactobacillus rhamnosus TISTR 541 strains was most effective in initially binding and also retaining AFB1, AFB2, AFG1 and AFG2, suggesting that the complexes formed with these strains were the most stable. Several strategies for the elimination or inactivation of mycotoxins have been reported in the literature, nevertheless, only few of them have been accepted for practical use such as ammonia treatment (Galvano et al., 2001), and none is entirely effective. Some specialists are of the opinion that the best approach for decontamination of mycotoxins should be degradation by selected microorganisms (Bata & Lastztity, 1999).

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Table 2. Percentage AFB1 and AFB2 bound on exposure to viable and heat treated bacteria and remaining bound after up to five washes with deionized sterile water. a Bacteria were incubated in PBS (4 ml) at 37C for 1 h (viable), boiled in PBS (4 ml) for 1 h (heat treated). Viable Bacteria Strains AFB1 Initial AFB2 AFB1 Final AFB2 AFB1 Initial AFB2 AFB1 30.9 2.7 72.7 2.9 16.6 2.3 33.1 1.7 Heat treated Final AFB2 35.6 3.6 68.99 1.9 11.5 1.4 36.4 3.1

Lactobacillus acidophilus 53.7 1.9 51.9 2.1 24.3 2.3 19.8 4.1 73.8 0.9 69.9 2.3 ATCC 20552 Lactobacillus rhamnosus 79.4 3.4 69.6 3.1 35.8 3.7 34.1 1.3 84.7 2.3 80.1 1.8 TISTR 541 Lactobacillus anfrasiscensis 22.6 1.7 21.6 4.1 1.9 3.6 2.7 2.4 44.5 2.1 35.41.9 DSM20451 Bifidobacterium angulatum 49.8.1.8 50.8 2.2 16.8 2.9 18.9 2.1 69.5 1.7 62.9 2.8 DSMZ 20098 b 8 Initial, percentage of AFB1 removed after 10 bacteria/ml were incubated with aflatoxins,. Final, percentage to the bacteria after five washes (1.5 ml each) with deionized sterile water. Results are the mean SD for triplicate samples.

of aflatoxins remaining bound

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Table 3. Percentage AFG1 and AFG2 bound on exposure to viable and heat treated bacteria and remaining bound after up to five washes with deionized sterile water. Viable Microorganism Initial AFG1 AFG2 AFG1 Initial Final AFG2 AFG1 Initial AFG1 AFG1 Heat treated Final AFG2

Lactobacillus acidophilus 50.7 2.1 53.5 2.3 21.4 2.2 18.6 3.7 71.7 1.3 67.9 21 31.9 2.2 34.6 3.3 ATCC 20552 Lactobacillus rhamnosus 69.4 2.3 65.7 3.1 34.9 3.5 36.1 1.3 86.2 2.1 81.4 1.6 74.7 2.6 65.18 2.3 TISTR 541 Lactobacillus sanfrasiscensis 23.6 1.9 21.8 2.6 2.7 3.3 4.7 2.1 41.4 1.9 33.4 2.1 17.6 1.9 18.4 1.8 DSM20451 Bifidobacterium angulatum 44.8.2.5 52.8 2.4 16.8 2.9 15.9 3.1 66.8 1.9 61.7 2.3 35.4 1.8 32.2 2.1 DSMZ 20098 a Bacteria were incubated in PBS (4 ml) at 37C for 1 h (viable), boiled in PBS (4 ml) for 1 h (heat treated). b Initial, percentage of AFB1 removed after 108 bacteria/ml were incubated with aflatoxins,. Final, percentage of aflatoxins remaining bound to the bacteria after five washes (1.5 ml each) with deionized sterile water. Results are the mean SD for triplicate samples.

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Treatment of bacteria with heat may affect their AFB1, AFB2, AFG1 and AFG2 binding ability. Heat treatments also resulted in unstable bacteria-AF complexes. Similar results were obtained by Haskard et al., (2001), El-Nezami et al. (1998) and Peltonen et al., (2001) who showed that the relative amounts of AFB 1 removed by viable and heat--treated bacteria depended on initial Aflatoxin concentrations. These results were also corroborated by the findings of Hwang et al. (2005), Zinedine et al. (2005) and Shahin (2007). They reported a wide range of genus, species and strains with specific binding capacities. Heat-killed bacteria have also been reported to bind aflatoxin B 1 in a strain specific manner (Oatley et al., 2000). It appears that AFB1 is bound to the surface components of lactic acid bacteria (Haskard et al., 2001). Other studies reported that other mycotoxins have been be removed by specific strains of lactic acid bacteria. El- Nezami et al., (2002) demonstrated that strains of L. rhamnosus GG and L. rhamnosus Lc-705 have the ability to rapidly remove zearalenone and its derivative -zearalenol (55%) after mixing with the bacteria. Turbic et al. (2002) showed that AFB1 (77- 99%) and ochratoxin A (36 76%) were removed by L. rhamnosus strains in high and moderate amounts. In addition, only minimal amounts of other aromatic dietary substances such as caffeine, vitamin B 12 and

folic acid were removed (9-28%). The destruction of specific components of the bacterial cell wall, such as carbohydrates and proteins, resulted in reduction in AFB 1 binding. The differences in AFB1 binding by the strains are probably due to different bacterial cell wall structures (Pierides et al., 2000, Haskard et al., 2001). Effect of lactic acid bacteria on the aflatoxins reduction in dough and different samples collected during baladi bread making process. The results in Tables 4 show the aflatoxin contents (ppb) of contaminated flour and the effect of fermenting with yeast alone, yeast + lactic acid bacteria and lactic acid bacteria alone during the balady bread making process. The results indicate that there were decreasing aflatoxins levels at all stages of the bread making process (P<0.05). Fermentation with yeast + lactic acid acid was most effetctive in reducing all aflatoxins tested. These reductions were however significant (P<0.05) only with AFB1 and AFG1. This shows the ability of Lactobacillus rhamnosus TISTR 541 to bind the aflatoxins from the contaminated flour. Similar results were obtained by Haskard et al., (2001), and El-Nezami et al. (1998), Peltonen et al. (2001) and Shahin (2007).

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Table 4. Levels of aflatoxins AFB1, AFB2, AFG1 and AFG2 (ppb) in aflatoxin contaminated flour, dough and samples collected at different stages of balady bread making process *Treatment (A): Baladi bread fermented by using the traditional backers yeast. Treatment A* Treatment B** Treatment C*** AFB 1 AFB2 AFG1 AFG2 AFB 1 AFB2 AFG1 AFG2 AFB 1 29.8 0.9 20.6 2.4 14.1 2.9 12.4 3.2 10.0 2.6 AFB2 4.3 1.2 3.0 0.4 2.0 0.3 AFG1 40.3 1.6 25.9 1.6 17.0 2.1 12.7 1.3 10.1 2.2 AFG2 5.2 0.9 3.6 1.2 2.4 0.6

Contaminated 29.8 40.3 29.8 40.3 4.3 1.2 5.2 0.9 4.3 1.2 5.2 0.9 Flour 0.9 1.6 0.9 1.6 Dough after 23.5 28.4 18.3 19.9 3.5 0.8 3.9 1.2 2.6 1.1 3.5 0.9 mixing directly 1.8 2.2 1.4 2.4 Dough after 20.0 12.6 14.2 fermentation 20.7 1.6 18.6 2.4 2.9 1.1 1.9 0.7 2.1 1.1 1.4 2.2 2.3 for1h. Dough after 16.3 15.2 10.1 11.0 fermentation for 2 1.9 2.1 2.0 0.6 1.3 0.2 1.8 0.3 2.1 1.7 2.1 2.1 h. Bread after 13.1 12.8 baking and 1.4 1.3 1.5 0.6 9.1 2.1 1.0 0.2 9.0 1.2 1.1 0.2 1.2 2.4 aeration **Treatment (B): Fermented by the mixture of yeast and Lactobacillus rhamnosus TISTR 541. ***Treatment (C): Fermented by Lactobacillus rhamnosus TISTR 541.

1.1 0.2 1.1 0..3

1.5 0.4

1.3 0.2

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Conclusion The results obtained demonstrate the ability of some viable and heat treated strains of lactic acid bacteria and bifidobacteria such as Lactobacillus acidophilus ATCC 20552, Lactobacillus sanfrasiscensis DSM20451, Lactobacillus rhamnosus TISTR 541 and Bifidobacterium angulatum DSMZ 20098 to bind aflatoxins. Lactobacillus rhamnosus TISTR 541 strain had highest binding ability and therefore could be chosen for the elimination or reduction of aflatoxins AFB1, AFB2, AFG1 and AFG2 B1 in bread produced from aflatoxincontaminated wheat. The present study also showed that lactic acid bacteria have the ability to remove aflatoxins from contaminated wheat flour during the balady bread making process. ACKNOWLEDGMENTS The authors are greatly indebted to Department of Bread and Pasta, Food Technology Research Institute, Regional Center for Food and Feed, Agricultural Research Center, Ministry of Agriculture, Egypt for supporting this research. REFERENCES Anon., (1962): Approved Methods of American Association of Cereal Chemists, 7th ed. The American Association, St. Paul Minn. (C. F. Egypt. J. Food Sci., 10(1-2): 3, 1982). Bata, A. and R. Lastztity, (1999). Detoxification of mycotoxin contaminated food and feed by microorganisms. Trends in Food Science and Technology. 10: 223228. Bhat, R.V., Shetty, P.H., Amruth, R.P., Sudershan, R.V. (1997). A foodborne disease outbreak due to the consumption of moldy sorghum and maize containing fumonisin mycotoxins. Clinical Toxicology 35: 249 255. Chao, T.C., Maxwell, S.M., Wong, S.Y. (1991). An outbreak of aflatoxicosis and boric acid poisoning in Malaysia: a clinicopathological study. Journal of Pathology 164: 225233. Charmley, L.L., Prelusky, D.B. (1995). Decontamination of Fusarium mycotoxins. Applied and Environmental Microbiology 6: 421 435.

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