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Invited review: Microbe-mediated aflatoxin decontamination of dairy products and feeds

      ABSTRACT

      Aspergillus flavus, Aspergillus parasiticus, and Aspergillus nomius contaminate corn, sorghum, rice, peanuts, tree nuts, figs, ginger, nutmeg, and milk. They produce aflatoxins, especially aflatoxin B1, which is classified as a Group 1 carcinogen by the International Agency for Research on Cancer. Many studies have focused on aflatoxin removal from food or feed, especially via microbe-mediated mechanisms—either adsorption or degradation. Of the lactic acid bacteria, Lactobacillus rhamnosus GG efficiently binds aflatoxin B1, and a peptidoglycan in the bacterium cell wall plays an important role. This ability of L. rhamnosus GG should be applied to the removal of aflatoxin B1. Aflatoxin can be removed using other aflatoxin-degrading microorganisms, including bacterial and fungal strains. This review explores microbe-associated aflatoxin decontamination, which may be used to produce aflatoxin-free food or feed.

      Key words

      INTRODUCTION

      Mycotoxins produced by fungi contaminate 25% of the cereals and grains marked for human consumption; of these, aflatoxins are among the most toxic types (
      • Wild C.P.
      • Turner P.C.
      The toxicology of aflatoxins as a basis for public health decisions.
      ;
      • CAST (Council for Agricultural Science and Technology)
      ). Aspergillus flavus, Aspergillus parasiticus, and Aspergillus nomius, which are known to contaminate corn, sorghum, rice, peanuts, tree nuts, figs, ginger, nutmeg, and milk, produce aflatoxins that are carcinogenic to the liver (
      • Ellis W.O.
      • Smith J.P.
      • Simpson B.K.
      • Oldham J.H.
      Aflatoxins in food: Occurrence, effects on organisms, detection, and methods of control.
      ;
      • FDA (Food and Drug Administration)
      Bad Bug Book: Foodborne Pathogenic Microorganisms and Natural Toxins. 2nd ed.
      ). Aflatoxins are secondary metabolites of low molecular weight that are synthesized by some aspergilli. Four major aflatoxins are aflatoxin B1 (most carcinogenic), aflatoxin B2, aflatoxin G1, and aflatoxin G2, and they have half-maximal lethal dose (LD50) values varying from 0.3 mg/kg of BW in rabbits to 18 mg/kg of BW in rats (
      • Moss M.O.
      Recent studies of mycotoxins.
      ;
      • IARC, Working Group on the Evaluation of Carcinogenic Risks to Humans
      Some traditional herbal medicines, some mycotoxins, naphthalene and styrene.
      ;
      • FDA (Food and Drug Administration)
      Bad Bug Book: Foodborne Pathogenic Microorganisms and Natural Toxins. 2nd ed.
      ). Aflatoxins are classified by the International Agency for Research on Cancer in 2012 as Group 1 carcinogens (i.e., carcinogenic to humans;
      • IARC (International Agency for Research on Cancer)
      Agents classified by the IARC monographs, volumes 1–113.
      ).
      The occurrence of aflatoxins in foods and feeds has been frequently reported in many countries. For instance, many reports have shown that raw agricultural products—including nuts, cereals, fruits, vegetables, herbs and spices—were contaminated with aflatoxin B1 at high levels, exceeding the maximum permissible limit (
      • Chen Y.C.
      • Liao C.D.
      • Lin H.Y.
      • Chiueh L.C.
      • Shih D.Y.C.
      Survey of aflatoxin contamination in peanut products in Taiwan from 1997 to 2011.
      ;
      • Guchi E.
      Implication of aflatoxin contamination in agricultural products.
      ;
      • Waliyar F.
      • Osiru M.
      • Ntare B.R.
      • Vijay Krishna Kumar K.
      • Sudini H.
      • Traore A.
      • Diarra B.
      Post-harvest management of aflatoxin contamination in groundnut.
      ), In addition, contamination with aflatoxin M1 has occurred in milk and milk products, including cheese, yogurt, and cream, and it remains even after milk pasteurization (
      • Yitbarek M.B.
      • Tamir B.
      Mycotoxines and/or aflatoxines in milk and milk products.
      ). Moreover, high levels of aflatoxin have been found in milk and dairy feed products, at contamination levels ranging from 0.028 to 4.98 μg/L and 7 to 419 μg/L, respectively, in a Greater Addis Ababa milk shed (
      • Gizachew D.
      • Szonyi B.
      • Tegegne A.
      • Hanson J.
      • Grace D.
      Aflatoxin contamination of milk and dairy feeds in the Greater Addis Ababa milk shed, Ethiopia.
      ).
      Many physicochemical technologies have been developed to decontaminate food or feed containing aflatoxin B1, but most of them also cause unwanted alteration of food properties, such as decreases in safety and sensory quality, and unsatisfactory applicability and practicability. To prevent aflatoxin B1 contamination in food, agricultural practices and storage conditions need to be improved (
      • Wu Q.
      • Jezkova A.
      • Yuan Z.
      • Pavlikova L.
      • Dohnal V.
      • Kuca K.
      Biological degradation of aflatoxins.
      ;
      • Gonçalves B.L.
      • Rosim R.E.
      • de Oliveira C.A.F.
      • Corassin C.H.
      The in vitro ability of different Saccharomyces cerevisiae-based products to bind aflatoxin B1.
      ). Therefore, chemical, physical, and biological treatments have been suggested to minimize toxin production and eliminate mycotoxins in food and feed (
      • Faucet-Marquis V.
      • Joannis-Cassan C.
      • Hadjeba-Medjdoub K.
      • Ballet N.
      • Pfohl-Leszkowicz A.
      Development of an in vitro method for the prediction of mycotoxin binding on yeast-based products: case of aflatoxin B1, zearalenone and ochratoxin A.
      ). Both chemical and physical approaches have drawbacks, including inefficient removal, lack of cost-effectiveness, or nutritional loss (
      • El-Nezami H.
      • Kankaanpää P.
      • Salminen S.
      • Ahokas J.
      Ability of dairy strains of lactic acid bacteria to bind a common food carcinogen, aflatoxin B1.
      ). Adsorbents as physical treatments have been widely used, and silicates, clays, and activated carbons are extensively available, but their efficacy depends on the chemical structure of the adsorbent: that is, the total charge and charge distribution, the size of the pores, and the accessible surface area (
      • Kabak B.
      • Dobson A.D.W.
      • Var I.
      Strategies to prevent mycotoxin contamination of food and animal feed: A review.
      ;
      • Di Natale F.
      • Gallo M.
      • Nigro R.
      Absorbents selection for aflatoxins removal in bovine milks.
      ). In addition, these nonedible materials need to be eliminated after aflatoxin decontamination from foods or feeds. Therefore, the use of probiotic strains has been suggested as a better technique for removing aflatoxin B1 through adsorption, especially using Lactobacillus rhamnosus GG. Additionally, many other microorganisms have been reported to convert aflatoxin into less toxic substances. Therefore, the objective of this article was to review the published literature on aflatoxin B1 decontamination by microbiological action, and to propose the applicability of microbes as additives for aflatoxin decontamination from dairy products and feeds.

      BACTERIA-BASED PHYSICAL ADSORPTION

      Yeast and a number of lactic acid bacteria can bind aflatoxins, decreasing aflatoxin bioavailability in feed or food. Because lactic acid bacteria prevent the growth of pathogenic bacteria by producing pathogen-inhibitory substances, and because most are used as probiotics and generally regarded as safe, they are considered a desirable method for aflatoxin removal (
      • Hernandez-Mendoza A.
      • Guzman-de-Peña D.
      • Garcia H.S.
      Key role of teichoic acids on aflatoxin B1 binding by probiotic bacteria.
      ). Among lactic acid bacteria, physical adsorption by L. rhamnosus GG has been extensively studied. Therefore, this review focuses more on describing the interaction between aflatoxin and L. rhamnosus GG.

      Removal of Aflatoxin B1 by L. rhamnosus GG

      The application of lactic acid bacteria to remove aflatoxin B1 is important for making food safer without changing its properties. Furthermore, lactic acid bacteria strains are known to be nonpathogenic and safe, and they function as natural agents and probiotics.
      • El-Nezami H.
      • Kankaanpää P.
      • Salminen S.
      • Ahokas J.
      Ability of dairy strains of lactic acid bacteria to bind a common food carcinogen, aflatoxin B1.
      examined the abilities of L. rhamnosus GG (ATCC53103), L. rhamnosus LC-705, Lactobacillus acidophilus ATCC4356, Lactobacillus gasseri ATCC33323, and Lactobacillus casei Shirota (YIT9018) to remove aflatoxin B1. One of the strains, L. rhamnosus GG, was more efficient than L. gasseri, L. acidophilus, and L. casei (
      • El-Nezami H.
      • Kankaanpää P.
      • Salminen S.
      • Ahokas J.
      Ability of dairy strains of lactic acid bacteria to bind a common food carcinogen, aflatoxin B1.
      ;
      • Oatley J.T.
      • Rarick M.D.
      • Ji G.E.
      • Linz J.E.
      Binding of aflatoxin B1 to bifidobacteria in vitro.
      ;
      • Haskard C.A.
      • El-Nezami H.S.
      • Kankaanpää P.E.
      • Salminen S.
      • Ahokas J.T.
      Surface binding of aflatoxin B1 by lactic acid bacteria.
      ). Indeed, L. rhamnosus GG was found to be capable of removing 80% of the aflatoxin B1 from contaminated media (
      • El-Nezami H.
      • Kankaanpää P.
      • Salminen S.
      • Ahokas J.
      Ability of dairy strains of lactic acid bacteria to bind a common food carcinogen, aflatoxin B1.
      ). Lactobacillus rhamnosus GG is a gram-positive bacterium that was isolated in 1983 by Barry R. Goldin and Sherwood L. Gorbach (hence the letters GG;
      • Silva M.
      • Jacobus N.V.
      • Deneke C.
      • Gorbach S.L.
      Antimicrobial substance from a human Lactobacillus strain.
      ). It has been used as a probiotic bacterium due to its resistance to gastric acid and bile and its great avidity for human intestinal mucosal cells, but it is a transient inhabitant (
      • Conway P.L.
      • Gorbach S.L.
      • Goldin B.R.
      Survival of lactic acid bacteria in the human stomach and adhesion to intestinal cells.
      ;
      • Walter J.
      The ecological role of lactobacilli in the gastrointestinal tract: Implication for fundmental and biomedical research.
      ). It has powerful adhesive properties and can exclude or reduce pathogenic adherence, as well as produce substances antagonistic to foodborne pathogens (
      • Gorbach S.L.
      Probiotics and gastrointestinal health.
      ). Many human trials have shown that L. rhamnosus GG reduced diarrhea in children and adults, including rotavirus diarrhea, traveler's diarrhea, and Clostridium difficile diarrhea (
      • Oksanen P.J.
      • Salminen S.
      • Saxelin M.
      • Hämäläinen P.
      • Ihantola-Vormisto A.
      • Muurasniemi-Isoviita L.
      • Nikkari S.
      • Oksanen T.
      • Pörsti I.
      • Salminen E.
      • Siitonen S.
      • Stuckey H.
      • Toppila A.
      • Vapaatalo H.
      Prevention of travellers' diarrhoea by Lactobacillus GG.
      ;
      • Oberhelman R.A.
      • Gilman R.H.
      • Sheen P.
      • Taylor D.N.
      • Black R.E.
      • Cabrera L.
      • Lescano A.G.
      • Meza R.
      • Madico G.
      A placebo-controlled trial of Lactobacillus GG to prevent diarrhea in undernourished Peruvian children.
      ;
      • Vanderhoof J.A.
      • Whitney D.B.
      • Antonson D.L.
      • Hanner T.L.
      • Lupo J.V.
      • Young R.J.
      Lactobacillus GG in the prevention of antibiotic-associated diarrhea in children.
      ;
      • Guandalini S.
      • Pensabene L.
      • Zikri M.A.
      • Dias J.A.
      • Casali L.G.
      • Hoekstra H.
      • Kolacek S.
      • Massar K.
      • Micetic-Turk D.
      • Papadopoulou A.
      • de Sousa J.S.
      • Sandhu B.
      • Szajewska H.
      • Weizman Z.
      Lactobacillus GG administered in oral rehydration solution to children with acute diarrhea: A multicenter Euopean trial.
      ). For this reason, many in vitro studies have suggested the use of this strain as a mycotoxin-removal agent in food.
      • Pierides M.
      • El-Nezami H.
      • Peltonen K.
      • Salminen S.
      • Ahokas J.
      Ability of dairy strains of lactic acid bacteria to bind aflatoxin in a food model.
      found that L. rhamnosus GG efficiently removed aflatoxin B1 from PBS by 65 to 77%, and from skim milk and full-cream milk by 26.6 and 36.6%, respectively. A study by
      • Vosough P.R.
      • Sani A.M.
      • Mehraban M.
      • Karazhyan R.
      In vitro effect of Lactobacillus rhamnosus GG on reduction of aflatoxin B1.
      also found that L. rhamnosus GG removed aflatoxin B1 from de Man, Rogosa and Sharpe broth medium by 50%. The differences in removal efficiencies between these studies may have been due to the different matrices contaminated with aflatoxin.
      • Bovo F.
      • Franco L.T.
      • Rosim R.E.
      • Trindade C.S.F.
      • de Oliveira C.A.F.
      The ability of Lactobacillus rhamnosus in solution, spray-dried or lyophilized to bind aflatoxin B1.
      found no difference in aflatoxin elimination between live and lyophilized L. rhamnosus GG cells. Therefore, lyophilized L. rhamnosus GG can be considered a practical alternative for aflatoxin B1 decontamination in food.
      The effect of L. rhamnosus GG on aflatoxin removal has also been confirmed in host cells and in animal models.
      • Gratz S.
      • Wu Q.K.
      • El-Nezami H.
      • Juvonen R.O.
      • Mykkänen H.
      • Turner P.C.
      Lactobacillus rhamnosus strain GG reduces aflatoxin B1 transport, metabolism, and toxicity in Caco-2 cells.
      evaluated the potential of L. rhamnosus GG to reduce aflatoxin B1 availability in vitro using Caco-2 cells, and found that treatment with the bacteria reduced aflatoxin B1 uptake, resulting in the protection of Caco-2 cells from both membrane and DNA damage. This result suggested a beneficial role for L. rhamnosus GG upon dietary exposure to aflatoxin.
      • Deabes M.M.
      • Darwish H.R.
      • Abdel-Aziz K.B.
      • Farag I.M.
      • Nada S.A.
      • Tawfek N.S.
      Protective effects of Lactobacillus rhamnosus GG on aflatoxins-induced toxicities in male Albino mice.
      evaluated whether L. rhamnosus GG could remove aflatoxin in vivo and showed that oral administration of L. rhamnosus GG at 1 × 10 cfu for 7 d to male albino mice significantly decreased aflatoxin-induced toxicity (0.7 mg/kg of BW) by preventing oxidative stress, and by maintaining glutathione levels and superoxide dismutase activity. Another group assessed the activity of L. rhamnosus GG in vivo and demonstrated that rats fed aflatoxin B1 (4.8 μmol/kg of BW) along with L. rhamnosus GG were safer from the hazardous effects of aflatoxin B1 (
      • Gratz S.
      • Taubel M.
      • Juvonen R.O.
      • Viluksela M.
      • Turner P.C.
      • Mykkanen H.
      • El-Nezami H.
      Lactobacillus rhamnosus strain GG modulates intestinal absorption, fecal excretion, and toxicity of aflatoxin B1 in rats.
      ).
      Taken together, these findings show that L. rhamnosus GG can be considered as a dietary supplement for effective aflatoxin removal from contaminated hosts, including humans and livestock.

      Mechanism of Aflatoxin B1 Decontamination by L. rhamnosus GG

      To determine the mechanism of aflatoxin B1 decontamination,
      • El-Nezami H.
      • Kankaanpää P.
      • Salminen S.
      • Ahokas J.
      Physico-chemical alterations enhance the ability of dairy strains of lactic acid bacteria to remove aflatoxin from contaminated media.
      evaluated the binding of aflatoxin B1 by both viable and nonviable (heat- and acid-treated) L. rhamnosus GG, revealing that even nonviable L. rhamnosus GG had the ability to bind aflatoxin B1.
      • Haskard C.A.
      • El-Nezami H.S.
      • Kankaanpää P.E.
      • Salminen S.
      • Ahokas J.T.
      Surface binding of aflatoxin B1 by lactic acid bacteria.
      also showed that the binding of aflatoxin B1 by viable and heat-treated L. rhamnosus GG strains appeared to be predominantly extracellular. Furthermore, the ability of L. rhamnosus GG to adsorb aflatoxin B1 is enhanced by the physical and chemical conditions of the medium. When bacterial cells were treated with acid or heat, the ability to bind to aflatoxin was slightly increased (
      • Haskard C.
      • Binnion C.
      • Ahokas J.
      Factors affecting the sequestration of aflatoxin by Lactobacillus rhamnosus strain GG.
      ), and in a study from
      • El-Nezami H.
      • Kankaanpää P.
      • Salminen S.
      • Ahokas J.
      Ability of dairy strains of lactic acid bacteria to bind a common food carcinogen, aflatoxin B1.
      , heat and acid treatments caused even more significant increases.
      • Vosough P.R.
      • Sani A.M.
      • Mehraban M.
      • Karazhyan R.
      In vitro effect of Lactobacillus rhamnosus GG on reduction of aflatoxin B1.
      also showed that the aflatoxin B1-binding capacity of viable (43%), heat-killed (49%), and acid-killed bacteria (50%) was not different. These results indicate that aflatoxin B1 is not removed by metabolism, but because it becomes physically bound to molecular components of the bacterium.
      • Haskard C.
      • Binnion C.
      • Ahokas J.
      Factors affecting the sequestration of aflatoxin by Lactobacillus rhamnosus strain GG.
      showed that the addition of protease or lipase to L. rhamnosus GG did not affect aflatoxin B1 binding, suggesting that binding is related to the carbohydrate components of the bacterial species and is involved in the hydrophobic and electrostatic interactions between the bacterial component and the toxin. Carbohydrates exist in 3 major forms in the bacterial cell wall: exopolysaccharides, teichoic or lipoteichoic acids, and peptidoglycans (
      • Lahtinen S.J.
      • Haskard C.A.
      • Ouwehand A.C.
      • Salminen S.J.
      • Ahokas J.T.
      Binding of aflatoxin B1 to cell wall components of Lactobacillus rhamnosus strain GG.
      ).
      • Lahtinen S.J.
      • Haskard C.A.
      • Ouwehand A.C.
      • Salminen S.J.
      • Ahokas J.T.
      Binding of aflatoxin B1 to cell wall components of Lactobacillus rhamnosus strain GG.
      extracted exopolysaccharides and a cell wall isolate containing peptidoglycans from bacteria, to establish which components of the L. rhamnosus GG cell envelope are involved in aflatoxin B1 binding. They suggested that peptidoglycans are associated with aflatoxin-B1 binding and ruled out the involvement of exopolysaccharides. They also suggested that there was no evidence of the involvement of teichoic acids, citing a study by
      • Knox K.W.
      • Wicken A.J.
      Immunological properties of teichoic acids.
      that showed no change in bacterial aflatoxin binding ability despite the removal of teichoic acid by trichloroacetic acid treatment.
      Besides L. rhamnosus GG, other lactic acid bacteria have also displayed the ability to bind aflatoxin B1.
      • Lee J.
      Adhesion of kimchi Lactobacillus strains to Caco-2 cell membrane and sequenstration of aflatoxin B1.
      showed that both heat- and acid-treated Lactobacillus plantarum KTCC3099, isolated from kimchi, also removed 49.8% of the aflatoxin B1.
      • Hernandez-Mendoza A.
      • Guzman-de-Peña D.
      • Garcia H.S.
      Key role of teichoic acids on aflatoxin B1 binding by probiotic bacteria.
      recently examined the binding of aflatoxin B1 by Lactobacillus reuteri NRRL14171, L. casei Shirota, Bifidobacterium bifidum NCFB2715, Lactobacillus johnsonii NCC533, and L. casei DN-114-001, finding that L. reuteri NRRL14171 and L. casei Shirota were the most efficient strains. The authors suggested that teichoic acids in the peptidoglycans may have been responsible for this binding, in contrast to the suggestion provided by
      • Lahtinen S.J.
      • Haskard C.A.
      • Ouwehand A.C.
      • Salminen S.J.
      • Ahokas J.T.
      Binding of aflatoxin B1 to cell wall components of Lactobacillus rhamnosus strain GG.
      . In summary, aflatoxin B1 binding by L. rhamnosus GG is clearly related to the bacterial peptidoglycans, but it has not yet been established which components are involved in the process.

      Other Microbial Binders of Aflatoxin

      Aflatoxin removal from foods or feeds can be accomplished with another lactic acid bacterium, Enterococcus faecium, which is naturally found in food and is added to dairy products such as cheese (
      • Giraffa G.
      Functionality of enterococci in dairy products.
      ;
      • Topcu A.
      • Bulat T.
      • Wishah R.
      • Boyaci I.H.
      Detoxification of aflatoxin B1 and patulin by Enterococcus faecium strains.
      ). Furthermore, E. faecium strains isolated from the feces of healthy dogs have been shown to reduce the levels of aflatoxin B1. The isolates were suggested as promising pet feed additives for aflatoxin decontamination. Similar to the findings of other studies on the aflatoxin-decontaminating capability of L. rhamnosus, nonviable and viable E. faecium strains show similar levels of decontamination, suggesting that the removal effect is again attributable not to metabolic transformation of the toxin, but to the bacterial physical adsorption (
      • Fernández Juri M.G.
      • Dalcero A.M.
      • Magnoli C.E.
      In vitro aflatoxin B1 binding capacity by two Enterococcus faecium strains isolated from healthy dog faeces.
      ).
      Saccharomyces cerevisiae also has the ability to adsorb aflatoxin to its cell wall components, as well as resistance to salivary and gastrointestinal environmental conditions. These benefits may increase the possibility of its use as feed additive for livestock such as poultry and ruminants (
      • Dogi C.A.
      • Armando R.
      • Ludueña R.
      • de Moreno de LeBlanc A.
      • Rosa C.A.
      • Dalcero A.
      • Cavaglieri L.
      Saccharomyces cerevisiae strains retain their viability and aflatoxin B1 binding ability under gastrointestinal conditions and improve ruminal fermentation.
      ;
      • Pizzolitto R.P.
      • Armando M.R.
      • Combina M.
      • Cavaglieri L.R.
      • Dalcero A.M.
      • Salvano M.A.
      Evaluation of Saccharomyces cerevisiae strains as probiotic agent with aflatoxin B1 adsorption ability for use in poultry feedstuffs.
      ). Furthermore, its aflatoxin-binding capability was not altered by co-contamination with another mycotoxin, fumonisin, produced by Fusarium spp., whose removal is also achieved by binding to the cell wall components of the microorganism (
      • Pizzolitto R.P.
      • Armando M.R.
      • Combina M.
      • Cavaglieri L.R.
      • Dalcero A.M.
      • Salvano M.A.
      Evaluation of Saccharomyces cerevisiae strains as probiotic agent with aflatoxin B1 adsorption ability for use in poultry feedstuffs.
      ).
      Another aflatoxin-binding bacterial strain, Brevibacillus lacterosporus, has been isolated from the gastrointestinal tract of Japanese quails (
      • Bagherzadeh Kasmani F.
      • Karimi Torshizi M.A.
      • Allameh A.
      • Shariatmadari F.
      A novel aflatoxin-binding Bacillus probiotic: Performance, serum biochemistry, and immunological parameters in Japanese quail.
      ). Because many Bacillus species are usually regarded as excellent probiotics for animal feeds due to their long shelf life and resistance to a number of stress conditions (
      • Shivaramaiah S.
      • Pumford N.R.
      • Morgan M.J.
      • Wolfenden R.E.
      • Wolfenden A.D.
      • Torres-Rodríguez A.
      • Hargis B.M.
      • Téllez G.
      Evaluation of Bacillus species as potential candidates for direct-fed microbials in commercial poultry.
      ), these bacteria are also prospective beneficial additives for controlling aflatoxin contamination in feeds.
      Aflatoxin M1, which is found in milk, can be removed by Lactobacillus and Bifidobacterium strains through reversible binding, but a low level of aflatoxin M1 is discharged back into the milk (
      • Kabak B.
      • Var I.
      Factors affecting the removal of aflatoxin M1 from food model by Lactobacillus and Bifidobacterium strains.
      ). Likewise, because adsorption-based aflatoxin removal is reversible, aflatoxin can be released into the gastrointestinal tracts of humans or animals that consume feeds or foods treated with aflatoxin-adsorbing microorganisms. Therefore, continuous administration of these microbial agents is needed to ensure that they reside as gut commensal flora, and to decrease aflatoxin availability inside the host.

      BIODEGRADATION

      Physical and chemical methods lead to unwanted side effects, and adsorption-associated technologies still have limitations, such as aflatoxin residues in foods, feeds, and hosts. For this reason, aflatoxin would best be removed by detoxifying action for successful decontamination. Accordingly, to compensate for these drawbacks, microbe-mediated biodegradation methods have been suggested for effective control of aflatoxin contamination at a higher rate. Indeed, many studies have reported aflatoxin-degrading fungal and bacterial strains isolated from soil, feces, and crops (Table 1, Table 2). Many aflatoxin-degrading microorganisms cleave the lactone ring of coumarin, a key aflatoxin structure responsible for its toxicity (
      • Lee L.S.
      • Dunn J.J.
      • DeLucca A.J.
      • Ciegler A.
      Role of lactone ring of aflatoxin B1 in toxicity and mutagenicity.
      ;
      • Guan S.
      • Ji C.
      • Zhou T.
      • Li J.
      • Ma Q.
      • Niu T.
      Aflatoxin B(1) degradation by Stenotrophomonas maltophilia and other microbes selected using coumarin medium.
      ). However, the use of living microorganisms as a food additive may raise safety issues and consumers may be reluctant to eat microbe-supplemented foods. The use of aflatoxin-degrading enzymes, produced by fungi and bacteria, may be able to overcome those drawbacks.
      Table 1Aflatoxin-degrading fungal strains
      NameSourceEnzymeActivity
      Percentage of aflatoxin removal (%) or specific enzyme activity (units) indicate the activity of the fungal strain in aflatoxin degradation. Conditions of activity were different for each study.
      MechanismTarget typeReference
      Armillariella tabescensNA
      NA = not available.
      Aflatoxin-oxidase3.72 U/mLOpening of bisfuran ringB1
      • Liu D.L.
      • Yao D.S.
      • Liang R.
      • Ma L.
      • Cheng W.Q.
      • Gu L.Q.
      Detoxification of aflatoxin B1 by enzymes isolated from Armillariella tabescens.
      ,
      • Liu D.L.
      • Yao D.S.
      • Liang Y.Q.
      • Zhou T.H.
      • Song Y.P.
      • Zhao L.
      • Ma L.
      Production, purification, and characterization of an intracellular aflatoxin-detoxifizyme from Armillariella tabescens (E-20).
      ;
      • Cao H.
      • Liu D.
      • Mo X.
      • Xie C.
      • Yao D.
      A fungal enzyme with the ability of aflatoxin B1 conversion: Purification and ESI-MS/MS identification.
      ;
      • Guan L.Z.
      • Sun Y.P.
      • Cai J.S.
      • Wu H.D.
      • Yu L.Z.
      • Zhang Y.L.
      • Xi Q.Y.
      The aflatoxin-detoxifizyme specific expression in mouse parotid gland.
      Aspergillus nigerGrainsLaccase118 U/L (55%)Cleavage of lactone ringB1
      • Alberts J.F.
      • Gelderblom W.C.
      • Botha A.
      • van Zyl W.H.
      Degradation of aflatoxin B(1) by fungal laccase enzymes.
      ;
      • Zhang W.
      • Xue B.
      • Li M.
      • Mu Y.
      • Chen Z.
      • Li J.
      • Shan A.
      Screening a strain of Aspergillus niger and optimization of fermentation conditions for degradation of aflatoxin B1.
      PeniophoraNALaccase496 U/L (40.45%)Cleavage of lactone ringB1
      • Alberts J.F.
      • Gelderblom W.C.
      • Botha A.
      • van Zyl W.H.
      Degradation of aflatoxin B(1) by fungal laccase enzymes.
      Phanerochaete sordidaNAManganese peroxidase86%Cleavage of lactone ringB1
      • Wang J.
      • Ogata M.
      • Hirai H.
      • Kawagishi H.
      Detoxification of aflatoxin B1 by manganese peroxidase from the white-rot fungus Phanerochaete sordida YK-624.
      Phoma glomerataBeans of Phaseolus vulgaris L.NA78%NAB1
      • Shcherbakova L.
      • Statsyuk N.
      • Mikityuk O.
      • Nazarova T.
      • Dzhavakhiya V.
      Aflatoxin B1 degradation by metabolites of Phoma glomerata PG41 isolated from natural substrate colonized by aflatoxigenic Aspergillus flavus.
      Pleurotus ostreatusDecomposing tree trunk, laboratory collectionManganese peroxidase, laccase416.39 U/L (35.90%)Cleavage of lactone ringB1
      • Motomura M.
      • Toyomasu T.
      • Mizuno K.
      • Shinozawa T.
      Purification and characterization of an aflatoxin degradation enzyme from Pleurotus ostreatus.
      ;
      • Alberts J.F.
      • Gelderblom W.C.
      • Botha A.
      • van Zyl W.H.
      Degradation of aflatoxin B(1) by fungal laccase enzymes.
      ;
      • Das A.
      • Bhattacharya S.
      • Palaniswamy M.
      • Angayarkanni J.
      Biodegradation of aflatoxin B1 in contaminated rice straw by Pleurotus ostreatus MTCC 142 and Pleurotus ostreatus GHBBF10 in the presence of metal salts and surfactants.
      ;
      • Yehia R.S.
      Aflatoxin detoxification by manganese peroxidase purified from Pleurotus ostreatus.
      ;
      • Ginterová A.
      • Polster M.
      • Janotková O.
      The relationship between Pleurotus ostreatus and Aspergillus flavus and the production of aflatoxin.
      Rhizopus oligosporusNANA64.50%NAB1
      • Kusumaningtyas E.
      • Widiastuti R.
      • Maryam R.
      Reduction of aflatoxin B1 in chicken feed by using Saccharomyces cerevisiae, Rhizopus oligosporus and their combination.
      Rhizopus oryzaeRice huskPeroxidase100%Cleavage of lactone ringB1, B2, G1, G2, M1
      • Hackbart H.C.
      • Machado A.R.
      • Christ-Ribeiro A.
      • Prietto L.
      • Badiale-Furlong E.
      Reduction of aflatoxins by Rhizopus oryzae and Trichoderma reesei.
      Saccharomyces cerevisiaeNANA70%HydrationB1
      • Inoue T.
      • Nagatomi Y.
      • Uyama A.
      • Naoki M.
      Degradation of aflatoxin B1 during the fermentation of alcoholic beverages.
      Trametes versicolorNALaccase1 U/mL (87.34%)Cleavage of lactone ringB1
      • Alberts J.F.
      • Gelderblom W.C.
      • Botha A.
      • van Zyl W.H.
      Degradation of aflatoxin B(1) by fungal laccase enzymes.
      Trichoderma reeseiCulture collectionPeroxidase100%Cleavage of lactone ringB1, B2, G1, M1
      • Hackbart H.C.
      • Machado A.R.
      • Christ-Ribeiro A.
      • Prietto L.
      • Badiale-Furlong E.
      Reduction of aflatoxins by Rhizopus oryzae and Trichoderma reesei.
      1 Percentage of aflatoxin removal (%) or specific enzyme activity (units) indicate the activity of the fungal strain in aflatoxin degradation. Conditions of activity were different for each study.
      2 NA = not available.
      Table 2Aflatoxin-degrading bacterial strains
      NameSourceEnzymeActivity
      Percentage of aflatoxin removal (%) or specific enzyme activity (units) indicate the activity of the bacterial strain in aflatoxin degradation. Conditions of activity were different for each study.
      MechanismTarget typeReference
      Bacillus licheniformisThai fermented soybeanNA
      NA = not available.
      74%NAB1
      • Petchkongkaew A.
      • Taillandier P.
      • Gasaluck P.
      • Lebrihi A.
      Isolation of Bacillus spp. from Thai fermented soybean (Thua-nao): Screening for aflatoxin B1 and ochratoxin A detoxification.
      Bacillus stearothermophilusNANA87%NAB1
      • Smith J.E.
      • Harran G.
      Microbial degradation of mycotoxins.
      Bacillus subtilisFish gut, Thai fermented soybeanNA85%Cleavage of lactone ringB1, G1, M1
      • Petchkongkaew A.
      • Taillandier P.
      • Gasaluck P.
      • Lebrihi A.
      Isolation of Bacillus spp. from Thai fermented soybean (Thua-nao): Screening for aflatoxin B1 and ochratoxin A detoxification.
      ;
      • Ma Q.G.
      • Gao X.
      • Zhou T.
      • Zhao L.H.
      • Fan Y.
      • Li X.Y.
      • Lei Y.P.
      • Ji C.
      • Zhang J.Y.
      Protective effect of Bacillus subtilis ANSB060 on egg quality, biochemical and histopathological changes in layers exposed to aflatoxin B1.
      ;
      • Fan Y.
      • Zhao L.
      • Ma Q.
      • Li X.
      • Shi H.
      • Zhou T.
      • Zhang J.
      • Ji C.
      Effects of Bacillus subtilis ANSB060 on growth performance, meat quality and aflatoxin residues in broilers fed moldy peanut meal naturally contaminated with aflatoxins.
      ;
      • Gao X.
      • Ma Q.
      • Zhao L.
      • Lei Y.
      • Shan Y.
      • Cheng J.
      Isolation of Bacillus subtilis: Screening for aflatoxins B1, M1, and G1 detoxication.
      Brachybacterium sp.Rabbit fecesNA74.83%Cleavage of lactone ringB1
      • Guan S.
      • Ji C.
      • Zhou T.
      • Li J.
      • Ma Q.
      • Niu T.
      Aflatoxin B(1) degradation by Stenotrophomonas maltophilia and other microbes selected using coumarin medium.
      Brevundimonas sp.Yellow cheek feces, goral fecesNA78.10%Cleavage of lactone ringB1
      • Guan S.
      • Ji C.
      • Zhou T.
      • Li J.
      • Ma Q.
      • Niu T.
      Aflatoxin B(1) degradation by Stenotrophomonas maltophilia and other microbes selected using coumarin medium.
      Cellulosimicrobium funkeiSoil around coal factoriesNA97%Cleavage of lactone ringB1
      • Sun L.H.
      • Zhang N.Y.
      • Sun R.R.
      • Gao X.
      • Gu C.
      • Krumm C.S.
      • Qi D.S.
      A novel strain of Cellulosimicrobium funkei can biologically detoxify aflatoxin B1 in ducklings.
      Enterobacter sp.Hog deer fecesNA75.92%Cleavage of lactone ringB1
      • Guan S.
      • Ji C.
      • Zhou T.
      • Li J.
      • Ma Q.
      • Niu T.
      Aflatoxin B(1) degradation by Stenotrophomonas maltophilia and other microbes selected using coumarin medium.
      Klebsiella sp.Rabbit fecesNA77.57%Cleavage of lactone ringB1
      • Guan S.
      • Ji C.
      • Zhou T.
      • Li J.
      • Ma Q.
      • Niu T.
      Aflatoxin B(1) degradation by Stenotrophomonas maltophilia and other microbes selected using coumarin medium.
      Mycobacterium  fluoranthenivorans sp.Soil of a former coal gas plantNA>90%NAB1
      • Teniola O.D.
      • Addo P.A.
      • Brost I.M.
      • Färber P.
      • Jany K.D.
      • Alberts J.F.
      • van Zyl W.H.
      • Steyn P.S.
      • Holzapfel W.H.
      Degradation of aflatoxin B(1) by cell-free extracts of Rhodococcus erythropolis and Mycobacterium fluoranthenivorans sp. nov. DSM44556(T).
      ;
      • Hormisch D.
      • Brost I.
      • Kohring G.W.
      • Giffhorn F.
      • Kroppenstedt R.M.
      • Stackebrandt E.
      • Färber P.
      • Holzapfel W.H.
      Mycobacterium fluoranthenivorans sp. nov., a fluoranthene and aflatoxin B1 degrading bacterium from contaminated soil of a former coal gas plant.
      Mycobacterium  smegmatisATCC 700084 (type strain)F420H2-dependent reductase83,000 nmol·min−1·μmol−1 enzymeReduction of α,β-unsaturated double bond C2–C6B1, B2, G1, G2
      • Taylor M.C.
      • Jackson C.J.
      • Tattersall D.B.
      • French N.
      • Peat T.S.
      • Newman J.
      • Briggs L.J.
      • Lapalikar G.V.
      • Campbell P.M.
      • Scott C.
      • Russell R.J.
      • Oakeshott J.G.
      Identification and characterization of two families of F420 H2-dependent reductases from Mycobacteria that catalyse aflatoxin degradation.
      ;
      • Lapalikar G.V.
      • Taylor M.C.
      • Warden A.C.
      • Scott C.
      • Russell R.J.
      • Oakeshott J.G.
      F420H2-dependent degradation of aflatoxin and other furanocoumarins is widespread throughout the actinomycetales.
      Myxococcus fulvusDeer fecesNA96.96%NAB1, G1, M1
      • Zhao L.H.
      • Guan S.
      • Gao X.
      • Ma Q.G.
      • Lei Y.P.
      • Bai X.M.
      • Ji C.
      Preparation, purification and characteristics of an aflatoxin degradation enzyme from Myxococcus fulvus ANSM068.
      Flavobacterium aurantiacumNANA>90%NAB1
      • Ciegler A.
      • Lillehoj E.B.
      • Peterson R.E.
      • Hall H.H.
      Microbial detoxification of aflatoxin.
      ;
      • Smiley R.D.
      • Draughon F.A.
      Preliminary evidence that degradation of aflatoxin B1 by Flavobacterium aurantiacum is enzymatic.
      ;
      • D'Souza D.H.
      • Brackett R.E.
      Aflatoxin B1 degradation by flavobacterium aurantiacum in the presence of reducing conditions and seryl and sulfhydryl group inhibitors.
      ;
      • Teniola O.D.
      • Addo P.A.
      • Brost I.M.
      • Färber P.
      • Jany K.D.
      • Alberts J.F.
      • van Zyl W.H.
      • Steyn P.S.
      • Holzapfel W.H.
      Degradation of aflatoxin B(1) by cell-free extracts of Rhodococcus erythropolis and Mycobacterium fluoranthenivorans sp. nov. DSM44556(T).
      Pseudomonas aeruginosaGrain kernels and soilsNA82.80%NAB1, B2, M2
      • Sangare L.
      • Zhao Y.
      • Folly Y.M.
      • Chang J.
      • Li J.
      • Selvaraj J.N.
      • Xing F.
      • Zhou L.
      • Wang Y.
      • Liu Y.
      Aflatoxin B1 degradation by a Pseudomonas strain.
      Pseudomonas stutzeriBudorcas taxicolor fecesNA90.03%NAB1
      • Li C.
      • Li W.
      • Yang W.
      • Li H.
      • Liu X.
      • Cao Y.
      Isolation and characterisation of an aflatoxin B1-degrading bacterium.
      Pseudomonas putidaNANA100%NAB1
      • Samuel M.S.
      • Sivaramakrishna A.
      • Mehta A.
      Degradation and detoxification of aflatoxin B1 by Pseudomonas putida.
      Rhodococcus erythropolisNANA4.16 mU/mLCleavage of lactone ringB1
      • Alberts J.F.
      • Engelbrecht Y.
      • Steyn P.S.
      • Holzapfel W.H.
      • van Zyl W.H.
      Biological degradation of aflatoxin B1 by Rhodococcus erythropolis cultures.
      ;
      • Teniola O.D.
      • Addo P.A.
      • Brost I.M.
      • Färber P.
      • Jany K.D.
      • Alberts J.F.
      • van Zyl W.H.
      • Steyn P.S.
      • Holzapfel W.H.
      Degradation of aflatoxin B(1) by cell-free extracts of Rhodococcus erythropolis and Mycobacterium fluoranthenivorans sp. nov. DSM44556(T).
      ;
      • Kong Q.
      • Zhai C.
      • Guan B.
      • Li C.
      • Shan S.
      • Yu J.
      Mathematic modeling for optimum conditions on aflatoxin B1 degradation by the aerobic bacterium Rhodococcus erythropolis.
      ;
      • Eshelli M.
      • Harvey L.
      • Edrada-Ebel R.
      • McNeil B.
      Metabolomics of the bio-degradation process of aflatoxin B1 by actinomycetes at an initial pH of 6.0.
      ;
      • Cserháti M.
      • Kriszt B.
      • Krifaton C.
      • Szoboszlay S.
      • Háhn J.
      • Tóth S.
      • Nagy I.
      • Kukolya J.
      Mycotoxin-degradation profile of Rhodococcus strains.
      Streptomyces aureofaciensLaboratory collectionNA87.95%NAB1
      • Eshelli M.
      • Harvey L.
      • Edrada-Ebel R.
      • McNeil B.
      Metabolomics of the bio-degradation process of aflatoxin B1 by actinomycetes at an initial pH of 6.0.
      Streptomyces lividansLaboratory collectionNA86.10%NAB1
      • Eshelli M.
      • Harvey L.
      • Edrada-Ebel R.
      • McNeil B.
      Metabolomics of the bio-degradation process of aflatoxin B1 by actinomycetes at an initial pH of 6.0.
      Stenotrophomonas  maltophiliaSouth American tapir fecesNA84.80%Cleavage of lactone ringB1
      • Guan S.
      • Ji C.
      • Zhou T.
      • Li J.
      • Ma Q.
      • Niu T.
      Aflatoxin B(1) degradation by Stenotrophomonas maltophilia and other microbes selected using coumarin medium.
      1 Percentage of aflatoxin removal (%) or specific enzyme activity (units) indicate the activity of the bacterial strain in aflatoxin degradation. Conditions of activity were different for each study.
      2 NA = not available.

      Enzymes of Fungal Origin

      Aspergillus flavus secretes aflatoxin to thrive in the presence of other microorganisms in the same ecological niches (
      • Shcherbakova L.
      • Statsyuk N.
      • Mikityuk O.
      • Nazarova T.
      • Dzhavakhiya V.
      Aflatoxin B1 degradation by metabolites of Phoma glomerata PG41 isolated from natural substrate colonized by aflatoxigenic Aspergillus flavus.
      ). Some fungi such as Phoma glomerata PG41, a fungus pathogen causing fiber spoilage, survive and coexist with aflatoxigenic A. flavus by producing aflatoxin-degrading enzymes (
      • Shcherbakova L.
      • Statsyuk N.
      • Mikityuk O.
      • Nazarova T.
      • Dzhavakhiya V.
      Aflatoxin B1 degradation by metabolites of Phoma glomerata PG41 isolated from natural substrate colonized by aflatoxigenic Aspergillus flavus.
      ). Interestingly, other Aspergillus species are involved in aflatoxin degradation. For example, Aspergillus niger, an isolate from feed samples, can biodegrade aflatoxin B1, potentially serving as a feed additive (
      • Zhang W.
      • Xue B.
      • Li M.
      • Mu Y.
      • Chen Z.
      • Li J.
      • Shan A.
      Screening a strain of Aspergillus niger and optimization of fermentation conditions for degradation of aflatoxin B1.
      ).
      Among fungal strains belonging to the phylum Basidiomycota, white-rot fungi such as Peniophora, Pleurotus ostretus, and Trametes versicolor convert aflatoxin B1 into less toxic substances (
      • Guan S.
      • Ji C.
      • Zhou T.
      • Li J.
      • Ma Q.
      • Niu T.
      Aflatoxin B(1) degradation by Stenotrophomonas maltophilia and other microbes selected using coumarin medium.
      ;
      • Alberts J.F.
      • Gelderblom W.C.
      • Botha A.
      • van Zyl W.H.
      Degradation of aflatoxin B(1) by fungal laccase enzymes.
      ). These fungal strains secrete oxidative enzymes such as laccase and manganese peroxidase, which contribute to detoxifying aflatoxin (
      • Alberts J.F.
      • Gelderblom W.C.
      • Botha A.
      • van Zyl W.H.
      Degradation of aflatoxin B(1) by fungal laccase enzymes.
      ;
      • da Luz J.M.
      • Nunes M.D.
      • Paes S.A.
      • Torres D.P.
      • de Cássia Soares da Silva M.
      • Kasuya M.C.
      Lignocellulolytic enzyme production of Pleurotus ostreatus growth in agroindustrial wastes.
      ). Laccase, a copper-containing oxidase, is known to degrade various xenobiotics, including aflatoxin, due its to low substrate specificity (
      • Alberts J.F.
      • Gelderblom W.C.
      • Botha A.
      • van Zyl W.H.
      Degradation of aflatoxin B(1) by fungal laccase enzymes.
      ). The activity of these enzymes is attributed to cleavage of the aflatoxin lactone ring, abolishing or decreasing fluorescence (
      • Lee L.S.
      • Dunn J.J.
      • DeLucca A.J.
      • Ciegler A.
      Role of lactone ring of aflatoxin B1 in toxicity and mutagenicity.
      ;
      • Motomura M.
      • Toyomasu T.
      • Mizuno K.
      • Shinozawa T.
      Purification and characterization of an aflatoxin degradation enzyme from Pleurotus ostreatus.
      ). Among these strains, the edible mushroom Pleurotus ostretus can use a variety of crop wastes, including corn cobs, rice straw, rye, and sawdust as a nutrient for growth (
      • Sánchez C.
      Cultivation of Pleurotus ostreatus and other edible mushrooms.
      ). Aflatoxin B1 in contaminated rice straw was successfully degraded by P. ostretus, and the activity was enhanced in the presence of metal salts and surfactants, suggesting the usefulness of this fungus as an aflatoxin-biodegradable agent in agronomy and animal husbandry (
      • Das A.
      • Bhattacharya S.
      • Palaniswamy M.
      • Angayarkanni J.
      Biodegradation of aflatoxin B1 in contaminated rice straw by Pleurotus ostreatus MTCC 142 and Pleurotus ostreatus GHBBF10 in the presence of metal salts and surfactants.
      ). Manganese peroxidase purified from Phanerochaete sordida was capable of removing 70% of aflatoxin in vitro, increasing to 100% with multiple additions of the enzyme (
      • Wang J.
      • Ogata M.
      • Hirai H.
      • Kawagishi H.
      Detoxification of aflatoxin B1 by manganese peroxidase from the white-rot fungus Phanerochaete sordida YK-624.
      ).
      Producers of enzymes that are generally recognized as safe, such as Rhizopus oryzae and Trichoderma reesei, are also associated with detoxification of aflatoxins B1, B2, G1, G2, and M1 (
      • Hackbart H.C.
      • Machado A.R.
      • Christ-Ribeiro A.
      • Prietto L.
      • Badiale-Furlong E.
      Reduction of aflatoxins by Rhizopus oryzae and Trichoderma reesei.
      ). These strains reduced aflatoxin B1 in contaminated defatted rice bran by 80% (
      • Cacciamani J.L.M.
      • Garda-Buffon J.
      • Badiale-Furlon E.
      Fungal fermentation: Proteic enrichment and mycotoxins degradation in cereal bran contaminated by aflatoxin B1 and ochratoxin A.
      ). Other Rhizopus species, including Rhizopus oligosporus, Rhizopus arrhizus, and Rhizopus stolonifer, have displayed aflatoxin-degrading ability. Of the Rhizopus species, R. oligosporus had the most powerful activity, exhibiting the best ability in combination with Saccharomyces cerevisiae, an aflatoxin-biotransforming yeast (
      • Kusumaningtyas E.
      • Widiastuti R.
      • Maryam R.
      Reduction of aflatoxin B1 in chicken feed by using Saccharomyces cerevisiae, Rhizopus oligosporus and their combination.
      ). In fact, the aflatoxin B1 contaminating the raw materials used for beer and wine can be converted into a less toxic substance, a hydrated form of aflatoxin B1, during fermentation with Saccharomyces species, Saccharomyces pastorianus or S. cerevisiae (
      • Inoue T.
      • Nagatomi Y.
      • Uyama A.
      • Naoki M.
      Degradation of aflatoxin B1 during the fermentation of alcoholic beverages.
      ).
      Armillariella tabescens, an edible mushroom, is a natural treatment agent for several diseases, including appendicitis, cholecystitis, hepatitis, and otitis media (
      • JSNMC (JiangSu Medical Colleage)
      ), and it is a powerful degrader of aflatoxin. Specifically, the fungal strain detoxifies aflatoxin by producing enzymes exerting dual reactions: epoxide formation and epoxide hydrolysis to dihydrodiol (
      • Liu D.L.
      • Yao D.S.
      • Liang R.
      • Ma L.
      • Cheng W.Q.
      • Gu L.Q.
      Detoxification of aflatoxin B1 by enzymes isolated from Armillariella tabescens.
      ). The detoxifying enzyme is called aflatoxin-oxidase, unlike other aflatoxin-degrading enzymes, such as fungal laccase and horseradish peroxidase (
      • Li S.C.
      • Chen J.H.
      • Cao H.
      • Yao D.S.
      • Liu D.L.
      Amperometric biosensor for aflatoxin B1 based on aflatoxin-oxidase immobilized on multiwalled carbon nanotubes.
      ). The dual reactions result in cleavage of the bisfuran ring, a primary toxic structure of the aflatoxin (
      • Liu D.L.
      • Yao D.S.
      • Liang R.
      • Ma L.
      • Cheng W.Q.
      • Gu L.Q.
      Detoxification of aflatoxin B1 by enzymes isolated from Armillariella tabescens.
      ;
      • McKean C.
      • Tang L.
      • Tang M.
      • Billam M.
      • Wang Z.
      • Theodorakis C.W.
      • Kendall R.J.
      • Wang J.S.
      Comparative acute and combinative toxicity of aflatoxin B1 and fumonisin B1 in animals and human cells.
      ;
      • Cao H.
      • Liu D.
      • Mo X.
      • Xie C.
      • Yao D.
      A fungal enzyme with the ability of aflatoxin B1 conversion: Purification and ESI-MS/MS identification.
      ). Opening of the bisfuran ring does not cause a change in fluorescence, a physicochemical property of aflatoxin (
      • Cao H.
      • Liu D.
      • Mo X.
      • Xie C.
      • Yao D.
      A fungal enzyme with the ability of aflatoxin B1 conversion: Purification and ESI-MS/MS identification.
      ).

      Enzymes of Bacterial Origin

      Aflatoxin-detoxifying enzymes from fungi are usually more stable than those from bacteria, but enzymes of certain bacterial origin seem to work more rapidly than those of fungal origin (
      • Praveen Rao J.
      • Sashidhar R.B.
      • Subramanyam C.
      Inhibition of aflatoxin production by trifluoperazine in Aspergillus parasiticus NRRL 2999.
      ).
      Actinomycetales of the suborder Corynebacterineae, including Mycobacterium, Rhodococcus, and Norcardia genera, are known to biodegrade aflatoxin (
      • Taylor M.C.
      • Jackson C.J.
      • Tattersall D.B.
      • French N.
      • Peat T.S.
      • Newman J.
      • Briggs L.J.
      • Lapalikar G.V.
      • Campbell P.M.
      • Scott C.
      • Russell R.J.
      • Oakeshott J.G.
      Identification and characterization of two families of F420 H2-dependent reductases from Mycobacteria that catalyse aflatoxin degradation.
      ). It has been reported that Mycobacterium fluoranthenivorans sp. nov., which metabolized polycyclic aromatic hydrocarbons in the soil of a former coal gas plant, could biotransform aflatoxin B1 into a less toxigenic substance, suggesting its use for decontamination of aflatoxin in foods and livestock feeds (
      • Hormisch D.
      • Brost I.
      • Kohring G.W.
      • Giffhorn F.
      • Kroppenstedt R.M.
      • Stackebrandt E.
      • Färber P.
      • Holzapfel W.H.
      Mycobacterium fluoranthenivorans sp. nov., a fluoranthene and aflatoxin B1 degrading bacterium from contaminated soil of a former coal gas plant.
      ;
      • Teniola O.D.
      • Addo P.A.
      • Brost I.M.
      • Färber P.
      • Jany K.D.
      • Alberts J.F.
      • van Zyl W.H.
      • Steyn P.S.
      • Holzapfel W.H.
      Degradation of aflatoxin B(1) by cell-free extracts of Rhodococcus erythropolis and Mycobacterium fluoranthenivorans sp. nov. DSM44556(T).
      ). Furthermore, cofactor F420-dependent reductase from Mycobacterium smegmatis has been shown to detoxify aflatoxin by catalyzing reduction of the α,β-unsaturated lactone moiety and its subsequent hydrolysis (
      • Taylor M.C.
      • Jackson C.J.
      • Tattersall D.B.
      • French N.
      • Peat T.S.
      • Newman J.
      • Briggs L.J.
      • Lapalikar G.V.
      • Campbell P.M.
      • Scott C.
      • Russell R.J.
      • Oakeshott J.G.
      Identification and characterization of two families of F420 H2-dependent reductases from Mycobacteria that catalyse aflatoxin degradation.
      ;
      • Lapalikar G.V.
      • Taylor M.C.
      • Warden A.C.
      • Scott C.
      • Russell R.J.
      • Oakeshott J.G.
      F420H2-dependent degradation of aflatoxin and other furanocoumarins is widespread throughout the actinomycetales.
      ). Rhodococcus erythropolis, a gram-positive bacterium closely related to Mycobacterium, has also been extensively studied for its effectiveness in aflatoxin B1 degradation and optimization of the degradation conditions (
      • Alberts J.F.
      • Engelbrecht Y.
      • Steyn P.S.
      • Holzapfel W.H.
      • van Zyl W.H.
      Biological degradation of aflatoxin B1 by Rhodococcus erythropolis cultures.
      ;
      • Kong Q.
      • Zhai C.
      • Guan B.
      • Li C.
      • Shan S.
      • Yu J.
      Mathematic modeling for optimum conditions on aflatoxin B1 degradation by the aerobic bacterium Rhodococcus erythropolis.
      ). Extracellular extracts of R. erythropolis liquid culture were responsible for the bioconversion of aflatoxin by an enzymatic process (
      • Alberts J.F.
      • Engelbrecht Y.
      • Steyn P.S.
      • Holzapfel W.H.
      • van Zyl W.H.
      Biological degradation of aflatoxin B1 by Rhodococcus erythropolis cultures.
      ). The conditions showing the best degrading efficiency were 23°C and pH 7.0 (
      • Kong Q.
      • Zhai C.
      • Guan B.
      • Li C.
      • Shan S.
      • Yu J.
      Mathematic modeling for optimum conditions on aflatoxin B1 degradation by the aerobic bacterium Rhodococcus erythropolis.
      ). However, another research group had different findings for optimal conditions, suggesting they were 30°C and pH 6.0 (
      • Eshelli M.
      • Harvey L.
      • Edrada-Ebel R.
      • McNeil B.
      Metabolomics of the bio-degradation process of aflatoxin B1 by actinomycetes at an initial pH of 6.0.
      ). Living cells of Norcardia corynebacterioides (formerly Flavobacterium aurantiacum) biodegraded aflatoxin B1 in aqueous foods such as peanut milk, and the degradation was attributed to an enzyme that is maximally active at neutral pH (
      • Hao Y.Y.
      • Brackett R.E.
      Removal of aflatoxin B1 from peanut milk inoculated with Flavobacterium aurantiacum.
      ;
      • Smiley R.D.
      • Draughon F.A.
      Preliminary evidence that degradation of aflatoxin B1 by Flavobacterium aurantiacum is enzymatic.
      ). Streptomyces, which belong to the family of Actinomycetales, are also involved in aflatoxin bioconversion (e.g., Streptomyces lividans and Streptomyces aureofaciens), and the activity of biodegradation was maximal in acidic conditions, at pH 5.0 (
      • Eshelli M.
      • Harvey L.
      • Edrada-Ebel R.
      • McNeil B.
      Metabolomics of the bio-degradation process of aflatoxin B1 by actinomycetes at an initial pH of 6.0.
      ).
      Interestingly, several bacteria harboring aflatoxin-degrading activity have been isolated from feces, with coumarin as their carbon source. The culture supernatant of Stenotrophomonas maltophilia was capable of degrading aflatoxin effectively at 37°C, pH 8.0 with the addition of ions Mg2+ and Cu2+, at the highest degradation efficiency (
      • Guan S.
      • Ji C.
      • Zhou T.
      • Li J.
      • Ma Q.
      • Niu T.
      Aflatoxin B(1) degradation by Stenotrophomonas maltophilia and other microbes selected using coumarin medium.
      ). Unlike S. maltophilia, another Stenotrophomonas sp., an isolate from a soil sample, inhibited aflatoxin production and possibly prevented aflatoxin contamination in the presence of aflatoxin-producing Aspergillus strains (
      • Jermnak U.
      • Chinaphuti A.
      • Poapolathep A.
      • Kawai R.
      • Nagasawa H.
      • Sakuda S.
      Prevention of aflatoxin contamination by a soil bacterium of Stenotrophomonas sp. that produces aflatoxin production inhibitors.
      ). Furthermore, other bacteria such as Bacillus spp., Brachybacterium spp., Enterobacter spp., Brevundimonas spp., Klebsiella spp., and Rhodococcus spp., have been isolated from animal feces as metabolic degraders of aflatoxin (
      • Guan S.
      • Ji C.
      • Zhou T.
      • Li J.
      • Ma Q.
      • Niu T.
      Aflatoxin B(1) degradation by Stenotrophomonas maltophilia and other microbes selected using coumarin medium.
      ).
      Myxococcus fulvus, an isolate obtained from deer feces, possesses an extracellular enzyme that is responsible for the degradation-mediated removal of aflatoxins B1, G1, and M1 in solution (
      • Zhao L.H.
      • Guan S.
      • Gao X.
      • Ma Q.G.
      • Lei Y.P.
      • Bai X.M.
      • Ji C.
      Preparation, purification and characteristics of an aflatoxin degradation enzyme from Myxococcus fulvus ANSM068.
      ). Furthermore, the enzyme activity was maintained at a wide range of pH values (5–7) and temperatures (30–45°C), which may be beneficial for the efficient degradation of aflatoxin, especially in the animal digestive system (
      • Zhao L.H.
      • Guan S.
      • Gao X.
      • Ma Q.G.
      • Lei Y.P.
      • Bai X.M.
      • Ji C.
      Preparation, purification and characteristics of an aflatoxin degradation enzyme from Myxococcus fulvus ANSM068.
      ). Similar to enzymes from Norcadia corynebacterioides and Stenotrophomonas maltophilia, the activity of the enzyme was enhanced in the presence of Mg2+, although it was reduced in the presence of Zn2+, suggesting that these enzymes are from the same family (
      • D'Souza D.H.
      • Brackett R.E.
      The influence of divalent cations and chelators on aflatoxin B1 degradation by Flavobacterium aurantiacum.
      ;
      • Guan S.
      • Ji C.
      • Zhou T.
      • Li J.
      • Ma Q.
      • Niu T.
      Aflatoxin B(1) degradation by Stenotrophomonas maltophilia and other microbes selected using coumarin medium.
      ;
      • Zhao L.H.
      • Guan S.
      • Gao X.
      • Ma Q.G.
      • Lei Y.P.
      • Bai X.M.
      • Ji C.
      Preparation, purification and characteristics of an aflatoxin degradation enzyme from Myxococcus fulvus ANSM068.
      ). However, it seems difficult for bacteria to produce these enzymes on an industrial scale, because they can be easily contaminated with other bacteria during fermentation, and the production yield of the enzyme is low (
      • Zhao L.H.
      • Guan S.
      • Gao X.
      • Ma Q.G.
      • Lei Y.P.
      • Bai X.M.
      • Ji C.
      Preparation, purification and characteristics of an aflatoxin degradation enzyme from Myxococcus fulvus ANSM068.
      ).
      Several studies have reported that Bacillus subtilis is also capable of detoxifying aflatoxins B1, M1, and G1 (
      • Petchkongkaew A.
      • Taillandier P.
      • Gasaluck P.
      • Lebrihi A.
      Isolation of Bacillus spp. from Thai fermented soybean (Thua-nao): Screening for aflatoxin B1 and ochratoxin A detoxification.
      ;
      • Gao X.
      • Ma Q.
      • Zhao L.
      • Lei Y.
      • Shan Y.
      • Cheng J.
      Isolation of Bacillus subtilis: Screening for aflatoxins B1, M1, and G1 detoxication.
      ;
      • Ma Q.G.
      • Gao X.
      • Zhou T.
      • Zhao L.H.
      • Fan Y.
      • Li X.Y.
      • Lei Y.P.
      • Ji C.
      • Zhang J.Y.
      Protective effect of Bacillus subtilis ANSB060 on egg quality, biochemical and histopathological changes in layers exposed to aflatoxin B1.
      ;
      • Fan Y.
      • Zhao L.
      • Ma Q.
      • Li X.
      • Shi H.
      • Zhou T.
      • Zhang J.
      • Ji C.
      Effects of Bacillus subtilis ANSB060 on growth performance, meat quality and aflatoxin residues in broilers fed moldy peanut meal naturally contaminated with aflatoxins.
      ). The B. subtilis ANSB060 strain, an isolate from fish gut, inhibited the growth of Aspergillus flavus, degraded aflatoxin, and was resistant to adverse conditions such as simulated gastric and intestinal environments. More importantly, its detoxification effect was confirmed in a chicken broiler fed peanuts naturally contaminated with aflatoxins and in laying hens exposed to certain levels of aflatoxins, strengthening the case for its utility as a feed additive (
      • Gao X.
      • Ma Q.
      • Zhao L.
      • Lei Y.
      • Shan Y.
      • Cheng J.
      Isolation of Bacillus subtilis: Screening for aflatoxins B1, M1, and G1 detoxication.
      ;
      • Ma Q.G.
      • Gao X.
      • Zhou T.
      • Zhao L.H.
      • Fan Y.
      • Li X.Y.
      • Lei Y.P.
      • Ji C.
      • Zhang J.Y.
      Protective effect of Bacillus subtilis ANSB060 on egg quality, biochemical and histopathological changes in layers exposed to aflatoxin B1.
      ). Furthermore, B. subtilis and Bacillus licheniformis isolated from Thai fermented soybean biotransformed aflatoxins (
      • Petchkongkaew A.
      • Taillandier P.
      • Gasaluck P.
      • Lebrihi A.
      Isolation of Bacillus spp. from Thai fermented soybean (Thua-nao): Screening for aflatoxin B1 and ochratoxin A detoxification.
      ).
      It was recently reported that Pseudomonas strains also act as aflatoxin-degrading agents. Pseudomonas stutzeri, an isolate from Budorcas taxicolor feces, has been suggested as a good aflatoxin-degrader (
      • Li C.
      • Li W.
      • Yang W.
      • Li H.
      • Liu X.
      • Cao Y.
      Isolation and characterisation of an aflatoxin B1-degrading bacterium.
      ). Furthermore, Pseudomonas putida has been shown to convert aflatoxin B1 into the less toxic metabolite, aflatoxin D (
      • Samuel M.S.
      • Sivaramakrishna A.
      • Mehta A.
      Degradation and detoxification of aflatoxin B1 by Pseudomonas putida.
      ). Pseudomonas aeruginosa N17-1, isolated from grain kernels and soils, displayed enzyme-based degradation of aflatoxins B1, B2, and M1. The highest ratio of degradation was observed at 55°C and in the presence of metal ions such as Mn2+ and Cu2+, but not Mg2+, Li2+, Zn2+, Se2+, or Fe2+ (
      • Sangare L.
      • Zhao Y.
      • Folly Y.M.
      • Chang J.
      • Li J.
      • Selvaraj J.N.
      • Xing F.
      • Zhou L.
      • Wang Y.
      • Liu Y.
      Aflatoxin B1 degradation by a Pseudomonas strain.
      ).
      Cellulosimicrobium funkei, a novel strain known as an opportunistic pathogen (
      • Petkar H.
      • Li A.
      • Bunce N.
      • Duffy K.
      • Malnick H.
      • Shah J.J.
      Cellulosimicrobium funkei: First report of infection in a nonimmunocompromised patient and useful phenotypic tests for differentiation from Cellulosimicrobium cellulans and Cellulosimicrobium terreum.
      ), reduced the amount of aflatoxin B1 (97%) in ducklings by detoxifying the mycotoxin (
      • Sun L.H.
      • Zhang N.Y.
      • Sun R.R.
      • Gao X.
      • Gu C.
      • Krumm C.S.
      • Qi D.S.
      A novel strain of Cellulosimicrobium funkei can biologically detoxify aflatoxin B1 in ducklings.
      ). More desirably, the administration of the bacterial strain to ducklings, even at high levels, was found to be non-toxigenic, thus suggesting that C. funkei could be used as a feed additive, playing a role in diminishing aflatoxicosis in ducklings (
      • Sun L.H.
      • Zhang N.Y.
      • Sun R.R.
      • Gao X.
      • Gu C.
      • Krumm C.S.
      • Qi D.S.
      A novel strain of Cellulosimicrobium funkei can biologically detoxify aflatoxin B1 in ducklings.
      ).
      Many bacterial strains are associated with aflatoxin degradation and may be applicable as feed additives, especially for cows. However, because the effects of these bacterial strains vary according to environmental conditions such as temperature, pH, and the presence of metal ions as enzyme cofactors, further studies are needed to determine their practical use.

      CONCLUSIONS

      Live or dead microorganisms can decontaminate aflatoxin produced by A. flavus contamination in foods or feeds, either by binding the toxin to their cell wall components or by degrading the toxin into less toxic or nontoxic compounds. Yeast and lactic acid bacteria are involved in aflatoxin binding. Among these, L. rhamnosus GG efficiently binds aflatoxin B1 in a process involving peptidoglycans, but further research is needed to establish which components of the peptidoglycan of L. rhamnosus GG take part in the binding of aflatoxin B1. The aflatoxin-B1-binding capability of L. rhamnosus GG should be applied to the practical decontamination of aflatoxin B1. A variety of microorganisms, including bacterial and fungal species, contribute to aflatoxin detoxification by bioconversion. These microorganisms use degradation machinery to remove xenobiotics such as aflatoxin to survive in the same ecological niches as aflatoxigenic Aspergillus species. All of these strains execute bioconversion via an enzymatic process. Because many bacterial strains isolated from animal feces or guts are capable of degrading aflatoxin, they can be sustained for a long period in animal gastrointestinal tracts and continuously degrade aflatoxin in the gut, making them a promising feed additive for aflatoxin decontamination. These microorganisms should also be further researched for commercial application in dairy products and feeds.

      ACKNOWLEDGMENTS

      This research was supported by the Sookmyung Women's University Research Grants 1-1403-0133.

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