WO1998034503A1

WO1998034503A1 - Removal of mycotoxin contaminants from biological products

Info

Publication number: WO1998034503A1
Application number: PCT/AU1998/000063
Authority: WO
Inventors: Hani El-Nezami; Pasi KANKAANPÄÄ; Seppo Salminen; Jorma Ahokas
Original assignee: Royal Melbourne Institute Of Technology
Priority date: 1997-02-07
Filing date: 1998-02-06
Publication date: 1998-08-13
Also published as: AUPO500597A0

Abstract

A method of treatment of a mycotoxin-contaminated biological product comprises contacting said contaminated biological product with mycotoxin-binding lactic acid or propionic acid bacteria to bind said mycotoxin to said bacteria.

Description

REMOVAL OF MYCOTOXIN CONTAMINANTS FROM BIOLOGICAL PRODUCTS

FIELD OF THE INVENTION

This invention relates generally to the removal of mycotoxin contaminants from biological products. More particularly, the invention relates to the removal of mycotoxins and especially aflatoxins from, or the binding of mycotoxins and especially aflatoxins in, biological products. The present invention also extends to a method of treatment of a human or other animal subject to bind mycotoxins and especially aflatoxins to prevent or inhibit absorption thereof by the subject.

BACKGROUND OF THE INVENTION

Aflatoxins are a group of a growing list of fungal secondary metabolites which are recognised as being of economic and health importance. They are produced by two toxic strains of Aspergillus namely Aspergillus flavus (A. flavus) and Aspergillus parasiticus (A. parasiticus). They are potent hepatocarcinogens in several species of animals (Eaton and Callagher, 1994) and epidemiological studies have implicated them as acute toxicants as well as human class I hepatocarcinogens in man (IARC, 1993). The occurrence of aflatoxin in feedstuff is quite common all over the world (Wood, 1992). Aflatoxins have been detected as contaminants of crops before harvest, between harvesting and drying, in storage, and after processing and manufacturing (Ellis et αi, 1991). Accumulation of AFB., and aflatoxin M ₁ into eggs, and milk from food producing animals after ingestion of aflatoxin contaminated feeds has been described (Allcroft and Carnaghan, 1963; Rodricks and Stoloff, 1977). Even at low concentrations aflatoxins may result in decreased milk yield and egg production in farm animals. In experimental animals such low levels have been shown to cause cancer and liver damage, have mutagenic and teratogenic activity and to be immunosuppressive (Robens and Richard, 1992; Eaton and Callagher, 1994). Thus chronic exposure to aflatoxins may not only significantly alter food production and animal farming efficiency but direct exposure to aflatoxin-contaminated food commodities may also constitute a great risk to the consumer. It is therefore important to reduce and/or prevent human exposure by developing practical and effective methods to detoxify aflatoxin-contaminated feedstuffs.

Once food is contaminated with mycotoxins, especially aflatoxin, there are two options if the food is to be used: either the toxin is removed or the toxin is degraded into less toxic or non toxic compounds. The first option is only viable when aflatoxin or other mycotoxin is present in identifiable pieces of food which can be removed from the remainder of the lot, or if a solvent system can be used to extract aflatoxin or other mycotoxin without leaving unwanted residues or markedly altering the composition of the food. As for the second option a variety of methods have been developed. Aflatoxin may be degraded by physical, chemical or biological methods (Park, 1993). Physical approaches to aflatoxin destruction involve treating with heat, ultraviolet light, or ionising radiation, none of which are entirely effective. Chemical degradation of aflatoxin is usually carried out by the addition of chlorinating, oxidising or hydrolytic agents. Chemical treatments require expensive equipment and may result in losses of nutritional quality of treated commodities. In addition, the undesirable health effects of such treatments have not been fully evaluated (Samarajeewa et al , 1990; Phillips et α/. ,1994).

It has been observed that many microorganisms, including bacteria, yeasts, moulds, actinomycetes and algae are able to remove or degrade aflatoxin in foods and feeds (Ciegler et α/. , 1996; Marth and Doyle, 1979), but biological detoxification of aflatoxin has not been established in practice. Flavobacterium aurantiacum is the only microorganism which has been reported to significantly remove aflatoxin from liquid media and food products without the production of toxic by-products (Ciegler et α/., 1966; Hao and Bracket, 1988; Line and Bracket, 1995). So far the mechanism by which this organism detoxifies aflatoxins is not known. Line and Bracket (1995) examined different factors which may affect the removal process and concluded that the bacterial population and viability greatly affected uptake of aflatoxin by cells. Strains of lactic acid bacteria have been reported to be effective in inhibiting aflatoxin biosynthesis, however none were efficient in removing aflatoxin from contaminated media (Coallier-Ascah and Idziak, 1985; Thyagaraja and Hosono, 1994).

During the last two decades, several studies have suggested that lactic acid bacteria and fermented dairy products possess anticarcinogenic activity (Goldin and Gorbach, 1984). Lactic acid bacteria are noted for their ability to bind mutagens (Hosono et al. , 1990), and as a result attention has been focussed on the binding ability of lactic acid bacteria with food carcinogens. A potential role these bacteria may play is the capacity to reduce the carcinogenic or toxic effect of food carcinogens or mutagens by binding to them or metabolically transforming them into less toxic and less carcinogenic forms.

In work leading to the present invention, it has been shown that selected strains of lactic acid bacteria, Propionibacteria and Bifidobacteria have the ability to bind to aflatoxins in contaminated biological products , and are able, for example, to remove aflatoxin B^ (AFB_T ) from contaminated liquid media, and to form complexes which are stable in vivo.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a method of treatment of a mycotoxin-contaminated biological product, which comprises contacting said contaminated biological product with mycotoxin-binding lactic acid or propionic acid bacteria to bind said mycotoxin to said bacteria. In another aspect, the present invention relates to a method for removal of mycotoxin from a mycotoxin-contaminated biological product, which comprises contacting said contaminated biological product with mycotoxin-binding lactic acid or propionic acid bacteria, to bind said mycotoxin to said bacteria, and recovering biological product from which mycotoxin has been removed.

The present invention is particularly, but not exclusively, directed to the removal of aflatoxin(s) from a contaminated biological product.

The present invention also extends to a method of treatment of a human or other animal subject to prevent or inhibit in vivo absorption of mycotoxin by the subject, which comprises administration to the subject of an effective amount of mycotoxin-binding lactic acid or propionic acid bacteria.

In another aspect, the invention extends to the use of mycotoxin-binding lactic acid or propionic acid bacteria in the prevention or inhibition of, or in the manufacture of a preparation for the prevention or inhibition of, in vivo absorption of mycotoxin by a human or animal subject.

Such in vivo absorption of mycotoxin may, for example, arise or be likely to arise from ingestion of a mycotoxin-contaminated biological product, particularly a foodstuff.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. DETAILED DESCRIPTION OF THE INVENTION

The methods of the present invention extend to the removal of mycotoxins, and in particular aflatoxins, such as aflatoxin B^ (AFB, ), from contaminated biological products, that is, biological products which are contaminated by the presence of such aflatoxins or other mycotoxins.

The term "biological product" is used herein in its broadest sense to include any product which is subject to contamination by the presence of aflatoxins or other mycotoxins. Such products include, but are not restricted to, food products for humans and animal feeds. The products include liquid media including water and liquid foodstuffs such as milk, as well as semi-solid foodstuffs such as yoghurt and the like. The present invention also extends to solid foodstuffs, particularly animal feeds. Any such "biological product" may be treated in accordance with the present invention.

The term "mycotoxin-binding lactic acid or propionic acid bacteria" is used herein to include not only gram positive lactic acid bacteria such as Lactobacillus spp and Bifidobacterium spp which are capable of binding to aflatoxins and other mycotoxins, but also gram positive propionic acid bacteria such as Propionibacterium spp which are similarly capable of binding to aflatoxins and other mycotoxins. Particularly preferred bacteria for use in accordance with the present invention are bacteria of the species Lactobacillus rhamnosus. However, the present invention also extends to the use of bacteria of other Lactobacillus species particularly dairy strains such as L. acidophilus, L gasseri, L. casei and . reuteuri, as well as Propionibacterium species such as P. freudenreichii and Bifidobacterium species such as B. animalis.

In accordance with the present invention, the mycotoxin-binding lactic or propionic bacteria may be contacted with the contaminated biological product in lyophilized form, however it is preferred that they are used as bacteria or, even more preferably, in a pretreated form. A number of forms of pretreatment of the bacteria have been found to modify the bacteria so as to enhance the binding of aflatoxins and other mycotoxins to the bacteria. In particular, heat treatment of the bacteria, for example either autoclaving of lyophilized bacterial cells or boiling of a bacterial cell suspension, has been found to be an effective form of pretreatment. Another form of pretreatment of the bacteria which has been found to be particularly effective in enhancing the mycotoxin-binding ability of the bacteria is acid treatment, for example treatment with hydrochloric acid. Accordingly, the references herein to "mycotoxin-binding lactic or propionic bacteria" are to be understood to include references to pretreated bacteria as described above.

Whilst it is not intended to be bound by any theoretical considerations, the bacteria are believed to bind aflatoxin in a contaminated biological product by binding to the bacterial cell wall or to some other component of the bacteria. This binding, which can be enhanced by acid or other pretreatment of the bacteria, takes place with sufficient binding strength that the bacteria may, for example, be subsequently separated from the biological product, thereby removing aflatoxin therefrom. Alternatively, the aflatoxin may be sufficiently strongly bound to the bacteria that bioavailability of the aflatoxin is markedly reduced and the treated, contaminated biological product may be used as a human or animal foodstuff without aflatoxin being absorbed within the gastro-intestinal tract of the human or animal.

Bacteria of the species Lactobacillus rhamnosus (L.rhamnosus) are well known to persons in the field as dairy strains of lactic acid bacteria. Particularly suitable strains of L. rhamnosus for use in the method of the present invention include strains LBGG and LC705, described herein. Lactobacillus rhamnosus strain GG is disclosed in European Patent Publication No. EP 0199535 and was deposited as the GG strain of Lactobacillus acidophilus in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 02852, United States of America, under ATCC Accession No. 53103, on 15 April 1985. This strain was later reclassified within a new species, Lactobacillus rhamnosus.

In tests carried out to examine the ability of lactic acid bacteria to remove aflatoxin B^ (AFB., ) from liquid media, both Lactobacillus rhamnosus strain GG (LBGG) and Lactobacillus rhamnosus strain LC705 (LC705) were found to significantly (p<0.05) remove AFB_Ϊ when compared to other strains of either gram positive and gram negative bacteria. Removal of AFB_! by LBGG and LC705 was a rapid process, and approximately 80% of AFB., was removed immediately on contact with the bacteria. The in vitro removal of AFB_T from liquid media was not a result of metabolic conversion of AFB., by bacteria since no evidence of AFB_, metabolites was detected and bacteria subjected to heat and acid treatments prior AFB incubation were also capable of binding AFB_Ϊ . One of these strains has previously been classified as a probiotic, Lactobacillus rhamnosus strain GG, with beneficial health effects toward human or animal hosts (Gorbach, 1996). They are currently used by both the food and pharmaceutical industries.

In a particularly preferred embodiment of this invention, the L. rhamnosus bacteria are pretreated before being brought into contact with the contaminated biological product so as to enhance their capacity/affinity to bind mycotoxins and especially aflatoxins. As previously described, particularly preferred pretreatments include heat treatment and acid treatment.

The work leading to the present invention has clearly shown that both the LBGG and LC705 strains of L. rhamnosus have a significant effect in reducing levels of aflatoxins in liquid media. The potential of using these two strains in the removal of aflatoxin is promising, and a significant reduction in the bioavailability of aflatoxin, and hence a reduction in the risk of exposure of both humans and animals to this highly toxic compound, is possible. Further features of the present invention are more fully described in the following Example(s). It is to be understood, however, that this detailed description is included solely for the purposes of exemplifying the present invention, and should not be understood in any way as a restriction on the broad description of the invention as set out above.

In the accompanying drawings:

Figure 1 shows the effect of different bacterial concentrations on removal of AFB_! by LBGG and LC705. Error bars represent standard deviations.

Figure 2 Shows the effect of different temperatures on removal of AFB_T by LBGG and LC705. Error bars represent standard deviations. Significance (*) p<0.05.

EXAMPLE 1

Materials and Methods

Bacterial strains, cultivation conditions and estimation of bacterial concentration. Bacterial strains, cultivation conditions and the media used are listed in Table 1. For the strain selection experiments, all the strains used in this study were precultured in the appropriate media at 37°C until a concentration of 10⁹ bacteria/mL was obtained.

Counting of viable bacteria was performed both by using colony forming units (CFU) and by flow cytometry (FCM) methods. £. coli and lactic acid bacteria were grown at 37°C for 1 and 2 days, respectively, whereas Propionibacterium freudenreichii ssp. shermanii JS was grown in anaerobic conditions on yeast extract - sodium lactate (YEL) agar at 30 °C for 5 days (Malik et al. , 1968). A method for enumeration of bacterial concentrations was developed using a Coulter Electronics EPICS Elite ESP cytometer equipped with an air-cooled 488 nm argon- ion laser at 15 mW. Direct counts were enumerated by using Fluoresbrite™ Beads (2.0 μm, Polysciences Inc.) as an internal calibration. Viability of bacterial populations was assessed by using SYTOX ® Green Nucleic Acid Stain (Molecular Probes, S-7020) at 1 μM/10⁶-10⁷ bacteria to detect non-viable bacteria. A band pass filter of 525 nm was used to collect the emission for green SYTOX®.

AFB₁ removal assay.

AFB_T (Sigma, St. Louis, MO) was suspended in benzene:acetonitrile (97:3). An amount equivalent to 10 μg AFB^mL phosphate buffered saline (PBS, pH 7.3) was prepared and the benzene:acetonitrile evaporated by heating in a water bath at 80°C for 10 min. Cells of the precultured strains named in Table 1 were pelleted by centrifugation at 4000 rpm for 15 min and resuspended in 1.5 mL of PBS containing AFB After suitable period of incubation, the cells were again pelleted by centrifugation and samples of the supernatant fluid were analysed by high performance liquid chromatography (HPLC).

To examine the effect of heat treatment on the ability of the selected strains to remove AFB^ lyophilised cells were resuspended in PBS and autoclaved at 121 °C for 20 min. Following heat treatment, cell pellets were suspended in PBS containing AFB_! and incubated for a suitable period and the unbound aflatoxin was measured. Lyophilised cells of selected strains were also examined for their ability to remove AFB_! at different conditions (temperature, bacterial concentration and AFB_T concentration). In all cases, positive and negative controls were included: for the positive control, PBS was substituted for the bacterial cells and for the negative control PBS was substituted for AFB_!.

Quantification of AFB ₁ by HPLC. The HPLC procedure used for the analysis of AFB_! was similar to that of El- Nezami et al. (1995) with slight modifications. No extraction of AFBi from samples of supernatant fluid was required, 70 μL of supernatant was injected directly into HPLC. The HPLC system (Applied Biosystem, CA) was fitted with a dual pump model 400 solvent delivery system, a model 980 programmable fluorescence detector and a 220 mm x 4.6 mm, 5 μm, 0DS Spheri-5 Brownlee column fitted with C₁₈ guard column. Water-acetonithle-methanol (60:30:10, vol:vol:vol) was used as the mobile phase with a flow rate of 1 mL/min. Detection was by excitation at 365 nm and emission at 418 nm. The retention time was 9.5 min. Chromatograms were recorded at chart speed of 0.3 cm/min and peak width of 0.4 min. The residue percentage was calculated using the formula: 100 x (Peak area of AFB in the supernatant/peak area of AFB_! 'ⁿ the positive control).

Statistical analysis.

The results of the removal assays were subjected to student's t-test to test if there is significant difference between strains and different conditions.

Table 1 Culture media and culture conditions for each test strain.

lyophilised stock, Valio Ltd., Finland.

CFU were determined at 30 °C in anaerobic conditions.

Results and Discussion

The strains tested in this study have not been tested previously for their ability to remove aflatoxins. Strain differences in the removal of AFB_! ^ere noted (Table 3). The ability of LBGG and LC705 to remove AFBi from the media was demonstrated, both of them show a significant ability (p<0.05) to remove AFB_! when compared to other strains used in this study. LBGG, LC705, L. gasseri, L acidophilus, L. Shirota and PJS (gram positive) significantly (p<0.0001) removed more AFB_! from media when compared to £. coli (gram negative). At 0 hour the % of AFB_! residue is not significantly different from that at 72 hour (except L. Shirota) suggesting that the elimination of aflatoxin is a rapid process involving the removal of approximately 80% of AFBi in case of LBGG and LC705. An interesting finding was that all the strains except L. Shirota released some AFB_! back into the media after 24 hour (not statistically significant) but the binding ability is gained back again at 48 hour and the bacterial ability to remove aflatoxin increased. This was not the case for PJS and £. coli where release of AFBi back to the media continues (Table 3). Line and Brackett (1995) reported that the 24 hour-old cultures Flavobacterium aurantiacum removed about 19% of AFB_! within 24 hour while the 72 hour-old cultures (late stationary phase) were the most effective and removed about 33% of AFB_! within 24 hour. In the present study, the 24 hour-old cultures of both LBGG and LC705 removed about 80% of AFBi within 24 hour. There was no significant difference between bacterial concentrations (Table 2) of the strains used in this study either when quantitated by CFU or FCM. Positive controls did not show a decrease in AFB_! content over time.

Based on the significant ability of LBGG and LC705 to remove AFB_{1 τ} both strains were selected to examine their performance under various conditions. The removal of AFB_! ^was both concentration and temperature dependent. Cell population was a statistically significant factor affecting aflatoxin removal (Figure 1). Approximately a minimum of 5 x 10⁹ CFU/mL is required for a significant AFB_! removal, this is in accordance with the findings of Line and Brackett (1995) which indicate that viable cell populations of 1 x 10⁹ CFU/mL or greater were necessary for significant removal of AFBi. A CFU of 2 x 10¹⁰ CFU/mL of LBGG and LC705 is capable of reducing the AFBi level to less than 0.1% and 13%, respectively. These figures are comparatively lower than that reported by Ciegler et αl. (1966) and Line and Brackett (1995) for Flavobacterium aurantiacum where a CFU of 10¹⁰ was reported to remove about 74% and 80%, respectively.

The removal of AFB_! by both LBGG and LC705 (Figure 2) was temperature sensitive with a maximal removal occurring at 37°C; this was significantly different (p<0.05) from the amount removed at 25°C and 4°C. This finding is similar to that of Lillehoj et al. (1967) where the maximal removal of AFBi by Flavobacterium aurantiacum occurs at 35 °C.

The amount of AFB_! removed increased with increasing concentration of AFB_! (Table 4) but the percentage removed was not significantly different. This is in contrast to that reported by both Line and Brackett (1995) and Ciegler et αl. (1966) where the percent removal of AFBi decreased as toxin levels increased.

On investigating the factors that may contribute to the disappearance of AFBi, pH across the range 4 to 6 did not appear to contribute to the disappearance of AFB This is in contrast to the finding of Megalla and Hefez (1982) who concluded that the pH may contribute to the transformation of AFBi to the non toxic AFB_2a in acidogenous yoghurt. Similar results were also reported by Rasic et αl. (1991) who reported that the fermentation of yoghurt and acidified milk containing AFB_! greatly reduced the amount of the toxin.

Interestingly, heat treatment (Table 5) of both LBGG and LC705 significantly enhances the removal of AFB_! (p<0.05) when compared to precultured and lyophilised bacteria. Both Thyagaraja and Hosono (1994) and Ciegler et αl. (1966) reported the ability of heat inactivated cells of Lactobacillus casei and Flavobacterium aurantiacum, respectively to remove AFBi from solution, a finding which was contradicted by Line and Brackett (1995) using Flavobacterium aurantiacum. The present study has also examined the effect of preculturing on the ability of LBGG and LC705 to remove AFB_!. Precultured viable cells of both LBGG and LC705 significantly removed AFBi compared to lyophilised cells (<p0.05) (Table 5), this may reflect that preculturing may induce changes in the cell wall components which enhance the ability of the bacteria to remove AFB_!. 'ⁿ addition, a significant difference between the ability of lyophilised cells of LBGG and LC705 has also been noticed (p<0.05) (Table 5); such a difference did not exist when precultured cells were used.

Table 2 Comparison of bacterial concentrations using in strain screening experiments. Each value is a mean of two determinations using flow cytometry (direct and viable counts) or traditional plate counting method. No significant difference was detected between bacterial concentrations estimated by plate counting and flow cytometry using the Student's t-test.

Concentration are means of two sets of analysis with two plates for each CFU calculation and two assessments by flow cytometry. ( ) Percentage of Coefficient of Variation. ^a SYTOX® could not be used to determine the viability of PJS due to high autofluorescence of this particular strain.

Table 3 AFBi concentration (percent of original) in the liquid culture following

0, 4, 24, 48 and 72 hours of incubation with each test bacterial strain. Each value is a mean ± SD for two samples.

Means sharing the same letter do not differ significantly at P<0.05.

Table 4 Ability of LBGG and LC705 to remove various AFBi concentrations.

Values are means of duplicate analyses. Table 5 Effect of bacterial viability on AFB_! removal.

Values are means of duplicate analyses.

EXAMPLE 2

Bacterial pellets (10¹⁰ bacteria) were suspended in 5 mL of 1 M HCI and incubated for 2 hrs at 37°C. After incubation, the suspension was centrifuged at 3400 rpm for 15 min. The supernatant was removed and the pellet washed twice with PBS. The washed pellets were then suspended in 1.5 mL PBS containing 5 μg AFB.,/mL. The suspension was incubated at 37°C for 2 hrs, then centrifuged and 100 μL of the supernatant injected into HPLC for aflatoxin analysis (as described in Example 1).

The results are set out in Table 6.

Table 6 Effect of acid treatment on AFBi removal.

EXAMPLE 3

This example demonstrates (1 ) the ability of Lactobacillus rhamnosus strains

GG and LC705 to bind AFB_! ^{ιn VIV0} inside the gastrointestinal tract and (2) the in vivo stability of the complex formed either in vitro or in vivo between these two strains and AFB A strain of Propionibacterium which is a weaker in vitro binder has also been tested.

Materials and Methods

Bacterial strains and determination of bacterial concentration. Bacteria used were Lactobacillus rhamnosus strain GG (ATCC 53103),

Lactobacillus rhamnosus strain LC705 and Propionibacterium freudenreichii ssp. shermanii JS. All three strains were obtained from Valio Ltd. (Helsinki, Finland) as a freeze-dried powder. The concentration of a lyophilised bacteria ( number of bacteria/g of powder) was measured by flow cytometer using a Coulter Electronics EPICS XL cytometer equipped with an air-cooled 488 nm argon-ion laser operating at 15 mW. Viability was assessed using method described in Example 1.

In vivo assays.

Experiments were approved by the University of Kuopio Animal Ethics Committee. One-week old broiler chicks (Vilppulan Hybrid Ltd., Vilppula, Finland) were used in this experiment. Chicks were anaesthetised by an intraperitoneal injection with 0.1 mL/100g body weight of Mebunat® solution (Orion, Finland) containing 60 mg/mL of sodium solution pentabarbitol. This dose was sufficient to keep the animal under anaesthesia for one hour. A 2-3 cm cross-sectional cut in the lower abdominal region was made and a 5-7 cm long segment of the duodenum around the pancreas was separated by two ligatures. Before tightening of the second ligature the test solutions were injected into the loop. For the positive control group, the dose was 0.5 mL of phosphate buffered saline (PBS, pH 7.4) containing the 1.5 μg of AFBi (Signa Aldrich, Mo, USA). To examine for the ability of the bacterial strains to bind AFB_! ^{ιn VIV0} _^ 0-1 9 of lyophilised bacteria (10¹⁰ viable bacteria/0.1 g) was suspended in 0.25 mL of PBS and injected into the duodenal loop immediately followed by an injection of 0.25 mL of PBS containing 3 μg AFB_! (to reach a final concentration of 1.5 μg/0.5 mL). The chicks were killed either within 1 min after injection or after 1 hour. As for the negative control, one group of chicks was injected with 0.5 mL of bacterial suspension (10¹⁰ bacteria) and killed after 1 hour.

The stability of the complex formed in vitro between AFBi and each of the bacterial strains was also examined. For this purpose, 10¹⁰ lyophilised bacteria were incubated in vitro in 1.5 mL of PBS containing 1.5 μg AFB_!. The mixture was incubated at 37°C for 1 hour after which the suspension was centrifuged and the amount of AFBi in the supernatant was quantified to verify that AFB_! was bound by the bacteria. Almost 80% of AFBi present in the media was bound to both Lactobacillus rhamnosus strain GG and LC705 compared to 40% that was removed by Propionibacterium freudenreichii ssp. shermanii JS. The bacterial pellets with the bound AFBi were then resuspended in 0.5 mL of PBS and injected into the duodenal loop of chicks which were either killed one minute or one hour after injection.

After the chicks were killed, the contents of the loop were rinsed out with 5 mL of PBS, centrifuged (4°C, 2000 g for 15 min) and the supernatant fraction was extracted twice with 2 mL chloroform. The two extracts were then pooled and evaporated till dryness and the residue was reconstituted in methanol.

The ability of the bacterial strains to inhibit the binding of AFB_! onto the intestinal tissue was also investigated. The duodenal tissue was homogenised twice with 2 mL chloroform and the combined chloroform layers were evaporated and reconstituted in methanol.

All samples were analysed with HPLC using the conditions described in Example 1. The volume of samples injected into the HPLC was 50 μL.

Statistical Analysis.

The Mann-Whitney test was used to test for statistical differences for AFB_! binding and release between different bacterial strains.

Results and Discussion

This study demonstrates the ability of selected bacterial strains to bind AFB_! in vivo. Previous in vitro results have shown that both Lactobacillus rhamnosus strain GG and LC705 can remove significantly more AFBi than Propionibacterium freudenreichii ssp. shermanii JS. In this study Lactobacillus rhamnosus GG decreased AFBi by 54% in the soluble fraction of the luminal fluid in one minute (Table 7). This strain was more efficient in binding AFBi compared with two other strains tested (p<0.05). Both Lactobacillus rhamnosus strain LC705 and Propionibacterium freudenreichii ssp. shermanii JS were only able to reduce AFB ^ by 44% and 36% in the soluble fractions, respectively. Although the Lactobacillus rhamnosus strain LC705 was able to bind significantly greater amount of AFB., in vitro than Propionibacterium freudenreichii ssp. shermanii JS, such a difference was not seen in vivo (Table 7). The reduction in the efficiency of the two Lactobacillus strains to bind AFBi in vivo when compared to their binding efficiency in vitro (nearly 80% AFBi removed by the two strains) may be result of the dilution of the bacterial suspension when mixing within the intestinal contents.

The disappearance of 19% (0.28 + 0.09 μg) of intraduodenally injected aflatoxin within one minute probably reflects the fraction of aflatoxin absorbed, since according to earlier studies, approximately 20% of aflatoxin is absorbed within short time after entering the intestinal lumen (Hsieh and Wong, 1994). In the present study, only 3% of the injected AFB_! was recovered in the intestinal fluid after one hour while no residues from tissue extracts were found, indicating that AFB_! was normally absorbed. The rapid bacterial binding of AFB_! reduced AFB_! absorption and hence its possible toxic systemic effects (Table 7). The most significant finding of this study is the ability of specific bacterial strains to bind AFB_T in vivo thereby reducing the amount of free aflatoxin in the intestinal lumen and tissue. This phenomenon is the result of binding of AFBi by bacteria, since the difference in the amounts of AFBi between the amount added and that in the tissue and the intestinal contents can be recovered from bacterial pellets. With Lactobacillus rhamnosus strain GG, the amount of AFB_! bound by the tissue compared to positive control was reduced from 0.28 μg to 0.08 μg (one minute), indicating 71% reduction in binding of AFB_! by the intestinal tissue. Comparable values for Lactobacillus rhamnosus strain LC705 and Propionibacterium freudenreichii ssp. shermanii OS were 39 and 71 %, respectively. The present findings indicate that specific bacteria can reduce the bioavailability of AFBi in the intestinal tract and thus prevent the AFB_! absorption.

The complexes formed either in vivo (in the intestinal lumen) or in vitro (in the test tube) between Lactobacillus rhamnosus strain GG and AFB_! were stable in the luminal conditions for the period of one hour (Table 8). This was not the case for the complexes with Lactobacillus rhamnosus strain LC705 and Propionibacterium freudenreichii ssp. shermanii . The complexes formed with these organisms were stable, in vitro, while those formed in vivo were partly disassociated after one hour of incubation.

The present study is of importance for the feed and food industry since aflatoxins are responsible for significant material losses encompassing a broad spectrum of crops and animal husbandry, and extending through the food chain to the consumer. Aflatoxins also decrease the profitability of animal production through decreased growth, impaired feed conversion efficiency, and reduced reproductive potential in herds, flocks and ponds. Successful application of the approach of probiotic protection against AFB_! will not only ensure the safety of the agricultural and meat supply, but also result in increased profits for the animal and agricultural industries.

Table 7 AFBi recovered from the intestinal tissue and luminal fluid of one week old chicks after intraduodenal injection of AFB_! ith or without bacteria.

The number of animals per group is given in the parentheses.

¹ Intraduodenal dose; 10¹⁰ bacteria + 1.5 μg of AFBi in 0.5 mL of PBS

² The luminal contents were rinsed out with 5 mL of PBS, centrifuged and the supernatant fraction was extracted with 2x2 mL of chloroform.

³ Below the detection limit of the HPLC method used. Table 8 Stability of the complex formed in vitro or in vivo between the bacteria strains used and AFB_! after 60 min incubation in duodenum of one- week old chicks.

% AFB_! released into intestinal contents from the complex formed within one minute.

Below the detection limit of the HPLC method used.

REFERENCES:

Allcroft, R. and Carnaghan, R.B.A. (1963). Groundnut toxicity: an examination for toxin in human food products from animals fed toxic groundnut meal. Vet. Res. , 75:259-263.

Ciegler, A., Lillehoj, B., Peterson, R.E., and Hall, H.H., (1966). Microbial detoxification of aflatoxin. Applied Microbiology, 14:934-939.

Coallier-Ascah, J., Idziak, E.S., (1985). Interaction between Streptococcus lactis and Aspergillus flavus on the production of aflatoxin. Applied and Environmental Microbiology, 49:163-167.

Eaton, D.L., and Callagher, E.P., (1994). Mechanisms of aflatoxin carcinogenesis. Annual Review in Pharmacology and Toxicology, 34:135-172.

Ellis, W.O., Smith, J.P. and Simpson, B.K., (1991). Aflatoxins in food: occurrence, biosynthesis, effects on organisms, detection, and methods of control. Critical Review in Food Science and Nutrition, 30:403-439.

El-Nezami, H.S., Nicoletti, G., Neal, G.E., Donohue, D.C., Ahokas, J.T., (1995). Aflatoxin Mi in human breast milk samples from Victoria, Australia and Thailand. Food and Chemical Toxicology, 33:173-179.

Goldin, B.R. and Gorbach, S.L., (1984). The effect of milk and Lactobacillus feeding on human intestinal bacterial enzyme activity. Journal of Clinical Nutrition, 39:756-761.

Gorbach, S. (1996). The discovery of Lactobacillus GG. Nutrition Today Supplement, 31:25-45.

Hao, Y., Brackett, R.E., (1988). Removal of aflatoxin Bi from peanut milk inoculated with Flavobacterium aurantiacum. Journal of Food Science, 53:1384- 1386.

Hosono, A., Wardojo, R. and Otani, H., (1990). Binding of amino acid pyrolyzates by lactic acid bacteria isolated from 'Dadih'. Lebensmittel-Wissenschaft und- Technologie, 23:149-153.

Hsieh, P.H. and Wong, J.J. Pharmacokinetics and excretion of aflatoxins, pp.73- 88. In Eaton, D.L and J.D. Groopman (eds.). The toxicology of aflatoxins: Human health, veterinary, and agricultural significance. Academic Press Inc., San Diego, California, USA. International Agency for Research on Cancer, (1993). Some naturally occurring substances: Food items and constituents, heterocyclic aromatic amines and mycotoxins. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Vol. 56 (Lyon, IARC).

Lillehoj, E.B., Ciegler, A. and Hall, H.H., (1967). Aflatoxin B, uptake by Flavobacterium aurantiacum and resulting toxic effects. Journal of Bacteriology, 93:464-471.

Line, J.E. and Brackett, R.E., (1995). Factors affecting aflatoxin B, removal by Flavobacterium aurantiacum. Journal of Food Protection, 58:91-4.

Malik, A.C., Reinbold, G.W. and Vedamuthu, E.R. (1968). An evaluation of the taxonomy of Propionibacterium. Canadian Journal of Microbiology, 14: 1185-1191.

March, E.H. and Doyle, M.P., (1979). Update on molds: Degradation of aflatoxin. Food Technology, January, 81-87.

Megalla, S.E. and Hefaz, A.H., (1982). Detoxification of aflatoxin Bi acidogenous yoghurt. Mycopathologia, 77:89-91.

Park, D.C., (1993). Controlling aflatoxin in food and feed. Food Technology, October, 92-96.

Phillips, T.D., Clement, B.V. and Park, D.L. (1994). Approaches to reduction of aflatoxins in foods and feeds. The Toxicology of Aflatoxins: Human Health, Veterinary and Agricultural Significance, edited by D.L. Eaton and J.D. Groopman (London, Academic Press), pp.383-406.

Rasic, J.L., Skrinjar, M. and Markov, S. (1991). Decrease of aflatoxin B, in yoghurt and acidified milk. Mycopathologia, 113:117-119.

Robens, J.F. and Richard, J.L. (1992). Aflatoxins in animal and human health. Rev. Environ. Contam. Toxicol, 127:69-74.

Rodricks, J.V. and Stoloff, L. (1977). Aflatoxin residues from contaminated feed in edible tissues of food producing animals, pp.75-89. In Rodricks, J.V., Hesseltine, C.W. and Mehlman, M.A. (eds.). Mycotoxins in human and health. Pathotox. Publ. Inc., Park Forest South, Illinois, USA.

Samarajeewa, U., Sen, A.C., Cohen, M.D. and Wei, C.I., (1990). Detoxification of aflatoxins in foods and feeds by physical and chemical methods. Journal Food Protection, 53:489-501. Thyagaraja, N. and Hosono, A., (1994). Binding properties of lactic acid bacteria from 'Idly" towards food-borne mutagens. Food and Chemical Toxicology, 32:805- 809.

Wood, G.E., (1989). Aflatoxins in domestic and imported foods and feeds. Journal of the Association of Official Analytical Chemists, 72:543-548.

Claims

CLAIMS:

1. A method of treatment of a mycotoxin-contaminated biological product, which comprises contacting said contaminated biological product with mycotoxin-binding lactic acid or propionic acid bacteria to bind said mycotoxin to said bacteria.

2. A method for removal of mycotoxin from a mycotoxin-contaminated biological product, which comprises contacting said contaminated biological product with mycotoxin-binding lactic acid or propionic acid bacteria, to bind said mycotoxin to said bacteria, and recovering biological product from which mycotoxin has been removed.

3. A method according to claim 1 or claim 2, wherein said contaminated biological product is an aflatoxin-contaminated product.

4. A method according to claim 3, wherein said contaminated biological product is an aflatoxin B^AFB^-contaminated product.

5. A method according to any of claims 1 to 4, wherein said biological product is a liquid, semi-solid or solid, human or animal foodstuff.

6. A method of treatment of a human or other animal subject to prevent or inhibit in vivo absorption of mycotoxin by the subject, which comprises administration to the subject of an effective amount of mycotoxin-binding lactic acid or propionic acid bacteria.

7. A method according to any of claims 1 to 6, wherein said mycotoxin-binding lactic acid or propionic acid bacteria are selected from the genera Lactobacillus, Bifidobacterium and Propionibacterium.

8. A method according to claim 7, wherein said mycotoxin-binding lactic acid or propionic acid bacteria are selected from L. rhamnosus, L. acidophilus, L. casei, L. reuteri and P. freudenreichii and B. animalis.

9. A method according to claim 8, wherein said mycotoxin-binding lactic acid or propionic acid bacteria are selected from L. rhamnosus strains LBGG and LC705.

10. A method according to any of claims 1 to 9, wherein said mycotoxin-binding lactic acid or propionic acid bacteria are used in lyophilized form.

11. A method according to any of claims 1 to 10, wherein said mycotoxin-binding bacteria are pretreated prior to use to enhance the binding of mycotoxins to the bacteria.

12. A method according to claim 11 , wherein said pretreatment comprises heat treatment of the bacteria.

13. A method according to claim 12, wherein said heat treatment comprises autoclaving of lyophilized bacterial cells or boiling of a bacterial cell suspension.

14. A method according to claim 11 , wherein said pretreatment comprises acid treatment of the bacteria.

15. A method according to claim 14, wherein said acid treatment comprises treatment with hydrochloric acid.

16. Use of mycotoxin-binding lactic acid or propionic acid bacteria in the prevention or inhibition of, or in the manufacture of a preparation for the prevention or inhibition of, in vivo absorption of mycotoxin by a human or other animal subject.