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Review

Comprehensive Review of Dietary Probiotics in Reducing Aflatoxin B1 Toxicity

by
Dasol Choi
1,
Xingrui Fan
2,3 and
Jae-Hyuk Yu
3,4,*
1
Department of Materials Science & Engineering, University of California, Los Angeles, CA 90095, USA
2
Department of Food Science, University of Wisconsin-Madison, Madison, WI 53706, USA
3
Food Research Institute, University of Wisconsin-Madison, Madison, WI 53706, USA
4
Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA
*
Author to whom correspondence should be addressed.
Toxins 2025, 17(10), 482; https://doi.org/10.3390/toxins17100482
Submission received: 27 August 2025 / Revised: 22 September 2025 / Accepted: 23 September 2025 / Published: 26 September 2025
(This article belongs to the Section Mycotoxins)
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Abstract

Aflatoxin B1 (AFB1), the most potent and widespread mycotoxin produced by Aspergillus flavus and Aspergillus parasiticus, poses a significant global threat to food safety and human health, with chronic exposure strongly linked to hepatocellular carcinoma (HCC). While physical and chemical detoxification approaches exist, their limitations have led to an increased interest in biological strategies, particularly probiotic interventions. In this review, we synthesize current in vivo and clinical evidence on the ability of probiotic lactic acid bacteria—including Lactobacillus casei Shirota, Lactobacillus rhamnosus GG, Lactobacillus rhamnosus LC705, Lactococcus lactis, and selected Bifidobacterium species—to reduce AFB1 absorption and toxicity. We summarize mechanistic insights into cell wall adsorption, gut microbiota modulation, intestinal barrier protection, and antioxidant enhancement. Clinical trials have shown reductions in AFB1 biomarkers following probiotic supplementation, supporting their translational potential for human health. However, clinical evidence remains limited by small sample sizes, short intervention periods, and variability in endpoints. Collectively, this review consolidates mechanistic, preclinical, and clinical findings to position probiotic lactic acid bacteria as promising biological countermeasures against AFB1-induced hepatocellular carcinoma.
Keywords:
aflatoxins; probiotic lactic acid bacteria (LAB); anti-carcinogenic effect; hepatocellular carcinoma; bio-adsorbent
Key Contribution: This review synthesizes in vivo evidence demonstrating that probiotic lactic acid bacteria can detoxify aflatoxin B1, reduce its absorption, and serve as practical dietary strategies to lower hepatocellular carcinoma risk.

1. Introduction

Filamentous fungi, including Aspergillus, Penicillium, and Fusarium, are prevalent in soils, farms, and other agricultural environments. Many species produce mycotoxins, which were defined by Bennett [1] as “natural products produced by fungi that evoke a toxic response when introduced in low concentration to higher vertebrates and other animals by a natural route.” As low-molecular-weight bioactive molecules, mycotoxins frequently infiltrate human food systems and animal feed supplies, establishing themselves as persistent threats to global health security [2]. The magnitude of mycotoxin contamination across global food systems represents a substantial crisis. Earlier estimates by the United Nations Food and Agriculture Organization (UN FAO) and World Health Organization (WHO) placed global mycotoxin contamination in crops at around 25%, but more recent assessment by Eskola et al. [3] suggests that the actual prevalence is considerably higher across food crops worldwide [3,4]. While the scientific literature documents nearly 400 distinct mycotoxin compounds, only a select group demonstrates the combination of extreme toxicity, environmental persistence, and widespread distribution that creates significant public health concerns. This critical subset encompasses aflatoxins (AF), ochratoxins (OT), zearalenone (ZEA), T-2 toxin, fumonisins (FB), deoxynivalenol (DON), and related trichothecene compounds [5]. The inherent chemical and thermal stability of these molecules renders conventional food processing techniques largely ineffective for their elimination [6,7].
The discovery of AF emerged from tragedy when an enigmatic illness, subsequently termed “Turkey X disease,” devastated British poultry in 1960, claiming approximately 100,000 turkey lives [8]. AF-producing fungi, including Aspergillus flavus and Aspergillus parasiticus, are common in crops including maize, corn, wheat, and sorghum [9,10,11,12]. Contemporary risk assessments indicate that between 4.5 and 5.5 billion individuals globally face ongoing AF exposure through dietary consumption [10,13]. Within the AF family, aflatoxin B1 (AFB1) stands as the most biologically potent member, capable of inducing genotoxicity, oncogenesis, and immunological dysfunction [14,15]. Therefore, the U.S. Food and Drug Administration (FDA) imposes an action level of 20 parts per billion (ppb) for total AF in foods. The carcinogenic potential of AFB1 has earned it a classification as a Group I carcinogen by the International Agency for Research on Cancer (IARC), with hepatic malignancy representing its primary oncological consequence [16,17]. Upon consumption, AFB1 undergoes hepatic biotransformation through cytochrome P450 enzymatic pathways, specifically involving CYP1A2 and CYP3A4 isoforms, resulting in the formation of aflatoxin-8,9-exo-epoxide, a highly reactive intermediate [17,18,19]. This metabolic intermediate demonstrates high affinity with DNA, RNA, and protein molecules [20,21]. The predominant DNA lesion involves adduct formation at guanine N7 positions, which is recognized as a biomarker for hepatocarcinogenic risk assessment [19,22]. These DNA modifications frequently compromise p53 tumor suppressor function through characteristic G→T transversion mutations occurring at codon 249, establishing conditions conducive to hepatocellular carcinoma (HCC) development (see Figure 1) [11,20,23,24,25]. Numerous studies have conclusively established chronic AF exposure as a principal etiological factor in HCC development [26,27,28,29,30,31]. Beyond direct genotoxic effects, aflatoxicosis initiates inflammation by damaging the liver tissue and results in elevated levels of apoptosis and carcinogenic activities [32,33,34].
HCC represents a central global malignancy, ranking fifth among all cancer types and third in cancer-associated mortality worldwide [35]. Current estimates attribute 4.6–28.2% of the global liver cancer burden directly to AF exposure [36,37]. Therefore, scientists are actively looking for strategies to mitigate the impact of AF on human health. In recent years, the human gut microbiome and probiotics have been extensively studied for potential anticarcinogenic functions, and interest has been raised in modifying the gut microbiome to reduce AFB1 bioavailability and toxicity [38]. The relationship between gut microbiota and AFB1 uptake has attracted considerable scientific attention and has been widely investigated. This review provides a critical synthesis of mechanistic, preclinical, and clinical data on probiotic interventions for AFB1 toxicity. It specifically evaluates the molecular mechanisms of action, summarizes key findings from animal models and human intervention studies, and discusses both the therapeutic potential and current limitations of probiotic strategies for AFB1 detoxification.

2. AFB1-Induced Alterations of Gut Microbiota

2.1. Adsorption of AFB1 in the GI Tract

The gastrointestinal tract (GI) serves as a crucial barrier, offering multifaceted defense mechanisms against pathogenic microorganisms, environmental toxins, and harmful xenobiotics. However, exposure to AFB1 disrupts this protective interface. Studies demonstrate that AFB1 exposure leads to a dose-dependent increase in intestinal crypt depth, even when villus length remains unchanged, indicating structural alterations that undermine normal mucosal function. These morphological changes, combined with reductions in intestinal weight that impaired nutrient absorption and diminished mucosal health, facilitated greater susceptibility to toxins like AFB1 [39,40]. Moreover, in vivo studies have shown that AFB1 exposure increases the plasma lactulose-to-rhamnose ratio, a marker of intestinal permeability since lactulose absorption is enhanced when tight junction integrity is compromised [41]. This disruption is accompanied by leukocyte and lymphocyte infiltration into the lamina propria [2], suggesting acute inflammation and impaired gut–liver axis function. The cytotoxic effects of AFB1 are primarily attributed to the excessive generation of intracellular reactive oxygen species (ROS), which triggers the release of high levels of lactate dehydrogenase (LDH) and consequently damages cell membranes and DNA integrity [42]. Within the GI tract, AFB1 has been linked to intestinal barrier disruption, altered cell proliferation, and increased apoptosis [11]. While detoxification of AFB1 primarily occurs in the large intestine through microbial biotransformation into less toxic derivatives, more than 80% of the toxin is rapidly absorbed in the duodenum by passive diffusion [11,42]. Therefore, restoration of barrier function and protection against these adverse effects can be achieved by modulating the gut microbiota, highlighting its critical role in maintaining intestinal health.

2.2. Modulation of Gut-Health-Induced Microbiota

Exposure to AFB1 induces significant changes in gut microbiota composition, and how microbiota reacts to AFB1 depends on the exposure level, as described in Table 1. A consistent observation across multiple studies is that AFB1 exposure modulates gut microbiota composition, characterized by a reduction in Bacteroidetes and a concomitant increase in Firmicutes [43,44,45,46]. These two phyla represent the dominant constituents of the gut microbiome, and their imbalance reflects a significant shift in microbial homeostasis [44]. This shift suggests that members of Firmicutes may possess greater tolerance to AFB1, enabling them to outcompete other taxa under toxin stress. Firmicutes comprise several Gram-positive genera, including Lactobacillus and Streptococcus, which belong to the LAB group. LAB can remove AFB1 through binding to cell wall structures [47,48]. Nevertheless, the impact of AFB1 on specific Firmicutes taxa remains inconsistent across studies. For instance, Streptococcus spp. and Lactococcus spp. showed pronounced declines at AFB1 concentrations ranging from 5 to 75 ppb [49], and Lactobacillus spp. abundance decreased by 50.5% (p < 0.05) in piglets when exposed to 320 ppb of AFB1 [50]. Conversely, other investigations have reported increased total LAB populations at 1500–2000 ppb AFB1 (p < 0.05), while a significant reduction was observed in broilers exposed to 1000 ppb [51]. Such discrepancies suggest that LAB responses to AFB1 are highly dose-dependent and possibly influenced by host species and diet. In addition to compositional changes, exposure to AFB1 affects microbial metabolism. At 2500 ppb AFB1, reductions in short-chain fatty acids (SCFAs) were observed, alongside depletion of SCFA-producing LAB strains [3,52]. This decline in SCFAs, critical for maintaining intestinal homeostasis, highlights the potential for AFB1 to disrupt gut metabolic activity. Interestingly, some taxa appear to proliferate under high toxin exposure, such as Bifidobacterium spp. abundance increased significantly at 10,000 ppb AFB1 (p = 0.001), accompanied by elevated xylanase (p = 0.005) and cellulase (p = 0.002) activities, suggesting an enzymatic adaptation to counter intestinal microecological imbalance [53]. Collectively, these findings indicate that AFB1 exerts profound, dependent effects on gut microbiota composition, toxin’s exposure level and metabolism. Shifts in the LAB populations highlight the dynamic interactions between probiotics and AFB1 stress, offering new insights into the role of beneficial microbes in AFB1 detoxification.

3. Methods for Detoxifying AFB1 in the GI Tract

A variety of physical, chemical, and biological approaches have been explored to inactivate and detoxify AFB1 in food and feed [54,55,56,57]. For example, clay minerals are widely used to selectively or nonspecifically adsorb mycotoxins in the GI tract [55,58]. Inorganic adsorbents, such as aluminosilicates, have also demonstrated a strong binding capacity for AFB1 in animal studies [59,60,61,62,63]. While they are generally recognized as safe (GRAS) for dietary inclusion, no adsorbent has yet been approved by the U.S. Food and Drug Administration (FDA) for clinical treatment of aflatoxicosis. Moreover, these strategies are often costly and impractical for widespread application. Despite the benefits of existing physical and chemical methods, there remains an urgent need for more effective, safe, and affordable detoxification strategies. In recent years, biological processes, particularly probiotic interventions, have gained increasing attention as promising alternatives. These approaches utilize the natural binding and detoxifying capacities of the gut microbiota to mitigate AFB1 toxicity in the gastrointestinal tract.

Probiotics LAB as Potential Detoxifiers of AFB1

According to the World Health Organization (WHO), probiotics are defined as “live microorganisms that, when administered in adequate amounts, provide a health benefit to the host” [64]. Beyond their well-recognized role in gut health and microbiota restoration, probiotics have also been investigated for their ability to detoxify AF in food and the GI tract. Some strains are capable of modifying the chemical structure of AFB1, converting it into less toxic or non-toxic metabolites. However, these transformations do not always eliminate toxicity entirely, as certain products, such as aflatoxicol, may retain harmful effects [65,66]. An alternative mechanism involves direct binding of AFB1 to microbial cells, which reduces its intestinal absorption and subsequent systemic toxicity. Among probiotics, LAB have shown particularly strong binding affinities toward AFB1 [48,67,68,69]. Both viable and non-viable LAB cells can effectively adsorb AFB1, indicating that the detoxification mechanism is linked to cellular structural components rather than active metabolism [70]. Supporting this, Haskard et al. [71] demonstrated that periodate treatment significantly reduced the AFB1-binding capacity of Lactobacillus rhamnosus GG, implicating carbohydrate structures in the cell wall as the key binding sites. In contrast, treatments with proteases or lipases exerted minimal effects, suggesting that proteins and lipids play a negligible role in AFB1 adsorption [70]. LAB are Gram-positive bacteria characterized by a thick peptidoglycan cell wall, a carbohydrate-rich structure regarded as the principal component responsible for sequestering AF. This was confirmed by Lahtinen et al. [72], who found that cell wall extracts of L. rhamnosus GG retained a binding capacity of 81% for AFB1, comparable to that of intact viable cells (84%), whereas purified exopolysaccharides bound less than 1%. Similarly, Zhu et al. [73] reported that highly purified (97.75%) peptidoglycan isolated from Limosilactobacillus reuteri adsorbed 64.3–75.9% of AFB1 in vitro. These findings indicate that peptidoglycan and related polysaccharide structures are the primary contributors to AFB1 adsorption onto LAB. The formation of AFB1–LAB complexes prevents toxin absorption via paracellular diffusion, thereby reducing the risk of hepatocarcinogenesis. Among probiotic LAB, Lactobacillus, Bifidobacterium, and Lactococcus are the most widely studied, with reported AFB1-binding capacities ranging from 5.6% to 59.7% [48]. In particular, Lactobacillus spp. consistently demonstrate high efficacy in sequestering AF, highlighting their potential as promising biological strategies for AFB1 mitigation [68,69,71].
Not all LAB strains exhibit the same capacity to bind AFB1. In a screening of 20 LAB strains conducted by Peltonen, el-Nezami, Haskard, Ahokas and Salminen [48], binding efficiencies ranged widely, from 5.6% in Lactococcus lactis ssp. cremoris MK4 to 59.7% in Lactobacillus amylovorus CSCC 5160. Even within the same species, significant variation was observed: Lactobacillus rhamnosus strain Lc 1/3 bound 54.6% of AFB1, while strain E-97800 bound only 22.7%. These differences suggest that factors beyond peptidoglycan, such as cell surface components, contribute to binding efficacy. Binding is believed to occur via weak, noncovalent interactions involving hydrophobic pockets, as well as through glycopolymers such as teichoic acids embedded in the cell wall [5,48,70,71,72,74,75]. Teichoic acids, in particular, influence adsorption efficiency under varying pH conditions [5]. In addition, extrinsic parameters such as probiotic cell density, initial AF concentration, and temperature play a crucial role in determining the binding efficiency of probiotic strains [76]. Given the variability in AFB1-binding affinity across species and strains, certain LAB strains have been studied in greater detail for their probiotic value and detoxification potential. Among them, Lactobacillus rhamnosus GG and LC705 demonstrated particularly strong and stable binding to AFB1 across 12 LAB strains tested (p < 0.05). Moreover, heat and acid treatments further enhanced their binding capacity. Even after washing steps, viable L. rhamnosus GG and LC705 retained 50% and 38% of their bound AFB1, respectively [71]. Similarly, Lactobacillus casei L30 exhibited high binding affinity and stability among eight L. casei strains, maintaining AFB1 binding after washing and exposure to bile salts. Interestingly, the presence of bile salts increased the proportion of AFB1–L. casei complexes, suggesting that modifications to the bacterial cell envelope may improve binding interactions with AFB1 [75]. Due to their superior binding efficiency, strains such as L. casei and L. rhamnosus have been widely investigated for their potential role in preventing AFB1-induced hepatocarcinogenesis.

4. Anticarcinogenic Effect of Probiotics LAB on AFB1-Induced Liver Carcinogenesis

4.1. Probiotic Lactobacillus casei Shirota (Lcs)

The potential of probiotic LAB as a dietary strategy to mitigate HCC risk associated with AFB1 exposure has been demonstrated in both animal studies and human clinical trials (see Table 2). Probiotic Lactobacillus casei Shirota (Lcs) has been comprehensively studied due to its outstanding efficacy in detoxifying AFB1. Supplementation with Lcs significantly lowered systemic AFB1 levels in contaminated feed models and improved liver function biomarkers, such as alanine transaminase (ALT) and aspartate transaminase (AST), which are typically elevated during aflatoxicosis [77,78]. For example, ALT and AST levels rose to 108 U/L and 124 U/L, respectively, in AFB1-exposed animals without probiotics, but decreased to 75 U/L and 100 U/L in the Lcs-fed group [77]. Another study demonstrated that the Lcs treatment reduced AFB1 in the blood from 88 ng/mL to 50 ng/mL (p < 0.05) in AFB1-exposed rats [79]. These hepatoprotective effects were further supported by reductions in lipid peroxidation and improvements in histological outcomes [80]. In parallel, Lcs enhanced the activities of key antioxidant enzymes, including glutathione peroxidase (GPx), glutathione-S-transferase (GST), superoxide dismutase (SOD), and catalase (CAT), which counteract AFB1-induced oxidative stress [80,81,82]. Notably, GST facilitates detoxification by conjugating the reactive AFB1-8,9-epoxide intermediate with glutathione, producing the excretable metabolite AFB1-mercapturic acid in urine [81]. Together, these findings underscore the dual role of probiotics in lowering systemic toxin burden and reinforcing antioxidant defenses to preserve hepatic integrity [80,82]. Beyond enzymatic activity, bacterial cell wall structures contribute to AFB1 detoxification (see Figure 2). Serrano-Niño et al. [83] reported that teichoic acids, which constitute ~30% of the Lcs cell wall, undergo conformational changes upon AFB1 binding, as visualized by scanning electron microscope (SEM) [79]. The teichoic acid backbone, which is composed of glycerol and ribitol linked by phosphodiester bonds and decorated with glucose and D-alanine, provides hydroxyl groups capable of hydrogen bonding with AFB1 carbonyls (Figure 2) [83]. Strains deficient in teichoic acids exhibited markedly lower AFB1-binding efficiency, showing their importance in the detoxification process [5]. Other cell wall constituents also contribute; for example, β-D-glucans can capture AFB1 via hydrogen bonding and van der Waals interactions, particularly at the C(6)-hydroxyl group of glucopyranose residues [84]. Surface proteins likewise serve as binding sites, with heat-denatured Lcs showing enhanced AFB1 binding to unfolded proteins [79,85,86]. Collectively, these mechanisms demonstrate that AFB1-binding capacity is strongly strain-dependent and mediated by multiple structural components of the Lcs cell wall.
Building on animal studies, several clinical trials have assessed the efficacy of Lcs in reducing AFB1 absorption in humans. A prospective, randomized clinical trial recruited 71 healthy university employees with urinary aflatoxin M1 (AFM1) levels above 0.005 ng/mL, exploring whether fermented milk containing Lactobacillus casei probiotics could prevent AFB1 absorption in the GI tract [87]. According to the study, the concentrations of AFB1-lys in blood serum were reduced from 6.24 pg/mg to 5.48 pg/mg (p = 0.035) during the 4-week intervention period. Furthermore, a significant difference of 13.7% was observed between the placebo drink and milk with Lcs, at 6.35 pg/mg and 5.48 pg/mg, respectively, after 4 weeks of intervention (p = 0.005) [87]. Later, this research group recruited a broader range of participants (n = 174) from Selangor, Malaysia. After 12 weeks of intervention, the Lcs probiotics treatment group has a 23% reduction in AFM1 in excreted urine as compared to the placebo group [88]. These results provide compelling evidence that Lcs can be used as a dietary intervention to reduce AFB1 absorption and thereby lower the risk of long-term AF exposure in humans.

4.2. Probiotic Lactobacillus rhamnosus

Studies have also demonstrated that L. rhamnosus strains can substantially reduce the gastrointestinal absorption of AFB1. In chickens, duodenal uptake of AFB1 decreased by over 70% with L. rhamnosus GG (LGG) and by 37% with L. rhamnosus LC705, indicating that LGG exhibits stronger binding capacity than LC705 despite both belonging to the same species [89]. Similar findings were reported in rats: fecal excretion of AFB1 increased significantly within 24 h of LGG administration, and ALT activity was reduced, suggesting mitigation of AFB1-induced hepatotoxicity [90]. Subsequent experiments confirmed that LGG promoted the excretion of AFB1 in feces by forming stable LGG–AFB1 complexes within the GI tract, thereby reducing systemic toxicity [91]. As with other LAB strains (Section 4.1), the protective action of L. rhamnosus is also largely dependent on cell wall-mediated binding of AFB1 within the GI tract. Beyond adsorption, L. rhamnosus exhibits a distinct anti-inflammatory mechanism: it suppresses NF-κB signaling in AFB1-exposed liver tissue, thereby reducing the expression of proinflammatory cytokines (IL-1β, TNF-α, IL-6) and mitigating hepatotoxic responses [92].
The protective effects of L. rhamnosus have also been evaluated in clinical settings. In a randomized trial, El-Nezami et al. [93] showed that supplementation with L. rhamnosus LC705 significantly reduced urinary excretion of the DNA adduct AFB-N7-guanine. Levels decreased from 0.42 ng/mL to 0.27 ng/mL after 3 weeks (36% reduction) and to 0.19 ng/mL after 5 weeks (55% reduction) compared with placebo (p < 0.05). However, urinary AFB-N7-guanine levels returned to baseline after the intervention period, indicating that sustained probiotic intake may be necessary to maintain the detoxification effect [93]. All things considered, L. rhamnosus has an anti-inflammatory role and protective effects that attenuate the proinflammatory effects caused by AFB1 [90,92]. While clinical studies consistently show the potential of probiotics to reduce AFB1 absorption and related biomarkers, several limitations must be acknowledged. Many trials are restricted by relatively small sample sizes, short intervention durations (weeks rather than months), and variability in measured endpoints (e.g., urinary metabolites vs. serum adducts). These factors may limit generalizability and long-term risk assessment. Future research should focus on larger, multi-center trials with standardized protocols and extended follow-up to strengthen clinical evidence.

4.3. Mixture of Probiotic LABs

In addition to individual strains, mixtures of probiotic LABs have been investigated for their synergistic protective effects against AFB1-induced HCC. One study examined fermented milk containing a combination of L. rhamnosus GG (LGG) and L. casei Shirota (Lcs) in a 1:2 ratio. After 25 weeks of supplementation, this probiotic mixture markedly reduced both tumor incidence and tumor size in AFB1-exposed animals. At the molecular level, expression of oncogenes and proliferation-related factors including c-myc, bcl-2, cyclin D1, and ras p21 was significantly downregulated. Since these genes are central to tumor progression and ROS-mediated pathways, the findings highlight the anti-hepatocarcinogenic potential of LGG–Lcs co-administration [94]. Another investigation evaluated a broader probiotic mixture composed of L. reuteri, L. plantarum, L. pentosus, L. rhamnosus, and L. paracasei. Chickens supplemented with this formulation exhibited a significant reduction in AFB1-induced liver enlargement, measured as relative liver weight (% EBW). Moreover, dietary supplementation decreased AFB1 accumulation in liver tissue by ~58% in the low-dose group (1000 ppb) and ~50% in the high-dose group (5000 ppb) (p < 0.05). Consistently, excretion of AFB1 in feces increased by 67% and 46% at the respective dose levels compared to unsupplemented controls [95]. Mixtures of probiotics may offer a more practical dietary approach than single strains, reflecting the diversity of probiotics naturally present in fermented foods. By combining different binding capacities, enzymatic defenses, and immunomodulatory functions, such formulations could provide broader protection against AFB1 and hold promise as feasible interventions for populations with regular dietary exposure.
Table 2. In vivo experiments: Anticarcinogenic effect of probiotic LABs on AFB1-induced liver carcinogenesis.
Table 2. In vivo experiments: Anticarcinogenic effect of probiotic LABs on AFB1-induced liver carcinogenesis.
SubjectsDose of AFB1Treatment PeriodAnti-Hepatocarcinogenic FunctionsLAB StrainsRef
Male Sprague Dawley
rats (7–8 weeks old)		25 ppbDaily for 20 daysALT & AST ↓
Serum AFB1 ↓		L. casei Shirota[77]
Male Sprague Dawley
rats (7–8 weeks old)		25 ppbDaily for 5 daysSerum AFB1 ↓L. casei Shirota[79]
Male Wistar rats
(4 weeks old)		450 ppbTwice/week for 6 weeksTBARS ↓
Antioxidant enzymes ↑		L. casei Shirota
L. rhamnosus GG		[80]
71 employees in UPMUrinary AFM1 > 0.005 ppb4 weeks of interventionSerum AFB1 ↓L. casei Shirota[87]
Broiler chickens
(1 week old)		3000 ppbSingle injectionAFB1 in duodenal tissue & luminal fluid ↓L. rhamnosus LC705
L. rhamnosus GG		[89]
Han-Wistar rats
(5 weeks old)		1500 ppbDaily for 3 daysALT ↓
AFB1 in feces ↑		L. rhamnosus GG[90]
Male Holstein calves (120 days old)38 ppbSingle oral AFB1 in feces ↑L. rhamnosus GG[91]
Male Kunming mice
(5 weeks old)		300 ppbTwice/day for 8 weeksInflammatory factors ↓
ALT & AST ↓		L. rhamnosus[92]
90 male students at Sun Yat-Sen UniversityUrinary AFM1 > 0.008 ppbTwice/day for 5 weeks of interventionUrinary AFB-N7-guanine ↓L. rhamnosus LC705[93]
Male Wistar rats
(4 weeks old)		450 ppbTwice/week for 25 weeksc-myc, bcl-2, cyclin D1 & rasp-21 ↓
Tumor incidence ↓		Mixture of L. casei Shirota &
L. rhamnosus GG		[94]
Male Ross broiler chicks (1 day old)Low (1000 ppb)
High (5000 ppb)		Daily for 35 daysLiver EBW ↓
AFB1 in liver tissue ↓ AFB1 in excreta ↑		Mixture of LAB[95]
ALT: alanine transaminase; AST: aspartate transaminase; EBW (empty body weight) = body weight before sacrifice—weight of alimentary tract filled with chime; ppb: part per billion (1 ppb = μg/kg), ↑: indicates increase, ↓: indicates decrease.

5. Conclusions

AFB1 remains a significant global challenge to food safety and public health, with chronic dietary exposure strongly associated with hepatocellular carcinoma (HCC). Probiotic lactic acid bacteria (LAB) have emerged as promising biological countermeasures, supported by growing evidence from mechanistic studies, animal experiments, and human clinical trials. These probiotics, including Lactobacillus casei Shirota, Lactobacillus rhamnosus GG, Lactobacillus rhamnosus LC705, Lactococcus lactis, and selected Bifidobacterium species, mitigate AFB1 toxicity through multiple complementary mechanisms. The most compelling evidence comes from clinical intervention trials, which consistently demonstrate reductions in urinary AFM1 and DNA adducts following probiotic supplementation, highlighting clear translational potential. Moreover, findings from mechanistic and animal studies—such as enhanced antioxidant activity, NF-κB pathway modulation, intestinal barrier restoration, and shifts in gut microbiota—provide valuable insight but still require confirmation in long-term human populations. Looking ahead, approaches such as engineered probiotics, probiotic–prebiotic combinations, and standardized multi-strain formulations represent exciting future directions, though they remain largely hypothetical and demand rigorous evaluation. At the molecular level, probiotic protection is largely mediated by cell wall components such as peptidoglycans, teichoic acids, β-D-glucans, and surface proteins, which adsorb AFB1 and limit its intestinal absorption. These interactions emphasize the importance of strain selection and mechanistic characterization, particularly given the variability in binding efficiency across strains. To advance translation, further biochemical studies are needed to clarify structural mechanisms, and large-scale randomized controlled trials in high-risk populations are required to establish efficacy, optimize dosing regimens, and evaluate the synergistic effects of probiotic mixtures. From a practical perspective, probiotics are generally safe, affordable, and widely accepted within dietary contexts, making them attractive candidates for large-scale food safety interventions. Nonetheless, regulatory approval, product standardization, and quality control can remain critical challenges to their widespread application. In summary, probiotic LABs represent safe, cost-effective, and scalable interventions for reducing AFB1 exposure and preventing HCC. Their multifaceted mechanisms of action highlight their promise as a cornerstone of global mycotoxin management, provided that ongoing mechanistic research and clinical validation bridge the gap between laboratory potential and real-world implementation.

Author Contributions

Conceptualization: D.C.; Investigation: D.C., and X.F.; Writing—Original Draft: D.C.; Writing-Review & Editing: D.C.; X.F., and J.-H.Y.; Funding Acquisition: J.-H.Y.; Supervision: J.-H.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the Hatch Project (No. 7000326) from the National Institute of Food and Agriculture, U.S. Department of Agriculture, awarded to J-H.Y., and by the Food Research Institute at the University of Wisconsin–Madison.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

No new data were created or analyzed in this study.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Pathway of AFB1 metabolism leading to hepatocellular carcinoma (HCC). Created with Biorender.com.
Figure 1. Pathway of AFB1 metabolism leading to hepatocellular carcinoma (HCC). Created with Biorender.com.
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Figure 2. Proposed interactions between AFB1 and probiotic cell wall components in the duodenum of the small intestine. This schematic (not to scale) illustrates potential mechanisms by which probiotics reduce AFB1 adsorption and thereby lower the risk of HCC. AFB1 can interact with four major bacterial cell wall constituents: (1) peptidoglycans, (2) surface proteins, (3) wall teichoic acids, and (4) β-D-glucans. Variations in teichoic acid structures are represented for Lactobacillus casei Shirota and Lactobacillus rhamnosus GG as proposed by [83]. The suggested mechanism involves hydrogen bonding between hydroxyl groups of ribitol phosphate or glucose residues in teichoic acids and carbonyl oxygens of AFB1. Although these interactions are supported by experimental observations, the precise molecular mechanisms remain to be fully elucidated. AFB1–LAB complexes have been detected in both the duodenum and feces, supporting the concept that binding to probiotic surfaces inhibits intestinal absorption and reduces the likelihood of AFB1-induced carcinogenesis. Figure created with BioRender.com; chemical structures were prepared using ChemDraw v18.0.0.231.
Figure 2. Proposed interactions between AFB1 and probiotic cell wall components in the duodenum of the small intestine. This schematic (not to scale) illustrates potential mechanisms by which probiotics reduce AFB1 adsorption and thereby lower the risk of HCC. AFB1 can interact with four major bacterial cell wall constituents: (1) peptidoglycans, (2) surface proteins, (3) wall teichoic acids, and (4) β-D-glucans. Variations in teichoic acid structures are represented for Lactobacillus casei Shirota and Lactobacillus rhamnosus GG as proposed by [83]. The suggested mechanism involves hydrogen bonding between hydroxyl groups of ribitol phosphate or glucose residues in teichoic acids and carbonyl oxygens of AFB1. Although these interactions are supported by experimental observations, the precise molecular mechanisms remain to be fully elucidated. AFB1–LAB complexes have been detected in both the duodenum and feces, supporting the concept that binding to probiotic surfaces inhibits intestinal absorption and reduces the likelihood of AFB1-induced carcinogenesis. Figure created with BioRender.com; chemical structures were prepared using ChemDraw v18.0.0.231.
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Table 1. In vivo experiments: Gut-health induced microbiota alteration caused by AFB1.
Table 1. In vivo experiments: Gut-health induced microbiota alteration caused by AFB1.
SubjectsDose of AFB1Treatment PeriodMicrobial Community AlterationRef
Dorper Mutton Sheep1000 ppb (1/2 LD50)One timeFirmicutes ↑
Spirochaetes ↑
Proteobacteria ↑
Actinobacteria ↓
Bacteroidetes ↓		[43]
Male Balb/c mice25 ppb (1/192 LD50)Daily for 28 daysParabacteroides ↑
Escherichia-Shigella ↑
Lactobacillus ↑
Alistipes ↓
Bacteroidetes ↓		[44]
Dorper Mutton Sheep1000 ppb (1/2 LD50)One timeFirmicutes ↑
Spirochaetes ↑
Verrucomicrobia ↑
Proteobacteria ↑
Bacteroidetes ↓		[45]
Male Balb/c mice25 ppb (1/192 LD50)Daily for 28 daysFirmicutes ↑
Lactobacillus ↑		[46]
Male Fischer 344
rats (5 weeks old)		Low (5 ppb)
Medium (25 ppb)
High (75 ppb)		5 days/week for 4 weeksStreptococcus spp.
& Lactococcus spp. ↓		[49]
Crossbred TOPIGS-40 hybrid piglets320 ppbDaily for 30 daysLactobacillus ↓[50]
Male broiler chicks (1-day old)Low (1000 ppb)
Medium (1500 ppb)
High (2000 ppb)		Daily for 21 daysTotal LAB ↓ with low AFB1
Total LAB ↑ with medium and high dosage of AFB1		[51]
Kunming miceLow (2500 ppb)
Medium (4000 ppb)
High (10,000 ppb)		Twice/day for 62 daysBifidobacterium spp. ↑[53]
LD50: median lethal dose, ppb: part per billion (1 ppb = μg/kg), ↑: indicates increase, ↓: indicates decrease.
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Choi, D.; Fan, X.; Yu, J.-H. Comprehensive Review of Dietary Probiotics in Reducing Aflatoxin B1 Toxicity. Toxins 2025, 17, 482. https://doi.org/10.3390/toxins17100482

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Choi D, Fan X, Yu J-H. Comprehensive Review of Dietary Probiotics in Reducing Aflatoxin B1 Toxicity. Toxins. 2025; 17(10):482. https://doi.org/10.3390/toxins17100482

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Choi, Dasol, Xingrui Fan, and Jae-Hyuk Yu. 2025. "Comprehensive Review of Dietary Probiotics in Reducing Aflatoxin B1 Toxicity" Toxins 17, no. 10: 482. https://doi.org/10.3390/toxins17100482

APA Style

Choi, D., Fan, X., & Yu, J.-H. (2025). Comprehensive Review of Dietary Probiotics in Reducing Aflatoxin B1 Toxicity. Toxins, 17(10), 482. https://doi.org/10.3390/toxins17100482

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