Aflatoxin binding / absorption by microorganisms. Note: PBS, Phosphate-Buffered Saline; cfu, colony formingunit.
1. Introduction
Nowadays, about 100,000 fungi have already been identified. From these, more than 400 may be considered potentially toxigenic, and about 5% are known to produce toxic compounds or classes of compounds that cause adverse effects in animals and humans in several parts of the world [1]. These compounds, called mycotoxins, are secondary metabolites of low molecular weight produced by mycelia or spores of filamentous fungi [2]. It is suggested that mycotoxin production is generally limited to a relatively small number of mold species, and that toxin may be produced by the whole species or just one specific strain [3]. The more complex the synthesis pathway of a mycotoxin, the lesser the number of mold species that produce it.
The term “mycotoxin” originates from the Greek word "Mykes”, meaning fungus, and from the Latin word “Toxicum”, meaning poison or toxin [2]. Mycotoxins are classified as the most important chronic and noninfectious foodborne risk factor, more important than synthetic contaminants, plant toxins, food additives, and pesticide residues. Both humans and animals may show acute or chronic intoxication caused by mycotoxin ingestion, and the pathological condition that results from this ingestion is called mycotoxicosis [4]. Some factors affect the magnitude of toxicity in humans or animals, including the animal species, mechanism of action, metabolism and defense mechanisms [5].
About 400 types of mycotoxins have been already discovered, and they are generally divided into groups based on structural similarities and most important toxic effects [6]. From all mycotoxins that have been isolated, aflatoxin is one of the most well-known and widely distributed in foodstuffs, with proven and marked toxic properties. Aflatoxins are predominantly produced by
Aflatoxins are distributed worldwide.
Other factors may also influence aflatoxin production: substrate composition, water activity, pH, atmosphere (concentration of oxygen and carbon dioxide), microbial competition, mechanical damage to the seeds, mold lineage, strain specificity and variation, instability of toxigenic properties, plant stress, insect infestation, and use of fungicides or fertilizers [2, 5, 11]. It is important to remember that aflatoxin contamination is cumulative, and the moment of harvesting and drying, and storage conditions may also play an important role in aflatoxin production [12].
Concerns related to the negative impacts of aflatoxins on health led to the study of strategies to prevent toxin formation in foodstuffs, as well as to eliminate, inactivate or reduce toxin bioavailability in contaminated products [13]. Contamination may be prevented by improved agricultural practices, antifungal agents, genetic engineering, and control of storage conditions [2]. Bioavailability may be reduced by enterosorption, which is done by adding nutritionally inert adsorbent compounds to the diet. These compounds are mycotoxin sequestrants, and prevent the toxin from being absorbed in the gastrointestinal tract of the animals, making its distribution to the target organs impossible [14]. This method has limited practical use, due to the safety of the adsorbent agents used, and the difficulty in applying them to human foods [15]. Elimination or inactivation, that is, decontamination, may be achieved by physical, chemical, and biological methods, which have to present the following characteristics: complete inactivation; destruction or removal of the toxin; no production or toxic residues in foods or no remainders of them; preservation of nutritional value and palatability of the food; destruction of fungal spores and mycelia to prevent production or re-appearance of the toxin; no significant changes in the physical properties of the food; low cost and ease of use [1,11].
Physical methods for mycotoxin decontamination involve procedures such as thermal inactivation, ultraviolet light, ionizing radiation, or extraction with solvents. Chemical methods are based on agents that break mycotoxin structure, such as chlorine treatment (sodium hypochlorite or chlorine gas), oxidizing agents (hydrogen peroxide, ozone and sodium disulfide), or hydrolytic agents (acids, alkalis and ammonia). However, both chemical and physical methods have disadvantages, either because removal is not efficient, or because of high costs or nutritional losses to the product [16,17]. Biological methods are based on the action of microorganisms on mycotoxins. These microorganisms may be yeasts, filamentous fungi, bacteria, algae, among others, and their mechanisms of action is based on competition by nutrients and space, interactions, and antibiosis, among others [18].
Biodegradation of aflatoxins by microorganisms offers an attractive alternative for the control or elimination of aflatoxins in foods and animal feed, preserving their quality and safety [19]. Besides, their use have a more "natural" appeal, given the ever-growing resistance of the consumer to chemical treatments [1]. Biological decontamination methods are being widely studied and may be a very promising choice, provided they show to be efficient, specific, cost-effective, and are environmentally friendly [20]. Among the types of microorganisms available and that may be used to remove aflatoxins from a contaminated medium, lactic acid bacteria (LAB) and yeasts are the most studied ones, showing the most promising results.
Therefore, the objective of this chapter was to present results of studies on microbiological methods for aflatoxin decontamination, more specifically on the ability of LAB and yeasts to degrade or sequestrate this mycotoxin.
2. Toxicological Properties of Aflatoxins
Nowadays, there are 18 similar compounds called aflatoxins. However, the most important types in terms of health and medical interest are identified based on their fluorescence under ultraviolet light (B = Blue and G = Green), such as aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2). From these compounds, AFB1 is the most prevalent and toxic one [21]. When AFB1 is ingested by domestic animals in contaminated feed or foodstuffs, such as by dairy cows, the toxin undergoes liver biotransformation and is converted into aflatoxin M1 (AFM1), becoming the hydroxilated form of AFB1, which is excreted in milk, tissues and biological fluids of these animals [22-24]. It was reported that of all AFB1 ingested in feed, about 0.3% to 6.2% is transformed in AFM1 in milk and that there is a linear relationship between the concentration of AFM1 in milk and the concentration of AFB1 in contaminated feeds consumed by the animals [25,26].
Chronic exposure to low levels of aflatoxins represents a serious risk to economy, and mainly to health [21]. Economic losses are related to decreased efficiency in industrial or agricultural production, with loss in quality, lower yield, and defective product [27]. It was also reported that in some states of the USA, economic losses to agriculture amount to 100 million dollars [19]. On the other hand, these losses caused by mold contamination and mycotoxins are greater than 1.6 billion dollars in the US, and African feeds lose about 670 billion dollars a year due to barriers to the trade of aflatoxin-contaminated foodstuffs [28].
As for human and animal health, biological effects of aflatoxins may be carcinogenic, mutagenic, teratogenic, hepatotoxic, and immunosuppressive [29]. The International Agency for Research on Cancer classifies AFB1 and AFM1 as Group 1 human carcinogens, even though AFM1 is about 10 times less carcinogenic than AFB1 [30]. All these aflatoxin effects are influenced by variations according to the animal species, sex, age, nutritional status, and effects of other chemical products, besides the dose of toxin and the length of exposure of the organism to it [31].
Aflatoxicosis is the poisoning caused by the ingestion of moderate to high levels of alfatoxin in contaminated foods. Acute aflatoxicosis causes quick and progressive jaundice, edema of the limbs, pain, vomiting, necrosis, cirrhosis and, in severe cases, acute liver failure and death, caused by the ingestion of about 10 to 20 mg of aflatoxin in adults. Aflatoxin LD50 shows the following order of toxicity: AFB1> AFM1> AFG1> AFB2> AFG2 [4, 32]. Chronic aflatoxicosis causes cancer, immunosuppression and other pathological conditions, having the liver as the primary target organ [4].
The greatest risk presented by aflatoxins for human beings is chronic exposure causing hepatocellular carcinoma, which may be made worse by hepatitis A virus [5]. It was also report that aflatoxins were found in the tissues of children affected by Reye syndrome (encephalopathy with serious lesions in liver and kidneys after influenza or chickenpox), and Kwashiorkor (protein-energy malnutrition). Aflatoxicosis is considered, then, a contributing factor to these diseases.
AFB1 is metabolized in the liver by the cytochrome P450 system, generating its most carcinogenic metabolite, AFB1-8,9-epoxide (AFBO), or other less mutagenic forms, such as AFM1, Q1 or P1. There are several pathways for AFBO after it is metabolized, with one of them leading to cancer, another to toxicity and another one, to excretion. AFBO exo-form easily binds to cell macromolecules, including genetic material such as DNA proteins, producing adducts. Formation of these DNA adducts leads to genetic mutations and cancer, and their excretion in the urine of infected people is not only a proof that humans have the necessary biochemical pathways for carcinogenesis, but also offers a reliable biomarker for AFB1 exposure [24].
Potential risk to human health caused by aflatoxins has led to surveillance programs for the toxin in different raw materials, as well as regulations determined by almost every country in the world [9]. A study carried out by the Food and Agriculture Organization of the United Nations (FAO) in 2002 pointed out that about 100 countries had specific regulations for the presence of aflatoxin in foods, dairy products and animal feed, and that the total population of these countries amounted to 90% of the world population. The same study showed that regulations for aflatoxin are getting more diverse and detailed, including sampling methods and methods of analysis [33].
In countries where a regulation for aflatoxin exists, tolerance levels for the total aflatoxin (sum of aflatoxins B1, B2, G1 and G2) ranges from 1 to 35 µg/kg for foods, with an average of 10 g/kg; and from zero to 50 µg/kg for animal feed, with an average of 20 µg/kg. For AFM1 in milk, tolerance levels are between 0.05 and 0.5 µg/kg, with most countries adopting a threshold of 0.05 µg/kg [10].
3. Decontamination of Aflatoxins by Lactic Acid Bacteria
LAB is a large group of genetically different bacteria that, besides producing lactic acid as the main product of their metabolism, have similar characteristics: they are all gram-positive, non-sporoformers, non-motile, and catalase, and oxidase negative. They are, therefore, aerotolerant anaerobes. Besides, they mandatorily ferment sugars and tend to be nutritionally fastidious, frequently requiring specific amino acids and B-complex vitamins as growth factors [34]. Several LAB genera, such as
Fermentation enables longer shelf life and improves sensory and nutritional properties of the product, as sugar fermentation lowers pH and inhibits growth of spoilage and pathogenic microorganisms. Fermentation is also responsible for other reactions, such as proteins hydrolysis, improving texture and flavor; synthesis of aromatic components and texturizers, affecting the consistency of the product; and production of inhibitory components [35,36]. This inhibition is, in part, caused by the final products of fermentation, such as lactic acid, diacetyl, acetaldehyde and acetic acid, which may accumulate in inhibitory concentrations in certain foods and drinks. In other cases, inhibition may also be caused by secondary by-products of metabolism, such as hydrogen peroxide or bacteriocins [37].
Therefore, two aspects may be considered when LAB are used: fermentation and antibiosis ability. In the first case, the starter culture added to the food acts on the substrate, causing advantages to the food. In the second case, the starter culture has to inhibit the development of undesirable microorganisms that may spoil the product or be hazardous to human health. In reference [38], authors state that one of the effects that were identified in LAB was protection against toxins found in foods, such as heterocyclic amines, polycyclic aromatic hydrocarbons, reactive oxygen species, and mycotoxins. In the latter case, studies have demonstrated that LAB have the ability to inhibit aflatoxin biosynthesis, or that they have the ability to remove mycotoxins from the medium, reducing their effects.
It should be emphasized that with increased interest in probiotic food production all over the world, selection of LAB cultures with probiotic characteristics and greater ability to remove mycotoxins may help to reduce risk of exposure to these toxins in foodstuffs, which is a very promising line of research in mycotoxicology. Yeast and LAB strains have great ability to remove mycotoxins, and may be used as part of starter cultures in the fermentation of foods and drinks [39]. These microorganisms have, thus, ability to ferment and decontaminate the medium, and purified components of these strains may be used in small amounts as food additives without compromising the characteristics of the final product.
One of the first studies in this area was carried out in the 1960s, when these authors evaluated the ability of about 1,000 types of microorganisms to degrade aflatoxins [40]. Yeasts, filamentous fungi, bacteria, actinomycetes, algae, and fungal spores were among the organisms studied. From these, only the bacterium
After this study, many others followed. However, the most significant ones started to appear after the 1990s. Table 1 presents the most relevant studies carried out with bacteria for aflatoxin decontamination. The action of 7 different types of bacteria on AFB1 was evaluated and it was found that some strains of
Most assays on aflatoxin removal in the studies cited above were carried out in phosphate-buffered saline (PBS). In reference [42], besides testing the ability of 27 strains of
AFB1 was added to yogurt and acidified milk in concentrations ranging from 1,000 to 1,400 g/kg, and a reduction of AFB1 in yogurt (pH 4.0), ranging from 97.8% to 90% was obtained [43]. Maximum decrease in AFB1 was observed during milk fermentation. As for milk acidified with citric, lactic, and acetic acid (pH 4.0) AFB1 reduction (concentration of 1,000 μg/Kg) was 90%, 84% and 73%, respectively. The ability of probiotic bacteria (
Toxin polarity has an important role in the binding mechanism. The percentage of aflatoxin removed by LAB decreases in the following order: AFB1> AFB2> AFG1> AFG2. This observation correlates with the decrease in the polarity of these toxins, and is consistent with hydrophobic reactions, which may also have a role in the binding mechanism [45]. AFM1 is less efficiently removed than AFB1. However, scientific literature has few studies on the ability of LAB to remove AFM1.
In reference [46], authors examined the ability of 4 strains of
The ability of
Some physical, chemical, and enzymatic treatments may increase the ability of LAB to bind to aflatoxin in the medium. In reference [48] authors studied the ability of
Comparing the ability of viable and heat-treated bifidobacteria cells, it was observed that viable cells removed 4 to 56% AFB1 from the medium, whereas non-viable cells removed 12 to 82% [23]. Evaluating the influence of the inactivation treatment on the ability of 4 types of
Removal of AFM1 with 8 LAB strains showed that heat-treated cells bound more efficiently (25.5 to 61.5%) to the toxin than viable bacterial cells (18.1 to 53.8%) [29]. In reference [50] it was observed that heat-treated cells removed greater percentages of AFM1 (12.4% to 45.7%) in PBS compared with viable cells (5.6% to 33.5%), with no significant differences between 15 minutes or 24 hours of contact. Similar results were found in [51], because viable cells of
In [53] authors observed that
These examples show that both viable and non-viable cells are able to remove aflatoxin from aqueous solutions. As non-viable cells are also able to remove the toxin, it is supposed that cells are physically bound to the toxin, that is, components of the bacterial cell wall adhere to it, mainly polysaccharides and peptidoglycans, taking into account the possibility of a covalent bond or degradation caused by bacterial metabolism [1, 55, 56].
Both polysaccharides and peptidoglycans of the bacterial cell wall may be extremely affected by heat and acid treatment, once heat may denature proteins or form Maillard reaction products. Besides, acid treatment may break glycosidic bonds of polysaccharides, releasing monomers that may be further broken into aldehydes, also degrading proteins to smaller components, such as peptides and amino acids. Thus, acid treatment may break the peptidoglycan structure, compromising its structural integrity, that is, decreasing the thickness of this layer, reducing cross links and increasing the size of the pores. These changes caused by the treatments cited above enable AFB1 to bind to the bacterial cell wall and to the components of the plasmatic membrane that were not available when the bacterial cell was intact [27].
In reference [57] authors explained that the integrity of the bacterial cell wall is important in the process of toxin removal by both viable and non-viable cells. In their study of AFB1, they observed that both the bacterial cell wall and its purified fragments were able to remove aflatoxin from the medium. However, when the cell wall was lost or destroyed (totally or partially) by enzymatic treatment, there was a significant decrease in the ability to remove the toxin. It was observed, using atomic force microscopy, that the bond between AFB1 and
The ability of
In the use of pronase E, lipase and periodate, treatment with periodate led to significant reduction in the ability to remove the toxin, both by viable and non-viable cells, once it oxidizes the -OH cis groups in aldehyde and carboxylic acid groups, suggesting that the bonds involve predominantly bacterial polysaccharides. Treatment with pronase E caused the same significant reduction in AFB1 removal, evidencing that proteins may also be involved in the process. Thus, the fact that pronase E and periodate both have a significant reduction on AFB1 removal indicates that binding sites are made of protein. Treatment with lipase, on its turn, did not cause any significant reduction in AFB1, showing that lipids, such as lypoteichoic acid probably do not have a role in the process. Although the treatments decreased AFB1 removal, it was still substantial in all cases, possibly showing the involvement of multiple components in the bond with mycotoxin [48].
However, not only the type of bacterial strain and the inactivation treatment used may influence formation and stability of the LAB/aflatoxin complex, but also of other factors, such as bacterial counts, specificity of the bacteria, pH, incubation temperature, addition of nutrients, and the solvents used, among others [23, 27, 48].
As for the number of bacterial cells in the medium, it has been concluded that there was a significant decrease in the amount of AFM1 removed when cell counts changed from 107 CFU/mL (0 to 5.02%) to 108 CFU/mL (10.22 to 26.65%), indicating that bacterial counts are critical factors in the removal of AFM1 by LAB [46]. In reference [59] authors observed that no less than 5 x 109 CFU/mL of
In reference [17] authors reported that, for
Assays with AFB1 and
The effect of washing on the stability of the LAB/aflatoxin complex was analyzed [47]. They observed that after the first washing of bacterial pellets with PBS, the proportion of AFM1 released by the bacteria was 87.3% for
Thus, the LAB/aflatoxin complex seems to be unstable, once part of the aflatoxin, both for AFB1 and AFM1, is released from the complex after washing, and gradually returns to the aqueous solution. Therefore, the greater the number of washings, the greater the amount of aflatoxin released back into the solution. This shows that the bond is not a strong one, suggesting it is a weak non-covalent bond and an association with hydrophobic sites on the surface of the bacteria [23, 48].
Different from this hypothesis, in reference [61], performing the same washings on a complex between
The stability of the LAB/aflatoxin complex in a wide range of pH is an important factor in the use of these microorganisms to remove aflatoxin from foods, once gastric release of the toxin would have negative health implications. Therefore, the complex formed has to resist environmental stress caused by the gastrointestinal tract, such as low pH and presence of bile. When the influence of the presence of bile on the LAB/aflatoxin complex was analyzed, it was observed that
The ability of
Rats treated with feed added of aflatoxin (3 mg/kg of feed) presented a significant decrease in the feed intake compared with the control group, different from the animals fed diets containing
In Egypt, a pilot study investigated the effect of the addition of
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|
|
|
|
B1 | |||
Viable cells Freeze-dried cells Heat-treated cells Viable cells Freeze-dried cells Heat-treated cells |
5 µg/mL | 78.4 65 81 78.8 50 82 58.1 67.4 33.2 16.3 |
2 x 1010 cfu/mL, 0h, 37 °C, PBS 4h, 37 C, PBS 4h, 37 °C, PBS 2 x 1010 cfu/mL, 0h, 37 °C, PBS 4h, 37 °C, PBS 4h, 37 °C, PBS 2 x 1010 cfu/mL, 0h, 37 C, PBS 7 x 109 cfu/mL, 0h, 37 °C, PBS 1 x 1010 cfu/mL, 0h, 37 C, PBS 5 x 1010 cfu/mL, 0h, 37 °C, PBS |
[17] |
|
B1, B2, G1, G2 4 or 40 µg/kg |
18-33 27-50 |
6h, 37 °C , barley flour (50%), wheat flour (45%) and corn flour (5%) mixed with water in 1:1.5 proportion 24h, 37 °C , barley flour (50%), wheat flour (45%) and corn flour (5%) mixed with water in 1:1.5 proportion |
[44] |
B1
5 µg/mL |
5.6 17.3 18.2 22.7 28.4 31.6 18.0 34.2 41.1 54.6 20.7 22.6 30.1 33.1 57.8 59.7 34.7 37.5 45.7 48.7 |
1 x 1010 cfu/mL, 24h, 37 °C, PBS | [23] | |
Viable cells Heat-treated cells Acid-treated cells Viable cells Heat-treated cells Acid-treated cells Viable cells Heat-treated cells Acid-treated cells Viable cells Heat-treated cells Acid-treated cells Viable cells Heat-treated cells Acid-treated cells Viable cells Heat-treated cells Acid-treated cells Viable cells Heat-treated cells Acid-treated cells Viable cells Heat-treated cells Acid-treated cells Viable cells Heat-treated cells Acid-treated cells Viable cells Heat-treated cells Acid-treated cells Viable cells Heat-treated cells Acid-treated cells Viable cells Heat-treated cells Acid-treated cells |
B1 5 µg/mL | 78.9 84.1 86.7 76.5 87.8 88.3 59.7 74.7 84.2 59.0 58.1 69.5 48.3 69.7 81.3 29.9 35.5 62.7 21.8 41.5 32.3 15.6 33.7 75.8 17.5 29.8 58.1 22.3 67.3 82.5 26.9 40.1 43.7 32.7 42.0 63.8 |
1 x 1010 cfu/mL, 1h, 37 °C, PBS 1 x 1010 cfu/mL, 1h, 37°C, PBS 1 x 1010 cfu/mL, 1h, 37 °C, PBS 1 x 1010 cfu/mL, 1h, 37 °C, PBS 1 x 1010 cfu/mL, 1h, 37 °C, PBS 1 x 1010 cfu/mL, 1h, 37 °C, PBS 1 x 1010 cfu/mL, 1h, 37 °C, PBS 1 x 1010 cfu/mL, 1h, 37 °C, PBS 1 x 1010 cfu/mL, 1h, 37 °C, PBS 1 x 1010 cfu/mL, 1h, 37 °C, PBS 1 x 1010 cfu/mL, 1h, 37 °C, PBS 1 x 1010 cfu/mL, 1h, 37 °C, PBS |
[27] |
B1 2 µg/mL |
37 37 46 25 31 46 37 37 41 |
30 min, 37 °C, PBS | [22] | |
Living cells Dead cells by boiling Dead cells by autoclaving Living cells Dead cells by boiling Dead cells by autoclaving |
B1 0.5 µg/mL |
54.8 86.7 82.3 71.0 81.0 100 80.0 81.0 91.5 90.7 66.5 100.0 96.8 81.7 96.0 83.0 |
107-108 cfu/mL,30 min, 37 °C, in: PBS maize oil sunflower oil soybean oil PBS maize, sunflower or soybean oil PBS PBS maize oil sunflower oil soybean oil PBS maize oil sunflower oil soybean oil PBS |
[42] |
Yoghurt Culture | B1 0.6mg/kg1 mg/kg 1.4mg/kg 1 mg/kg |
97 91 90 90 84 73 |
42 °C/3h, pH 4.0, overnight, milk milk acidified with citric acid milk acidified with latic acid milk acidified with acetic acid |
[43] |
Living cells Heated cells Living cells Heated cells Living cells Heated cells Living cells Heated cells Living cells Heated cells Living cells Heated cells |
M1 5, 10 and 20 ng/mL |
14.9-20.2 14.4-15.4 17.0-24.9 16.6-19.0 20.4-25.3 21.8-22.7 22.1-26.8 23.7-25.1 10.2-16.0 7.8-10.5 14.0-21.8 12.8-15.9 23.5-26.6 24.0-25.9 24.3-28.9 25.4-27.4 16.6-22.1 15.5-18.3 17.4-23.5 17.1-22.2 20.1-24.0 20.4-22.2 23.4-27.8 22.9-26.3 |
108 cfu/mL, 0, 4, 24 h, 37 °C, PBS 108 cfu/mL, 4h, 37 °C, milk 0, 4 , 24 h, 37 °C, PBS 4h, 37 °C, milk 108 cfu/mL, 0, 4, 24 h, 37 °C, PBS 108 cfu/mL, 4h, 37 °C, milk 0, 4, 24 h, 37 °C, PBS 4h, 37 °C, milk 108 cfu/mL, 0, 4, 24 h, 37 °C, PBS 108 cfu/mL, 4h, 37 °C, milk 0, 4, 24 h, 37 °C, PBS 4h, 37 °C, milk 108 cfu/mL, 0, 4, 24 h, 37 °C, PBS 108 cfu/mL, 4h, 37 °C, milk 0, 4,24 h, 37 °C, PBS 4h, 37 °C, milk 108 cfu/mL, 0, 4,24 h, 37 °C, PBS 108 cfu/mL, 4h, 37 °C, milk 0, 4 and 24 h, 37 °C, PBS 4h, 37 °C, milk 108 cfu/mL, 0, 4, 24 h, 37 °C, PBS 108 cfu/mL, 4h, 37 °C, milk 0, 4 and 24 h, 37 °C, PBS 4h, 37 °C, milk |
[46] |
|
AFM1 |
9.4-73.1 4.5-38.3 7.8-41.6 73 41.6 64-80.5 46.0-68.5 67.0-72.5 80.5 73 |
96 h, 37 °C , PBS 96 h, 37 °C , PBS 96 h, 37 °C , PBS 96 h, 37 °C , PBS 96 h, 37 °C , PBS 96 h, 37 °C ,milk 96 h, 37 °C , milk 96 h, 37 °C , milk 96 h, 37 °C , milk 96 h, 37 °C , milk |
[47] |
(pre-cultured) Viable cells Heat-killed cells (pre-cultured) Viable cells Heat-killed cells (lyophilized) Viable cells Heat-killed cells (lyophilized) Viable cells Heat-killed cells Viable cells Heat-killed cells Viable cells Heat-killed cells Viable cells Heat-killed cells Viable cells Heat-killed cells |
M1 0.15 µg/ml |
50.7 18.8 26.0 57.8 26.6 36.6 46.3 69.6 27.4 51.6 63.6 30.1 53.8 56.2 45.7 57.4 40.4 38.9 30.8 61,5 18,3 25,5 18,1 39,9 |
5.3 x 108, 15 - 16h, , 37 °C, in: PBS skim milk full cream milk PBS skim milk full cream milk PBS skim milk full cream milk PBS skim milk full cream milk 1.0 x 1010, 15-16h, 37 °C, PBS 1.0 x 1010, 15-16h, 37 °C, PBS 2.9 x 108, 15-16h, , 37 °C, PBS 3.9 x 108, 15-16h, , 37 °C, PBS 1.7 x 109, 15-16h, , 37 °C, PBS 3.9 x 108, 15-16h, , 37 °C, PBS |
[29] |
Pre-treatment: Pronase E Viable cells Heat-treated cells Acid-treated cells Lipase Viable cells Heat-treated cells Acid-treated cells Phosphate Buffer Viable cells Heat-treated cells Acid-treated cells m-Periodater Viable cells Heat-treated cells Acid-treated cells Iodate Viable cells Heat-treated cells Acid-treated cells Urea Viable cells Heat-treated cells Acid-treated cells Water (Milli Q) Viable cells Heat-treated cells Acid-treated cells |
B1
5 µg/mL |
66 72 85 76 74 89 86 85 91 60 49 36 83 84 80 64 60 50 76 83 84 |
1h, 37 °C, 5% CO2, PBS Boiled for 1h, PBS 2 mol/L HCl, 1h, 37 °C, 5% CO2 1h, 37 °C, 5% CO2, PBS Boiled for 1h, PBS 2 mol/L HCl, 1h, 37 °C, 5% CO2 1h, 37 °C, 5% CO2, PBS Boiled for 1h, PBS 2 mol/L HCl, 1h, 37 °C, 5% CO2 1h, 37 °C, 5% CO2, PBS Boiled for 1h, PBS 2 mol/L HCl, 1h, 37 °C, 5% CO2 1h, 37 °C, 5% CO2, PBS Boiled for 1h, PBS 2 mol/L HCl, 1h, 37 °C, 5% CO2 1h, 37 °C, 5% CO2, PBS Boiled for 1h, PBS 2 mol/L HCl, 1h, 37 °C, 5% CO2 1h, 37 °C, 5% CO2, PBS Boiled for 1h, PBS 2 mol/L HCl, 1h, 37 °C, 5% CO2 |
[48] |
Pre-treatment: None Heat Ethanol Acid Alkaline None Heat Ethanol Acid Alkaline None Heat Ethanol Acid Alkaline None Heat Ethanol Acid Alkaline |
B1 5 µg/mL |
56.6 71.9 46.5 87.0 27.4 22.4 41.8 21.8 43.1 12.0 17.8 28.5 18.0 56.3 9.1 16.3 33.5 15.9 586 8.3 |
4h, 37 °C, PBS |
[49] |
Viable cells Heat-killed cells Viable cells Heat-killed cells Viable cells Heat-killed cells Viable cells Heat-killed cells Viable cells Heat-killed cells Viable cells Heat-killed cells Viable cells Heat-killed cells |
M1 0.15 µg/mL PBS solution 0.5 µg/mL skimmed milk |
5.6 8.1 7.4 6.6 8.7 7.8 21.4 22.8 30.2 33.5 33.5 17.1 27.8 24.5 16.9 23.6 32.5 |
1010 cfu/mL, 15 min, 37°C, in: PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS skimmed milk PBS PBS skimmed milk PBS PBS skimmed milk |
[50] |
|
M1 10 ng/mL |
18.7 27.6 29.4 39.2 14.8 |
4h, 37 °C, PBS 4h, 42 °C, milk 4h, 37 °C, PBS 4h, 42 °C, milk Yoghurt |
[51] |
|
M1 10 µg/mL |
100 | 5 x 1010 cfu/mL, 30 °C, 4h, PBS and milk | [52] |
Viable cells Cell Free Supernatant |
B1 2.5 µg/mL |
85.7 95 78.4 |
96h, 30 °C, nutrient broth culture 108 cfu/ml, 120 h, 30 °C, pistachio nuts 120 h, 35 °C, nutrient broth culture |
[53] |
“Inocula” suspension Cell Cell extract Culture Supernatant |
B1
G1 M1 (0.5 µg/mL) B1 |
81.5 80.7 60 10.5 9.6 78.7 |
72h, 37 °C, Luria-Bertani medium 72h, 37 °C, PBS |
[54] |
“In vivo” “In vitro” “In vivo” “In vitro” “In vivo” “In vitro” |
B1 3 µg/mL |
51 92 80 36 71 77 37 82 22 |
1010cfu/mL: 1 min, duodenum of chicks 1 h, duodenum of chicks 37 °C, 1h , pH 7.3 1 min, duodenum of chicks 1 h, duodenum of chicks 37 °C, 1h , pH 7.3 1 min, duodenum of chicks 1 h, duodenum of chicks 37 °C, 1h , pH 7.3 |
[62] |
4. Decontamination of Aflatoxins by Yeasts
Yeasts are non-photosynthetic organisms with a separate nucleus and complex life cycle. They are larger than bacteria, normally spherical, non-motile, and reproduce by budding. Although their main function is alcoholic fermentation, these organisms are also capable of producing enzymes and vitamins. The primary substrates for yeasts are fermentable sugars, which are mainly transformed in ethanol, carbon dioxide, and biomass under oxygen-limited conditions. Under adequate oxygen supply, yeast produces carbon dioxide, water, and biomass [65].
As it occurs with LAB, SC cells have been studied to evaluate their ability to remove aflatoxins from contaminated media. The most important results obtained until now are summarized in Table 2. Products based on SC (cell wall from baker and brewer yeasts, inactivated baker yeast, or alcohol yeast) was studied, and it was observed that in pH 3, 37 °C and 15 minutes of contact, AFB1 removal ranged from 2.5% to 49.3%, depending on the concentration of the toxin in the medium, and on the yeast-based products used [66]. These authors also observed a decrease in toxin adsorption as the initial concentration increased, and concluded that adsorption is not a linear phenomenon. Similar results with a SC strain and AFB1 concentration ranging from 1 to 20 µg/mL was also reported [56]. At the 1 µg/mL concentration, 69.1% AFB1 was removed; at 5 g/mL, removal rate was 41%; and at 20 µg/mL, 34%.
The ability of SC (0.1%, 0.2%, and 0.3%) to adsorb AFB1 in contaminated corn (150, 300, 450 and 800 µg/kg corn was analyzed [68]. The adsorption process showed an inversely proportional relationship with the concentration, that is, the greater the AFB1 concentration in the medium, the lower the efficiency of AFB1 removal by SC (16% to 66% for 800 µg/kg AFB1 vs. 40% to 93% for 150 µg/kg AFB1). The authors concluded, using densitogram analysis, that the adsorption process did not change the molecular structure of the mycotoxin, and that the decreased AFB1 adsorption rates observed as the toxin concentration increased may possibly be caused by saturation of the adsorption sites on the SC cell. Other factors, such as length of incubation, pH, method of biomass purification, and methods of analysis, may also influence this process.
Immobilized SC cells (ATTC 9763) was investigated for their ability to remove AFB1 from pistachio seeds, and it was observed that the amount of toxin removed was dependent on its concentration in the medium (40% and 70% of removal for concentrations of 10 ng/mL and 20 ng/mL AFB1, respectively) [69]. The authors also concluded that this ability to remove the toxin was greater in SC exponential growth phase, and that the process was a quick one, being saturated after 3 hours of contact. Besides, the ability of SC cells to remove toxin was increased after treatment with acid (60% and 73% for 10 ng/mL and 20 ng/mL AFB1, respectively) and heat (55% and 75%, respectively). In another study, authors also concluded that the treatment of SC cells with heat at 60 °C and 120 °C, and with chloric acid (2 mol/L) increased their ability to remove AFB1 from the medium to 68.8%, 79.3%, and 72.1%, respectively, against 38.7% when viable yeast cells were used [56].
Heat treatment may increase the permeability of the external layer of the cell wall due to the suspension of some mannanes on the cell surface, leading to increased availability of previously hidden binding sites. Besides, countless physical-chemical changes take place on the cell wall during heat treatment, leading to more exposed binding sites. On the other hand, acid conditions may affect polysaccharides by releasing monomers, which are further fragmented in aldehydes after glycosidic bonds are broken. Continuous removal of aflatoxin, even after use of acid and heat treatments, confirms that yeast cell viability is not a significant factor for the removal of aflatoxin from the medium [69].
During the fermentation of broiler feed using LAB (3 strains of
In a study with mice, it was observed that the addition of AFB1 to the diet (0.4 and 0.8 mg/kg) caused a significant reduction in weight gain, and an increase of 85% (0.8 mg/kg) in the rate of micronucleated normochromatic erythrocytes (MNE) after 3 weeks of ingestion, compared with the control group [68]. When diets containing AFB1 and SC (0.3%) were administered, weight gain was twice greater than in diets that contained only the toxin, and the rate of MNE increased only 46% (0.8 mg/kg) The authors stated that reduced body weight is one of the most common consequences of AFB1 ingestion, because the toxin alters the activity of several digestive enzymes, giving rise to a malabsorption syndrome characterized by steatorrhea, hypovitaminosis A and a decrease in the levels of bile, pancreatic lipase, trypsin, and amylase. Besides, biotransformation of AFB1 gives rise to several metabolites, particularly AFB1-8,9-epoxide, which may bind covalently to DNA and proteins, changing enzymatic processes such as gluconeogenesis, Krebs cycle, and fatty acid synthesis [74]. MNE rate is used to determine the genotoxicity of AFB1, because it quantifies broken chromosomes and whole chromosomes that are abnormally distributed to daughter cells, showing thus, that AFB1 is a potent mutagenic agent.
A diet containing 5 g/g of aflatoxin (82.06% AFB1, 12.98% AFB2, 2.84% AFG1, and 1.12% AFG2) by female quails (49 to 84 days of age) led to decreased egg production, feed intake, and feed conversion (31%, 28%, and 47%, respectively) [75]. However, addition of SC (2 g/kg) significantly increased these parameters (16%, 4%, and 14%, respectively). They also observed that the diet with aflatoxins caused a marked decrease in weight gain and egg weight, besides increasing animal mortality (39%, 7%, and 50%, respectively), whereas addition of SC reverted the negative effect on these parameters (65%, 8%, and 50%, respectively). The authors stated that these negative effects of aflatoxins in egg production, feed intake, and feed conversion may have been caused by anorexia, apathy, and inhibition of protein synthesis and lipogenesis. Besides, affected liver function and mechanisms of use of protein and lipids may have affected performance criteria and the general health of the animals. In reference [76] authors reported that the components of the cells wall of SC are able to adsorb mycotoxins, stimulate the immune system, and compete for binding sites in the enterocytes, inhibiting intestinal colonization by pathogens.
SC cell wall is mainly made up of polysaccharides (80-90%), and its mechanical resistance is due to an inside layer composed of β-D-glucans, which are formed by a complex network of highly polymerized β-(1,3)-D-glucans, branched off as β-(1,6)-D-glucans, that have a low level of polymerization. This inside layer is firmly bound to the plasmatic membrane by linear chains of chitin, which have a significant role in the insolubility of the overall structure and packing of the branched β-D-glucans. Both chitin chains and β-D-glucans affect the plasticity of the cell wall. The external layer of the yeast cell wall is formed by mannoproteins, which have an important role in the exchanges with the external environment. This whole structure is highly dynamic and may vary according to the yeast strain, phase of the cell cycle, and culture conditions, such as pH, temperature, oxygenation rate, nature of the medium, concentration and nature of the carbon source. Thus, these differences in the composition of the cell wall among yeast strains are related with their ability to bind to the mycotoxin [77].
Studies have shown that the components of SC cell wall, called oligomannanes, after esterification, are able to bind more than 95% AFB1 [78]. Addition of 0.05% glucomannanes in the basal diet improved broiler performance [79].
The possible binding mechanisms between yeast cell wall and mycotoxins were studied, and authors suggested that β-D-glucans are the components of the cell wall that are responsible for forming the complex with the toxin, and that the reticular organization of β-D-glucans and their distribution in β-(1,3)-D-glucans and β-(1,6)-D-glucans have an important role in the efficiency of the bond [77]. Besides, studies have shown that weak hydrogen and van der Waals bonds are involved in the complex chemical connection between the mycotoxins and β-D-glucans, a chemical interaction that is much more "adsorption” than “bond”. As for AFB1, they observed that the aromatic ring, the lactone and ketone groups of the polar form of AFB1, or chemical bonds with glucose units in the single helix of the β-D-glucans, are what keep the toxin bound to the glucans.
It was demonstrated that yeast strains isolated from environments were animals are raised are able to bind to AFB1 in saline solution (PBS, pH 7) [67]. These strains presented other properties that were beneficial to the host, such as the inhibition of pathogenic bacteria. Therefore, SC strains acted both as probiotics (co-aggregation and inhibition of pathogenic bacteria), and as mycotoxin adsorbents.
In reference [72], SC was able to reduce the deleterious effects of AFB1 in the diet of broilers and in [68] authors replicated these findings in rats. Protective effect against aflatoxins produced by yeasts was confirmed in rats. However, when yeast cells were inactivated by heat, they were inefficient [80] but when glucomannanes extracted from the cell wall of yeasts were used, there was an increase in the efficiency of the bond with AFB1, OTA and T-2 toxin [81-84], individually or in combination [75, 79, 85, 86]. The addition of SC in the diet reduced AFB1 toxic effects in chickens [72, 87]. The ability of SC to reduce AFB1 toxic effects in quails was demonstrated, and this effect was apparently more efficient with the increase in inclusion rates [88].
In [89] authors obtained a significant reduction in AFB1 concentration during beer production, probably due to the bond between mycotoxins and SC cell. This hypothesis was supported by other studies [39, 90]. A 19% reduction in AFB1 during dough fermentation in bread production was observed [91].
|
|
|
|
|
|
B1
0.0058- 6.35 μg/mL |
7.6-49.3 7.6-29 10-24 4-29 17-44 3-44 23-35 27-44 |
15 min, 37 °C: YCW from brewer’s yeast YCW from brewer’s yeast Inactivated baker’s yeast YCW from baker’s yeast Inactivated baker’s yeast YCW from baker’s yeast YCW from baker’s yeast Alcohol yeast |
[66] |
Strain A18 Strain 26.1.11 Pre-treatment: Heated cells 52°C Strain A18 Strain 26.1.11 Heated cells 55 °C Strain A18 Strain 26.1.11 Heated cells at 60 °C Strain A18 Strain 26.1.11 Heat cells at 120 °C Strain A18 Strain 26.1.11 2 mol/L HCl / 1h Strain A18 Strain 26.1.11 |
B1 1 μg/mL 5 μg/mL 10 μg/mL 20 μg/mL 1 μg/mL 5 μg/mL 10 μg/mL 20 μg/mL 5 μg/mL |
69.1 41 33 34.2 65.1 37.2 31 32.6 58.8 56.5 64.5 64 68.8 67 79.3 77.7 72.1 69.3 |
3h, 25 C, PBS 3h, 25 C, PBS 3h, 25 C, PBS |
[56] |
Strain RC008 Strain RC009 Strain RC012 Strain RC016 |
B1 (ng/mL) 50 ng/mL 100 500 50 100 500 50 100 500 50 100 500 |
67.6 43.5 38.2 16.4 21.3 31.8 29.6 20.6 20.2 82.0 48.7 65.5 |
107 cells/mL, 1h, 37 °C, PBS |
[67] |
Yeast concentration: 0.1 % 0.2 % 0.3 % |
B1 (µg/kg) 150 300 450 800 150 300 450 800 150 300 450 800 |
40 25 17 16 88 76 64 51 93 86 81 66 |
37 °C, 24 h, corn 37 °C, 24 h, corn 37 °C, 24 h, corn |
[68] |
Pre-treatment: None Acid treated cells (2 mol/L / 90 min) Heat-treated cells (120 °C / 20 min) |
B1 (ng/mL) 10 20 10 20 10 20 |
40 70 60 73 55 75 |
3 h, 25 °C, pistachio nuts |
[69] |
|
B1
1 mg/kg 5 mg/kg |
55 39 |
37 °C, 6h fermentation in broiler feed |
[70] |
B1
1 µg /g 10 µg /g |
86 72 |
12 °C, 8 days, brewing process |
[89] |
5. Concluding Remarks
Considering the data from several studies carried out until now, it may be observed that microorganisms, among them lactic acid bacteria and yeasts, have a huge potential application in aflatoxin degradation in foodstuffs. However, new studies are necessary to identify bacterial species with greater binding potential with aflatoxins, once there are differences in sensitivity and selectivity, besides the influence of factors that are intrinsic and extrinsic to the bacteria in the decontamination process. After this step of choosing species with greater efficiency has been overcome, new production technologies that are economically viable to be applied to human and animal foods may be developed.
Several studies have demonstrated that the cell wall of SC and LAB and their components are responsible for binding with aflatoxins. However, the mechanisms by which this bond occurs remain unclear. Cell walls with glucomannanes and manno-oligosaccharides have been pointed out as the responsible elements for AFB1 bond with yeasts. The great advantage in the commercial use of these microorganisms as binding agents is that these strains are approved and already used in a wide range of fermented food products, being recognized as safe. However, aflatoxin may be released from the cell-aflatoxin complex with changes in the pH and temperature conditions. Therefore, further studies are necessary to determine the behavior of yeasts in the different environmental conditions before they are used commercially.
Acknowledgments
The authors wish to acknowledge the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) of Brazil for financial support and fellowships.
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