Saccharomyces Cerevisiae And Probiotic Bacteria Potentially Inhibit Aflatoxins Production In Vitro And In Vivo Studies
S Nada, H Amra, M Deabes, E Omara
a. flavus, aflatoxins, kidney, liver, probiotic bacteria, rat, saccharomyces cerevisiae
S Nada, H Amra, M Deabes, E Omara. Saccharomyces Cerevisiae And Probiotic Bacteria Potentially Inhibit Aflatoxins Production In Vitro And In Vivo Studies. The Internet Journal of Toxicology. 2009 Volume 8 Number 1.
Aflatoxins are group of closely related difuranocoumarin compounds produced by several fungi, mainly
Aflatoxin contamination occurs by colonization of the fungus on susceptible crop, or may arise during harvesting, drying, storage, or processing.
Human hepatocelluar carcinoma is the fifth most commonly occurring cancer in the world and the third greatest cause of cancer mortality (Anwar et al., 2008). Aflatoxin B1 (AFB1) is the most prevalent and carcinogenic of the aflatoxin, the International Agency for Research on Cancer reported that AFB1 is a carcinogen of group I (Bedard and Massey, 2006).
Filamentous moulds are common spoilage organisms of food products, e.g. fermented milk products, cheese, bread, as well as stored crops and feed such as hay and silage (Filtenborg, et al.,1996). It is estimated that between 5 and 10% of the world’s food production is lost annually due to fungal deterioration (Pitt and Hocking, 1997). In Western Europe, mould spoilage of bread alone is estimated to cause annual economical losses of more than £200 million (Legan, 1993).
The aim of our study was to investigate the efficacy of probiotic bacteria (LGG and LC705) and
Materials and Methods
Sep-pak silica cartridge C18 columns Sep-Pak silica cartridge, C18-E (55 µm, 70 A°) 500 mg/6 ml were obtained from Phenomenex Co., Torrance, CA, USA.
Chemical used in the analytical analysis. Chloroform, acetone, trifluroacetic acid (TFA), methanol, acetonitrile, diethylether and acetic acid, of HPLC grade were produced by BDH,Chemicals Ltd., Poole, England.
Aflatoxins and Cultures : (a) Potato dextrose agar (PDA) , (b) de`Mane- Regosa-Sharp (MRS), (c) Malt Extract Agar (MEA) and (d) Yeast extract-malt extract-sucrose broth medium were obtained from Sigma Chemical Company, St., Louis, MO, USA).
Diagnostic kits were purchased from Boehringer Mannheim GmbH Diagnostica, E.Merck, Postfach 4119, D-6100, and Darmstadt, Germany. All other chemicals were of highest quality available and were obtained from commercial sources.
Animal care and use: The experimental protocols were approved by The National Research Centre, Cairo, Egypt of Animal Care and Use Committee and were in accordance with the guidelines of the International Association for the Study of Pain Committee for Research and Ethical Issues (Zimmermann 1983).
The experiment was conducted using mature male rats weighing 120-130 g b.wt, purchased from Animal House colony. Animals were divided into equal groups (six rats each) housed under standard environmental conditions (23 ± 1◦C, 55 ± 5% humidity and a 12-h light: 12-h dark cycle) and maintained on a standard laboratory diet
(a): Preparation of
Cultures of toxic molds were grown on potato dextrose agar (PDA) slants for 7 days at 25 °C (Bullerman, 1974). The librated Aflatoxins were analyzed according to AOAC (2000) and quantified by HPLC technique (Sep-pak silica cartridge C18 columns) according to method’s described by Ferreira et al., (2005).
(d) Inhibition experiments:
Inhibition of mold growth in the presence of
Eight groups of normal rats (6 rats each) were used in this experiment, the first four groups (1, 2, 3, and 4) were orally administered
After 15 days of the daily treatment
Immediately after sacrifice, a sample of the liver was fixed in 10% formalin. The washed tissue was dehydrated in descending grades (70 % -100 %) of alcohol and finally cleared in zylene. The tissue was embedded in paraffin wax. Sections were cut at 5µm thickness and stained with haematoxylin and eosin (Drury et al., 1980). The sections were then, viewed under a light microscope for Histopathological examination. Histochemical examination of DNA content was performed using the Feulgen technique (Gardikas and Israels, 1948).
Statistical analysis: The obtained results were analyzed by ANOVA (one or two-way) using the Microsoft Excel ( Redmond, WA) software package.
It was clearly demonstrated that the addition of the tested biocontrol microorganisms to YES media containing
Table (1) shows that
Moreover, production of AFG1 & AFG2 were inhibited by all studied biocontrol microorganisms. Particularly, LGG at the concentration tested was the most potent inhibitor for AFG1 production when compared to SC or LC 705. The percentage of inhibition of AFG1 and AFG2 production were (
The dry weight of
It was found that
The different capital letters superscripts are significantly different between treatments at P< 0.05
Biochemical results (Table 2) showed the effect of different treatments on liver enzymes (ALT, AST and GGT), kidney function tests (BUN and creatinine) and on GSH level in the blood of all groups.
Single treatment with probiotic bacteria (LGG and LC705) showed no-significant changes in ALT, AST and GGT activities comparing with the control group. Liver enzymes were significantly inhibited by
Rats treated with AFB1 alone, showed significant elevation in amino-transferases activities. This elevation was normalized by co-administration with LGG, while the combined treatment with
AFB1-administration also affected the Kidney functions; BUN and creatinine values elevated significantly than other treatments (Table 2). Combined treatments with
GSH levels were depleted in rats administered AFB1 alone, and it was increased significantly by the combination with LGG, LC 705 and
ANOVA – one way.
The different capital letter superscripts are significantly different at P< 0.05
Histopathological results supported the obtained data from biochemical studies. Histopathological examination of the liver revealed the following changes. The control groups presented liver with normal architecture (Fig.1A). No histopathological changes were observed in the liver of rats treated with
Figure (2 A) shows normal histological structure of the kidney of control rat. Kidneys of groups treated with LGG, LC 705 and Yeast showed normal kidney histology. AFB1 treated group caused vacuolar degeneration, cellular swelling, and pyknotic nuclei were observed in the epithelial cells of proximal convoluted tubules. Dilated and congested blood vessels are a prominent feature, together with many large areas of interstitial hemorrhage, in AFB1-treated animals (Fig. 2 B). Microscopic examination of renal tissue in animals treated with LGG, LC 705 and Yeast plus AFB1 showed normal appearance of kidney tissue and degeneration of some proximal convoluted tubules. Mild congestion of blood vessels was also observed.
Histochemical staining revealed that liver sections stained with Feulgen stain showed normal content of DNA in the nucleus (Fig. 1 E). The liver of rats treated with AFB1 only showed decrease in DNA content (Fig. 1 F). Liver sections examined after treatment with LGG, LC 705 and Yeast plus AFB1 showed improvement in the DNA content as compared with AFB1-terated groups (Figs. 1 G and H).
Histochemical staining revealed normal DNA content in the kidney tubules and glomeruli. The DNA contents were decreased in the kidney tissue after treatment with AFB1. However, DNA content appeared relatively normal in the kidney tubules and glomerulus after treatment with LGG, LC 705 and
During the past decades, several studies have suggested that
Several investigators report that LGG and LC-705 significantly inhibit ~ 99% of AFB1 production
Reduced glutathione (GSH) is the main component of endogenous non-protein sulfhydryl pool that scavenges free radicals in the cytoplasm (Shaw et al., 1990). However, antioxidants restore the cellular defense mechanisms, block lipid peroxidation, and thus protect against the oxidative tissue damage (Toklu et al., 2006).
Many previous studies suggest that the presence
The potential function of the tested probiotec strains (LGG and LC705) may be due to their binding ability to the toxins or metabolically transforming them into non-toxic degradation products (Hooper et al., 2001; Zhou et al., 2001). Yan et al. (2007) found that LGG prevent cytokine-induced apoptosis in intestinal epithelial cells, the intestinal epithelial tight junction (TJ) prevents the diffusion of toxins from gastrointestinal lumen into the tissue (Anderson et al., 1995). Moreover, AFB1-oral administration disrupts TJ and adherents junction proteins due to the presence of free radicals and inflammatory process induced by the mycotoxin ingestion (Gratz et al., 2006, Seth et al., 2008, Shifflett et al., 2005).
Probiotic bacteria and
The authors gratefully acknowledge the financial support from the National Research Centre, Cairo, Egypt; the authors thank Prof. Dr. H. Amra for his kind donation of Probiotec organisms, and for production and estimation of Aflatoxins.