Mutagenic and antimutagenic activities of lactic acid bacteria (LAB) Lactobacillus plantarum isolated from the local fermented durian (tempoyak) was determined by Ames test (Salmonella/microsome mutagenicity assay). Our study also involved pre-incubation assay against Salmonella typhimurium TA 98 and TA 100 bacterial strain in the presence and absence of metabolic activator S9 system. It was found that the L. plantarum showed no mutagenic activity on both S. typhimurium strain TA 98 and TA 100 in the presence and absence of metabolic activator. Significant antimutagenic activity (p < 0.05) was observed in both cell-free supernatant and bacterial cell suspension of L. plantarum as compared to the mutagenicity induced by 2-Aminoanthracene in the presence of metabolic activator. Meanwhile, in the absence of metabolic activator, only the bacterial cells of L. plantarum showed antimutagenicity acitivity against Sodium Azide and 2-Nitrofluorene. In conclusion, L. plantarum could play a vital role as chemopreventive agent by binding to mutagens and suppressing mutagenesis. Thus, L. plantarum could be consider as a good candidate for functional food development as a supplement product to prevent development of colon cancer

Ahmad Rohi Ghazali,

Asmariah Ahmad,

Noorhisham Tan Kofli,

Safura Salik,

Yap, Wei Boon

UKM Journal Article Repository

23Jurnal Sains Kesihatan Malaysia Isu Khas 2018: 23-26DOI : http://dx.doi.org./10.17576/JSKM-2018-04Mutagenicity and Antimutagenic Activities of Lactic Acid Bacteria (LAB) Isolated from Fermented Durian (Tempoyak)(Aktiviti Mutagenik dan Antimutagenik Bakteria Asid Laktik yang Dipencilkan daripada Fermentasi Durian (Tempoyak))ASMArIAh AhMAD, SAFurA SALIK, YAp WeI BOOn, nOOrhIShAM TAn KOFLI & AhMAD rOhI GhAzALIABSTrACTMutagenic and antimutagenic activities of lactic acid bacteria (LAB) Lactobacillus plantarum isolated from the local fermented durian (tempoyak) was determined by Ames test (Salmonella/microsome mutagenicity assay). Our study also involved pre-incubation assay against Salmonella typhimurium TA 98 and TA 100 bacterial strain in the presence and absence of metabolic activator S9 system. It was found that the L. plantarum showed no mutagenic activity on both S. typhimurium strain TA 98 and TA 100 in the presence and absence of metabolic activator. Significant antimutagenic activity (p < 0.05) was observed in both cell-free supernatant and bacterial cell suspension of L. plantarum as compared to the mutagenicity induced by 2-Aminoanthracene in the presence of metabolic activator. Meanwhile, in the absence of metabolic activator, only the bacterial cells of L. plantarum showed antimutagenicity acitivity against Sodium Azide and 2-Nitrofluorene. In conclusion, L. plantarum could play a vital role as chemopreventive agent by binding to mutagens and suppressing mutagenesis. Thus, L. plantarum could be consider as a good candidate for functional food development as a supplement product to prevent development of colon cancer.Keywords: Mutagenicity; antimutagenicity; Lactobacillus plantarum; lactic acid bacteria; Ames test; tempoyakABSTrAKAktiviti mutagenisiti dan antimutagenisiti bakteria asid laktik (LAB) Lactobacillus plantarum yang dipencilkan daripada durian yang difermentasi (tempoyak) telah dinilai melalui Ujian Ames (Salmonella/asai mutagenisiti mikrosom). Kajian kami juga melibatkan asai pra-inkubasi dengan Salmonella typhimurium TA 98 dan TA 100 dengan keadaan kehadiran dan ketidakhadiran sistem pengaktifan metabolik S9. Hasil kajian kami menunjukkan L. plantarum tidak mempunyai sebarang kesan mutagenik terhadap kedua-dua TA 98 dan TA 100 dalam kedua-dua keadaan ujian. Aktiviti antimutagenik yang signifikan (p < 0.05) dilihat pada kedua-dua supernatan bebas sel dan juga suspensi sel bakteria L. plantarum berbanding mutagenisiti yang diaruh oleh 2-aminoanthracene pada asai dengan kehadiran sistem pengaktifan metabolik. Manakala pada keadaan ketidakhadiran sistem pengaktifan metabolic pula, hanya suspensi sel bakteria L. plantarum menunjukkan kesan antimutagenisiti terhadap Sodium Azide dan 2-Nitrofluorene. Sebagai kesimpulan, L. plantarum boleh memainkan peranan penting sebagai ejen kemoprevensi dengan mengikat mutagen dan menghalang mutagenesis. Oleh itu, L. plantarum berpotensi untuk dibangunkan sebagai makanan berfungsi seterusnya produk suplementasi bagi mencegah perkembangan kanser usus.Kata kunci: Mutagenisiti; antimutagenisiti; Lactobacillus plantarum; bakteria asid laktik; Ujian Ames; tempoyakInTrODuCTIOnCancer is the leading cause of death in economically developed countries and the second leading cause of death in developing countries (WhO 2008). Colon cancer is one of the leading causes of cancer death too. Dietary intake pattern that is high red meat and low in vegetables are highly associated with colon cancer (Slattery et al. 1998). The complex web of gut microbiota in the human intestines may play a crucial function for both human health and diseases. production of microbial enzymes such as azoreductase, nitroreductase and β-glucuronidase by the microbiota are capable to mediate carcinogen production in human colon through metabolic activation of dietary component (Commane et al. 2005). probiotic defined as live microbial food ingredients (supplements) confer beneficial health effects to the host upon ingestion in adequate amounts (Salminen & Wright 1998). They are beneficial bacteria that may alter the gut microbiota by promoting good digestion, inhibiting the growth of harmful bacteria as well as by reducing levels of microbial enzymes, boosting immune function and increase resistance towards food-borne infection (helland et al. 2004). previous in vitro studies suggested that some probiotic strain might exert potential to protect against colon cancer, through several mechanisms such as, modifications of metabolic activities of gut microbiota, alteration of gut microbiota, reduction of possible pro-carcinogens in colon, production of anti-tumor and anti-mutagenicity compounds (Kumar et al. 2010). Studies had reported the antimutagenic activities Chap 4.indd   23 31/05/2018   15:23:3924of lactic acid bacteria from various fermented food sources (Ahmadi et al. 2014; park et al. 1998; Asahara et al. 1992). The aim of the present study was to investigate the antimutagenic potential of Lactobacillus plantarum isolated from tempoyak (fermented Durio zibethinus) a traditional condiment in Malaysia against Sodium Azide, 2-Aminoanthracene and 2-nitroflourene in TA 98 and TA 100 strain of Salmonella typhimurium using Ames test.MATerIALS AnD MeThODSBACTerIAL STrAIn AnD GrOWTh COnDITIOnSLactobacillus plantarum was isolated from fermented durian (Durio zibethinus) at Department of Chemical & process engineering, Faculty of engineering & Built environment, universiti Kebangsaan Malaysia, Bangi Selangor, Malaysia. Strain was stored at -80°C in de Man rogosa (MrS) broth (Oxoid Ltd, hampshire, england), supplemented with 20% (v/v) glycerol. As the routine analysis, L. plantarum was subcultured twice in MrS broth (Oxoid Ltd, hampshire, england) and MrS agar (de Man rogosa, Merckmilipore, Darmstadt, Germany) at 37°C for 16 hours to get 8.0 log cfu /ml before every experiment was conducted. Bacterial cells were then harvested and washed by centrifugation (10000 rpm, 10 min) and then re-suspended in phosphate buffer saline (pBS, 2M, ph 7.4), while the cell-free supernatant (CFS) was filtered using filter membrane 0.22µM. These samples were then immediately subjected to mutagenicity and antimutagenicity assay.Salmonella typhimurium strain TA 98 and TA 100 were used for the Ames assay. Test of histidine requirement, rfa mutation, uvrB mutation and r-factor were also performed to confirm the genotypes of both as previously described by Maron and Ames (1983). Salmonella typhimurium TA 98 is a frame shift strain which contain the his3052 mutation and Salmonella typhimurium TA 100 contain the base-pair substitution mutation hisG46. prior to each mutagenicity and antimutagenicity test, Salmonella typhimurium strain TA 98 and TA 100 was grown in fresh nutrient broth no. 2 (Oxoid, Basingstoke, hamsphire, england) at 37°C for 12-16 hour.MuTAGenS AnD S9 MIXSodium Azide (nan3), 2-nitrofluorene (2-nF) and 2-aminoanthracene (2-AA) were purchased from Sigma-Aldrich Co. (St. Louis, MO, uSA) and dissolved in dimethyl-sulfoxide (DMSO). An S9 microsomal fraction (S9 mix) of rat liver obtained from MOLTOX, Inc. (uSA).MuTAGenICITY AnD AnTIMuTAGenICITY ACTIVITY ASSAYMutagenic and antimutagenic activities of L. plantarum were determined using the pre-incubation method of Maron and Ames (1983) with minor modification. For antimutagenicity assay, overnight culture of Salmonella typhimurium strain TA 98 or TA 100 (0.1 ml) in a tube was added with cell suspension or culture medium of L. plantarum (0.1 ml), pBS (0.5 ml) (without microsomal activation) or S9 mix (microsomal activation) following 20 minutes of incubation at 37°C in the rotary shaker. Then 2.0 ml top agar supplemented with 0.5 mM L-histidine/D-biotin and mutagen such as 2-AA, 2-nF and nan3 were mixed with the tube content. The tubes were vortexed and poured onto the minimal glucose agar plates and the reverted colonies were counted after 48 hours of incubation at 37°C.percent of inhibition of mutagenicity (%) was determined by the following (negi et al. 2003).Inhibition rate (%) = 1 – (T/M) × 100 %Where T, is the number of revertants per plate in the presence of mutagen and bacterial sample and M is the number of revertants per plate in positive control. no antimutagenic effect was considered to give a value smaller than 25%, a moderate effect value between 25 and 40% and a strong antimutagenicity value greater than 40%. Mutagenicity of L. plantarum was examined under the condition described for antimutagenicity testing except without the addition of mutagens.STATISTICAL AnALYSISThe results were expressed as mean ± SeM of three independent experiments. Data analysis was carried out by the Independent t-test. p values < 0.05 were considered to be significant.reSuLTS AnD DISCuSSIOnAs shown in Table 1, the number of revertants per plate with and without the presence of metabolic activation system were less than twice that of spontaneous revertants (negative control). Both bacterial cells and cell-free supernatant (CFS) of L. plantarum had no mutagenic effect on both Salmonella typhimurium TA 98 and TA 100. The absence of mutagenic activities from L. plantarum indicated that it would be developed further for the use in nutraceuticals.As shown in Table 2. The antimutagenic activity of L. plantarum with and without the presence of activation system against nan3, 2-nF and 2-AA. The percentage of antimutagenicity L. plantarum against 2-nF in TA 98 and nan3 in TA 100 on assay without the presence of metabolic activation were 28.2% and 72% for bacterial cells, while there was no inhibition by CFS in TA 98 and only 18% inhibition rate in TA 100. Meanwhile, in the presence of metabolic activation, the antimutagenic activity against 2-AA in both TA 98 and TA 100 were 93% and 50% for bacterial cells and 91.7% and 36% for CFS.Chap 4.indd   24 31/05/2018   15:23:3925Colon carcinogenesis is a multistep process which starts from initiation process with the occurrence of DnA mutation (Cooper 2000). DnA mutation either happens spontaneously or is chemically induced and leads to development of carcinogenesis if left untreated (Carr 1948). Ames mutagenicity assay is the basic test or model used to detect the mutagenic properties of a chemical or drug of interest. The bacterial strain of Salmonella typhimurium used in the assay carried mutation in histidine operon, thus are histidine dependent. upon introduction of mutagenic substances, this bacterial strain will revert back into the wildtype strain. Meanwhile, non-mutagenic substances would not be able to revert back those strains. The strains used both carry different type of mutation. TA 98 carried frameshift mutation while TA 100 carried base pair mutations (Mortelmans & zeiger 2000). In the present study, the mutagenicity and antimutagenicity activity of isolated lactic acid bacteria isolated from the fermented durian (tempoyak) had been reported. Our study was the first finding on the potential chemopreventive mechanism involved in lactic acid bacteria isolated from fermented durian (tempoyak).Mutagenicity and carcinogenicity are often correlated (Griffiths 2000). Thus, mutation and cancer occurrence that arises from diet are actually highly preventable by consumption of dietary antimutagens (Ferguson 1994). probiotic are considered as a potential antimutagen diet to help prevent colon carcinogenesis (Lim 2014). Mechanisms of antimutagenic activity of probiotic remain unclear. previous study reported that the antimutagenic activity of LAB are highly related to the binding interaction between cell wall and cells’ components with the mutagen and its metabolites (Lo et al. 2004). In Lactobacillus plantarum KLAB21, the antimutagenic activities was mediated by its extracellular excretion which consist of glycoproteins (rhee & park 2001). Meanwhile, LAB isolated from fermented cabbage (kimchi) a type of Korean traditional dish showed that the antimutagenic activities were mainly contributed to cell wall fraction rather than the cytosolic fraction (park et al. 1998). The antimutagenic activities of LAB was also dependent to bacterial strain and mutagen used. Some Lactobacillus sp. exhibit inhibitory effect on 2-amino-3-methylimidazo[4,5-f]quinoline compared to Streptococcus thermophilus and Bifidobacterium adolescentis while in other experiment Lactobacillus alimentarius has a percentage of antimutagenicity of 65% against SA and 41% against benzo[a]pyrene (Tavan et al. 2002; Apas et al. 2014).TABLe 2. Antimutagenic activities of L. plantarum without metabolic activation S9 (- S9) and with metabolic activation S9 (+ S9) Strain   Samples Mean ± SeM (-S9) Inhibition rate (%) Mean ± SeM (+S9) Inhibition rate (%) TA 98 positive Control 374 ± 12 - 332 ± 13 - TA 98 Bacterial Cells 263 ± 19a 28.2 23 ± 5a 93 TA 98 positive Control 335 ± 42 - 340 ± 8 - TA 98 CFS 409 ± 38 nD 28 ± 5a 91.7 TA 100 positive Control 471 ± 9 - 245 ± 16 - TA 100 Bacterial Cells 413 ± 20a 72 121 ± 4a 50 TA 100 positive Control 478 ± 14 - 236 ± 18 - TA 100 CFS 203 ± 9 18 150 ± 17a 36results are presented as mean ± SeM for three plates.(n=3) a Statistically significant differences compared to positive control value at p < 0.05, nD; not detectedTABLe 1. Mutagenic activities of L. plantarum without metabolic activation S9 (- S9) and with metabolic activation S9 (+ S9) Strain  Samples          number of revertants per plate   Mean ± SeM (-S9) Mean ± SeM (+S9) TA 98 Control (pBS) 15.33 ± 1.45  16.44 ± 2.62 TA 98 Bacterial Cells 19 ± 2.08 13.33 ± 0.33 TA 98 Control (MrS) 25.33 ± 4.25 23.44 ± 0.22 TA 98 CFS 39.67 ± 0.88 25.11 ± 3.63 TA 100 Control (pBS) 68.67 ± 4.33 169.22 ± 9. 63 TA 100 Bacterial Cells 50.15 ± 1.15 170.33 ± 1.07 TA 100 Control (MrS) 84 ± 7.23 234.11 ± 2.38 TA 100 CFS 55.33 ± 3.71 205.11  17. 46 results are presented as mean ± SeM for three plates. (n = 3)Chap 4.indd   25 31/05/2018   15:23:3926COnCLuSIOnOur results demonstrated that Lactobacillus plantarum isolated from fermented durian (tempoyak) did not exerts any mutagenic effect towards both Salmonella typhimurium strains. It also had strong antimutagenic activity when co-incubated with 2-AA and nan3. The antimutagenicity of probiotic Lactobacillus plantarum observed in the present study implied its potential to be developed as a putative functional food as a chemopreventive agent.ACKnOWLeDGMenTSThis study was supported by Fundamental research Grant Scheme (FrGS/1/2014/SG03/uKM/02/2) granted by Ministry of higher education Malaysia (MOhe).reFerenCeSAhmadi, M.A., ebrahimi, M.T., Mehrabian, S., Tafvizi, F., Bahrami, h. & Dameshghian, M. 2014. Antimutagenic and Anticancer effects of Lactic Acid Bacteria Isolated from Tarhana through Ames Test and phylogenetic Analysis By 16S rDnA. Nutrition and cancer 0(0): 1-8.Apas, A.L., Gonzales, S. n. & Arena, M.D. 2014. potential of goat probiotic to bind mutagens. Anaerobe 28: 8-12.Asahara, n., zhang, X.B. & Ohta, Y. 1992. Antimutagenicity and Mutagen-binding Activation of Mutagenic pyrolyzates by microorganisms Isolated from Japanese Miso. Journal of Science Food Agriculture 58: 395-401.Carr, B.J. 1948. Chemically induced mutation. British Journal of Cancer 2(2): 132-134.Commane, D., hughes, r., Shortt, C. & rowland, I. r. 2005. The potential Mechanisms involved in the anti-carcinogenic action of probiotics. Mutation Research 591: 276-289.Cooper, G.M. 2000. The Cell: A Molecular Approach. 2nd edition. Sinauer Associates, Inc. Available from: https://www.ncbi.nlm.nih.gov/books/nBK9963/Ferguson, L.r. 1994. Antimutagens as cancer chemopreventive agents in the diet. Mutation Research 307: 395-410.Griffiths, A.J.F., Miller, J., Suzuki, D.T., Lewontin, r.C. & Gelbart, W.M. 2000. relation between mutagens and carcinogens. An Introduction to Genetic Analysis. 7th edition. new York: W. h. Freeman. Available from: https://www.ncbi.nlm.nih.gov/books/nBK21788/helland, M.h., Wicklund, T. & narvhus, J.A. 2004. Growth and metabolism of selected strains of probiotic bacteria, in maize porridge with added malted barley. International Journal of Food Microbiology 91(3): 305-313.Kumar, M., Kumar, A., nagpal, r., Dheeraj, M., Behare, p., Verma, V., Kumar, p., poddar, D., Aggarwal, p.K., henry, C.J.K., Jain, S. & Yadav, h. 2010. Cancer-preventing attributes of probiotics: an update. International Journal of Food Sciences and Nutrition 61(5): 473-496.Lim, S.M. 2014. Antimutagenicity Activity of the putative probiotic strain Lactobacillus paracasei subsp. tolerans JG22 isolated from pepper leaves Jangajji. Food Science Biotechnology 23(1): 141-150. Lo, p-. r., Yu, r-.C., Chou, C-.C. & huang, e-.C. 2004. Determinations of the antimutagenic activities of several probiotic bifidobacteria under acidic and bile conditions against benzo[α] pyrene by a modified Ames test. International Journal of Food Microbiology 93: 249-257.Maron, D.M. & Ames, B.n. 1983. revised methods for the Salmonella mutagenicity test. Mutation Research 113(3-4): 173-215.Mortelmans, K. & zeiger, e. 2000. The Ames Salmonella/microsome mutagenicity assay. Mutation Research 455: 29-60.negi, p., Jayaprakasha, G. & Jena, B.S. 2003. Antioxidant and antimutagenic activities of pomegranate peel extract. Food Chemistry 80(3): 393-397.park, K-.K., Kim, S-.h. & Son, T-.J. 1998. Antimutagenic Activities of Cell Wall and Cytosol Fractions of Lactic Acid Bacteria Isolated from Kimchi. Journal Food Science Nutrition 3(4): 329-333.rhee, C-.h. & park, h-.D. 2001. Three glycoproteins with antimutagenic activity identified in Lactobacillus plantarum KLAB21. Applied and Environmental Microbiology 67(8): 3445-3449.Salminen, S. & Von Wright, A. 1998. Current probiotics-safety assured. Microbiology Ecology Health Disease 10: 68-77.Slattery, M.L., Boucher, K.M., Caan, B.J., potter, J.D. & Ma, K.n. 1998. eating patterns and risk of Colon Cancer. American Journal of Epidemiology 148(1): 4-16.Tavan, e., Cayuela, C., Antoine, J-.M. & Cassand, p. 2002. Antimutagenic activities of various lactic acid bacteria against food mutagens: heterocyclic amines. Journal of Diary Research 69: 335-341.World health Organization. The Global Burden of Disease: 2004 update. Geneva: World health Organization; 2008.Asmariah AhmadSafura SalikYap Wei BoonAhmad rohi Ghazaliprogramme of Biomedical ScienceSchool of Diagnostic & Applied health SciencesFaculty of health Scienceuniversiti Kebangsaan Malaysia.noorhisham Tan Kofli Department of Chemical & process engineeringFaculty of engineering & Built environmentuniversiti Kebangsaan Malaysia.Corresponding author: Ahmad rohi Ghazaliemail: rohighazali@ukm.edu.my. Tel: +60392897618Fax: +60326938717received: August 2017Accepted for publication: January 2018Chap 4.indd   26 31/05/2018   15:23:40

Mutagenicity and antimutagenic activities of lactic acid bacteria (LAB) isolated from fermented durian (tempoyak)

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Mutagenicity and antimutagenic activities of lactic acid bacteria (LAB) isolated from fermented durian (tempoyak)

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