Wageningen Academic P u b l i s h e r s
Beneficial Microbes, June 2010; 1(2): 149-154
In vitro binding of mutagenic heterocyclic aromatic amines by Bifidobacterium pseudocatenulatum G4 F. Faridnia1, A.S.M. Hussin1, N. Saari2, S. Mustafa3, L.Y. Yee4 and M.Y.A. Manap1 1Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM Serdang,
Selangor, Malaysia; 2Department of Food Science, Faculty of Food Science and Technology, 43400 UPM, Selangor, Malaysia; 3Halal Products Research Institute, Universiti Putra Malaysia, Selangor, Malaysia; 4Department of Medicine, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Selangor, Malaysia;
[email protected] Received: 24 July 2009 / Accepted: 5 April 2010 © 2010 Wageningen Academic Publishers
Abstract Consumption of probiotics has been associated with decreased risk of colon cancer and reported to have antimutagenic/anti-carcinogenic properties. One possible mechanism for this effect involves physical binding of the mutagenic compounds, such as heterocyclic amines (HCAs), to the bacteria. Therefore, the objective of this study was to examine the binding capacity of bifidobacterial strains of human origin on mutagenic heterocyclic amines which are suspected to play a role in human cancers. In vitro binding of the mutagens Trp-p-2, IQ, MeIQx, 7,8DiMeIQx and PhIP by three bacterial strains in two media of different pH was analysed using high performance liquid chromatography. Bifidobacterium pseudocatenulatum G4 showed the highest decrease in the total HCAs content, followed by Bifidobacterium longum, and Escherichia coli. pH affects binding capacity; the highest binding was obtained at pH 6.8. Gram-positive tested strains were found to be consistently more effective than the gram-negative strain. There were significant decreases in the amount of HCAs in the presence of different cell concentrations of B. pseudocatenulatum G4; the highest decrease was detected at the concentration of 1010 cfu/ml. The results showed that HCAs were able to bind with all bacterial strains tested in vitro, thus it may be possible to decrease their absorption by human intestine and increase their elimination via faeces. Keywords: heterocyclic aromatic amines, anti-mutagenic, anti-carcinogenic, bacterial binding, Bifidobacterium
1. Introduction
A group of carcinogens of dietary origin are the heterocyclic aromatic amines (HCAs), formed in protein-rich muscle foods during high temperature of cooking or grilling
(Armindo et al., 2008; Cheng et al., 2006; Melo et al., 2008). HCAs were first discovered in cooked foods by Sugimura and his collaborators more than 25 years ago (Sugimura et al., 1977, 2004). More than 20 HCAs have since been isolated and identified in cooked foods. Their formation is dependent upon the type of meat, cooking method, temperature and cooking time. Several HCAs have been classified as possible/probable carcinogens by the International Agency for Research on Cancer (IARC, 1977), thus reducing human exposure to these compounds is highly recommended. Recently a correlation between pancreatic cancer and meat intake, especially those cooked at high temperature, was shown, suggesting the involvement of HCA consumption (Smith et al., 2008).
ISSN 1876-2833 print, ISSN 1876-2891 online, DOI 10.3920/BM2009.0035
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Colorectal cancer is the second major cause of death from cancer in Europe and the USA. Dietary factors and colonic microbiota seem to play a crucial role in colorectal carcinogenesis (Capurso et al., 2006; Gooderham et al., 2007). The molecular genetic mechanisms of colorectal cancer are well established, but environmental factors such as diet also play a major role in the development of sporadic colon cancer.
F. Faridnia et al.
On the other hand, intestinal bacteria could play a part in initiating colon cancer through the production of carcinogens, co-carcinogens, or pro-carcinogens. Some intestinal microorganisms strongly increase damage to DNA in colon cells induced by heterocyclic amines, whereas other intestinal bacteria can take up and detoxify such compounds (Wollowski et al., 2001). Bacteria of the Bacteroides and Clostridium genera increase the incidence and growth rate of colonic tumours induced in animals, whereas other genera such as Lactobacillus and Bifidobacterium prevent tumorigenesis (O’Mahony et al., 2001; Onoue et al., 1997). There is a growing interest in trying to improve gastrointestinal (GI) health with functional foods including probiotics and prebiotics. Evidence for the protective effects of pro- and prebiotics against cancer was derived from in vitro studies, animal models, epidemiology and human intervention studies (Rafter, 2003). Several bifidobacteria and lactic acid bacteria exhibit anti-mutagenic activities against HCAs, N-nitroso compounds, benzo[a]pyrene and aflatoxin B (Lo et al., 2004). Bifidobacteria are natural inhabitants of the human GI tract, in which they occur at concentrations of 109 to 1011cfu/g (Collado et al., 2007; Trejo et al., 2006). Bifidobacteria also have a long history of use in foods and fermented products due to their health-promoting effects. In fact, these bacteria have a ‘generally regarded as safe’ (GRAS) status. They are known and frequently used as probiotic organisms because of their potentially beneficial role in the intestinal tract of humans including balancing of intestinal microbiota, increasing protein digestion, human immune system activation, amelioration of diarrhoea or constipation, inhibition of the growth of potential pathogens, reduction of serum cholesterol, and prevention of colon cancer induction (Delgado et al., 2005; Hughes and Hoover, 1991). Reddy and Rivenson in 1993 have shown that lyophilised cultures of Bifidobacterium longum administered in the diet to rats inhibited liver, colon and mammary tumours induced by the cooked food mutagen 2-amino-3-methyl3H-imidazo[4,5-f ]quinoline (IQ). It is possible that the protective effects are due to direct binding, which appears to be the most important detoxification mechanism (Salminen et al., 1998). Evidence from human studies also demonstrates that the consumption of lactobacilli by healthy volunteers reduces the mutagenicity of urine and faeces associated with the ingestion of carcinogens in cooked meat. Epidemiological studies have shown a link between consumption of fermented dairy products which contain Bifidobacterium and lower incidence of colon cancer. Another epidemiological study performed in Finland demonstrated that, despite high fat intake in that country, the colon cancer incidence there was lower than in other countries due to the high consumption of milk, yoghurt and other dairy products (IARC, 1977; Malhotra, 1977).
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Bifidobacterium pseudocatenulatum is one common species which has been isolated from breast-fed infants’ stool in Malaysia. It is particularly well adapted to the gastro-intestinal tract (GIT) environment, as reflected by its ability to adapt to low pH and a high concentration of bile salts (Shuhaimi et al., 1999, 2002; Yazid et al., 2000). Preliminary investigations have shown that the probiotic candidate strain G4, which has been recently identified as a safe probiotic for incorporation into functional food for human consumption (Kabeir et al., 2008; Stephenie et al., 2007), possesses the required criteria of successful probiotic microorganisms (Ali et al., 2009; Kabeir et al., 2008). Early research conducted in our laboratory spurred interest in this group of species (Mariam et al., 2004; Stephenie et al., 2007;Wong et al., 2006). The aims of this study were to evaluate the binding ability of Bifidobacterium strains; B. pseudocatenulatum G4, a species which has not yet been explored as a commercial probiotic, B. longum BB536, and Escherichia coli ATCC 25922, to mutagenic HCAs under different pH levels. The binding ability of bacteria to the selected mutagens in different concentrations of bacterial cells was also examined to identify the most effective concentration of probiotic bacteria.
2. Material and methods Preparation of bacterial cells The Bifidobacterium strains used in this study were obtained from the collection of Food Biotechnology and Functional Food Laboratory, University Putra, Malaysia. B. pseudocatenulatum G4 (used as test strain) and B. longum BB536 (a commercial probiotic used as reference strain) were grown in TPY and MRS broth, respectively, to stationary phase from stock cultures stored at -40 °C in 20% glycerol for 12-16 h under anaerobic conditions. E. coli ATCC 25922 was grown in Brain Heart Infusion (BHI) broth at 37 °C for 24 h. Cells were harvested by centrifugation 4,000 rpm at 4 °C for 20 min, washed twice with 0.15 mol/l phosphate buffers (pH 6.2) and lyophilised. The content of 1 mg of freeze-dried bacteria was 108 cells. One mol/l HCL solution was used to prepare phosphate buffer saline at two different pH levels of 5.6 and 6.8. One mol/l NaOH solution was also used to adjust the pH levels of the solutions. The solutions were prepared in 100 ml volumes using screw-capped glass bottles, autoclaved at 121 °C for 15 min and stored at room temperature.
Binding of heterocyclic amines to cells The mutagenic compounds to be tested were 2-amino-3,8dimethylimidazo[4,5-f ]quinoxaline (MeIQx), 2-amino1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), Beneficial Microbes 1(2)
Ability of probiotic to bind cooked food mutagens
3,7,8-trimethyl-3H-imidazo[4,5-f ]quinoxalin-2-amine (7,8-DiMeIQx), 2-amino-3-methyl-3-imidazo[4,5-f ] quinoline (IQ and 3-amino-1-methyl-5H-pyrido[4,3-b] indole (Trp-p-2) and IQ. All these HCAs were obtained from Toronto Research Chemicals Inc., Ontario, Canada. One mg of lyophilised bacterial cells (108 cfu/ml) was suspended in 0.950 ml of phosphate buffer. 50 µl of methanolic solutions (100 µg/0.05 ml) of mutagenic compounds were added to the suspensions. The mixtures were incubated at 37 °C for 30 min with rotation. 0.950 ml phosphate buffed saline was mixed with 50 µl of each mutagen solution as a control. All assays were performed in triplicate. After incubation, the samples were centrifuged at 6,000×g for 10 min. The supernatant was filtered using a micro filter (pore size 0.22 µm) and subjected to high-performance liquid chromatographic analysis (HPLC) to determine the amount of unbound mutagen.
HPLC assay The chromatographic separation of HCAs was achieved using a reversed phase TSK-Gel ODS-80 TM column (25 cm × 4.6 mm, 5 µm) from Tosoh Bioscience, Japan. The column temperature was set at 40 °C. The HPLC system used in this study consisted of a LC-20AT Liquid Chromatography with SPD-M20A detector (Shimadzu, Japan) set at 190300 nm range. Optimal separation was achieved with a binary mobile phase at a flow rate of 1 ml/min. The linear gradient system was used for the HPLC separation with: 0.01 mol/l triethylamine in water (pH 3.3 adjusted with 1 mol/l phosphoric acid; solvent A) and acetonitrile 100% (solvent B). The gradient elution program consisted of 95% solvent A : 5% solvent B to 70% solvent A : 30% solvent B. The retention time was developed from 0 to 42 min. The loading volume was 20 µl and samples were injected using
a SGE syringe. All the solutions were passed through a 0.22 µm nylon filter before injection into the HPLC.
Statistical analysis The Statistical Analysis System (SAS) (version 9.1, SAS Inst. Inc., Cary, NC, USA, 2002) was used for the analysis of the data. The experimental design was a completely randomised design using full factorial design. One-way analysis of variance (ANOVA) was performed to determine the significant differences among treatments at P
