Probiotic Lactobacillus casei Shirota (LcS) has been extensively reported to have aflatoxin-binding ability. Here, we discuss the available evidence and mechanism of action for aflatoxin-reducing properties. The possible factors affecting the probiotic efficacy in removing aflatoxin are highlighted.
by Chang Wei Lin and Dr Mohd Redzwan Sabran
Probiotics are live microorganisms that have health benefits beyond basic nutrition (when consumed in adequate amounts). Lacticaseibacillus casei, more commonly known as Lactobacillus casei, is one of the most consumed and best characterised probiotics.
The name Lactobacillus casei has been officially reclassified into Lacticaseibacillus casei in April 2020, where its new name was derived from the combination of Latin words: “lacti” means milk, “casei” means cheese, and “bacillus” means small rod.1 It implies that the probiotic is a small rod-shaped microorganism that is often found in fermented dairy products.
The research into L. casei strain Shirota (LcS) began in the 1930s when the strain was isolated and cultivated by a Japanese scientist, Dr. Minoru. The Shirota strain was later developed into a new fermented milk drink named Yakult after five years of discovery.2
Aflatoxins and Its Toxic Effects
Aflatoxins are a family of toxins produced by certain moulds, particularly Aspergillus species. These moulds are abundant in warm and humid areas. There are four major aflatoxins, including aflatoxin B1 (AFB1 ), aflatoxin B2 (AFB2 ), aflatoxin G1 (AFG1 ), and aflatoxin G2 (AFG2 ). These aflatoxins are commonly found in food and feed such as nuts, spices, grains, and dried fruits. Herbal and traditional medicine may also be affected by aflatoxin contamination.3 Besides the B and G aflatoxins, aflatoxin M1 (AFM1 ) is the hydroxylated metabolite of AFB1 and is mainly excreted in milk or urine. When lactating animals ingested feed containing AFB1, these toxins are metabolised and excreted as AFM1 in milk. The contamination of AFM1 is not limited to raw milk but the whole milk chain since it could be carried over to dairy products.4 Dairy products such as cheese, cultured milk, and yoghurt have been detected with aflatoxins.5 These toxins cannot be seen with the naked eyes since they are colourless, odourless, and tasteless.
Acute exposure to a large dose of aflatoxins can lead to toxicity. This acute effect is characterised by abdominal pain, nausea, vomiting, and other signs of acute liver injury.6 These effects may worsen with high carbohydrate intake and low protein intake.7 Long-term consumption of aflatoxin-contaminated foods is a risk factor for liver cancer.
Aflatoxins have been classified by the International Agency for Research on Cancer (IARC) as carcinogenic to humans (Group 1), the same category as smoking and eating processed meat.8 Agents in Group 1 are known to have strong and clear evidence of carcinogenicity in humans. AFB1 is the most potent among all the aflatoxins. The relative potency of aflatoxins is reported in the order of AFB1 > (AFG1 and AFM1) >> (AFG2 and AFB2).9 Apart from being carcinogenic, AFB1 is known to be mutagenic, genotoxic, and immunosuppressive. Liew et al. (2022) proposed that the adverse effects are induced by aflatoxins by impairing the gut microbiota stability and increasing inflammation.10
Probiotic LcS as Potential Aflatoxin Binder
The aflatoxin-reducing properties of probiotics including LcS have been extensively discussed.11 Most animal studies were in favour of LcS to reduce aflatoxin levels and/or modulate the adverse effects of aflatoxins.12,13,14,15 These protective effects exist either by the administration of LcS individually or jointly with other components including chlorophyllin16 and a high protein diet.15 Indeed, AFB1-lysine adduct level in the blood samples of rats was reduced even when LcS was given prior to the aflatoxin exposure.17 This implies that the effects of treating LcS on pre- or post-aflatoxins exposure may be similar. Indeed, LcS was recoverable in adults during the 14 day-treatment18 and six months of supplementation in children.19 Pre-exposure to probiotics may reduce the binding of aflatoxins with intestinal mucus, leading to faster removal.20 Thus, regular supplementation of probiotics may be of value for early prevention of aflatoxin toxicity, especially among those with a high risk of exposure.
A human interventional study exploring the effect of LcS in reducing aflatoxins is scarce. The first and only study was reported in a randomised, double-blind, cross-over, placebo-controlled trial, whereby 71 healthy adults were given fermented milk containing LcS followed by placebo drinks or vice versa for four weeks separated by a two-week washout period.21 The supplementation of LcS did not yield any conclusive findings on the reduction of serum AFB1-lysine adduct and urinary AFM1 due to several confounding factors. Nonetheless, findings from the literature based on in vitro and animal studies12,14,22 showed promising effects of LcS as aflatoxin binder.
Mechanism of Action
The aflatoxin-reducing properties of LcS are possibly attributed to its binding ability toward aflatoxin in the gut, which reduces the aflatoxin bioavailability.12 Aflatoxins adhere physically to the carbohydrate components of the probiotic cell wall by weak, non-covalent interactions corresponding to the formation of van der Waals interaction, hydrogen bonds, and electrostatic interactions.23 The aflatoxin-binding capacity of LcS is not limited to live cell but also other cell components although live cell was shown as the most efficient binder (98.0 per cent).12 The probiotics bound to aflatoxin are less likely to adhere to the intestinal wall, increasing the excretion of aflatoxin from the body.20 In addition to the aflatoxin-binding capacity, LcS may modulate the AFB1-induced gut microbiota imbalance and thus minimise the toxic effects of AFB1.22
The binding of probiotics towards aflatoxin is strain specific. AFB1-LcS complex was significantly more stable than other strains, retaining 93.8 per cent of AFB1 after four hours of incubation.24 The basis for the observed differences is unclear but it is speculated that the binding of aflatoxin may relate to the hydrophobicity of the cell surface.25 Gram-positive bacteria like LcS significantly removed more AFB1 than Gram-negative bacteria, indicating that the detoxicating effect depends on the structure of the cell wall.26 Under atomic force microscopy and scanning electron microscopy, aflatoxin binding has induced structural changes on the probiotic bacterial cell surface.12,17 The alteration in morphology may provide some hints that the binding of AFB1 occurs on the cell wall surface of probiotics. In addition, experimental data discovered teichoic acids as a key component of LcS cell wall structure that may be involved in complex binding.24 Further studies are needed to further elucidate these mechanisms.
Factors Affecting the Efficacy of Probiotic LcS
The binding activity of LcS towards aflatoxins can be affected by the gut condition, including gastric pH, digestive enzymes, and intestinal mucus. Probiotic LcS is highly sensitive to acidic conditions and does not show viability when immersed in the stomach under fasting conditions.27 The greatest extent of aflatoxin binding by LcS was at pH 7.2.24 Thus, it is strongly advised to consume probiotics after meals to maximise the beneficial effects.
Consuming a high intake of carbohydrates, fats, and/or proteins can trigger the secretion of digestive juices,28,29 which may not be adapted by all probiotics. However, a recent in vivo study by Nurul Adilah et al. (2018) did not agree as the authors demonstrated a protective effect against urinary AFM1 levels in rats who received LcS supplementation with a high protein diet.15 These findings lend for further studies to assess their interaction.
Probiotics may be less capable of binding aflatoxin in the presence of intestinal mucus.20 Despite that, their binding sites on probiotics were unlikely to be similar as AFB1 is known to bind to carbohydrates25 while mucus must adhere to proteins.30 The addition of proteins to mucus in the intestinal tract may minimise the reductive effects of AFB1 binding by probiotics.20 It also leads to the question of whether dietary fibre could mask the probiotic binding efficacy since dietary fibre is responsible for mucus production.31 The mechanisms of the interaction between probiotics, aflatoxins, and intestinal mucus deserved further investigation.
Probiotic consumption may be a practical dietary approach to prevent human exposure to aflatoxins. Future trials are warranted to explore the factors affecting probiotic efficacy as they may explain the conflict of results observed between in vitro and in vivo with human studies. [APBN]
About the Authors
Ms. Chang Wei Lin holds a MSc degree in Nutritional Science at the Universiti Putra Malaysia. She is studying her Ph.D under the main supervision of Dr. Mohd Redzwan Sabran. She is engaged in clinical trials, exploring the effects of probiotics in reducing aflatoxins.
Dr. Mohd Redzwan Sabran is a senior lecturer in Nutrition at the Universiti Putra Malaysia. Dr. Redzwan has been engaged in teaching and research for more than 7 years. His expertise includes Nutritional Sciences, Food Safety and Food Contaminants, as well as Probiotics.
Both authors are from Department of Nutrition, Faculty of Medicine and Health Sciences, University Putra Malaysia