Reduction of aflatoxin level in aflatoxin-induced rats by the activity of probiotic Lactobacillus casei strain Shirota

Authors


Correspondence

Rosita Jamaluddin, Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. E-mail: rosita@medic.upm.edu.my

Abstract

Aims

Aflatoxin B1 (AFB1) is considered as the most toxic food contaminant, and microorganisms, especially bacteria, have been studied for their potential to reduce the bioavailability of mycotoxins including aflatoxins. Therefore, this research investigated the efficacy of oral administration of Lactobacillus casei Shirota (LcS) in aflatoxin-induced rats.

Methods and Results

Sprague Dawley rats were divided into three groups of untreated control, the group induced with AFB1 only, and the group given probiotic in addition to AFB1. In the group induced with AFB1 only, food intake and body weight were reduced significantly. The liver and kidney enzymes were significantly enhanced in both groups induced with AFB1, but they were lower in the group given LcS. AFB1 was detected from all serum samples except for untreated control group's samples. Blood serum level of AFB1 in the group induced with AFB1 only was significantly higher than the group which received probiotic as a treatment (< 0·05), and there was no significant difference between the control group and the group treated with probiotic.

Conclusions

LcS supplementation could improve the adverse effect of AFB1 induction on rats' body weight, plasma biochemical parameters and also could reduce the level of AFB1 in blood serum.

Significance and Impact of the Study

This study's outcomes contribute to better understanding of the potential of probiotic to reduce the bioavailability ofAFB1. Moreover, it can open an opportunity for future investigations to study the efficacy of oral supplementation of probiotic LcS in reducing aflatoxin level in human.

Introduction

Toxins are regularly ingested. A number of toxins come from foods eaten every day. As one of the main groups of toxicants that naturally occur in food, mycotoxins are said to pose a considerable health risk (Shetty and Jespersen 2006). The most important mycotoxin is aflatoxin (Pitt 2000). Aflatoxin is a fungal toxin and commonly found to contaminate a variety of food commodities such as maize and other types of crop especially during the process of production, harvesting, storage or processing (Logrieco et al. 2003). It can often be found in staple food, including groundnuts and oil seeds, like maize. Aspergillus flavus and Aspergillus parasiticus, which are both from Aspergillus species of fungi and are both classified as group one carcinogen by the International Agency for Research on Cancer (IARC), can produce aflatoxin B1 (AFB1). The exposure to aflatoxin is linked to chronic disease such as liver cancer. Among many aflatoxin metabolites, AFB1 is a very strong carcinogen in many species, and exposure to AFB1 is known to cause both chronic and acute hepatocellular injury (Jakhar and Sadana 2004).

On the global scale, the prevalence and level of human exposure to aflatoxins have been reviewed, and it has been found that about 4·5 billion of the populations of developing countries are constantly exposed to uncontrollably considerable amounts of the toxin. Exposure to aflatoxin can negatively affect immunity and nutrition, as well as health factors (Mata et al. 2004).

On the other hand, there are several hypotheses, suggesting the mechanisms of probiotics and toxin. Fermentation of food has been used as a method of preservation for centuries, and lactic acid bacteria (LAB) are reported to reduce mould growth and aflatoxin production (Mokoena et al. 2006).

Several bacterial strains have been tested for their ability to bind aflatoxins and other mycotoxins to their surface (El-Nezami et al. 2002a,b; Styriak and Conkova 2002). El-Nezami et al. (1998) found that gram-positive bacteria (five strains of Lactobacillus and one Propionibacterium) were more efficient in removing aflatoxin from a liquid medium than gram-negative Escherichia coli (E. coli). In a study of a range of Lactobacilli and Bifidobacteria, Peltonen et al. (2001) reported significant differences in the binding abilities of AFB1, even in the closely related strains. The capability of some probiotic bacteria strains to bind with AFB1 strongly correlates with their ability to reduce AFB1 mutagenicity in the Ames assay for Lactobacilli and Bifidobacteria cultured in MRS broth (Lankaputhra and Shah 1998) or Lactobacilli cultured in milk (Hosoda et al. 1996).

In addition to bacterial strain specificity, the bacterial concentration can also affect the AFB1 removal. Different minimum concentrations have been reported such as 5 × 109 colony-forming unit (CFU) ml−1 of either Lactobacillus acidophilus or Bacterium longum to remove only 13% of the AFB1 within 1 h (Bolognani, Rumney, & Rowland, 1997) or 2 × 109 CFU ml−1 of Lactobacilli and Propionibacterium to remove 50% of free AFB1; however, higher binding occurred at 1010 CFU ml−1 (El-Nezami et al. 1998).

In their attempt to explain the nature of potential AFB1-binding sites on the surface of Lactobacilli, Haskard et al. (2001) subjected the bacteria to a number of different chemical, physical and enzymatic treatments as well as cell wall polysaccharides. They reported peptidoglycans as responsible for the binding of AFB1 to the surface of probiotic Lactobacillus rhamnosus strain GG and probiotic Lact. rhamnosus strain LC705. This was further confirmed by Lahtinen et al. (2004) who studied different cell wall components (exopolysaccharides, cell wall isolates and peptidoglycans) of GG and concluded that peptidoglycans are the most likely binding sites for AFB1.

Besides these in vitro studies, the binding ability of probiotics for AFB1 was tested ex vivo in the intestinal lumen of chicks (El-Nezami, Mykkänen, Kankaanpää, Salminen, & Ahokas, 2000). The authors reported that probiotic GG removed 54%, probiotic LC705 removed 44% and Propionibacterium freudenreichii ssp. Shermanii JS (PJS) removed 36% of the AFB1 from the soluble fraction of the luminal fluid within 1 min. As it can be inferred from these findings, bacterial AFB1 binding emerges in physiological conditions in animals, which may be proposed as a way to diminish aflatoxin bioavailability in the organism.

Under in vivo conditions, it is expected that probiotic LAB binds to AFB1 as soon as they interact in the intestinal tract, and also numerous studies reveal the benefits of probiotics to human health. Thus, the goal of this study is to identify the potential of a probiotic, specifically, Lactobacillus casei Shirota (LcS), on toxin reduction in rats.

Materials and methods

Chemicals and kit

AFB1 was purchased from Sigma-Aldrich. (Selangor, Malaysia). The probiotic drink, which is the commercialized Yakult drink, was purchased from a local market in Malaysia. MRS agar and MRS broth were purchased from Merck (Darmstadt, Germany); Glycerol solution from Sigma-Aldrich (St. Louis, MO, USA); and normal saline solution (sodium chloride) from Merck. Merck provided the acetonitrile (HPLC grade) and methanol (HPLC grade) used in this study. Benzene was purchased from Sigma-Aldrich and PBS from Applichem (Darmstadt, Germany). TMS company (Selangor, Malaysia) supplied the deionized water, amber glass vial and the cap; AflaTest WB Columns for HPLC, Immunoaffinity Columns was purchased from Vicam (New York, NY, USA). Vicam Fluted Filter Paper was purchased from Vicam. Pump stand with fish pump and glass syringe which were used in this study were purchased from TMS company, and RIDASCREEN aflatoxin kit was purchased from R-Biopharm (Darmstadt, Germany).

Probiotic bacteria

In this study, standard plate count method was used as a quantitative method for determination of bacterial population, and LcS was selected as the probiotic. For isolation of LcS, 1 ml of Yakult cultured milk, which contained only LcS (Hong-Lim et al. 2008), was cultured on MRS agar for 48 h at 37°C with 5% CO2. Later, a single colony of the bacteria was further grown in MRS broth for 24 h in an incubator shaker at 250 rev min−1 and temperature of 37°C. The growth curve of LcS during 24 h is shown in Fig. 1.

Figure 1.

Lactobacillus casei Shirota growth curve in MRS broth for 24 h. (…….) OD and (– – –) CFU.

The growth of LcS was monitored optically at 600 nm, and corresponding CFUs were checked every 2 h for 24 h. For each interval, 100 μl of the cultured medium containing LcS was withdrawn and spread onto MRS agar plate to obtain the corresponding CFU for the particular time. The response of optical density (OD) and CFU was recorded and plotted using Microsoft EXCEL.

According to the OD and CFU that was recorded in 24 h, in order for the CFU to reach 108, the cell must be incubated for at least 16 h. In other words, a single colony of bacteria required 16 h growth in MRS broth in incubator shaker at 250 rev min−1 and temperature of 37°C to achieve 108 CFU.

For storage purpose, the cultured medium was centrifuged at 19957 g for 10 min. The supernatants were discarded, and the pellet was re-suspended with 1 ml of 50% (v/v) glycerol solution and stored in −30°C until further usage. Upon usage, the culture stock (108 CFU) was centrifuged at 19957 g for 10 min to separate the cell from glycerol solution. After the supernatant (i.e. the glycerol solution) was discarded, the pellet was re-suspended with 1 ml normal saline before inducing to the rats.

Animals

Twenty-four male Sprague Dawley rats, 7–8 weeks of age and 170–190 g of weight, were provided from Chenur Supplier, Kajang, Selangor, Malaysia, and kept at room temperature under a photographic cycle of 12-h dark light in the animal research house of Faculty of Medicine and Health Sciences, Universiti Putra Malaysia. All rats were housed individually in plastic cages with wood chip bedding. The cleaning processes of the cages including checking of water supply were performed on a daily basis. The standard or basal commercial diet for rats was purchased from Chenur Supplier. The treatment was approved by Animal Care and Use Committee (ACUC) of the Faculty of Medicine and Health Sciences, Universiti Putra Malaysia with approval number UPM/FPSK/PADS/BR-UUH/00366.

Experimental study

Twenty-four rats were divided into three subgroups of as follows: untreated control under normal diet (Cc), the group induced with AFB1 without any treatment by probiotic (Bc), and the group treated with probiotic LcS (108 CFU) by oral gavage daily for 20 successive days and induced with AFB1 (Ac). Immediately after the fourth probiotic dose and from the 4th day, rats of both groups (groups Ac and Bc) were given multiple oral dose of AFB1 in amount of 25 μg kg−1 body weights daily for 5 days per week over the next 2 weeks to obtain chronic AFB1 exposure (Roebuck and Maxuitenko 1994). Body weight of all rats was recorded daily for 20 days using electronic balance (A&D Co., Ltd., Tokyo, Japan). Considering the half-life (T1/2) of AFB1 which is about 64 h (Firmin et al. 2010) and to avoid any loss of AFB1, blood sample of rats in all groups was taken on days 18, 19 and 20. Rats were anesthetized using diethyl ether, and blood sample was taken by cardiac puncture from the artery vessel.

Blood withdrawal

About 3–4 ml blood was drawn from the artery vessel and collected in vacutainers containing EDTA for liver and kidney function tests. In addition, about 3–4 ml blood was collected in plain tubes for analysis of AFB1 in blood serum samples. Blood plasma and serum were separated using Hitachi Universal 32R centrifuge (Hetichi Zentrifugen, Tuttlingen, Germany) at 4°C for 10 min at 4277 g. The plasma and serum were collected in Hitachi cups and stored at −80°C until analysis.

Determination of liver and kidney enzymes

Blood plasma was analysed colorimetrically using a Hitachi chemical automatic analyser (Roche Diagnostic Ltd., West Sussex, England) at the Pathology Laboratory, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia. The level of blood alanine transaminase (ALT) and aspartate transaminase (AST) was measured for liver function test, and blood creatinine (CREA) and urea level (UREA) were measured for kidney function test. All the results were compared with those of control group.

Determination of AFB1 blood serum level

For the analysis of the amount of AFB1 in blood serum, the extraction and purification of AFB1 in the serum samples was performed according to the R-Biopharm recommendation which is described in the following. AFB1 level was quantified by competitive enzyme immunoassay (ELISA) using a RidaScreen® Aflatoxin Total kit (R-Biopharm AG, Darmstadt, Germany) (Sayed et al. 2005). The procedure was carried out as described in the manual supplied by the manufacturer. The absorbance was read at 450 nm using a SIRIO micro-plate reader (Indonesia), and a six-point standard curve was prepared. Data were collected and analysed using the Rida® Soft Win software (R-Biopharm). The limit of detection (LOD) of the assay was 0·05 ppb with the recovery rate of 90% for sample buffer and about 70% for human blood serum. Results were expressed as ppb AFB1 per blood serum.

Extraction and purification of AFB1

Collected blood serum was centrifuged to eliminate interfering components that could clog the Immunoaffinity Columns. As AFB1 is a polar compound, it should dissolve in a polar solvent like acetonitrile, hexane and methanol. Between all polar solvents, methanol is environmental friendly; therefore, 4 ml methanol (100%) was added to 2 ml serum. Thus, AFB1 could dissolve in the methanol and bind with it. After that, the sample was vortexed to increase the bind between methanol and AFB1 and homogenized thoroughly and then again centrifuged for 10 min in 3000 g and 10°C. Following the second centrifugation, 3 ml supernatant was taken, and 20 ml PBS was added to increase the solubility and then was shaken. In this stage, the sample was passed through Immunoaffinity Aflatoxin Columns (without pressure, by gravity) to bind AFB1 and antibody inside the column. Afterwards, Immunoaffinity Aflatoxin Columns was washed with 20 ml PBS to remove the rest of interfering components, and the column was dried by passing air within the Immunoaffinity Aflatoxin Columns. Then, the amber vial was placed beneath the column, the toxin was eluted with 1·5 ml methanol, and the flow rate was adjusted to 1 drop per second. After that, 3 ml distilled water was passed through the column. Finally, the ELISA test was run.

Statistical analysis

Statistical analysis was performed using Statistical Package for Social Sciences (SPSS) software version 17.0 (SPSS Inc., Chicago, IL, USA). Differences for all tests were considered significant at  0·05. Data were expressed as mean ± SD (standard deviation). Descriptive statistics (mean and standard deviation) and two-way repeated measures anova followed by post hoc test were utilized to determine the effect of probiotic on the weight of rats dosed with AFB1. Descriptive statistics (mean and standard deviation), analysis of variance (anova) and post hoc test were used to investigate the effect of probiotic on hepatotoxic effect of AFB1 induction (through liver function test), effect of probiotic on the kidney function test and also to investigate the effect of probiotic on blood serum level of AFB1 in this study.

Results

Body weight of rats in the three groups of Ac, Bc and Cc were almost similar at the beginning of the study; Cc (190·05 ± 8·05), Bc (190·00 ± 13·40) and Ac (193·10 ±14·80). Prior to the start of multiple AFB1 induction, the daily body weight gain of treatment groups (Ac and Bc) was similar and not different from the body weight gain of untreated control group. Based on the two-way repeated measure anova, a significant difference was found on body weight of rats in group Bc and the other two groups (Cc and Ac) from day nine to the end of the study (day 20). However, there was no significant difference between groups Cc and Ac regarding rats' body weight. The comparison of rats' body weight gain between the three groups is shown in Fig. 2. According to the figure, animals in group Cc and Ac gained weight normally during the study, but food intake and body weight gain of animals of groups Bc decreased significantly compared with the two other groups.

Figure 2.

Comparison of rats' body weight gain between the three groups of Ac, Bc and Cc during 20 days in aflatoxin-induced rats. Ac is the group given probiotic Lactobacillus casei Shirota plus AFB1; Bc is the group induced only with AFB1; Cc is untreated control group in chronic aflatoxicosis. (– – –) Ac; (……) Bc and (––––) Cc.

The liver and kidney function tests are shown in Table 1. As demonstrated in the table, it was found that induction of aflatoxicosis elevated the liver and kidney enzymes. However, with the supplementation of probiotic, a significant reduction in liver enzymes and slight reduction in kidney enzymes were observed. The mean reduction percentage of ALT and AST is 1·43 and 1·25%, respectively, while the mean percentage reduction in CREA and UREA is 1·04 and 1·03%, respectively.

Table 1. Biochemistry analysis of rats' blood samples in chronic aflatoxicosis
GroupLiver function testKidney function testSerum AFB1 (ppb ml−1)P-value
Alanine transaminase (U l−1)P-valueAspartate transaminase (U l−1)P-valueCreatinine (mg dl−1)P-valueUrea (mmol l−1)P-value
  1. Ac is the group given probiotic Lactobacillus casei Shirota plus AFB1; Bc is the group induced only with AFB1; Cc is untreated control group in chronic aflatoxicosis. The P-value was obtained from analysis of variance (anova).

Cc46·76 ± 6·740·000 76·11 ± 14·100·00051·86 ± 7·000·0006·26 ± 0·540·0030·20 ± 0·0040·000
Bc107·57 ± 11·94124·42 ± 9·9969·60 ± 8·297·83 ± 0·871·38 ± 0·55
Ac74·91 ± 16·17 99·37 ± 16·5066·88 ± 3·497·54 ± 0·820·50 ± 0·15

To investigate the effect of probiotic LcS on the absorption of AFB1 in aflatoxin-induced rats, blood serum level of AFB1 was measured in all groups and compared together. Although there are many metabolites of AFB1 such as AFM1 and AFM2 (found in urine and milk samples) and AFB1-8,9 epoxide (bind to DNA and albumin), this study aimed to investigate the effectiveness of the treatment in reducing the AFB1 level. To do so, the metabolites can be identified with the use of HPLC with fluorescence detector and LC-MS/MS. As expected, AFB1 was detected from all serum samples except for serum samples in group Cc. Serum AFB1 level of all three groups of Cc, Bc and Ac is provided in Table 1. As results indicated, the mean of AFB1 level in the group induced with AFB1 only was greater than the group treated with LcS.

The comparison of AFB1 blood serum level in the three groups of Ac, Bc Cc is shown in Table 1. As shown in the table, there was a significant difference between control group (group Cc) and group Bc which is the group induced with AFB1 without LcS in this study. However, there is no statistically significant difference between groups Cc and Ac with regard to aflatoxin blood serum level, but the mean of AFB1 blood serum level in group Ac is higher than group Cc. Moreover, reduction percentage mean of AFB1 blood serum level was 2·76%.

Discussion

Based on the findings on body weight changes after treatment, it can be hypothesized that having probiotic LcS plus AFB1 could neutralize the adverse effect of AFB1 on feed intake and body weight gain of animals in the group which received probiotic and AFB1 (group Ac). In agreement with this finding, in a study by Hathout et al. (2011), rats fed aflatoxins-contaminated diet showed a significant difference in body weight compared with the control group or the group treated with probiotic Lact. casei or Lactobacillus reuteri. Furthermore, in a study by Abdel-Wahhab et al. (2006), rats were treated with AFB1 and shown low leptin levels which can affect the regulation of energy balance and body weight control (Yuan et al. 2004; Abdel-Wahhab et al. 2006). Moreover, Barber et al. (2004) reported a relationship between low leptin concentration with the high levels of cortisol and IL-6, which can affect the feeding response and contribute to weight loss. Thus, to prove the effect of LcS on neutralizing the adverse effect of AFB1 on body weight, it can be assumed that LcS might have contributions on the reduction in AFB1 absorption in bacteria-treated rats in group Ac therefore diminish its effect on leptin level in blood serum. Our observation also corroborated with the finding by Abdel-Wahhab and Aly (2003), and Mayura et al. (1998) as food consumption and body weight of rats induced with aflatoxin decreased throughout the experiment as compared with the control group.

Aflatoxin ingestion can also lead to body weight loss by changing the different digestive enzymatic activities that cause a mal-absorption syndrome, characterized by steatorhea as well as hypocarotenoidemy, and that lower the bile, pancreatic lipase, trypsin and amylase (Osborne et al. 1982). Additionally, AFB1 biotransformation causes different metabolites, like 8,9 epoxide, covalently binding to DNA as well as proteins, and then changes enzymatic processes like gluconeogenesis, Krebs cycle or fatty acid synthesis (Lesson et al. 1995).

The effect of LcS on rats' body weight could be attributed to probiotic bacteria function which reduces the quantity of free AFB1 within the intestinal tract, thereby reducing toxicity. High aflatoxin biomarker levels in children, who were naturally exposed to aflatoxins through diet, have reportedly been associated with growth faltering (Gong et al. 2002, 2003; Turner et al. 2003). Another hypothesis in line with the present findings is that aflatoxin-induced intestinal damage is also associated with growth faltering. Inasmuch as probiotic bacteria may also improve intestinal barrier integrity (Parvez et al. 2006); they are likely to protect the intestinal epithelium against AFB1 toxicity indirectly.

Liver is the main target organ for AFB1 toxicity, and liver-specific enzyme activities in plasma or liver function test have been often used as a toxicity marker. Given the result of this study, rats induced with AFB1 showed a significant increase in ALT and AST levels compared with rats of untreated control group. The increased level of ALT and AST may indicate degeneration change and hypofunction of the liver (Abdel-Wahhab et al. 2008). Based on these study outcomes, the level of these liver enzymes is higher in the group Bc compared with the group Ac. Therefore, it can be suggested that probiotic treatment may reduce hepatotoxic effects of AFB1 dosing in rats given probiotic.

The significant reduction in ALT and AST levels in rats treated by probiotic LcS in our experimental study is in agreement with a study by Hathout et al. (2011). In Hathout's study, treatment with two probiotics of Lact. casei and Lact. reuteri succeeded to prevent the liver injury resulted by aflatoxin as indicated by significant improvement in plasma biochemical parameters (ALT and AST), similar to this study.

The mechanism through which Lact. casei can induce its protective property could be attributed to its capability to bind aflatoxin in the gastrointestinal tract, which can eventually lead to a reduction in the bioavailability of aflatoxin. As Salminen et al. (2010) reported in a recent study, several bacteria strains, identified as mycotoxin binders, have been observed to decrease exposure to dietary mycotoxins. In this respect, research has proved the significant effect of specific strains of Lactobacillus on removing AFB1 in model system (Turbic et al. 2002).

The increased uric acid and creatinine levels may be a sign of protein catabolism and/or kidney dysfunction (Abdel-Wahhab et al. 2007). These results clearly express that AFB1 had stressful effects on renal tissues (Miller and Willson 1994; Abdel-Wahhab and Kholif 2008; Sherif et al. 2009). However, there is no statistically significant difference between the group induced only with AFB1 and the group given probiotic in addition to AFB1; nevertheless, as mentioned previously, the level of uric acid and creatinine is greater in the group induced with AFB1 only, and it can be assumed that treating with LcS may have effects to not significantly but slightly reduce kidney enzymes level.

These findings are in agreement with what has been reported by Hathout et al. (2011). In Hathout's study, treatment with two probiotics of Lact. casei and Lact. reuteri succeeded to prevent the elevation of uric acid and creatinine resulted by aflatoxin as indicated by improvement in plasma biochemical parameters (UREA and CREA), similar to this study.

As previously mentioned in the results, probiotic LcS treatment was successful to reduce the level of AFB1 significantly in rats of group Ac compared with group Bc. These finding clearly indicates that treating with probiotic LcS could be attributable to the ability of probiotic to bind aflatoxin and reduce its absorption in the intestinal tract and in aflatoxin-induced rats.

Based on extensive animal studies, the duodenum of the small intestine is the primary site for absorption of aflatoxin (Ramos and Hernández 1996). Previous research findings have indicated the ability of probiotic bacteria to survive at the gastrointestinal tract subsequent to oral intake (Taranto et al. 2000; Valeur et al. 2004). Therefore, it sounds logical to state that aflatoxin was exposed to the bacteria in the intestinal lumen, which before its natural process of absorption favored AFB1 binding by bacteria.

Upon absorption in the intestine, aflatoxin merges to the blood stream. The amount of adducts in blood samples of the group, which was treated only with AFB1, shows the cumulative dose of aflatoxin intake throughout the experimental period. This suggests that the decrease in the AFB1 blood serum level in the group treated with aflatoxin plus bacteria can be attributed to the potential of probiotics to bind AFB1 inside the intestinal lumen, thereby avoiding its passage into the blood stream. In support of this study finding, in a study by Hernandez-Mendoza et al. (2011), the same finding was observed with regard to AFB1 absorption. In Hernandez-Mendoza study, the content of AFB1-Lys adducts in animals receiving AFB1 plus probiotic Lact. reuteri was lower than those receiving only AFB1.

Other studies have shown that the presence of mucus can lower the ability of probiotics to bind AFB1. This is because mucus could interfere with the adsorption of AFB1 to the bacterial cell wall of probiotic (Gratz et al. 2004, 2005). In the present study, the interference of mucus was reduced with the supplementation of probiotic 4 days before the oral induction of AFB1. Besides that, regular administration of probiotic bacteria during the experimental period may reduce the effect of mucus. Thus, it can be postulated that as the number of CFU increased, the effect of mucus on the adsorption of AFB1 by bacterial cell wall will be reduced (Gratz et al. 2004).

To conclude, we could mention that probiotic LcS supplementation could improve the harmful effect of AFB1 induction on rats' body weight, blood liver and kidney enzymes and AFB1 blood serum level in aflatoxin-induced rats. The ability of probiotic bacteria to bind or remove aflatoxin has been experimented in a few animal studies, most of which have been conducted following ex vivo methods, which have the limitation of stimulating intestinal conditions. Some of them are with in vivo methods that restrict their results by the application of only a single dose of aflatoxin. These studies outcome contributes to better understanding of the potential of probiotic to reduce the bioavailability of AFB1 in animals by performing chronic exposure to AFB1. Nevertheless, the short experimental duration with limited number of animals could affect on the reliability of any experimental studies. Thus, many more studies with longer experimental period and more number of animals including more groups like the group which only given probiotic without any aflatoxin induction is suggested to be performed to confirm the ability of probiotic bacteria as a biological barrier to reduce the bioavailability of AFB1 that orally ingested in a single or multiple doses. On the other hand, the analytical method used in this study is not applicable to study the human exposure to aflatoxin. Given that humans might have low level of aflatoxin compared with the induced aflatoxicosis animals, more accurate and precise methods such as with HPLC, LC-MS/MS should be used to study the extent of human exposure to aflatoxin (Wang et al. 2008).

Acknowledgements

This research was funded by Fundamental Research Grant Scheme (FRGS)-grant number: 5524027 from the Ministry of Higher Education of Malaysia.

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