Mycotoxin detoxification of food by lactic acid bacteria

Abstract

Today, a few hundred mycotoxins have been identified and the number is rising. Mycotoxin detoxification of food and feed has been a technically uphill task for the industry. In the twenty-first century, the public demand is healthy food with minimum use of chemicals and preservatives. Among all the fungal inhibition and mycotoxin detoxification methods so far developed for food, biopreservation and biodetoxification have been found safe and reliable. Nowadays, lactic acid bacteria (LAB) are of great interest as biological additives in food owing to their Generally Recognized as Safe (GRAS) classification and mycotoxin detoxification capability. The occurrence of fungul growth in the food chain can lead to health problems such as mycotoxicosis and cancer to humans due to producing mycotoxins such as aflatoxins. Biopreservation is among the safest and most reliable methods for inhibition of fungi in food. This review highlights the great potential of LAB as biodetoxificant by summarizing various reported detoxification activities of LAB against fungal mycotoxins released into foods. Mechanisms of mycotoxin detoxification, also the inherent and environmental factors affecting detoxifying properties of LAB are also covered.

Introduction

Fungi are known as substantial microorganisms in spoilage of food, food products and feed. Spoiling fungi and their toxins contaminate much more than 25% of raw materials produced by agriculture worldwide (Eskola et al. 2020). Apart from economic loss, the presence of fungi in foodstuff could be along with the production of mycotoxins which are among the components of most concern to human health (Nguegwouo et al. 2018; Leiva et al. 2019). The Rapid Alert System for Food and Feed (RASFF) annual report listed mycotoxins among the 10 top hazards on food products in 2019 (RASFF annual report 2019). Fungal species of, Fusarium, Penicillium and Aspergillus genus which produce fumonisins, ochratoxins, trichothecenes, patulins, aflatoxins and zearalenones are considered as main producers of mycotoxins in food products (Magembe et al. 2017; Bhat et al. 2010).

The best strategy is to prevent fungal growth and combat mycotoxins before harvest and during the storage of corps. Mycotoxin contamination however is not ideally controlled at those stages, and alternatives for decontamination, inactivation and removal of mycotoxins in food should be taken to avoid spoilage and infections to humans (Shehata et al. 2019). On the other hand, public also demands minimum chemical preservatives/additives in food; simultaneously, high quality, safe and lease processed food with longest possible shelf life.

Biopreservation is defined as “the use of microorganisms or their metabolites to extend shelf-life and enhance the safety of food commodities” (Stiles 1996). Lactic acid bacteria (LAB) are a group of Gram-positive organisms, some of which have been classified as “Generally Recognized as Safe (GRAS)” by The European Food Safety Authority (EFSA) and The Food and Agricultural Organization of the United States (FAO). They are, therefore, qualified to be used as probiotics and biopreservatives in food products (Dalié et al. 2010; Salminen et al. 1998). Studies demonstrated that LAB cultures isolated from native fermented food products with probiotic attributes and mycotoxin binding could be of immense value in decontaminating mycotoxins in food (Drobna et al. 2017; Shetty and Jespersen 2006).

This review summarizes the potential of LAB supplementation as green preservatives in food by highlighting their capability in detoxification of foodborne mycotoxins in vitro and in situ. Different mechanisms of mycotoxin detoxification as well as various characteristics of the food and microorganisms (both fungi and LAB strains) contributing to the detoxification properties of LAB were also overviewed.

Foodborne mycotoxins and their effect on human health

Mycotoxins are toxic and thermostable secondary metabolites of fungi that can be transferred into food and feed and are able to withstand various food microbial stabilization processes such heating (Oliveira et al. 2014; Conte et al. 2020). Consequently, the contaminated food and feed consumed by humans and animals exposes them to the toxic effects of them (Abdallah et al. 2015). Mycotoxins can grow on many foodstuffs, such as cereals, crops, nuts, fruits and dried fruits, cheese and spices, during all storage, harvesting, and cultivation steps (Patel et al. 2021).

A few hundred mycotoxins mycotoxins have been so far identified, among which, about 30 occur in foods and feeds (Zhang et al. 2016). Aflatoxins, fumonisins, trichothecenes, ochratoxin A and zearalenone are the major foodborne mycotoxins of public health interest (Wu et al. 2014; Nguegwouo et al. 2018). Mycotoxins have been associated with some of mild and chronic human diseases. Their carcinogenic effects on different organs like liver, lung and kidney have been proven. Figure 1 depicts the harms caused by the most common foodborne mycotoxins to human organs.

Fig. 1

Harms caused by the most common food-born mycotoxins to human organs. The picture has been designed by Freepik.com

Mycotoxin contamination is widely reported in food products worldwide, especially in fermented and biologically ripened foods (Chelule et al. 2010a). The main substrates or crops best supporting the growth of fungi and release of their mycotoxins are cereals, milk, nuts, oilseeds, dried fruits, coffee and spices, flour and their by-products (Marín et al. 2018; Ofori et al. 2016). Penicillium is the predominant flora in cheese (Sengun et al. 2008). Patulin released by Penicillium expansum was found to pose more potent effects in comparison with other carcinogens such as nitrosamines, polycyclic aromatic hydrocarbons, citrinin and heterocyclic aromatic amines (Knasmüller et al. 2004). Maize and maize-based products are host for Fusarium species who are producer of fumonisins. Fumonisins are thought to be the cause of edema and esophageal cancer in humans (Smith 2018). Ochratoxins are another carcinogen and immunosuppressive mycotoxin happening mostly in cereals produced by Aspergillus and Penicillium species (Blanchard and Manderville 2016). Milk and milk products are another group of food substrate for Aspergillus flavus and Aspergillus parasiticus, the main producers of aflatoxins, one of the most hazardous mycotoxins (Makau et al. 2016). Table 1 illustrates most common food-born mycotoxins, their fungi producer and the main foods host for them.

Table 1 Most common food-born mycotoxins, their fungi producer and foods host for them

Mycotoxin detoxification in food systems

One of the most challenging tasks for the food industry is avoiding/minimizing mycotoxin occurrence and detoxification of mycotoxins in food. Since mycotoxins are mostly heat-stable, cooking and other typical heat processes used in the food industry are not efficient to remove mycotoxins from contaminated food (Chelule et al. 2010b). There are some alternatives for degradation of mycotoxins such as the use of alkaline ammonia as chemical treatment, though not yet favored as they potentially pose side effects to human health and might also alter the properties of the food (Chelule et al. 2010a).

The consequence demand for more natural alternatives for food preservation has driven experts to turn to biological approaches for combating fungal growth and mycotoxin contamination (Guimarães et al. 2018b). Natural preservatives, especially bio-based ones like bacteria and bacteriocins have been extensively studied. Among the different potential microorganisms, LAB have been considered as one of the oldest and most promising natural biopreservatives particularly in fermented foods (Shehata et al. 2019; Byakika et al. 2019).

The term “LAB” used for a wide range of Gram-positive, catalase-negative, non-motile and non-spore forming bacteria that produce lactic acid as the main ultimate product of fermentation (Holzapfel and Schillinger 2002). LAB, due to their GRAS status and Qualified Presumption of Safety (QPS) status provided by FDA and EFSA, have greatly appealed researchers to investigate their potential as biopreservatives (Fraqueza 2015; Byakika et al. 2019).

LAB as mycotoxin detoxificant in food

LAB have been used as mycotoxin detoxifiers in some foods including milk, grains and sorghum beverages for inhibiting aflatoxin M1, Fusarium mycotoxins and aflatoxin, respectively (Haskard et al. 2001; Juodeikiene et al. 2018; Byakika et al. 2019). Stability of mixture in the gastrointestinal tract and resident time are two substantial factors in choosing LAB aimed for mycotoxin in food, though binding strength is highly strain-specific (Shetty and Jespersen 2006).

LAB could enhance the mycotoxin combat in food via two assistances of being added either in heat treated form or as starter/adjunct cultures to fermented food and beverages; alternatively, by supplementing the food by their purified substances (Juodeikiene et al. 2012). Mainly, there are two mechanisms proposed for LAB mycotoxin detoxification action; physical binding to the cell wall of bacteria, and inhibition of mycotoxin biosynthesis (Dalié et al. 2010). Number of studies reporting the detoxification attribute of LAB species against mycotoxins are listed in Table 2.

Table 2 Detoxification extent of food mycotoxins by LAB

Reduction of mycotoxin biosynthesis and degradation of mycotoxins

LAB were found to contribute to reducing the biosynthesis of mycotoxin by fungi. Cell-free supernatant of Lb. plantarum UM55 and Lb. buchneri UTAD104 reduced the production of ochratoxin from P. nordicum by 60%. Phenyllactic (PLA), acetic acid and indole lactic (ILA) were found to be the most effective substances causing the effect (Guimarães et al. 2018a). In another study from the same group, it was shown that cell-free supernatant of Lb. plantarum UM55 inhibits the production of aflatoxins from A. flavus by 91%. They attributed the antiaflatoxigenic properties of LAB to the strain’s capability to produce lactic acid, PLA, OH-PLA and ILA. In their study, PLA exhibited a stronger activity, also 90% inhibitory concentration (IC90) for the aflatoxins was obtained 0.87 mg/ml. Aflatoxins IC90 for ILA, OH-PLA and lactic acid were of 1.47, 1.80, and 3.92 mg/ml, respectively (Guimarães et al. 2018b).

Concentration of supernatant was found influential in mycotoxin biosynthesis reduction of LAB. According to Shehata et al. (2019) cell free supernatant of Lactobacillus sp. RM1 at the concentration of 5 mg/ml, reduced the aflatoxin production of A. parasiticus ITEM 11 and A. flavus ITEM 698 whereas it stopped aflatoxin production of the strains at 15 and 10 mg/ml concentration. Similarly, the production of ochratoxin A by A. carbonarius ITEM 5010 reduced about 5-folds at a concentration supernatant of 10 mg/ml and stopped at the concentration of 15 mg/ml (Shehata et al. 2019). There are some mechanisms proposed for the reduction of mycotoxin biosynthesis by LAB. The first possibility is the release of molecules due to the natural lysis of LAB which inhibit fungal growth; as a result, lowering their mycotoxin production (Dalié et al. 2010). Later, it was also suggested that bioactive compounds secreted by LAB such as organic acids, PLA, reuterin, fatty acids, peptides and cyclic peptides are capable to interact with mycotoxins; consequently, reduce their amount in food (Muhialdin et al. 2020).

The capability of the main acids produced from LAB in the degradation of aflatoxin B1 was compared in a study. They heated aflatoxin B1 at 80 °C in the presence of lactic, acetic and citric acids for different times. Lactic acid was most efficient among all three in degrading aflatoxin B1 to less toxic aflatoxin B2a or aflatoxin B2 (Aiko et al. 2016). In another study, a cyclic dipeptide detected from P. pentosaceus supernatant was detected as the main substance responsible for anti-aflatoxigenic effect of the strain binding aflatoxin from A. niger (Ebrahimi et al. 2020). Juodeikiene et al. (2018), observed that treatment of malting grains with Lb. sakei KTU05–6, P. pentosaceus KTU05–8, P. acidilactici KTU05–7 and KTU05–09, and KTU05–10 strains reduced level of zearalenone, deoxynivalenol, T-2 and HT-2 toxin concentrations by 23, 34, 58 and 73%, respectively. They reported that the reduction was probably due to either physical binding or degradation of mycotoxins (Juodeikiene et al. 2018).

Mycotoxin binding

Since LAB bind mycotoxins in both dead and alive forms, the mechanism of mycotoxin removal in many reports was explained to be through the cell wall of LAB binding the toxins. Reduction of aflatoxin M1 in three different contaminated samples of phosphate buffer saline, skim and full cream milk was found independent of the viability of LAB cells. No significant difference between the binding ability of viable and heat-killed cells supports the mycotoxin removal activity of LAB through physical mechanism rather than the metabolic or covalent function of the cells (Pierides et al. 2000). Later also, heat-killed LAB cells removed aflatoxin M₁ more efficiently than viable cells in both contact times of 15 min and 24 h. The difference was explained because treatment exposes the binding spots of the cell walls which facilitates binding (Bovo et al. 2013). Heat and acid treatments affect the cell wall binding sites through two major constructive compounds of polysaccharides and proteins. The treatments denature proteins and degrade them to smaller peptides by breakage of protein ammonium sulfate bonds, they also break down polysaccharide glycosidic bond loosening peptidoglycan monosaccharide which leads to exposing more binding sites (Lili et al. 2018). Supporting this fact is the study of Haskard et al. (2001) where they observed that several times of washing with water left 38 and 50% of the bound toxins to the viable cells of Lb. rhamnosus strains LGG and LC105 retained, respectively. This was while heat and acid treated cells retained higher percentage of the toxin by 66–71% (Haskard et al. 2001). Another example is reduction of deoxynivalenol (DON) was reported through physical binding by Lactobacillus strains and the cell wall of the yeast Saccharomyces cerevisiae (Ghamsari et al. 2021). Moreover, DON was removed in liquid culture by 40.7% through cell wall binding with Lactobacillus paracasei LHZ-1 (Zhai et al. 2019).

Removal of mycotoxins was demonstrated to reduce the ability of the LAB strain to adhere which provides evidence for the cell wall binding mycotoxin detoxification mechanism of LAB. Kankaanpää et al. (2000) believe that employing LAB could reduce aflatoxins in the intestine since they observed that after Lb. rhamnosus GG being subjected to aflatoxin B1 experiment lost its adhesion ability from 30% to 5% indicating that aflatoxins may influence the adhesion properties of probiotics (Kankaanpää et al. 2000). Similarly, Gratz et al. (2004) observed that binding of Lb. rhamnosus GG to intestinal mucus reduced after pre-treatment of bacterial cells with aflatoxin B1, though the pre-incubation of the cells with intestinal mucus also its ability to bind aflatoxin B1. Mycotoxin binding by LAB was reported to be a rapid reaction and dependent on the bacterial density. Lb. rhamnosus LGG and LC 705 were shown to bind zearalenone and its derivative up to 55% (w/w, Gratz et al. 2004). This was while co-incubation of zearalenone and its derivative reduced the quantity of the toxins bound which indicates that the binding sites on the cell wall were shared between the toxins, thus less density of cells available for each one (El-Nezami et al. 2002b).

LAB cell wall, as Gram positive bacteria, consists of peptidoglycan network embedding teichoic acid, lipoteichoic acid and S-layer as other main substances present in the cell wall (Delcour et al. 1999). The binding function of the cell wall is directed by these major components, alone or in cooperation (Haskard et al. 2001). Studying the mechanism of aflatoxin cell wall binding to Lb. rhamnosus using enzyme treatments demonstrated that the binding occurred through carbohydrate and protein content of the cell wall. The same study highlighted the contribution of hydrophobic interactions in binding since treatment with urea in their experiments decreased the binding (Haskard et al. 2000). Lahtinen et al. (2004), however, emphasized the greater role of carbohydrates of peptidoglycan or the structures closely associated with peptidoglycan in the Aflatoxin B1 binding process (Lahtinen et al. 2004).

Mycotoxin binding properties of LAB are highly strain-specific. Many studies have demonstrated that LAB strains bind mycotoxins with different strengths. Lb. rhamnosus LGG and LC 705 and Propionibacterium freudenreichii were shown to effectively bind some of common Fusarium toxins such as deoxynivalenol, nivalenol, 3-acetyldeoxynivalenol, diacetoxyscirpenol, fusarenon, T-2 toxin and HT-2 toxin. The strains bound the toxins in different levels, Lb. rhamnosus strains LGG and LC 705 presenting higher binding ability towards aflatoxin B1 than aflatoxin B2 and G1 (El-Nezami et al. 2002a). Shetty and Jespersen (2006) also reported the same fact where 15 strains of LAB were tested, and Lb. plantarum and Lb. fermentum among others were found to bind higher than 60% (w/w) of aflatoxin B1 (Shetty and Jespersen 2006). In another study, Drobna et al. (2017), reported that 5 LAB strains bound 10.8–66.7% of aflatoxin B1 present in the samples in vitro. The best results concerning aflatoxin B1 reduction and reproducibility of the reduction process were from Lb. reuteri KO4b strain (66.7% ± 1.0%) followed by Lb. plantarum KG4 (59.4% ± 1.6%) after an incubation of 24 h (Drobna et al. 2017).

Factors affecting mycotoxin binding activity of LAB

The effectiveness of LAB strains in binding mycotoxins is determined by multiple factors like cell density, concentration of toxins, pH value, viability and temperature. Haskard et al. (2001) investigated the binding ability of aflatoxin B1 by three forms of viable, heat-killed and acid-killed Lb. rhamnosus strain GG. They reported that there was no significant difference between all three forms of cells in terms of binding ability. Since treatment with periodate, resulted in the highest aflatoxin B1 binding, they concluded that binding occurs predominantly through carbohydrates of bacterial cells. Urea also caused a reduction in aflatoxin B1 binding for all bacteria, suggesting that hydrophobic interactions also largely contributes to binding. Finally, increasing NaCl or CaCl₂ concentrations did not significantly affect aflatoxin B₁ binding which implies mediation of electrostatic interactions (Haskard et al. 2001). Hydrogen bonding interactions were found to play role in binding ability of LAB as an increase in pH from 2.5 to 8.5 did not influence the aflatoxin B1 binding ability of Lactobacillus GG while decreased binding of aflatoxin B2a (Haskard et al. 2000).

The impact of temperature in detoxification of mycotoxins was highlighted by Bovo et al. (2013) where they reported that LAB strains of Lb. bulgaricus, Lb. rhamnosus and Bifidobacterium lactis bound aflatoxin M₁ in values of 13, 19 and 37% at 4 °C and 33, 24 and 32% at 37 °C, respectively. In another study, Shehata et al. (2019) increasing temperature from 28 to 37 °C caused a significant decrease in antifungal activity of Lactobacillus sp. RM1 against A. parasiticus (Bovo et al. 2013).

Incubation time also was found effective in the detoxification ability of LAB. Drobna et al. (2017) tested the ability of 5 LAB strains to bind AB1 in vitro. According to their results, the extent of binding ability was directly proportional to the length of incubation with aflatoxin B1 for all strains except Lb. mucosae D strain, which showed a decreased activity with time. They observed that aflatoxin B1 reduction and reproducibility of the reduction process were obtained with Lb. reuteri KO4b strain (66.7% ± 1.0%) followed by Lb. plantarum KG4 (59.4% ± 1.6%) after an incubation of 24 h (Drobna et al. 2017).

The role of pH in the binding ability of LAB also was revealed in another study. Guimarães et al. (2018b) reported that inhibition of Lb. plantarum UM55 against A. flavus was dependent on the pH of cell-free supernatant, increased with increasing concentrations of cell-free supernatant. Increasing the pH from 5 to 7 did not significantly affect the antifungal properties of Lacticaseibacillus sp. RM1 against A. parasiticus, A. flavus and A. carbonarius (Shehata et al. 2019). Raisin pH above 7, however, resulted in a drop of activity by 50% (Guimarães et al. 2018b).

Conclusion and future perspectives

Fungal spoilage of food is implicated in many food poisoning outbreaks in human and animals by producing mycotoxins with possible carcinogenic and teratogenic effects. Application of LAB species with mycotoxin detoxification properties in food was reported to reduce mycotoxins. Mycotoxin detoxification by LAB is mainly based on adhesion of mycotoxins to the cell wall peptidoglycan structure of LAB, also reducing mycotoxin synthesis and degradation of mycotoxins. Additionally, mycotoxin detoxification properties of LAB is strain-specific meaning that different LAB strains might or might not bind a particular mycotoxin, and binding strength varies from strain to strain. Binding activity and strength depends on pH of food, storage temperature, LAB cell density and concentration of mycotoxins in food. Therefore, multiple factors including the properties of the food itself and the potential mycotoxins and their fungi producer as well as LAB strains should be taken to account before employing LAB as mycotoxin detoxification agents in food.

Valuable advances in detection of LAB strains with antifungal activities and their active substances have been achieved in recent years. There is still, however, a need for further investigation to be pursued in the biochemical basis of the detoxification mechanism of LAB. A better understanding of detoxification LAB biochemical pathways will provide insights into the production of potential mycotoxin detoxifying bioactive compounds. Regarding that studies in this area are mostly lab-scale reports, a promising strategy at this stage might be directing studies more in line with industrializing these findings. The pathways and mechanism of action of LAB as detoxificant could be also investigated for targeted food poisoning therapy by clinicians associated with toxicology.