REVIEW ON BIO-DETOXIFICATION OF AFLATOXINS BASED ON LACTIC ACID BACTERIA: MECHANISM AND APPLICATIONS

considered, due to maximum efficiency, low cost, eco-friendly and non-degradation of nutritional quality. The protective effect of lactic acid bacteria as probiotic microorganisms against mutagenic factors, such as polycyclic amines, N-nitrosamine compounds, and mycotoxins has been proven. Therefore, in addition to the beneficial properties using these probiotic bacteria with the ability to remove AF can help to enhance food safety. Although some naturally-occurring bacteria in the intestine can be attached to harmful components such as toxins and prevent them from binding to the intestinal layer, but probiotics have the potential to inactivate toxins through surface binding, due to high adhesion properties in their cell wall proteins. Polysaccharides and peptidoglycans in the cell wall are two of the main ingredients for decomposition, bonding and binding of AFs to lactic acid bacteria. Therefore, this review examines the economic and health impacts of AF contamination in foods. Further, this review discusses how lactic acid bacteria are able to detoxify common food AFs.


MYCOTOXINS
Mycotoxinogenic fungi play an undeniable role in reducing food safety through the production of mycotoxins.These natural carcinogens (mycotoxins) are present in human diets due to the contamination of raw materials or the production of toxic substances during the processing or storage of food (Yang et al., 2020).Mycotoxins are toxic secondary metabolites of low molecular weight, which do not have antigenic properties alone and therefore cannot stimulate the host immune system (Santos et al., 2019).The importance of mycotoxins is due to their toxicity, carcinogenic, teratogenic, mutagenic, acute and long-term suppressive effects (Bakırdere et al., 2012).Specifically, they are common in tropical and subtropical countries and infect about 25% of human food and animal feed (Monda and Alakonya, 2016).Their prevalence may be by using toxic food or contaminated plant and animal products.Six groups of mycotoxins, including AFs, fumonisin, ochratoxin, patulin, tricotesen, and zearalenone, are often seen in various food systems (Huffman et al., 2010).The studies of the effect of microorganisms on mycotoxins have been begun decades ago.The surface binding of some microorganisms to various mycotoxins including AFs, ochratoxin and zearalenone has been reported.Many studies have been conducted to remove mycotoxins by lactic acid bacteria.The ability of LABs to remove mycotoxins greatly depends on the environmental conditions, type, composition of the amino acid and the peptidoglycan structure (Agriopoulou et al., 2020).The connection of ochratoxin and zearalenone is attributed to cell wall glucans.Hence, it can be concluded that the removal of mycotoxins from different matrices by living and non-living cells depends on their strain and species, and each strain and species behave in a different way (García-Béjar et al., 2021; Vartiainen et al., 2020).

Aflatoxins
AFs are a group of carcinogenic mycotoxins that cause acute or chronic poisoning and liver cancer in humans and animals.They also cause a lot of economic losses in the industry due to contamination of food and animal feed (Vartiainen et al., 2020).These toxins are secondary metabolites of some Aspergillus species, especially Aspergillus flavus and Aspergillus parasiticus and mainly found before harvesting or during storage in cereals, especially rice, corn, wheat, barley, sorghum, almonds, peanuts, Brazilian nuts, pistachios and oilseeds such as cottonseed (Liu et al., 2020).The production of this toxin can be affected by several factors, including water activity (aw), temperature, light, oxygen Access to healthy food, without any undesirable contamination, is one of fundamental human right.Some of mycotoxins, especially aflatoxins (AFs), contamination feed and food that cause problems such as acute damage liver, irritation, and cancer of the liver and teratogenic complications.Among the physical, chemical and biological methods used to prevent the production, reduction, elimination and deactivation of AF in contaminated food, biological methods have been considered, due to maximum efficiency, low cost, eco-friendly and non-degradation of nutritional quality.The protective effect of lactic acid bacteria as probiotic microorganisms against mutagenic factors, such as polycyclic amines, N-nitrosamine compounds, and mycotoxins has been proven.Therefore, in addition to the beneficial properties using these probiotic bacteria with the ability to remove AF can help to enhance food safety.Although some naturally-occurring bacteria in the intestine can be attached to harmful components such as toxins and prevent them from binding to the intestinal layer, but probiotics have the potential to inactivate toxins through surface binding, due to high adhesion properties in their cell wall proteins.Polysaccharides and peptidoglycans in the cell wall are two of the main ingredients for decomposition, bonding and binding of AFs to lactic acid bacteria.Therefore, this review examines the economic and health impacts of AF contamination in foods.Further, this review discusses how lactic acid bacteria are able to detoxify common food AFs.concentrations, pH, nutrients, storage time, mechanical/thermal damage, and competitive growth of other microorganisms.Among them, aw and temperature, the two key environmental factors during storage, have a strong impact on both fungal growth and secondary metabolite production.Also, mold growth is strongly influenced by humidity.The optimal growth temperatures for Aspergillus range from 28-40 °C with and aw 0.94-0.99,but the fungi can survive temperatures of 12-48 °C.Both A. flavus and A. parasiticus can grow over the pH range of 2.1 to 11.2, with an optimum between pH 3.5 and 8 (Lv et al., 2019).More than 20 types of AFs are known, of which Aflatoxin B1 (AFB1), Aflatoxin B1 (AFB2), Aflatoxin G1 (AFG1), Aflatoxin G1 (AFG2), Aflatoxin M1 (AFM1), and Aflatoxin M1 (AFM2) are biologically common (Miklós et al., 2020).which their structure is shown in Figure 1.Among AFs, AFB1 and AFB2 are the most important and type B1 is known as the most toxic AF.The liver is the main target organ for AF, although the tumor may also occur in other organs, such as the lung, kidney, and colon (Long et al., 2016).Aspergillus Flavus is known to produce AFB, while Aspergillus Parasiticus produces both AFB and G through numerous biochemical processes.Hydroxylation of AFB1 leads to the formation of AFM1.Receive and direct absorption of AFB1 by animal's produces and stores AFM1 in milk (Patyal et al., 2020).AFM1 is resistant to pasteurization and sterilization.The European Union and the International Codex have set the maximum level of 0.5 mg/L for AFM1 in milk, milk powder and other processed products (Fashandi et al., 2018).Chemical and physical methods may be subject to limitations in the areas of biological safety, the loss of quality and nutritional value, the limitations on the efficiency, effectiveness and cost of equipment.A variety of chemical methods are used in the food and feed industry to remove or reduce mycotoxin content, such as oxidation, alkalization, acidification, reduction, and ammoniation.The use of hundreds of chemicals, such as ammonia, calcium hydroxide monoethylamine, sodium hydroxide, ozone, chlorine, and calcium hydroxide, has shown promise in reducing mycotoxin levels in foods.It is possible to partially destroy mycotoxins by using chemical methods, such as oxidation and alkalization, but some nutrients are also destroyed (Jard et al., 2011;Karlovsky et al., 2016).The safety and harmless of lactic acid bacteria and Bifidobacteria as probiotics provide a good opportunity for application in biological reduction of AF in food (Lyagin and Efremenko, 2019).The ability and stability of microorganisms in binding toxins are very important for evaluating the ability of strains to reduce the biological effects of AFs.The release of toxin during transition through the stomach may result in harmful health effects.This limitation is reversible and its stability depends on the strain, formation conditions and method used to determine the stability (Ahlberg et al., 2015).
Figure 1 The structure of common Aflatoxins

PROBIOTIC BACTERIA
Probiotic is derived from the Greek word "for living".They are known as living organisms, which can have beneficial effects on the host by improving the balance of the microbial flora of the gut if used sufficiently (Kouhi et al., 2022).Probiotics are living organisms that confer health benefits on the host when administered in an adequate amount.Typically, they are available as either food products (fermentable or non-fermentable) or dietary supplements (capsule, tablet, or powder).A growing awareness among consumers is leading to an increase in consumption of probiotics.Probiotics are an innovative and inexpensive invention in society that can improve the health of people.Due to the accumulation of toxic substances during digestion, the presence of bad microorganisms causes many diseases and illnesses in the human body.During this time, the immune system begins to deteriorate (Fashandi et al., 2018).Probiotics in higher doses are an alternative method to treating such problems instead of taking antibiotics.Probiotics can be formulated in various products, especially foods, medicines and dietary supplements.This group of bacteria plays an important role in inhibiting infections in different parts of the body, especially the mouth, gastrointestinal and genitourinary tract (Picard et al., 2005).Among probiotics, Lactobacillus species and Bifidobacterium species are most used, but Saccharomyces cerevisiae and some Bacillus species are also used as probiotics.Therefore, it can be said that lactic acid bacteria, especially lactobacillus and Bifidobacterium, are the most important probiotic bacteria that are completely safe and non-pathogenic to humans.In order to provide health benefits, the end product population of viable bacteria must be greater than 10 7 CFU/mL.These probiotics can prevent the absorption of AFs in the gastrointestinal tract by binding to AFs in foods (Guarner et al., 2012).

Lactic acid bacteria and bifidobacteria
Lactic acid bacteria are Gram-positive, non-spore forming and catalase-negative bacteria and they mainly produce lactic acid by fermentation of carbohydrates.For the first time, they were isolated from milk and widely used as starting cultures in dairy, meat, vegetable and cereal industries (Valenzuela et al., 2019).They are mainly divided into four genera, such as Lactobacillus, Lactococcus, Leuconostoc, and Pediococcus.The term lactic acid bacteria are used for different species of Bifidobacterium, although they have unique carbohydrate fermentation pathways and have no physiological relationship (Hernandez-Mendoza et al., 2011).Lactobacillus species have strong antimicrobial activity against many pathogen microorganisms.Also, Enterococcus faecium is one of the lactic acid bacteria found in nature and some processed dairy products (Bartkiene et al., 2020).Lactic acid bacteria naturally exist in mucous membranes and skin of the human and animal's body.They have been widely used in fortified foods, due to their beneficial health effects and preservative properties.Most of their useful properties relate to the binding capacity and adhesion to the intestinal mucosa or epithelial cells.They also protect food from toxic substances and produce some antagonistic compounds that are capable of controlling pathogenic bacteria and spoilage (Alp and Kuleaşan, 2019; Mirlohi et al., 2008).Due to their efficiency, low cost, and nature-friendly properties, biological decontamination procedures are rapidly becoming an encouraging alternative to chemical methods.There is a significant efficacy of LAB for eradicating mycotoxins irreversibly, without leaving any toxic residues.In different food products such as dairy products, they can be used as starter cultures in fermentation processes (Sevim et al., 2019).It is possible to enjoy the benefits of LAB through consumption of probiotic-related food products.Most commonly, LABs preserve materials by producing organic acids, competing for nutrients, and ultimately releasing antimicrobials.As well as reducing and inactivating toxins, some antifungal metabolites released by LABs also suppress fungal growth.Metabolites such as phenolic compounds, fatty acids, hydrogen peroxide, and proteinaceous compounds are present in the body.Fermented dairy products are highly dependent on this fermentation procedure that controls the contamination of the product by pathogens by producing lactic acid from lactose.Two mechanisms are involved in mycotoxins' detoxification from food by the LAB.It is possible to detoxify food by LAB by using the viable cells of the microbes or by using the enzymes produced by some LAB strains (Muhialdin et al., 2020).Microorganisms, including fungi, have been a source of food spoilage for an extended period of time (Varga and Tóth, 2005).A number of bioactive metabolites produced by LAB limit the growth of fungi and prevent the formation of mycotoxins in food.The bioactive compounds contained in LAB include acids, hydrogen peroxide, carbon dioxide, phenyllactic acid, and low molecular weight peptides.The LAB produces a number of proteolytic enzymes capable of hydrolyzing proteins.These enzymes include cell-wall bound proteinases, which break down proteins into polypeptides, peptide transporters that carry peptides into cells, and intracellular peptidases which degrade them into amino acids.As a result of LAB proteolytic enzymes, mycotoxins in food are detoxified most effectively (Alberts et al., 2009).Generally, the antimicrobial activity of LAB can be explained by three mechanisms such as organic acid function, competition for nutrients and the production of antagonistic compounds (Chen et al., 2019).Antifungal metabolites of lactic acid bacteria include sugary catabolites (such as organic acids, acetic acid, and formic acid), oxygenated catabolites (such as hydrogen peroxide), and protein compounds (such as low molecular weight peptides, hydroxylic fatty acids, and phenolic compounds) (Servin, 2004).Antifungal activity of LAB is affected by some processing parameters including temperature, incubation time, pH and nutritional factors (Dalié et al., 2010).Using a combination of certain strains of probiotic bacteria may be more effective than using a single strain, however, it may reduce the toxin removal capacity (Liu et al., 2019).Therefore, to remove a single compound, an efficient strain and to remove several compounds, a mixture of the best strains is used (Khorshidian et al., 2020).

Cell wall of acidic lactic acid bacteria
The LAB has a peptidoglycan matrix, which forms the main constituent of the cell wall structure.Teichoic Acid, Lipoteichoic Acid, protein layers, and neutral polysaccharides are the other constituents of the cell wall that have different functions Frequently it has been recognized that lipoteichoic acid is poly (glycerol phosphate) LAB that is almost similar to the poly (glycerol phosphate) teichoic acids, which differ only in the chirality of glycerol.Similar to teichoic acids, the lipoteichoic acid also have glycerol and di-alanine, which are replaced by hydroxyl groups in glycerol.It has been reported that many LAB of the genus Lactobacillus produce s-layer protein.These proteins are noncovalent and have a size of 25-50 KDa (Silhavy et al., 2010).LAB that cannot produce s-layer proteins has a negative surface charge at neutral pH.Despite the fundamental nature of the surface layer protein, it is reported that surface charge on the surface layer which produced by Lactobacillus also have been reported negative (Ventura et al., 2002).A hypothesis for this mechanism may be the involvement of positive-charge regions in surface-layer proteins on binding to peptidoglycans.Injury or destruction in the cell wall of the bacteria in Comparison to a completely healthy and without attachment sites can reveal unattended connection sites and may allow the attachment of AF to the cell wall and plasma membrane compounds (Macek et al., 2019;Smit et al., 2001).The use of acidic and heat treatments is likely to disrupt the integrated structural form, and thus allow AF to be incorporated into intracellular compounds.This mechanism was identified during the use of antibody to analyze AFB1 in acidtreated bacteria that prevents antibody entry, because the size of the antibody molecule is large and could not cross the cell wall of the bacterium and bind to the AFB1 molecule in the inner wall.According to a study by Haskard et al. (2001b), Lactobacillus rhamnosus was exposed to enzymatic treatment and its effect on the binding of AFB1 was investigated.The results showed that there was no evidence of exopolysaccharide involvement, but cell wall proteins, calcium ions and magnesium ions played a decisive role in this regard.Adsorption to surface components of different nature has been reported as the major mechanism for binding, mycotoxins.Figure 2 shows the cell wall structure in LAB and proposed processes involved in the interaction with AFs.
Figure Schematic representation of the cell wall structure in LAB and proposed processes involved in the interaction with AFs.Adsorption to surface components of different nature has been reported as the major mechanism for binding, mycotoxins.EPS, exopolysaccharides; SLP, S-layer proteins; PG, peptidoglycan; TA, teichoic acids; LTA, lipoteichoic acids; M, cytoplasmic membrane.

The function of lactic acid bacteria in aflatoxin detoxification
According to previous studies, microorganisms such as Saccharomyces cerevisiae, Flavobacterium and Lactobacillus species, including Lactobacillus acidophilus and Lactobacillus rhamnosus, and other subtypes, could decompose and bind AFs through their cell walls (Shetty and Jespersen, 2006).Injury or destruction in the cell wall of the bacteria in Comparison to a completely healthy and without attachment sites can reveal unattended connection sites and may allow the attachment of AF to the cell wall and plasma membrane compounds (Gao et al., 2021;Haskard et al., 2001b).According to previous studies, polysaccharides and peptidoglycans in the cell wall are two main elements in LAB, and both of these compounds are strongly affected by acidic and heat treatment in order to effectively interact with mutagens (Sezer et al., 2013).Heat causes the denaturation of protein or formation of maillard reaction products between polysaccharides and proteins or peptides.Also, acid can break glycosidic bonds in polysaccharides, and release monomer, which later turns into aldehyde and then breaks down peptide bonds and releases amino acids.Although the peptidoglycan layer is quite thick in these microorganisms, but in this case there may be a reduction in thickness and transverse joints in it (El-Nezami et al., 1998; Gao et al., 2021).It is noted that AFB1, connects to the superficial compounds of LAB.Destruction of these specific compounds of the cell wall, such as carbohydrates and proteins, reduces AFB1 bonding by Lactobacillus rhamnosus GG, and it is stated that these compounds are important for the binding of AFB1.According to a study by Haskard, El-Nezami, Kankaanpää, Salminen, and Ahokas (2001a), the bacterium Lactobacillus rhamnosus was exposed to enzymatic treatment and its effect on binding of AFB1 was investigated and the results showed that there was no evidence of exopolysaccharides involvement, but the proteins in the cell wall,  Attaching of LAB to AF is fast (more than 1 minute) and reversible (Bueno et al., 2007).Pre-treatment of LAB with heat or acid increases the AF decontamination, while treatment with some of the main compounds such as sodium hydroxide, sodium carbonate and isopropanol have a negative effect on this connection.The total AFB1 molecules, which can be connected to a live bacteria is estimated to be more than 107 molecules (Gonçalves et al., 2020).More over AFB1, these bacteria also bind to other AFs, such as B2a, G1, G2, M1, M2, B2, as well as other mycotoxins, but not as much as AFB1 (Bueno et al., 2007;Salminen et al., 2010).It has been confirmed that the addition of metal ions, for example, sodium chloride and calcium chloride, and a wide range of pH (from 2.5 to 8.5), did not significantly affect the binding of AFB1, which means that electrostatic interactions and hydrogen bonding do not play an important role in bonding (Vázquez-Durán et al., 2021).

CONCLUSION
Biological control methods based on LAB provide a promising approach to controlling the growth of mycotoxins in the food chain and hence reduce the health risks of fungal toxins.Many studies have shown different efficacy of LAB from a variety of matrices in the removal of mycotoxins (especially AF).Mainly, the detoxification relies on the binding of mycotoxin to the lactic acid bacteria and inactivation by antifungal products such as acetic acid.Most likely, it is related to the cell wall components (mainly peptidoglycan and exopolysaccharides) of LAB.In addition, studies have shown that treatment of cells with acid and heat can detoxify them.Therefore, removal and reduction can be demonstrated by the connection of toxin to the bacterial cell wall.However, the exact mechanism for bonding AF to probiotics is not known, but studies have shown that AF binding is a reversible reaction which occur in the surface of the bacteria and it includes interactions with carbohydrates, peptidoglycans and to some extent the protein structure.It seems that AF binding extremely related to strain, matrix, temperature, and pH and incubation time.There for, most probably, specific strains LAB is required against fungal contamination in different applications.Especially, strains of Lactobacillus genus have been studied and according to reports, potentially could reduce the risks of AFs hazards associated with food matrix and contaminated Animal feed.But still further research is needed to identify the potential species LAB from traditional fermented products, which produced in different parts of the world and testing their functionality in different matrices.The optimum conditions for mycotoxin detoxification and the mechanisms involved in mycotoxin degradation still require further research.There is a need to determine the diversity and suitability of LAB for different food applications based on their detoxification activity.It is important for future studies to combine LAB strains with different detoxification mechanisms in order to further enhance the efficiency of mycotoxin degradation.
(Niderkorn et al., 2009).Adhesion and biding to macromolecules are the function of the fiber network in Teichoic Acid and polysaccharides such as cell wall polysaccharides and exopolysaccharides.Cell wall polysaccharides of lactic acid bacteria are one of the most commonly used polysaccharides with a wide variety in the compounds containing rhamnose (Chapot-Chartier and Kulakauskas, 2014).Exopolysaccharides are either transmitted to the extracellular medium or attached to a surface to form a capsular polysaccharide (Liu et al., 2019).Some genera of Lactic acid bacteria, including Lactobacillus, Enterococcus and Streptococcus, as well as the genera used in the dairy industry, such as Bifidobacteria and Propionic bacteria, produce exopolysaccharides as glucose, galactose, rhamnose, mannose, N-acetylglucosamine and N-acetyl galactosamine.Peptidoglycan in LAB is composed of N-acetylglucosamine and N-acetylmuramic acid disaccharide polymers that are arranged side by side with beta-1, 4 glycosylated bonds and these chains are bonded by transverse pentapeptide bridges (Sánchez et al., 2006).The peptidoglycan disaccharide units have three different modes.Acetyl groups may be broken down in both N-acetylglucosamine disaccharides and N-acetyl muramic acid (DeMeester et al., 2019).An extra acetyl group is added to the oxygen number 6 of N-acetyl-muramic acid and the position of carbon-6 of N-acetylmuramic acid is replaced by teichoic acid.The peptide segment consists of 3 amino acids that are linked to muramic acid.The amino acids of the penta-peptide bridge in N-acetyl-muramic acid usually contain one of the two compounds, L-alanine, D-glutamine, Diaminopimelic acid, or Lalanine, D-glutamine, and L-Lysine.Although at least five different subtypes have been identified for peptidoglycans (Delcour et al., 1999).A di-peptide residue from D-alanine is directly or via a bridge attached to this peptide.The amino acid D-alanine in some lactic acid bacteria that are resistant to vancomycin, such as Enterococcus faecium, Pediococcus Pentosaceus, Lactobacillus plantarum and Lactobacillus casei replace the d-lactate in penta peptide bridge, but in the bacteria Enterococcus faecium, Pediococcus Pentosaceus, Lactobacillus plantarum, Lactobacillus casei and Enterococcus Gallinarum, D-serine is replaced (Barbieri et al., 2019).Teichoic acids are anionic polymers in the cell wall that covalently bond to the peptidoglycan layer and are the cause of the serological difference is the presence of several gram-positive bacteria (Brown et al., 2013).Teichoic acids are composed of glycerol phosphate or ribitol phosphate polymers and carbohydrates that are bonded to each other by a phosphodiester bond.The composition of the structural unit in Lactobacillus plantarum has been reported as glycerol phosphaten-acetyl manozamin with glycosylated beta-1 and 4 grafts (Seltmann and Holst, 2013).The lipoteichoic acid is structurally similar to teichoic acids, but they attach to the cytoplasmic membrane glycolipid, instead of peptidoglycans.Generally, the glycolipid consists of diacylglycerol bonded to di-, or disaccharide units (Hynönen and Palva, 2013).
calcium and magnesium ions play a decisive role in this regard (Fochesato et al., 2019; Haskard et al., 2001a).It is likely that the use of acid and heat treatment eliminate the structural integrity and there for AF could bond to intracellular compounds.This mechanism was identified during the use of antibodies to analyze AFB1 in acid-treated bacteria, which prevented the entry of antibodies, because the size of the antibody molecule is large and does not have the ability to cross the cell wall of the bacterium and connect to the AFB1 molecule in the inner wall (Ahlberg et al., 2015).Some of the different types of LAB and Bifidobacteria have been investigated for the removing AFB1 from aqueous buffer solution, in vitro, and it has been said that their elimination is by binding of bacteria to toxic compounds and is not related to bacterial metabolism (Haskard et al., 2001b; Hernandez-Mendoza et al., 2009).Table 1 lists the LAB that can remove AFs.Muaz et al. (2021) investigated the in vitro ability of L. rhamnosus, L. lactis ssp.lactis and one L. lactis ssp.cremoris at 10 10 cells/mL to bind to AFM1 in skimmed milk, and showed higher binding capacities by 81.4,56.8, and 50.8%, respectively.This binding is due cell wall isolates and exopolysaccharide compounds.Liew et al. (2018) studied the ability of L. casei Shirota (live cell, heat-treated, and cell wall compounds) to bind AFB1, the binding capacity of L. casei Shirota was dependent on concentration of AFB1.They found that live cells had maximum binding capacity (98%).In vivo studies, L. casei Shirota neutralized toxicity of AFB1 on body weight and intestine by binding process.Chlebicz and Śliżewska (2020) performed in vitro study on the binding properties of AFB1 by 12 strains of lactic acid bacteria, which its concentration was decreased on average by 60% after 24 h incubation.Ben Salah-Abbes et al. (2020) have investigated the ability of L. paracasei BEJ01 to bind AFM1 in vitro and have revealed this strain was very effective for eliminating AFM1 with more than 95% of the toxin with concentration of 100 µg/ml.

Table 1
The ability of lactic acid bacteria to reduction of Aflatoxins.