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NFS 44,1

32 Received 8 November 2012 Revised 8 November 2012 Accepted 11 November 2012

In vitro effect of Lactobacillus rhamnosus GG on reduction of aflatoxin B1 Parya Rahnama Vosough and Ali Mohamadi Sani Department of Food Science and Technology, Quchan Branch, Islamic Azad University, Quchan, Iran

Masoumeh Mehraban ACECR Mashhad Branch, Islamic Azad University, Mashhad, Iran, and

Reza Karazhyan Food Science and Technology Institute, ACECR Mashhad Branch, Mashhad, Iran Abstract Purpose – Since a sound detoxification method is needed for controlling aflatoxin B1 (AFB1), as one of the most harmful mycotoxins in animal production and food industry, this study was performed. The paper aims to discuss these issues. Design/methodology/approach – This study was conducted to examine the ability of Lactobacillus rhamnosus strain GG to remove AFB1 from liquid media. The binding of AFB1 to Lb. rhamnosus GG was studied for viable, heat-killed and acid-killed bacteria. AFB1 at concentrations (5, 10 and 20 mg/l) was added to the bacterial culture (109 cfu/ml) in MRS broth medium and incubated at 258C for 4, 12 and 24 h. The aflatoxin-binding capacity of the strain was quantified by the amount of unbound AFB1 using ELISA technique. Findings – Results showed the AFB1-binding capacity of viable, heat-killed and acid-killed bacteria was about 43, 49 and 50 percent, respectively. The percentage of AFB1 removed was the highest amount in low (5 mg/l) and high (20 mg/l) concentrations, and there was no significant difference between them ( p ¼ 0.05). These findings suggest that lactic acid bacteria can be exploited as an approach to detoxification of aflatoxins from foods. Practical implications – This method is safe because non-viable bacteria have more ability to remove toxin than viable bacteria, and also it is an effective method with 50 percent approximately toxin removal. Originality/value – Since there has been no research on the ability of this strain on the removal of AFB1, the authors assessed the ability of the strain in high levels of AFB1. Keywords Food safety, Starter culture Paper type Research paper

Nutrition & Food Science Vol. 44 No. 1, 2014 pp. 32-40 q Emerald Group Publishing Limited 0034-6659 DOI 10.1108/NFS-11-2012-0122

Introduction Aflatoxins are a group of toxic, mutagenic and carcinogenic compounds. They are synthesized as secondary metabolites of toxigenic Aspergillus flavus, Aspergillus parasiticus and Aspergilus nomius strains (Wood, 1989). Four principal aflatoxins are produced by these fungi: AFB1, AFB2, AFG1 and AFG2. The positive correlation The authors are greatly indebted to the Food Science and Technology Institute, ACECR for supporting this research (Project No. 304).

between the consumption of AFB1-contaminated foods and the increased incidence of liver cancer in several Asian and African populations has led to the classification of AFB1 as a group 1A carcinogen by the International Agency for Research on Cancer (IARC, 1993). Aflatoxins have been found in many foods and animal feeds and their production in such commodities can be influenced by several factors, including temperature, water activity, pH, available nutrients and competitive growth of other micro-organisms (Ellis et al., 1991). The frequent incidence of these toxins in agricultural commodities has a potential negative impact on the economies of the affected regions, especially in the developing countries where harvest and post-harvest techniques adequate for the prevention of mold growth are seldom practiced (Massey et al., 1995). Commodities and products frequently contaminated with mycotoxins include corn, wheat, barley, rice, oats, nuts, peanuts and cottonseed (Council for Agricultural Science and Technology (CAST, 2003)). Various physical and chemical methods have been developed and tested for controlling AFB1. However, disadvantages of these methods, such as nutritional loss, sensory quality reduction and high cost of equipment, have limited their practical applications. It is expected that progress in the control of mycotoxin contamination will depend on the introduction of technologies for specific, efficient, and environmentally sound detoxification (Fanelli et al., 2003; Gowda et al., 2007; Mishra and Das, 2003; Zhou et al., 2008). Previous studies have clearly shown that many strains of lactic acid bacteria and their fractions inhibit the mutagenicity and carcinogenicity of a range of chemical compounds found in the human diet (Rafter, 1995). This study focused on the binding of AFB1 by Lactobacillus rhamnosus strain GG. Materials and methods Bacterial strain, culture condition and estimation of bacterial concentration Lactobacillus rhamnosus strain GG was tested for aflatoxin B1-binding capacity. The strain was obtained from Iranian Research Organization for Science and Technology (IROST), Tehran (Iran). Lyophilized bacteria was pre-cultured in de Man, Rogosa, Sharp (MRS) broth (Merck, Germany) at 378C. Bacterial samples were prepared using a 1 percent inoculum of pre-culture and incubating samples for 20 h. Bacterial pellets (about 109 cells) were collected by centrifugation (3,400g, 48C, 10 min) and washed twice with phosphate-buffered saline (PBS). Counting of viable bacteria was performed by both traditional plate counting and spectrophotometry methods using 7 Mc Farland solution and by measuring the optical density at 600 nm (OD600 nm) of samples (Lahtinen et al., 2004). Bacteria (109 cfu/ml) were either used as viable, heat treated (autoclaved at 1218C in PBS for 15 min) and acid treated (incubated at 378C in 10 ml 2 M HCl for 1 h). Acid treated bacteria were washed twice with (PBS) prior to use to prevent formation of aflatoxin B2a through reaction of AFB1 with remaining acid (Haskard et al., 2000). AFB1 binding assay AFB1 (Sigma, Merck) was dissolved in benzene-acetonitrile (97:3, vol/vol) to obtain initial AFB1 concentration of 2 mg/ml. To prepare a solution containing 100 mg/ml of AFB1, benzene-acetonitrile was evaporated in a waterbath at 808C for 10 min and methanol was added (Haskard et al., 2001). The bacterial pellets of the strain either viable or non-viable were suspended in 10 ml of PBS containing 5, 10 and 20 mg/l AFB1, then incubated at 258C for 4, 12 and 24 h. After incubation, samples were centrifuged (5,000g, 258C, 10 min) and the supernatant was analysed by ELISA method

In vitro effect of Lactobacillus rhamnosus GG 33

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(Hernandez-Mendoza et al., 2009). In all cases; positive and negative controls were included: for the negative control, PBS was substituted for the bacterial cells and for the positive control PBS was substituted for AFB1. ELISA assay Microtitre plate wells, were coated with rabbit antibodies to mouse IgG. According to Europroxima aflatoxin B1 (Art no. 5121) test kit manual, 50 ml aflatoxin standard solutions and 50 ml prepared test samples were added into separate wells of microtiter plate, in duplicate. Then, 25 ml of the diluted conjugate (aflatoxin-HRP) and 25 ml of the antibody solution were added to each well, mixed gently and incubated for 1 h at 378C in the dark. The liquid was then removed completely from the wells, the each well was washed with rinsing buffer. The washing procedure was repeated for three times in ELISA washer (ELX 50, Bio-Tek Inst., USA). After the washing step, 100 ml substrate solution was added to each well and incubated for 30 min at room temperature in the dark. Finally, 100 ml of the stop solution was added to each well and the absorbance was measured at 450 nm in ELISA plate reader (ELX 808, Bio-Tek Inst., USA). Statistical analysis All experiments were carried out as duplicates. SPSS 16 for windows was used for statistical analysis. Data were subjected to analysis of mean. Significant differences in the mean values were reported at p # 0.05. Result and discussion Various food commodities may be contaminated with aflatoxins, which even in small quantities have been detrimental effects on human and animal health. Several strategies including physical, chemical and biological methods have been investigated to eliminate, inactivate or reduce the bioavailability of aflatoxins (Cai et al., 2007; FAO, 2002). Probiotic bacteria have been studied intensively for their potential to bind food grade carcinogens, including aflatoxins. A series of studies has identified Lactobacillus rhamnosus strain GG as a very potent AFB1 binding strain (Haskard et al., 2001; El-Nezami et al., 1997). The ability of this strain to bind AFB1 in PBS as viable and non-viable preparations and at different AFB1 concentrations and different incubation times are summarized in Tables I-III. Effect of heat and acid on AFB1 binding ability The ability of Lactobacillus rhamnosus strain GG to remove AFB1 from the medium was demonstrated (Figure 1). Viable and non-viable (acid- and heat-treated) bacteria showed a significant ability ( p # 0.05) to remove AFB1. The most efficient removal of AFB1 was achieved by acid killed bacteria. It was 49.62 percent. This was statistically

Table I. Percentage of AFB1 binding on exposure to viable bacteria (mean ^ SD)

AFB1 concentration (mg/l) 5 10 20

0 28.6 ^ 1.3 27.5 ^ 0.76 28.6 ^ 0.68

AFB1 binding (%) Incubation time (h) 4 12 66.4 ^ 0.2 50.7 ^ 0.35 64.7 ^ 0.35

70.6 ^ 0.3 70.3 ^ 1.7 70.6 ^ 0.3

24 70 ^ 2.5 68 ^ 0.51 69.5 ^ 1.01

significant ( p # 0.05) in comparision with removal percentage of heat killed and viable bacteria that was 48.86 and 42.23 percent. Positive controls did not show a decrease in AFB1 content. The treatments (heat and acid) were found to markedly increase the bacterial AFB1 binding ability (Peltonen et al., 2001). Heat treated LAB have previously been shown to effectively bind aflatoxins (El-Nezami et al., 1998; Thyagaraja and Hosono, 1994). Haskard et al. (2001) revealed that heat and acid treatments also significantly enhanced the ability of Lactobacillus rhamnosus strain GG (A53103) and L. rhamnosus strain LC-705 (DSM7061) to remove AFB1 from liquid media, acid treatment being more effective than heat treatment in most cases (Haskard et al., 2001). It is reported by El-Nezami et al. (1997) that heat-treated dairy strains of lactic acid bacteria has the same ability to remove AFB1 as viable bacteria (El-Nezami et al., 1997).


32.8 ^ 0.4 32 ^ 0.51 32.8 ^ 1.4


0

0 35.4 ^ 1.7 34.6 ^ 0.3 35.3 ^ 1.4

AFB1 binding (%) Incubation time (h) 4 12 74.8 ^ 0.2 74.5 ^ 0.35 74.8 ^ 0.35

79 ^ 0.3 78.8 ^ 1.7 79 ^ 0.3

AFB1 binding (%) Incubation time (h) 4 12 77 ^ 1.01 75.8 ^ 0.66 75.5 ^ 0.76

79 ^ 1.5 78.8 ^ 0.4 79 ^ 0.51

Notes: Control: no bacteria; the ability of treated and untreated bacteria to remove different concentration of AFB1 was assessed in mentioned incubation times for 64 sample

24 78 ^ 2.5 78 ^ 0.51 78 ^ 1.01

24 78 ^ 0.51 77 77.5 ^ 0.25


Table II. Percentage of AFB1 binding on exposure to heat-treated bacteria (mean ^ SD)

Table III. Percentage of AFB1 binding on exposure to acid-treated bacteria (mean ^ SD)

Figure 1. Effect of bacterial heat and acid treatment on the removal of AFB1

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In another study, El-Nezami et al. (1998) reported that the binding ability increased in acid treatment in PBS experimentally. Effect of AFB1 concentration on the reduction of AFB1 The effect of various AFB1 concentrations on AFB1 removal was also tested (Figure 2). The percentage of AFB1 removed was the highest amount in low (5 mg/l) and high (20 mg/l) concentrations and it was not significantly different. Decrease in binding was also observed for concentration of 10 mg/l of AFB1. According to El-Nezami et al. (1998) the amount of AFB1 removed increased with increasing concentration of AFB1 but the percentage removed was not significantly different. It contrasted with findings by Line and Brackett (1995) where the percentage removal of AFB1 decreased as toxin levels increased. Also Pizzolitto et al. (2011) resulted that L. rhamnosus1, L. acidophilus24 and L. casei subsp. rhamnosus were the best binder at 50, 100 and 500 ng ml-1 AFB1, respectively. Lee et al. (2003) refer to AFB1 binding as a process of very high-affinity, linear relation with the toxin concentration used, and therefore, the amount of AFB1 bound should be limitless; in other words they concluded that the bacterial surface does not have a defined number of binding sites. Effect of incubation time on the reduction of AFB1 After incubation period of 12 h, the percentage of AFB1 removed was significantly different from that at 0, 4 and 24 h (Figure 3). Bacterial ability to remove aflatoxin increased with increasing incubation time from 0 h (17.95 percent) to 12 h (42.65 percent) but binding ability did not resume after 12 h and decreased to 43 percent within 24 h. Peltonen et al. (2001) reported that the AFB1 binding of L. amylovorus CSCC 5160 was increased significantly ( p , 0.05) with extended incubation time from

Figure 2. Effect of AFB1 concentration on the removal of AFB1

Note: Different concentration of toxin was examined to test the ability of used bacteria in viable and non-viable form in different incubation times


Note: Four incubation time was concerned to test the ability of used bacteria in viable and non-viable form in appearance of different concentration of toxin

52.6 percent (0 h) to 73.2 percent (72 h). El-Nezami et al. (1997) reported at 24 h the AFB1 binding of Lb. rhamnosus strain Lc1/3 was significantly ( p , 0.05) reduced compared with the initial binding at 0 h. The binding of AFB1 for Lb. amylovorus strain CSCC5197 increased after 24 h, but remained constant for Lb. rhamnosus strain Lc1/3. They resulted the removal of AFB1 was also a rapid process, with no significant difference observed between different incubation periods, this findings by El-Nezami et al. (1997) are different from our conclusions. The loss of AFB1, in both case of low (5 mg/l) and high (20 mg/l) concentrations of AFB1 in PBS for viable bacteria was the same, but for non-viable bacteria there was no significant difference ( p , 0.05) and percentage of binding was great and equaled about 66 percent (Figure 4). Other studies (Haskard et al., 2000, 2001) have shown that the relative amounts of AFB1 removed by viable and heat and acid treated bacteria depend on initial AFB1 concentrations. Results showed the binding ability of heat-killed and acid-killed bacteria were not significant at 12 and 24 h (Figure 5). The maximum removal was found for non-viable bacteria after 12 and 24 h and for viable bacteria after 12 h. However, there was significant difference in binding ability between viable and non-viable bacteria after 12 h incubation. Figure 6 shows effect of different AFB1 concentrations in different incubation periods on reduction of AFB1. The most effective time for AFB1 removal were 12 and 24 h in concentrations of 10 and 20 mg/l and differences in the binding ability of bacteria during 12 and 24 h incubation time in high concentrations (10 and 20 mg/l) were not significant, although, in PBS containing 5 mg/l of AFB1, 12 h incubation time was effective. Conclusion In this study it was determined that extended incubation time is more effective in increasing the percentage of AFB1 removed from medium. Furthermore, it has been clearly shown that non-viable bacteria significantly reduced more AFB1 as compared

Figure 3. Effect of incubation time on the removal of AFB1

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Figure 4. Interaction effect of heat and acid treatment in different AFB1 concentration on the removal of AFB1

Figure 5. Interaction effect of bacterial heat and acid treatment in different incubation time on the removal of AFB1

Notes: Control: no bacteria; different incubation times was assessed and the number of samples was 64

Note: Control: no bacteria

to viable bacteria ( p , 0.05). The application of this phenomenon in the removal of aflatoxins from contaminated food is urgently needed to improve the safety of food supply. In addition, conducting the experiments in vivo will play an important role in determining the binding properties of bacteria.


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Figure 6. Interaction effect of AFB1 concentration in different incubation time on the removal of AFB1

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