**3. Decontamination of Aflatoxins by Lactic Acid Bacteria**

Research on Cancer classifies AFB1 and AFM1 as Group 1 human carcinogens, even though AFM1 is about 10 times less carcinogenic than AFB1 [30]. All these aflatoxin effects are influ‐ enced by variations according to the animal species, sex, age, nutritional status, and effects of other chemical products, besides the dose of toxin and the length of exposure of the or‐

Aflatoxicosis is the poisoning caused by the ingestion of moderate to high levels of alfatoxin in contaminated foods. Acute aflatoxicosis causes quick and progressive jaundice, edema of the limbs, pain, vomiting, necrosis, cirrhosis and, in severe cases, acute liver failure and death, caused by the ingestion of about 10 to 20 mg of aflatoxin in adults. Aflatoxin LD50 shows the following order of toxicity: AFB1> AFM1> AFG1> AFB2> AFG2 [4, 32]. Chronic afla‐ toxicosis causes cancer, immunosuppression and other pathological conditions, having the

The greatest risk presented by aflatoxins for human beings is chronic exposure causing hep‐ atocellular carcinoma, which may be made worse by hepatitis A virus [5]. It was also report that aflatoxins were found in the tissues of children affected by Reye syndrome (encephal‐ opathy with serious lesions in liver and kidneys after influenza or chickenpox), and Kwa‐ shiorkor (protein-energy malnutrition). Aflatoxicosis is considered, then, a contributing

AFB1 is metabolized in the liver by the cytochrome P450 system, generating its most carcino‐ genic metabolite, AFB1-8,9-epoxide (AFBO), or other less mutagenic forms, such as AFM1, Q1 or P1. There are several pathways for AFBO after it is metabolized, with one of them leading to cancer, another to toxicity and another one, to excretion. AFBO exo-form easily binds to cell macromolecules, including genetic material such as DNA proteins, producing adducts. Formation of these DNA adducts leads to genetic mutations and cancer, and their excretion in the urine of infected people is not only a proof that humans have the necessary biochemi‐ cal pathways for carcinogenesis, but also offers a reliable biomarker for AFB1 exposure [24].

Potential risk to human health caused by aflatoxins has led to surveillance programs for the toxin in different raw materials, as well as regulations determined by almost every country in the world [9]. A study carried out by the Food and Agriculture Organization of the Unit‐ ed Nations (FAO) in 2002 pointed out that about 100 countries had specific regulations for the presence of aflatoxin in foods, dairy products and animal feed, and that the total popula‐ tion of these countries amounted to 90% of the world population. The same study showed that regulations for aflatoxin are getting more diverse and detailed, including sampling

In countries where a regulation for aflatoxin exists, tolerance levels for the total aflatoxin (sum of aflatoxins B1, B2, G1 and G2) ranges from 1 to 35 µg/kg for foods, with an average of 10 g/kg; and from zero to 50 µg/kg for animal feed, with an average of 20 µg/kg. For AFM1 in milk, tolerance levels are between 0.05 and 0.5 µg/kg, with most countries adopting a

ganism to it [31].

liver as the primary target organ [4].

62 Aflatoxins - Recent Advances and Future Prospects

methods and methods of analysis [33].

threshold of 0.05 µg/kg [10].

factor to these diseases.

LAB is a large group of genetically different bacteria that, besides producing lactic acid as the main product of their metabolism, have similar characteristics: they are all gram-posi‐ tive, non-sporoformers, non-motile, and catalase, and oxidase negative. They are, therefore, aerotolerant anaerobes. Besides, they mandatorily ferment sugars and tend to be nutritional‐ ly fastidious, frequently requiring specific amino acids and B-complex vitamins as growth factors [34]. Several LAB genera, such as *Lactobacillus*, *Bifidobacterium* and *Lactococcus* are known for they ability to act as preserving agents in fermented foods, such as vegetables, cereals, dairy and meat products, actively inhibiting spoilage and growth of pathogenic bac‐ teria, besides increasing shelf life and sensory properties of these foods [ 23].

Fermentation enables longer shelf life and improves sensory and nutritional properties of the product, as sugar fermentation lowers pH and inhibits growth of spoilage and patho‐ genic microorganisms. Fermentation is also responsible for other reactions, such as proteins hydrolysis, improving texture and flavor; synthesis of aromatic components and texturizers, affecting the consistency of the product; and production of inhibitory components [35,36]. This inhibition is, in part, caused by the final products of fermentation, such as lactic acid, diacetyl, acetaldehyde and acetic acid, which may accumulate in inhibitory concentrations in certain foods and drinks. In other cases, inhibition may also be caused by secondary byproducts of metabolism, such as hydrogen peroxide or bacteriocins [37].

Therefore, two aspects may be considered when LAB are used: fermentation and antibiosis ability. In the first case, the starter culture added to the food acts on the substrate, causing advantages to the food. In the second case, the starter culture has to inhibit the development of undesirable microorganisms that may spoil the product or be hazardous to human health. In reference [38], authors state that one of the effects that were identified in LAB was protec‐ tion against toxins found in foods, such as heterocyclic amines, polycyclic aromatic hydro‐ carbons, reactive oxygen species, and mycotoxins. In the latter case, studies have demonstrated that LAB have the ability to inhibit aflatoxin biosynthesis, or that they have the ability to remove mycotoxins from the medium, reducing their effects.

It should be emphasized that with increased interest in probiotic food production all over the world, selection of LAB cultures with probiotic characteristics and greater ability to re‐ move mycotoxins may help to reduce risk of exposure to these toxins in foodstuffs, which is a very promising line of research in mycotoxicology. Yeast and LAB strains have great abili‐ ty to remove mycotoxins, and may be used as part of starter cultures in the fermentation of foods and drinks [39]. These microorganisms have, thus, ability to ferment and decontami‐ nate the medium, and purified components of these strains may be used in small amounts as food additives without compromising the characteristics of the final product.

One of the first studies in this area was carried out in the 1960s, when these authors evaluat‐ ed the ability of about 1,000 types of microorganisms to degrade aflatoxins [40]. Yeasts, fila‐ mentous fungi, bacteria, actinomycetes, algae, and fungal spores were among the organisms studied. From these, only the bacterium *Flavobacterium aurantiacum* B-184 (known today as *Nocardia corynebacterioides*) was able to irreversibly remove aflatoxins from the solution.

After this study, many others followed. However, the most significant ones started to appear after the 1990s. Table 1 presents the most relevant studies carried out with bacteria for aflatox‐ in decontamination. The action of 7 different types of bacteria on AFB1 was evaluated and it was found that some strains of *Lactobacillus* (*L. rhamnosus* GG and *L. rhamnosus* LC-705) were able to efficiently remove most mycotoxin from the medium, up to about 80% [17]. In refer‐ ence [27] authors analyzed 9 strains of *Lactobacillus* and achieved the same result, that *L. rhamnosus* GG and *L. rhamnosus* LC-705 were the most efficient strains in removing AFB1, with removal rates of 78.9% and 76.5%, respectively. Fifteen types of LAB, among them *Lactobacillus* and *Lactococcus*, and 5 types of bifidobacteria, were studied and it was observed that removal of AFB1 ranged from 5.6% to 59.7% [23]. Strains of *Lactobacillus amylovorus* (CSCC 5160 and CSCC 5197) and *L. rhamnosus* LC 1/3 showed the best results: 59.7%, 57.8%, and 54.6%, respectively. It was also observed that different strains of bifidobacteria removed from 37% to 46% AFB1, and that *Staphylococcus aureus* and *Escherichia coli* removed 46% and 37%, respectively [22]. It may be observed that among a given genus, and even a given species, not all the strains show equivalent toxin removal rates. On the contrary, the ability to remove aflatoxin is a character‐ istic of specific lineages, and efficiency varies widely [41].

In reference [46], authors examined the ability of 4 strains of *Lactobacillus* spp. and 2 strains of *Bifidobacterium* spp. to remove AFM1 in PBS and reconstituted skim milk. In PBS, viable cells of 6 strains were able to remove from 10.22 to 26.65% AFM1 in solution, depending on the level of contamination and the length of incubation, whereas non-viable cells removed from 14.04 to 28.97% of the toxin. In reconstituted skim milk incubated for 4 hours, 7.85 to 25.94% AFM1 were removed by viable cells, and 12.85 to 27.31% for cells rendered non-via‐ ble by heat treatment. These researchers concluded that the removal process was fast, with no differences between 0, 4, and 24 hours of contact, different from what was observed in [47] for strains of *Lactobacillus* spp., *Lactobacillus* spp*.* and *Bifidobacterium* spp., which showed removal rates ranging from 0 to 14.6% after 24 hours of contact, and from 4.5 to 73.1% after

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65

The ability of *L. rhamnosus* GG to remove AFM1 from reconstituted skim and whole milk was investigated and it was observed rates of 18.8% and 26.0%, respectively [29]. The au‐ thors concluded that the decrease in removal efficiency may be explained by the fact that AFM1 is possibly not accessible in milk, that is, it is associated with casein, and the interfer‐ ence of proteins in toxin removal may be the greatest responsible factor for the difference between skim milk and whole milk (approximately 10% lower), once powdered skim milk used in the study contained 37g of protein / 100 g, whereas protein content in powdered whole milk was 25g /100g. In the same study, AFM1 removal in buffered solution (50.7%) was compared with AFB1 removal by the same bacterial strain in the same solution (75.3%). It was concluded that AFM1 removal was less effective possibly due to the presence of an - OH group in the molecule, increasing its polarity and making it less hydrophilic, what in‐

Some physical, chemical, and enzymatic treatments may increase the ability of LAB to bind to aflatoxin in the medium. In reference [48] authors studied the ability of *L. rhamnosus* GG to bind to AFB1, observing little difference between aflatoxin removal by heat-treated and acid-treated cells (85% and 91%, respectively), compared with viable bacterial cells (86%). The use of physical and chemical treatments (chloric acid, and heat treatment in autoclave or boiling at 100 °C) on *L. rhamnosus* GG and LC-705 caused a significant increase in AFB1 re‐ moval, showing that metabolic degradation caused by viable bacterial cells may be ruled out

Comparing the ability of viable and heat-treated bifidobacteria cells, it was observed that viable cells removed 4 to 56% AFB1 from the medium, whereas non-viable cells removed 12 to 82% [23]. Evaluating the influence of the inactivation treatment on the ability of 4 types of *Lactoba‐ cillus* spp. to remove AFB1, it was observed that acid treatment (58.6 to 87.0%) and heat treatment (33.5 to 71.9%) increased the ability to remove the toxin, compared with viable cells in PBS (16.3 to 56.6%) [49]. On the other hand, alkali treatment (8.3 to 27.4%) and ethanol treatment

Removal of AFM1 with 8 LAB strains showed that heat-treated cells bound more efficiently (25.5 to 61.5%) to the toxin than viable bacterial cells (18.1 to 53.8%) [29]. In reference [50] it was observed that heat-treated cells removed greater percentages of AFM1 (12.4% to 45.7%) in PBS compared with viable cells (5.6% to 33.5%), with no significant differences between 15

(15.9 to 46.5%) decreased the amount of aflatoxin removed from the medium.

creases the tendency of the molecule to be retained in aqueous solutions.

as a possible mechanism of action [15-17].

96 hours of contact.

Most assays on aflatoxin removal in the studies cited above were carried out in phosphatebuffered saline (PBS). In reference [42], besides testing the ability of 27 strains of *Lactococcus* spp. and 15 strains of *Streptococcus* spp. isolated from yogurt, raw milk, and Karish cheese to remove AFB1 in buffered solution, observed that *Lactococcus L. lactis* and *Streptococcus ther‐ mophilus* presented the greatest rates of toxin removal (54.85% and 81.0%, respectively). They also tested the ability of viable and non-viable cells to remove AFB1 in different vegeta‐ ble oils, and observed that viable *L. lactis* cells removed from 71% to 86.7% AFB1, whereas non-viable cells removed 100% of the toxin in all the oils. Moreover, viable *S. thermophilus* cells removed from 66.5% to 91.5% of the toxin, and non-viable ones, from 81.7% to 96.8%.

AFB1 was added to yogurt and acidified milk in concentrations ranging from 1,000 to 1,400 g/kg, and a reduction of AFB1 in yogurt (pH 4.0), ranging from 97.8% to 90% was ob‐ tained [43]. Maximum decrease in AFB1 was observed during milk fermentation. As for milk acidified with citric, lactic, and acetic acid (pH 4.0) AFB1 reduction (concentration of 1,000 µg/Kg) was 90%, 84% and 73%, respectively. The ability of probiotic bacteria (*L. paracasei, L. casei, L. brevis* and *L. plantarum*) and the yeast *Saccharomyces cerevisiae* to remove a sum of aflatoxins (B1, B2, G1 and G2) during fermentation of dough made up of 50% barley flour, 45% wheat flour, and 5% corn flour was evaluated [44]. They observed that after 6 hours of fermentation, the amount of aflatoxin had decreased 18% and 33% for dough added of 4 and 40 µg of aflatoxin, respectively, and after 24 hours, the amount of aflatoxin decreased 27% and 50%, respectively.

Toxin polarity has an important role in the binding mechanism. The percentage of aflatoxin removed by LAB decreases in the following order: AFB1> AFB2> AFG1> AFG2. This observa‐ tion correlates with the decrease in the polarity of these toxins, and is consistent with hydro‐ phobic reactions, which may also have a role in the binding mechanism [45]. AFM1 is less efficiently removed than AFB1. However, scientific literature has few studies on the ability of LAB to remove AFM1.

In reference [46], authors examined the ability of 4 strains of *Lactobacillus* spp. and 2 strains of *Bifidobacterium* spp. to remove AFM1 in PBS and reconstituted skim milk. In PBS, viable cells of 6 strains were able to remove from 10.22 to 26.65% AFM1 in solution, depending on the level of contamination and the length of incubation, whereas non-viable cells removed from 14.04 to 28.97% of the toxin. In reconstituted skim milk incubated for 4 hours, 7.85 to 25.94% AFM1 were removed by viable cells, and 12.85 to 27.31% for cells rendered non-via‐ ble by heat treatment. These researchers concluded that the removal process was fast, with no differences between 0, 4, and 24 hours of contact, different from what was observed in [47] for strains of *Lactobacillus* spp., *Lactobacillus* spp*.* and *Bifidobacterium* spp., which showed removal rates ranging from 0 to 14.6% after 24 hours of contact, and from 4.5 to 73.1% after 96 hours of contact.

After this study, many others followed. However, the most significant ones started to appear after the 1990s. Table 1 presents the most relevant studies carried out with bacteria for aflatox‐ in decontamination. The action of 7 different types of bacteria on AFB1 was evaluated and it was found that some strains of *Lactobacillus* (*L. rhamnosus* GG and *L. rhamnosus* LC-705) were able to efficiently remove most mycotoxin from the medium, up to about 80% [17]. In refer‐ ence [27] authors analyzed 9 strains of *Lactobacillus* and achieved the same result, that *L. rhamnosus* GG and *L. rhamnosus* LC-705 were the most efficient strains in removing AFB1, with removal rates of 78.9% and 76.5%, respectively. Fifteen types of LAB, among them *Lactobacillus* and *Lactococcus*, and 5 types of bifidobacteria, were studied and it was observed that removal of AFB1 ranged from 5.6% to 59.7% [23]. Strains of *Lactobacillus amylovorus* (CSCC 5160 and CSCC 5197) and *L. rhamnosus* LC 1/3 showed the best results: 59.7%, 57.8%, and 54.6%, respectively. It was also observed that different strains of bifidobacteria removed from 37% to 46% AFB1, and that *Staphylococcus aureus* and *Escherichia coli* removed 46% and 37%, respectively [22]. It may be observed that among a given genus, and even a given species, not all the strains show equivalent toxin removal rates. On the contrary, the ability to remove aflatoxin is a character‐

Most assays on aflatoxin removal in the studies cited above were carried out in phosphatebuffered saline (PBS). In reference [42], besides testing the ability of 27 strains of *Lactococcus* spp. and 15 strains of *Streptococcus* spp. isolated from yogurt, raw milk, and Karish cheese to remove AFB1 in buffered solution, observed that *Lactococcus L. lactis* and *Streptococcus ther‐ mophilus* presented the greatest rates of toxin removal (54.85% and 81.0%, respectively). They also tested the ability of viable and non-viable cells to remove AFB1 in different vegeta‐ ble oils, and observed that viable *L. lactis* cells removed from 71% to 86.7% AFB1, whereas non-viable cells removed 100% of the toxin in all the oils. Moreover, viable *S. thermophilus* cells removed from 66.5% to 91.5% of the toxin, and non-viable ones, from 81.7% to 96.8%.

AFB1 was added to yogurt and acidified milk in concentrations ranging from 1,000 to 1,400 g/kg, and a reduction of AFB1 in yogurt (pH 4.0), ranging from 97.8% to 90% was ob‐ tained [43]. Maximum decrease in AFB1 was observed during milk fermentation. As for milk acidified with citric, lactic, and acetic acid (pH 4.0) AFB1 reduction (concentration of 1,000 µg/Kg) was 90%, 84% and 73%, respectively. The ability of probiotic bacteria (*L. paracasei, L. casei, L. brevis* and *L. plantarum*) and the yeast *Saccharomyces cerevisiae* to remove a sum of aflatoxins (B1, B2, G1 and G2) during fermentation of dough made up of 50% barley flour, 45% wheat flour, and 5% corn flour was evaluated [44]. They observed that after 6 hours of fermentation, the amount of aflatoxin had decreased 18% and 33% for dough added of 4 and 40 µg of aflatoxin, respectively, and after 24 hours, the amount of aflatoxin decreased

Toxin polarity has an important role in the binding mechanism. The percentage of aflatoxin removed by LAB decreases in the following order: AFB1> AFB2> AFG1> AFG2. This observa‐ tion correlates with the decrease in the polarity of these toxins, and is consistent with hydro‐ phobic reactions, which may also have a role in the binding mechanism [45]. AFM1 is less efficiently removed than AFB1. However, scientific literature has few studies on the ability

istic of specific lineages, and efficiency varies widely [41].

64 Aflatoxins - Recent Advances and Future Prospects

27% and 50%, respectively.

of LAB to remove AFM1.

The ability of *L. rhamnosus* GG to remove AFM1 from reconstituted skim and whole milk was investigated and it was observed rates of 18.8% and 26.0%, respectively [29]. The au‐ thors concluded that the decrease in removal efficiency may be explained by the fact that AFM1 is possibly not accessible in milk, that is, it is associated with casein, and the interfer‐ ence of proteins in toxin removal may be the greatest responsible factor for the difference between skim milk and whole milk (approximately 10% lower), once powdered skim milk used in the study contained 37g of protein / 100 g, whereas protein content in powdered whole milk was 25g /100g. In the same study, AFM1 removal in buffered solution (50.7%) was compared with AFB1 removal by the same bacterial strain in the same solution (75.3%). It was concluded that AFM1 removal was less effective possibly due to the presence of an - OH group in the molecule, increasing its polarity and making it less hydrophilic, what in‐ creases the tendency of the molecule to be retained in aqueous solutions.

Some physical, chemical, and enzymatic treatments may increase the ability of LAB to bind to aflatoxin in the medium. In reference [48] authors studied the ability of *L. rhamnosus* GG to bind to AFB1, observing little difference between aflatoxin removal by heat-treated and acid-treated cells (85% and 91%, respectively), compared with viable bacterial cells (86%). The use of physical and chemical treatments (chloric acid, and heat treatment in autoclave or boiling at 100 °C) on *L. rhamnosus* GG and LC-705 caused a significant increase in AFB1 re‐ moval, showing that metabolic degradation caused by viable bacterial cells may be ruled out as a possible mechanism of action [15-17].

Comparing the ability of viable and heat-treated bifidobacteria cells, it was observed that viable cells removed 4 to 56% AFB1 from the medium, whereas non-viable cells removed 12 to 82% [23]. Evaluating the influence of the inactivation treatment on the ability of 4 types of *Lactoba‐ cillus* spp. to remove AFB1, it was observed that acid treatment (58.6 to 87.0%) and heat treatment (33.5 to 71.9%) increased the ability to remove the toxin, compared with viable cells in PBS (16.3 to 56.6%) [49]. On the other hand, alkali treatment (8.3 to 27.4%) and ethanol treatment (15.9 to 46.5%) decreased the amount of aflatoxin removed from the medium.

Removal of AFM1 with 8 LAB strains showed that heat-treated cells bound more efficiently (25.5 to 61.5%) to the toxin than viable bacterial cells (18.1 to 53.8%) [29]. In reference [50] it was observed that heat-treated cells removed greater percentages of AFM1 (12.4% to 45.7%) in PBS compared with viable cells (5.6% to 33.5%), with no significant differences between 15 minutes or 24 hours of contact. Similar results were found in [51], because viable cells of *Lactobacillus delbrueckii* spp. *bulgaricus* CH-2 removed 29.42% AFM1 in PBS after 4 hours of contact at 37 C. These authors also analyzed the ability of *Streptococcus thermophilus* ST-36, observing that 18.70% AFM1 was removed from the medium. Until today, only one bacteri‐ um, *Flavobacterium aurantiacum* NRRL B-184, was able to remove 100% of AFM1 from conta‐ minated liquid medium, at a cell concentration of 5 x 1010 CFU/mL and 4 hours of contact [52].

found inside the cell wall in such a way that they produce no differences in the texture of

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The ability of *L. rhamnosus* GG to bind to AFB1 was studied, observing that the addition of urea - an anti-hydrophobic agent - to the medium, significantly decreased removal of the toxin by non-viable cells, from 85-91% to 50-60%, showing that hydrophobic interactions have a relevant role in the process [48]. Besides, addition of different concentrations of NaCl and CaCl2 (from 0.01 to 1 M), and pH variations from 2.5 to 8.5 had practically no effect on AFB1 removal by the bacterium, suggesting that hydrogen bonds and electrostatic interac‐

In the use of pronase E, lipase and periodate, treatment with periodate led to significant re‐ duction in the ability to remove the toxin, both by viable and non-viable cells, once it oxidiz‐ es the -OH cis groups in aldehyde and carboxylic acid groups, suggesting that the bonds involve predominantly bacterial polysaccharides. Treatment with pronase E caused the same significant reduction in AFB1 removal, evidencing that proteins may also be involved in the process. Thus, the fact that pronase E and periodate both have a significant reduction on AFB1 removal indicates that binding sites are made of protein. Treatment with lipase, on its turn, did not cause any significant reduction in AFB1, showing that lipids, such as lypo‐ teichoic acid probably do not have a role in the process. Although the treatments decreased AFB1 removal, it was still substantial in all cases, possibly showing the involvement of mul‐

However, not only the type of bacterial strain and the inactivation treatment used may influ‐ ence formation and stability of the LAB/aflatoxin complex, but also of other factors, such as bacterial counts, specificity of the bacteria, pH, incubation temperature, addition of nu‐

As for the number of bacterial cells in the medium, it has been concluded that there was a significant decrease in the amount of AFM1 removed when cell counts changed from 107

critical factors in the removal of AFM1 by LAB [46]. In reference [59] authors observed that no less than 5 x 109 CFU/mL of *Lactobacillus acidophilus* or *Bifidobacterium longum* are necessa‐

In reference [17] authors reported that, for *Lactobacillus rhamnosus* (strains GG and LC705), minimum counts of 2 x 109 CFU/mL were required to remove 50% AFB1, and greater remov‐ al rates were obtained when LAB concentration was increased to 1010 CFU/mL. In this same study, the authors observed that the process depended on the temperature, once the effi‐ ciency in aflatoxin removal was greater at 37 °C than at 4 and 25 °C. Besides, the authors observed that Gram-positive bacteria are better aflatoxin sequestrants than Gram-negative bacteria, with removal rates of 80% and 20%, respectively, suggesting the ability to remove the toxin depends on the structure of the cell wall. It has also been stated that aflatoxin con‐ centration in the medium also influences adsorption rates, leading to the conclusion that the greater its concentration in the medium, the greater the removal rate, both for viable and

CFU/mL (10.22 to 26.65%), indicating that bacterial counts are

the surface before the toxin was bound to it.

tions are not important in this process.

tiple components in the bond with mycotoxin [48].

trients, and the solvents used, among others [23, 27, 48].

ry to remove only 13% AFB1 in about one hour.

CFU/mL (0 to 5.02%) to 108

non-viable cells [60].

In [53] authors observed that *B. subtilis* UTBSP1 presented significant removal of AFB1 from a medium contaminated with 2.5 µg/g (52.67% and 80.53%, after 24 and 48 hours, respec‐ tively). After 72 and 96 hours, there was no significant increase in the amount of toxin re‐ moved from the medium. Strains of *B. subtilis* were analyzed and it was concluded that strain ANSB060 was the one that best removed AFB1, AFM1, and AFG1 from the medium (81.5%, 60%, and 80.7%, respectively) [54]. Results of this study also demonstrated that afla‐ toxin degradation is mainly observed in the supernatant culture, compared with cells or cell extracts. Besides, in assays that simulated the gastrointestinal environment (pH 2.0, and 0.3% of biliary salts), viable cells of the same strain were able to survive for 24 hours of incu‐ bation, and presented antimicrobial activity against *E. coli*, *S. typhimurium*, and *S. aureus*.

These examples show that both viable and non-viable cells are able to remove aflatoxin from aqueous solutions. As non-viable cells are also able to remove the toxin, it is supposed that cells are physically bound to the toxin, that is, components of the bacterial cell wall adhere to it, mainly polysaccharides and peptidoglycans, taking into account the possibility of a co‐ valent bond or degradation caused by bacterial metabolism [1, 55, 56].

Both polysaccharides and peptidoglycans of the bacterial cell wall may be extremely affect‐ ed by heat and acid treatment, once heat may denature proteins or form Maillard reaction products. Besides, acid treatment may break glycosidic bonds of polysaccharides, releasing monomers that may be further broken into aldehydes, also degrading proteins to smaller components, such as peptides and amino acids. Thus, acid treatment may break the peptido‐ glycan structure, compromising its structural integrity, that is, decreasing the thickness of this layer, reducing cross links and increasing the size of the pores. These changes caused by the treatments cited above enable AFB1 to bind to the bacterial cell wall and to the components of the plasmatic membrane that were not available when the bacterial cell was intact [27].

In reference [57] authors explained that the integrity of the bacterial cell wall is important in the process of toxin removal by both viable and non-viable cells. In their study of AFB1, they observed that both the bacterial cell wall and its purified fragments were able to remove aflatoxin from the medium. However, when the cell wall was lost or destroyed (totally or partially) by enzymatic treatment, there was a significant decrease in the ability to remove the toxin. It was observed, using atomic force microscopy, that the bond between AFB1 and *Lactobacillus casei* Shirota produced structural changes that modified the surface of the bacte‐ rial cell [58]. Before the toxin was bound to it, the surface was well-defined, smooth and ho‐ mogenous, and after AFB1 adsorption, there were changes in shape. These changes were probably caused by the bond between the toxin and the surface of the cell wall, which be‐ came very irregular and rough, with undefined edges. The authors suggest that changes in the shape of teichoic acids are responsible for these alterations, once these molecules are found inside the cell wall in such a way that they produce no differences in the texture of the surface before the toxin was bound to it.

minutes or 24 hours of contact. Similar results were found in [51], because viable cells of *Lactobacillus delbrueckii* spp. *bulgaricus* CH-2 removed 29.42% AFM1 in PBS after 4 hours of contact at 37 C. These authors also analyzed the ability of *Streptococcus thermophilus* ST-36, observing that 18.70% AFM1 was removed from the medium. Until today, only one bacteri‐ um, *Flavobacterium aurantiacum* NRRL B-184, was able to remove 100% of AFM1 from conta‐ minated liquid medium, at a cell concentration of 5 x 1010 CFU/mL and 4 hours of contact [52]. In [53] authors observed that *B. subtilis* UTBSP1 presented significant removal of AFB1 from a medium contaminated with 2.5 µg/g (52.67% and 80.53%, after 24 and 48 hours, respec‐ tively). After 72 and 96 hours, there was no significant increase in the amount of toxin re‐ moved from the medium. Strains of *B. subtilis* were analyzed and it was concluded that strain ANSB060 was the one that best removed AFB1, AFM1, and AFG1 from the medium (81.5%, 60%, and 80.7%, respectively) [54]. Results of this study also demonstrated that afla‐ toxin degradation is mainly observed in the supernatant culture, compared with cells or cell extracts. Besides, in assays that simulated the gastrointestinal environment (pH 2.0, and 0.3% of biliary salts), viable cells of the same strain were able to survive for 24 hours of incu‐ bation, and presented antimicrobial activity against *E. coli*, *S. typhimurium*, and *S. aureus*.

66 Aflatoxins - Recent Advances and Future Prospects

These examples show that both viable and non-viable cells are able to remove aflatoxin from aqueous solutions. As non-viable cells are also able to remove the toxin, it is supposed that cells are physically bound to the toxin, that is, components of the bacterial cell wall adhere to it, mainly polysaccharides and peptidoglycans, taking into account the possibility of a co‐

Both polysaccharides and peptidoglycans of the bacterial cell wall may be extremely affect‐ ed by heat and acid treatment, once heat may denature proteins or form Maillard reaction products. Besides, acid treatment may break glycosidic bonds of polysaccharides, releasing monomers that may be further broken into aldehydes, also degrading proteins to smaller components, such as peptides and amino acids. Thus, acid treatment may break the peptido‐ glycan structure, compromising its structural integrity, that is, decreasing the thickness of this layer, reducing cross links and increasing the size of the pores. These changes caused by the treatments cited above enable AFB1 to bind to the bacterial cell wall and to the components of the plasmatic membrane that were not available when the bacterial cell was intact [27]. In reference [57] authors explained that the integrity of the bacterial cell wall is important in the process of toxin removal by both viable and non-viable cells. In their study of AFB1, they observed that both the bacterial cell wall and its purified fragments were able to remove aflatoxin from the medium. However, when the cell wall was lost or destroyed (totally or partially) by enzymatic treatment, there was a significant decrease in the ability to remove the toxin. It was observed, using atomic force microscopy, that the bond between AFB1 and *Lactobacillus casei* Shirota produced structural changes that modified the surface of the bacte‐ rial cell [58]. Before the toxin was bound to it, the surface was well-defined, smooth and ho‐ mogenous, and after AFB1 adsorption, there were changes in shape. These changes were probably caused by the bond between the toxin and the surface of the cell wall, which be‐ came very irregular and rough, with undefined edges. The authors suggest that changes in the shape of teichoic acids are responsible for these alterations, once these molecules are

valent bond or degradation caused by bacterial metabolism [1, 55, 56].

The ability of *L. rhamnosus* GG to bind to AFB1 was studied, observing that the addition of urea - an anti-hydrophobic agent - to the medium, significantly decreased removal of the toxin by non-viable cells, from 85-91% to 50-60%, showing that hydrophobic interactions have a relevant role in the process [48]. Besides, addition of different concentrations of NaCl and CaCl2 (from 0.01 to 1 M), and pH variations from 2.5 to 8.5 had practically no effect on AFB1 removal by the bacterium, suggesting that hydrogen bonds and electrostatic interac‐ tions are not important in this process.

In the use of pronase E, lipase and periodate, treatment with periodate led to significant re‐ duction in the ability to remove the toxin, both by viable and non-viable cells, once it oxidiz‐ es the -OH cis groups in aldehyde and carboxylic acid groups, suggesting that the bonds involve predominantly bacterial polysaccharides. Treatment with pronase E caused the same significant reduction in AFB1 removal, evidencing that proteins may also be involved in the process. Thus, the fact that pronase E and periodate both have a significant reduction on AFB1 removal indicates that binding sites are made of protein. Treatment with lipase, on its turn, did not cause any significant reduction in AFB1, showing that lipids, such as lypo‐ teichoic acid probably do not have a role in the process. Although the treatments decreased AFB1 removal, it was still substantial in all cases, possibly showing the involvement of mul‐ tiple components in the bond with mycotoxin [48].

However, not only the type of bacterial strain and the inactivation treatment used may influ‐ ence formation and stability of the LAB/aflatoxin complex, but also of other factors, such as bacterial counts, specificity of the bacteria, pH, incubation temperature, addition of nu‐ trients, and the solvents used, among others [23, 27, 48].

As for the number of bacterial cells in the medium, it has been concluded that there was a significant decrease in the amount of AFM1 removed when cell counts changed from 107 CFU/mL (0 to 5.02%) to 108 CFU/mL (10.22 to 26.65%), indicating that bacterial counts are critical factors in the removal of AFM1 by LAB [46]. In reference [59] authors observed that no less than 5 x 109 CFU/mL of *Lactobacillus acidophilus* or *Bifidobacterium longum* are necessa‐ ry to remove only 13% AFB1 in about one hour.

In reference [17] authors reported that, for *Lactobacillus rhamnosus* (strains GG and LC705), minimum counts of 2 x 109 CFU/mL were required to remove 50% AFB1, and greater remov‐ al rates were obtained when LAB concentration was increased to 1010 CFU/mL. In this same study, the authors observed that the process depended on the temperature, once the effi‐ ciency in aflatoxin removal was greater at 37 °C than at 4 and 25 °C. Besides, the authors observed that Gram-positive bacteria are better aflatoxin sequestrants than Gram-negative bacteria, with removal rates of 80% and 20%, respectively, suggesting the ability to remove the toxin depends on the structure of the cell wall. It has also been stated that aflatoxin con‐ centration in the medium also influences adsorption rates, leading to the conclusion that the greater its concentration in the medium, the greater the removal rate, both for viable and non-viable cells [60].

Assays with AFB1 and *L. rhamnosus* GG and LC-705 at different incubation temperatures was also carried out, but it was not observed significant differences in the stability of the LAB/AFB1 complex formed in the temperatures range between 4 °C and 37 °C [27]. When pH of the medium was changed from 2 to 10, a range that includes the pH switch in the gas‐ trointestinal tract, only 10% AFB1 removed was released back into the solution, different from what happened when organic solvents were used. In this case, almost all AFB1 that was removed by the bacterial strains was released back into the medium, providing extra evidence that the process is based on a non-covalent bond. In this study, the release efficien‐ cy by solvents presented the following order: methanol < acetonitrile = benzene < chloro‐ form, which does not coincide with the order of decreasing polarity. This may be explained by the fact that AFB1 hydrophobicity is similar to that of the chloroform molecule. These re‐ sults show once more that hydrophobic interactions have an important role in the binding mechanism between LAB and the toxin.

the medium after the washings may be attributed to the interactions between aflatoxin mol‐ ecules retained on the cell wall of a bacterium and molecules retained on the cell wall of the adjacent bacterium, forming a kind of reticulated matrix that prevents aflatoxin release. It has also been suggested that the greater the number of molecules that are removed by the bacte‐

Recent Trends in Microbiological Decontamination of Aflatoxins in Foodstuffs

http://dx.doi.org/10.5772/51120

69

The stability of the LAB/aflatoxin complex in a wide range of pH is an important factor in the use of these microorganisms to remove aflatoxin from foods, once gastric release of the toxin would have negative health implications. Therefore, the complex formed has to resist environmental stress caused by the gastrointestinal tract, such as low pH and presence of bile. When the influence of the presence of bile on the LAB/aflatoxin complex was analyzed, it was observed that *Lactobacillus casei* removed more AFB1 when exposed to bile, suggesting that this exposure causes changes in the structure and composition of the bacterial cell wall, probably inducing the formation of new biding sites for aflatoxin, or increasing the size of

The ability of *L. rhamnosus* (strains GG and LC705) and *Propionibacterium freudenreichii* spp. *shermanii* JS to remove AFB1 from intestinal liquid medium extracted from the duodenum of chicks was investigated, and it was observed that AFB1 concentration was reduced in 54% in only 1 minute in the presence of *L. rhamnosus* GG, whereas it was reduced in only 44% in the presence of *L. rhamnosus* LC705, and 36% in the presence of *P. freudenreichii* spp. *shermanii* JS [62]. The authors observed that the accumulation of AFB1 in the intestinal tissue was re‐ duced in 74%, 63%, and 37%, respectively, for *L. rhamnosus* (strains GG and LC705) and *P. freudenreichii* spp. *shermanii* JS, showing that these bacteria may affect aflatoxin bioavailabili‐

Rats treated with feed added of aflatoxin (3 mg/kg of feed) presented a significant de‐ crease in the feed intake compared with the control group, different from the animals fed diets containing *Lactobacillus casei* and *Lactobacillus reuteri* (10 mL/kg of feed, with 1 x 1011 CFU/mL) and aflatoxin [63]. The second group did not show reduced feed intake. Conse‐ quently, animals treated with the diet containing only aflatoxin presented lower body weight, significant increase in serum levels of transaminase, alkaline phosphatase, choles‐ terol, triglycerides, total lipids, creatinine, uric acid, and nitric oxide; and in lipid peroxi‐ dation in the liver and kidneys, followed by a significant decrease in total antioxidant capacity. Treatment with bacteria was able to induce a significant improvement in all bio‐ chemical parameters and in the histological condition of the liver, with *L. reuteri* being

In Egypt, a pilot study investigated the effect of the addition of *L. rhamnosus* LC-705 and *P. freudenreichii* spp. *shermanii* JS in human diet on the levels of aflatoxin in feces samples. It was observed that from 11 of 20 volunteers, AFB1 ranged from 1.8 to 6 µg AFB1/kg feces, and after two weeks of supplementation with probiotic bacteria, there was a significant reduc‐ tion in the excretion rate, showing that these strains have the ability to influence the concen‐

rial cells, the longer these molecules remain adsorbed on the cell surface [60].

ty and be used to reduce its toxicity to humans and animals.

the sites available [13].

more efficient than *L. casei*.

tration of AFB1 in feces [64].

The effect of washing on the stability of the LAB/aflatoxin complex was analyzed [47]. They observed that after the first washing of bacterial pellets with PBS, the proportion of AFM1 released by the bacteria was 87.3% for *Lactobacillus* spp. strains; 85.7% for *Lactococcus* spp. strains, and 85.7% for strains of *Bifidobacterium* spp. They also observed that after the third washing, practically all bacteria had released adsorbed AFM1 back into the medium (92.0 a 100%). In reference [46] they concluded that AFM1 removal by bacteria was reversible, and that small amounts of toxin were released back to the PBS solution (5.62 to 8.54%). This find‐ ing is consistent with those observations of reference [27], who reported that *L. rhamnosus* GG, *L. rhamnosus* LC-705, and *Lactobacillus casei* Shirota released, respectively, 3.7%, 3.0% and 2.4% AFB1 back into the solution. Differently, in [23] authors showed that release of AFB1 back into the solution in the first washing was 48.6%, 30.7% and 26.5% for *L. amylovo‐ rus* (strains CSCC 5160 and CSCC 5197) and *L. rhamnosus* Lc 1/3, respectively. After 5 wash‐ ings, AFB1 adsorbed by *L. amylovorus* CSCC 5160 was almost completely released (94.4%), whereas *L. amylovorus* CSCC 5197 and *L. rhamnosus* Lc 1/3 retained, respectively, only 17.4% and 32.2% AFB1 found in the original solution.

Thus, the LAB/aflatoxin complex seems to be unstable, once part of the aflatoxin, both for AFB1 and AFM1, is released from the complex after washing, and gradually returns to the aqueous solution. Therefore, the greater the number of washings, the greater the amount of aflatoxin released back into the solution. This shows that the bond is not a strong one, sug‐ gesting it is a weak non-covalent bond and an association with hydrophobic sites on the sur‐ face of the bacteria [23, 48].

Different from this hypothesis, in reference [61], performing the same washings on a com‐ plex between *Flavobacterium aurantiacum* and AFB1, authors observed that aflatoxin was not released into the aqueous solution. Analyzing the stability of the complex formed between AFB1 and 8 strains of *Lactobacillus casei* after the washings, it was demonstrated that the amount of aflatoxin released ranged from practically zero and 9.2% [13]. Possible explanations for this variation in aflatoxin release include the differences in binding sites found in the different strains, or more probably, that these biding sites are similar, but that they present minimal differences depending on the strain. Authors explained that lower rate of toxin release into the medium after the washings may be attributed to the interactions between aflatoxin mol‐ ecules retained on the cell wall of a bacterium and molecules retained on the cell wall of the adjacent bacterium, forming a kind of reticulated matrix that prevents aflatoxin release. It has also been suggested that the greater the number of molecules that are removed by the bacte‐ rial cells, the longer these molecules remain adsorbed on the cell surface [60].

Assays with AFB1 and *L. rhamnosus* GG and LC-705 at different incubation temperatures was also carried out, but it was not observed significant differences in the stability of the LAB/AFB1 complex formed in the temperatures range between 4 °C and 37 °C [27]. When pH of the medium was changed from 2 to 10, a range that includes the pH switch in the gas‐ trointestinal tract, only 10% AFB1 removed was released back into the solution, different from what happened when organic solvents were used. In this case, almost all AFB1 that was removed by the bacterial strains was released back into the medium, providing extra evidence that the process is based on a non-covalent bond. In this study, the release efficien‐ cy by solvents presented the following order: methanol < acetonitrile = benzene < chloro‐ form, which does not coincide with the order of decreasing polarity. This may be explained by the fact that AFB1 hydrophobicity is similar to that of the chloroform molecule. These re‐ sults show once more that hydrophobic interactions have an important role in the binding

The effect of washing on the stability of the LAB/aflatoxin complex was analyzed [47]. They observed that after the first washing of bacterial pellets with PBS, the proportion of AFM1 released by the bacteria was 87.3% for *Lactobacillus* spp. strains; 85.7% for *Lactococcus* spp. strains, and 85.7% for strains of *Bifidobacterium* spp. They also observed that after the third washing, practically all bacteria had released adsorbed AFM1 back into the medium (92.0 a 100%). In reference [46] they concluded that AFM1 removal by bacteria was reversible, and that small amounts of toxin were released back to the PBS solution (5.62 to 8.54%). This find‐ ing is consistent with those observations of reference [27], who reported that *L. rhamnosus* GG, *L. rhamnosus* LC-705, and *Lactobacillus casei* Shirota released, respectively, 3.7%, 3.0% and 2.4% AFB1 back into the solution. Differently, in [23] authors showed that release of AFB1 back into the solution in the first washing was 48.6%, 30.7% and 26.5% for *L. amylovo‐ rus* (strains CSCC 5160 and CSCC 5197) and *L. rhamnosus* Lc 1/3, respectively. After 5 wash‐ ings, AFB1 adsorbed by *L. amylovorus* CSCC 5160 was almost completely released (94.4%), whereas *L. amylovorus* CSCC 5197 and *L. rhamnosus* Lc 1/3 retained, respectively, only 17.4%

Thus, the LAB/aflatoxin complex seems to be unstable, once part of the aflatoxin, both for AFB1 and AFM1, is released from the complex after washing, and gradually returns to the aqueous solution. Therefore, the greater the number of washings, the greater the amount of aflatoxin released back into the solution. This shows that the bond is not a strong one, sug‐ gesting it is a weak non-covalent bond and an association with hydrophobic sites on the sur‐

Different from this hypothesis, in reference [61], performing the same washings on a com‐ plex between *Flavobacterium aurantiacum* and AFB1, authors observed that aflatoxin was not released into the aqueous solution. Analyzing the stability of the complex formed between AFB1 and 8 strains of *Lactobacillus casei* after the washings, it was demonstrated that the amount of aflatoxin released ranged from practically zero and 9.2% [13]. Possible explanations for this variation in aflatoxin release include the differences in binding sites found in the different strains, or more probably, that these biding sites are similar, but that they present minimal differences depending on the strain. Authors explained that lower rate of toxin release into

mechanism between LAB and the toxin.

68 Aflatoxins - Recent Advances and Future Prospects

and 32.2% AFB1 found in the original solution.

face of the bacteria [23, 48].

The stability of the LAB/aflatoxin complex in a wide range of pH is an important factor in the use of these microorganisms to remove aflatoxin from foods, once gastric release of the toxin would have negative health implications. Therefore, the complex formed has to resist environmental stress caused by the gastrointestinal tract, such as low pH and presence of bile. When the influence of the presence of bile on the LAB/aflatoxin complex was analyzed, it was observed that *Lactobacillus casei* removed more AFB1 when exposed to bile, suggesting that this exposure causes changes in the structure and composition of the bacterial cell wall, probably inducing the formation of new biding sites for aflatoxin, or increasing the size of the sites available [13].

The ability of *L. rhamnosus* (strains GG and LC705) and *Propionibacterium freudenreichii* spp. *shermanii* JS to remove AFB1 from intestinal liquid medium extracted from the duodenum of chicks was investigated, and it was observed that AFB1 concentration was reduced in 54% in only 1 minute in the presence of *L. rhamnosus* GG, whereas it was reduced in only 44% in the presence of *L. rhamnosus* LC705, and 36% in the presence of *P. freudenreichii* spp. *shermanii* JS [62]. The authors observed that the accumulation of AFB1 in the intestinal tissue was re‐ duced in 74%, 63%, and 37%, respectively, for *L. rhamnosus* (strains GG and LC705) and *P. freudenreichii* spp. *shermanii* JS, showing that these bacteria may affect aflatoxin bioavailabili‐ ty and be used to reduce its toxicity to humans and animals.

Rats treated with feed added of aflatoxin (3 mg/kg of feed) presented a significant de‐ crease in the feed intake compared with the control group, different from the animals fed diets containing *Lactobacillus casei* and *Lactobacillus reuteri* (10 mL/kg of feed, with 1 x 1011 CFU/mL) and aflatoxin [63]. The second group did not show reduced feed intake. Conse‐ quently, animals treated with the diet containing only aflatoxin presented lower body weight, significant increase in serum levels of transaminase, alkaline phosphatase, choles‐ terol, triglycerides, total lipids, creatinine, uric acid, and nitric oxide; and in lipid peroxi‐ dation in the liver and kidneys, followed by a significant decrease in total antioxidant capacity. Treatment with bacteria was able to induce a significant improvement in all bio‐ chemical parameters and in the histological condition of the liver, with *L. reuteri* being more efficient than *L. casei*.

In Egypt, a pilot study investigated the effect of the addition of *L. rhamnosus* LC-705 and *P. freudenreichii* spp. *shermanii* JS in human diet on the levels of aflatoxin in feces samples. It was observed that from 11 of 20 volunteers, AFB1 ranged from 1.8 to 6 µg AFB1/kg feces, and after two weeks of supplementation with probiotic bacteria, there was a significant reduc‐ tion in the excretion rate, showing that these strains have the ability to influence the concen‐ tration of AFB1 in feces [64].


B1

5 µg/mL 78.9

*L. rhamnosus* GG Viable cells Heat-treated cells Acid-treated cells *L. rhamnosus* LC-705

Viable cells Heat-treated cells Acid-treated cells *L. acidophilus* LC1 Viable cells Heat-treated cells Acid-treated cells *L. lactis* subsp. lactis Viable cells Heat-treated cells Acid-treated cells *L. acidophilus* ATCC 4356

Viable cells Heat-treated cells Acid-treated cells *L. plantarum* Viable cells Heat-treated cells Acid-treated cells *L. casei* Shirota Viable cells Heat-treated cells Acid-treated cells *L. delbrueckii* subsp.

*bulgaricus* Viable cells Heat-treated cells Acid-treated cells *L. helveticus* Viable cells Heat-treated cells Acid-treated cells *P. freudenreichii* subsp.

*shermanii* JS Viable cells Heat-treated cells **(%)**

84.1 86.7

76.5 87.8 88.3

59.7 74.7 84.2

59.0 58.1 69.5

48.3 69.7 81.3

29.9 35.5 62.7

21.8 41.5 32.3

15.6 33.7 75.8

17.5 29.8 58.1

22.3 67.3 **Conditions Ref.**

[27]

http://dx.doi.org/10.5772/51120

71

1 x 1010 cfu/mL, 1h, 37 °C, PBS

Recent Trends in Microbiological Decontamination of Aflatoxins in Foodstuffs

1 x 1010 cfu/mL, 1h, 37°C, PBS

1 x 1010 cfu/mL, 1h, 37 °C, PBS

1 x 1010 cfu/mL, 1h, 37 °C, PBS

1 x 1010 cfu/mL, 1h, 37 °C, PBS

1 x 1010 cfu/mL, 1h, 37 °C, PBS

1 x 1010 cfu/mL, 1h, 37 °C, PBS

1 x 1010 cfu/mL, 1h, 37 °C, PBS

1 x 1010 cfu/mL, 1h, 37 °C, PBS

1 x 1010 cfu/mL, 1h, 37 °C, PBS

Recent Trends in Microbiological Decontamination of Aflatoxins in Foodstuffs http://dx.doi.org/10.5772/51120 71


**Microorganism AF Bound**

*L. rhamnosus* GG B1

Viable cells Freeze-dried cells Heat-treated cells *L. rhamnosus* LC-705

70 Aflatoxins - Recent Advances and Future Prospects

Viable cells Freeze-dried cells Heat-treated cells *L. gasseri L. acidophilus L. casei Shirota E. coli*

*L. paracasei*, *L. casei*, *L. brevis*, *L. plantarum* and

*Lc. lactis ssp. cremoris Lactobacillus delbrueckii*

*Lb. acidophilus Lb. rhamnosus Lb. plantarum Lc. lactis ssp. lactis Bifidobacterium lactis Lb. helveticus Lc. lactis* ssp. *cremoris Lb. rhamnosus Lc Lb. acidophilus Lb. fermentum Lb. johnsonii Lb. rhamnosus Lb. amylovorus Lb. amylovorus Bb. lactis Bb. longum Bb. animalis Bb. lactis*

*S. cerevisiae*

**(%)**

78.8 50 82 58.1 67.4 33.2 16.3

18-33

27-50

5.6 17.3 18.2 22.7 28.4 31.6 18.0 34.2 41.1 54.6 20.7 22.6 30.1 33.1 57.8 59.7 34.7 37.5 45.7 48.7

5 µg/mL 78.4 65 81

B1, B2, G1, G2 4 or 40 µg/kg

B1 5 µg/mL **Conditions Ref.**

[17]

[44]

2 x 1010 cfu/mL, 0h, 37 °C, PBS

2 x 1010 cfu/mL, 0h, 37 °C, PBS

2 x 1010 cfu/mL, 0h, 37 C, PBS 7 x 109 cfu/mL, 0h, 37 °C, PBS 1 x 1010 cfu/mL, 0h, 37 C, PBS 5 x 1010 cfu/mL, 0h, 37 °C, PBS

6h, 37 °C , barley flour (50%), wheat flour (45%) and corn flour (5%) mixed with water in 1:1.5

24h, 37 °C , barley flour (50%), wheat flour (45%) and corn flour (5%) mixed with water in 1:1.5

1 x 1010 cfu/mL, 24h, 37 °C, PBS [23]

4h, 37 C, PBS 4h, 37 °C, PBS

4h, 37 °C, PBS 4h, 37 °C, PBS

proportion

proportion


0.6mg/kg 1 mg/kg 1.4mg/kg

1 mg/kg

M1 5, 10 and 20 ng/mL

Yoghurt Culture B1

*L. acidophilus* NCC12

*L. acidophilus* NCC36

*L. acidophilus* NCC68

Living cells

Heated cells

Living cells

Heated cells

Living cells

Heated cells

Heated cells

Heated cells

*L. rhamnosus* Living cells

Heated cells

*B. bifidum* NCC 381 Living cells

*B. bifidum* Bb13 Living cells

**(%)**

97 91 90

90 84 73

14.9-20.2 14.4-15.4 17.0-24.9 16.6-19.0

20.4-25.3 21.8-22.7 22.1-26.8 23.7-25.1

10.2-16.0 7.8-10.5 14.0-21.8 12.8-15.9

23.5-26.6 24.0-25.9 24.3-28.9 25.4-27.4

16.6-22.1 15.5-18.3 17.4-23.5 17.1-22.2

20.1-24.0 20.4-22.2

23.4-27.8 22.9-26.3

*Lactobacillus* strains AFM1 9.4-73.1 96 h, 37 °C , PBS [47]

**Conditions Ref.**

[43]

http://dx.doi.org/10.5772/51120

73

[46]

42 °C/3h, pH 4.0, overnight, milk

Recent Trends in Microbiological Decontamination of Aflatoxins in Foodstuffs

milk acidified with citric acid milk acidified with latic acid milk acidified with acetic acid

108 cfu/mL, 0, 4, 24 h, 37 °C, PBS 108 cfu/mL, 4h, 37 °C, milk 0, 4 , 24 h, 37 °C, PBS 4h, 37 °C, milk

108 cfu/mL, 0, 4, 24 h, 37 °C, PBS 108 cfu/mL, 4h, 37 °C, milk 0, 4, 24 h, 37 °C, PBS 4h, 37 °C, milk

108 cfu/mL, 0, 4, 24 h, 37 °C, PBS 108 cfu/mL, 4h, 37 °C, milk 0, 4, 24 h, 37 °C, PBS 4h, 37 °C, milk

108 cfu/mL, 0, 4, 24 h, 37 °C, PBS 108 cfu/mL, 4h, 37 °C, milk 0, 4,24 h, 37 °C, PBS 4h, 37 °C, milk

108 cfu/mL, 0, 4,24 h, 37 °C, PBS 108 cfu/mL, 4h, 37 °C, milk 0, 4 and 24 h, 37 °C, PBS

108 cfu/mL, 0, 4, 24 h, 37 °C, PBS 108 cfu/mL, 4h, 37 °C, milk

0, 4 and 24 h, 37 °C, PBS

4h, 37 °C, milk

4h, 37 °C, milk


B1 2 µg/mL

B1 0.5 µg/mL

Acid-treated cells *Lc. lactis* subsp. *cremoris*

72 Aflatoxins - Recent Advances and Future Prospects

Viable cells Heat-treated cells Acid-treated cells *S. thermophilus* Viable cells Heat-treated cells Acid-treated cells

*E. Coli*

*Lc. lactis* Living cells

*L. rhamnosus* GG *S. aureus*

*Bifidobacterium* sp. Bf6 *B. adolescentis* 14 *B. bifidum* BGN4 *Bifidobacterium* sp. CH4 *B. longum* JR20 *Bifidobacterium* sp. JO3

Dead cells by boiling

*S. thermophilus* Living cells

Dead cells by boiling

Dead cells by autoclaving

Dead cells by autoclaving

**(%)**

82.5

26.9 40.1 43.7

32.7 42.0 63.8

54.8 86.7 82.3 71.0 PBS maize oil sunflower oil soybean oil

PBS

PBS

PBS maize oil sunflower oil soybean oil PBS maize oil sunflower oil soybean oil

PBS

81.0 100

80.0

81.0 91.5 90.7 66.5 100.0 96.8 81.7 96.0

83.0

**Conditions Ref.**

1 x 1010 cfu/mL, 1h, 37 °C, PBS

1 x 1010 cfu/mL, 1h, 37 °C, PBS

30 min, 37 °C, PBS [22]

107-108 cfu/mL,30 min, 37 °C, in:

[42]

maize, sunflower or soybean oil


B1 5 µg/mL

Heat-killed cells *L. gasseri* (ATCC 33323)

Viable cells Heat-killed cells *L. acidophilus* strain LA1

Viable cells Heat-killed cells *L. rhamnosus* strain 1/3

Viable cells Heat-killed cells

Lipase Viable cells Heat-treated cells Acid-treated cells Phosphate Buffer Viable cells Heat-treated cells Acid-treated cells m-Periodater Viable cells Heat-treated cells Acid-treated cells

Iodate Viable cells Heat-treated cells Acid-treated cells

Urea Viable cells Heat-treated cells Acid-treated cells Water (Milli Q) Viable cells Heat-treated cells Acid-treated cells

*L*. *rhamnosus* strain GG Pre-treatment: Pronase E Viable cells Heat-treated cells Acid-treated cells

**(%)**

38.9

30.8 61,5

18,3 25,5

18,1 39,9

66 72 85

76 74 89

86 85 91

60 49 36

83 84 80

64 60 50

76 83 84 **Conditions Ref.**

Recent Trends in Microbiological Decontamination of Aflatoxins in Foodstuffs

[48]

http://dx.doi.org/10.5772/51120

75

3.9 x 108, 15-16h, , 37 °C, PBS

1.7 x 109, 15-16h, , 37 °C, PBS

3.9 x 108, 15-16h, , 37 °C, PBS

1h, 37 °C, 5% CO2, PBS Boiled for 1h, PBS

1h, 37 °C, 5% CO2, PBS Boiled for 1h, PBS

1h, 37 °C, 5% CO2, PBS Boiled for 1h, PBS

1h, 37 °C, 5% CO2, PBS Boiled for 1h, PBS

1h, 37 °C, 5% CO2, PBS Boiled for 1h, PBS

1h, 37 °C, 5% CO2, PBS Boiled for 1h, PBS

1h, 37 °C, 5% CO2, PBS Boiled for 1h, PBS

2 mol/L HCl, 1h, 37 °C, 5% CO2

2 mol/L HCl, 1h, 37 °C, 5% CO2

2 mol/L HCl, 1h, 37 °C, 5% CO2

2 mol/L HCl, 1h, 37 °C, 5% CO2

2 mol/L HCl, 1h, 37 °C, 5% CO2

2 mol/L HCl, 1h, 37 °C, 5% CO2

2 mol/L HCl, 1h, 37 °C, 5% CO2



M1 0.15

µg/ml 50.7

18.8 26.0 57.8 26.6 36.6 PBS skim milk full cream milk

PBS skim milk full cream milk

PBS skim milk full cream milk

PBS skim milk full cream milk

46.3 69.6 27.4 51.6 63.6 30.1

53.8 56.2

45.7 57.4

40.4

*Lactococcus* strains *Bifidobacterium* strains

74 Aflatoxins - Recent Advances and Future Prospects

*L. plantarum B. adolescentes Lactobacillus* strains *Lactococcus* strains *Bifidobacterium* strains

*L. bulgaricus B. adolescentes*

Heat-killed cells

*L. rhamnosus*strain

Heat-killed cells

(lyophilized) Viable cells Heat-killed cells *L. rhamnosus* strain

LC-705 (lyophilized) Viable cells Heat-killed cells *L. lactis* ssp. *cremoris* strain ARH74 Viable cells

*L. rhamnosus strain GG*

LC-705 (pre-cultured) Viable cells

*L. rhamnosus* strain GG (pre-cultured) Viable cells

**(%)**

96 h, 37 °C , PBS 96 h, 37 °C , PBS

96 h, 37 °C , PBS 96 h, 37 °C , PBS 96 h, 37 °C ,milk 96 h, 37 °C , milk 96 h, 37 °C , milk 96 h, 37 °C , milk 96 h, 37 °C , milk

5.3 x 108, 15 - 16h, , 37 °C, in:

1.0 x 1010, 15-16h, 37 °C, PBS

1.0 x 1010, 15-16h, 37 °C, PBS

2.9 x 108, 15-16h, , 37 °C, PBS

4.5-38.3 7.8-41.6

73 41.6 64-80.5 46.0-68.5 67.0-72.5 80.5 73

**Conditions Ref.**

[29]


M1 10 ng/mL

M1 10 µg/mL

B1 2.5 µg/mL

B1 G1 M1 (0.5 µg/mL) B1

B1

3 µg/mL 51

*L. rhamnosus* Viable cells Heat-killed cells

*B. lactis* Viable cells Heat-killed cells

*L. delbrueckii* subsp. *bulgaricus* CH-2

*S. thermophilus* ST-36

*F. aurantiacum* NRRL

*B. subtilis* UTBSP1 Viable cells

Cell Free Supernatant

*B. subtilis* ANSB060 "Inocula" suspension

Culture Supernatant

*L. rhamnosus* strain

*L. rhamnosus* strain GG

Cell Cell extract

"In vivo"

"In vitro"

LC-705

B-184

**(%)**

skimmed milk

skimmed milk

skimmed milk

4h, 37 °C, PBS 4h, 42 °C, milk

4h, 37 °C, PBS 4h, 42 °C, milk Yoghurt

and milk

nuts

culture

72h, 37 °C, PBS

1010cfu/mL:

1 min, duodenum of chicks

1 h, duodenum of chicks 37 °C, 1h , pH 7.3

100 5 x 1010 cfu/mL, 30 °C, 4h, PBS

96h, 30 °C, nutrient broth culture 108 cfu/ml, 120 h, 30 °C, pistachio

72h, 37 °C, Luria-Bertani medium

120 h, 35 °C, nutrient broth

PBS PBS

PBS PBS

33.5

17.1 27.8 24.5

16.9 23.6 32.5

18.7 27.6

29.4 39.2 14.8

85.7 95

78.4

81.5 80.7 60

10.5 9.6 78.7

92 80 **Conditions Ref.**

http://dx.doi.org/10.5772/51120

77

Recent Trends in Microbiological Decontamination of Aflatoxins in Foodstuffs

[51]

[52]

[53]

[54]

[62]


B1 5 µg/mL

*L. acidophilus* Pre-treatment:

76 Aflatoxins - Recent Advances and Future Prospects

*L. plantarum* Viable cells Heat-killed cells *E. avium* Viable cells Heat-killed cells *P. pentosaceus* Viable cells Heat-killed cells *L. gasseri* Viable cells Heat-killed cells *L. bulgaricus* Viable cells Heat-killed cells

None Heat Ethanol Acid Alkaline *L. casi* None Heat Ethanol Acid Alkaline *L. helveticus* None Heat Ethanol Acid Alkaline *L. bulgaricus* None Heat Ethanol Acid Alkaline

**(%)**

4h, 37 °C, PBS

56.6 71.9 46.5 87.0 27.4

22.4 41.8 21.8 43.1 12.0

17.8 28.5 18.0 56.3 9.1

16.3 33.5 15.9 586 8.3

5.6 8.1 PBS PBS

PBS PBS

PBS PBS

PBS PBS

PBS PBS

1010 cfu/mL, 15 min, 37°C, in:

7.4 6.6

8.7 7.8

21.4 22.8

30.2 33.5

M1 0.15 µg/mL PBS solution 0.5 µg/mL skimmed milk

**Conditions Ref.**

[49]

[50]


The ability of SC (0.1%, 0.2%, and 0.3%) to adsorb AFB1 in contaminated corn (150, 300, 450 and 800 µg/kg corn was analyzed [68]. The adsorption process showed an inversely propor‐ tional relationship with the concentration, that is, the greater the AFB1 concentration in the medium, the lower the efficiency of AFB1 removal by SC (16% to 66% for 800 µg/kg AFB1 vs. 40% to 93% for 150 µg/kg AFB1). The authors concluded, using densitogram analysis, that the adsorption process did not change the molecular structure of the mycotoxin, and that the decreased AFB1 adsorption rates observed as the toxin concentration increased may pos‐ sibly be caused by saturation of the adsorption sites on the SC cell. Other factors, such as length of incubation, pH, method of biomass purification, and methods of analysis, may also

Recent Trends in Microbiological Decontamination of Aflatoxins in Foodstuffs

http://dx.doi.org/10.5772/51120

79

Immobilized SC cells (ATTC 9763) was investigated for their ability to remove AFB1 from pistachio seeds, and it was observed that the amount of toxin removed was dependent on its concentration in the medium (40% and 70% of removal for concentrations of 10 ng/mL and 20 ng/mL AFB1, respectively) [69]. The authors also concluded that this ability to remove the toxin was greater in SC exponential growth phase, and that the process was a quick one, be‐ ing saturated after 3 hours of contact. Besides, the ability of SC cells to remove toxin was increased after treatment with acid (60% and 73% for 10 ng/mL and 20 ng/mL AFB1, respec‐ tively) and heat (55% and 75%, respectively). In another study, authors also concluded that the treatment of SC cells with heat at 60 °C and 120 °C, and with chloric acid (2 mol/L) in‐ creased their ability to remove AFB1 from the medium to 68.8%, 79.3%, and 72.1%, respec‐

Heat treatment may increase the permeability of the external layer of the cell wall due to the suspension of some mannanes on the cell surface, leading to increased availability of previ‐ ously hidden binding sites. Besides, countless physical-chemical changes take place on the cell wall during heat treatment, leading to more exposed binding sites. On the other hand, acid conditions may affect polysaccharides by releasing monomers, which are further frag‐ mented in aldehydes after glycosidic bonds are broken. Continuous removal of aflatoxin, even after use of acid and heat treatments, confirms that yeast cell viability is not a signifi‐

During the fermentation of broiler feed using LAB (3 strains of *Lactobacillus*) and SC strains resistant to gastric juices and bile, 55% AFB1 was removed when AFB1 concentration in the medium was 1 mg/kg, and 39% when concentration was 5 mg/kg AFB1, after 6 hours [70]. This tendency for removal was maintained as incubation continued, and after 24 hours, the amount of AFB1 removed was 73% and 53%, respectively, for the two concentrations of the toxin. The authors considered that, from a practical point of view, the most important factor was the 6-hour fermentation period, once the passage of feed through the gastrointestinal tract of broilers lasts from 4 to 8 hours. In reference [71], authors analyzed the ability of SC to remove AFB1 from a contaminated medium at different pH values (3.0, 6.0, and 8.0), and observed that the three strains analyzed showed great ability to remove the toxin (41.6% to 94.5%), and that after washing, only a small amount of AFB1 was released back into the me‐ dium. *In vitro* studies are not always good indications of the *in vivo* responses, as *in vivo*

tively, against 38.7% when viable yeast cells were used [56].

cant factor for the removal of aflatoxin from the medium [69].

influence this process.

**Table 1.** Aflatoxin binding / absorption by microorganisms. Note: PBS, Phosphate-Buffered Saline; cfu, colony formingunit.
