Probiotic potentials of Lactobacillus plantarum isolated from fermented durian (Tempoyak), a Malaysian traditional condiment

Abstract Lactic acid bacterium isolated from fermented durian (tempoyak) was investigated for its potentials as a probiotic strain. Bacterial tolerance toward gastrointestinal environment, adhesion, and cytotoxic activity in human colon adenocarcinoma cell line HT‐29 was evaluated. 16S rRNA sequencing identified the lactic acid bacterium as Lactobacillus plantarum. The bacterium demonstrated good tolerance toward gastrointestinal pH 2.0 and 0.3% bile salts. It showed strong adhesive capacity in human intestinal cell line, HT‐29, with an adhesion index of 159 ± 10. Cytotoxicity of L. plantarum was investigated using both live bacterial cells (BC) and cell‐free supernatant (CFS). Findings showed that both BC and CFS of L. plantarum reduced proliferation of HT‐29 colon adenocarcinoma cells using MTT assay. The results imply potential probiotic properties of L. plantarum isolated from tempoyak.


| INTRODUC TI ON
The idea of using probiotics to promote optimal health and help reduce risk of diseases has been an interesting subject for several decades. First postulated by Ellie Metchnikoff in early 1900s, a theory that bacillus isolated from Bulgarian kiselo mleko could colonize intestinal tract and contribute to the health of human beings was made following his observation on the longevity of Bulgarian peasants that was largely associated with the consumption of fermented milk products. The Nobel Laureate proposed that by manipulating the intestinal microbiome with host-friendly bacteria found in yogurt could lead to health-enhancing and delayed-senility effects (Mackowiak, 2013). Throughout the years, many definitions of probiotics have been proposed by researchers. Some defined probiotics as compounds that either stimulate bacterial growth or improve host immunity (Fujii & Cook, 1973) while other defined them as organisms and substances which contribute to intestinal microbial balance (Parker, 1974). FAO/WHO (2002) then suggested the definition of probiotics as live microorganisms that when administered adequately can boost its host's health.
Probiotics are beneficial bacteria that maintain intestinal microflora balance, inhibit the growth of harmful bacteria, promote good digestion, increase resistance to infection, and boost immune functions (Helland, Wicklund, & Narvhus, 2004). Other physiological benefits of probiotics include removal of carcinogens from the intestinal tract, immunomodulation of host immune system by reducing allergic reactions, and lactose intolerance, as well as to enhance nutrient bioavailability in hosts (Parvez, Malik, Ah, & Kim, 2006). Most probiotic microorganisms belong to the lactic acid bacteria (LAB) group, such as Lactobacillus spp. and Enterococcus spp. or genus Bifidobacterium (Klein, Pack, Bonaparte, & Reuter, 1998). LAB are commonly found in fermented products and have been widely used as starter cultures in food industry (Carr, Chill, & Maida, 2002). Some LAB such as Lactobacillus and Bifidobacterium strains are widely available as commercial probiotics (Jamaly, Benjouad, & Bouksaim, 2011). Given various members of LAB, thorough screening and characterization of LAB are, therefore, important before considering them as potential probiotic strains. The selection criteria for probiotic LAB include ability to survive in the gastrointestinal tract environment such as gastric and bile acid conditions, resistance to antibiotics, and adhesion to gastrointestinal epithelial lining (Pisano et al., 2014). Additionally, a good probiotic candidate must possess desirable physiological criteria such as antagonism toward microbial pathogens, cholesterol metabolism, antimutagenic, and anticarcinogenic properties in hosts (Vasiljevic & Shah, 2008).
Tempoyak is a traditional fermented condiment made from the pulp of durian in Malaysia and Indonesia. Durian flesh is mixed with salt (2.5%, w/v), placed in a sealed container, and allowed to ferment for 1 week. Tempoyak is often used as condiment with certain fish and vegetable dishes (Endo, Irisawa, Dicks, & Tamasupawat, 2014).
Tempoyak typically has long shelf life because it is preserved by lactic acid produced by LAB and salt added during processing to inhibit growth of food-spoiling bacteria (Amiza, Zakiah, Ng, & Lai, 2006).
Earlier analysis showed that LAB are the most predominant microorganisms that present in tempoyak. In addition, several organic acids such as acetic, lactic, and propionic acids were also detected (Chuah et al., 2016). Lactobacillus spp., Leuconostoc spp., and Fructobacillus durionis are among the known LAB that had been isolated from tempoyak (Endo et al., 2014) with Lactobacillus plantarum and F. durionis as the dominant LAB members (Chuah et al., 2016;Leisner et al., 2001). The chemical compositions of durian flesh with a total sugar content of 15%-20% and saccharose of 17% are nutritious enough to support the growth of LAB (Ketsa & Daengkanit, 1998;Leisner et al., 2001). Despite its popularity as one of the Malaysian traditional fermented condiments, very limited research has been conducted on the health benefits of tempoyak, especially its LAB content. Thus, the objective of our study was to identify and characterize LAB strain isolated from tempoyak in accordance with the established criteria and hence a potential probiotic strain in local food industry. Isolated colonies were subjected to Gram staining, catalase test, and genetic identification using 16S rRNA. The isolated colonies were further cultured in MRS broth (Laboratorios Conda, Spain) and kept in 20% (v/v) glycerol until further use. Bacterial isolates were grown on MRS agar and in MRS broth prior to assays. Bacteria were grown in MRS broth at 37°C for 16-18 hr. The whole culture (WC) bacterial samples were then prepared in two forms, bacterial cells (BC) and cell-free supernatant (CFS). Both BC and CFS were collected from freshly grown bacterial culture at 10,000 g for 10 min; the pellet was then washed, centrifuged, and resuspended in 0.1 mol/L phosphate buffer saline (PBS) as BC and CFS were obtained after supernatant from freshly grown culture was filter-sterilized (0.22 μmol/L pore size; Sartorius).

| Lactic acid bacteria identification by 16S rRNA sequences
Molecular identification of the bacterial isolates was made by 16s rRNA gene sequencing (First Base Laboratories SDN. BHD). Two universal primers of 518F (5′ CCAGCAGCCGCGGTAATACG 3′) and 800R (5′ TACCAGGGTATCTAATCC 3′) were used in order to amplify 16S rRNA gene in a polymerase chain reaction (PCR). The amplified 16S rRNA gene sequence was subsequently compared with the GenBank database using Basic Local Alignment Search Tool (BLAST).

| Antibacterial activity
The inhibitory potential of bacterial isolates against pathogenic bacteria was evaluated with well diffusion assay using WC, BC, and CFS. The pathogenic bacteria used as indicators strain were gram-negative Escherichia coli ATCC25922 and gram-positive Staphylococcus aureus ATCC25923. Briefly, 50 μl of bacterial suspension of indicator strains (10 8 CFU/ml, equivalent to 0.5 McFarland standard) was spread on Mueller Hinton agar plates (Oxoid Ltd, Hampshire, UK) and allowed to dry. Three wells (6 mm, in diameter, each) were made with sterile borer. Then, 20 μl of WC, BC, and CFS of L. plantarum was added separately into the wells. The plates were incubated at 37°C for 24 hr. The diameter of zone of inhibition was measured and expressed as mean ± standard deviation (SD).

| Acid tolerance activity
In acid tolerance assay, bacteria (100 μl) collected from overnightgrown cultures were transferred into 900 μl PBS adjusted to pH 1.5, 2, 3, and 4 and then incubated at 37°C. The numbers of viable bacteria were determined at 0 and 3 hr of incubation, on MRS agar plate.
The assay was performed in triplicate. Data obtained from the study were expressed in terms of log 10 CFU/ml and as mean ± SD.

| Bile salts tolerance activity
In the bile tolerance assay, 100 μl of overnight culture was transferred into a 900 μl of MRS broth containing 0.3% (w/v) bile salt (Oxoid, Thermo Fisher) and then incubated at 37°C. The number of viable bacteria was determined at 0 and 3 hr by incubating the cells on MRS agar. The assay was carried out in triplicates. Data were expressed in terms of log 10 CFU/ml and as mean ± SD.

| DPPH-free radical scavenging assay
Turbidity of WC and BC interferes with absorbance reading; therefore, CFS alone was tested in the assay. The DPPH-free radical scavenging assay of CFS of L. plantarum was measured according to Afify, Romeilah, Sultan, and Hussein (2012) with slight modifications.
Briefly, 1 mmol/L DPPH solution in ethanol was prepared, and 50 μl of the solution was added to 950 μl of CFS or ascorbic acid (positive control) and incubated for 30 min at room temperature. DPPH solution added with MRS broth served as negative control. The results were read at 517 nm. The percentage of radical scavenging activity was calculated according to the following equation; A0 = negative control, A1 = Sample

| Ferric-reducing ability of plasma (FRAP) assay
Ferric-reducing ability of plasma assay was conducted according to Benzie and Strain (1996)  Gibco, USA) at 37°C under 5% CO 2 environment until 80%-90% confluency. For MTT assays, cells were seeded in 96-well tissue culture plates (NEST, China), whereas for adhesion assays, cells were prepared on glass coverslips placed in six-well plates (NEST).

| Antiproliferation activity
Cell proliferation was assayed using MTT kit (Thirabunyanon & Hongwittayakorn, 2013) 3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyl tetrazolium (MTT; Sigma). Cells were seeded and cultured on 96-well tissue culture plates at 5 × 10 4 cells/ml, 37°C for 24 hr. In this assay, BC and CFS were tested separately in order to justify the biological active component of antiproliferative activity. After incubation, 100 μl of BC or CFS was added to each well and further incubated for 24 hr. Cells added with MRS and PBS were prepared as negative controls. Cells were then washed thrice with PBS. About 20 μl of MTT solution (5 mg/ml in PBS, pH 7.4) was added and incubated further for 4 hr. Formazan crystals were then solubilized with dimethyl sulfoxide (Thermoscientific) and further incubated for 15 min.
The intensity was determined with an ELISA reader (Bio-Rad, USA) at 570 nm. The percentage of viable cells was calculated by the following equation:

| In vitro adhesion assay
Adherence of bacteria to HT-29 was examined as described previously by Gopal, Prasad, Smart, and Gill (2001)

| Scanning electron microscopy (SEM)
For SEM analysis, HT-29 colon adenocarcinoma cell line was pre-

| Statistical analysis
All data were expressed as mean ± SD. For statistical analysis, SPSS version 20 (IBM, USA) was used. One-way ANOVA was used to analyze the data, followed by Tukey's test for post hoc comparisons. Data with p < .05 were considered statistically significant.

| RE SULTS AND D ISCUSS I ON
In the present study, probiotic and cytotoxic potentials of a lactic acid bacterium isolated from tempoyak (fermented durian) were in-   Rjiniemon, Hussain, and Rajamani (2015), the antioxidative properties of LAB isolated from traditional fermented foods could be potentially useful in treating chronic diseases such as cancer and diabetes. The study also found that L. plantarum isolated from fermented ragi malt was able to scavenge DPPH-free radicals.
According to Kim, Woo, Kim, Kim, and Lee (2003), different fractions of LAB, such as whole cells, heat-killed cells, the cell wall, peptidoglycan, and cytoplasmic fraction, have inhibitory effect against human cancer cell lines. Wang et al. (2014)  Bacterial cell and CFS of L. plantarum isolated from tempoyak showed a dose-dependent cytotoxicity in HT-29 cells as detected by the MTT assay ( Figure 3). CFS exhibited stronger inhibition on the cancer cells than BC. This is in parallel to the findings reported by Er, Koparal, and Kivanc (2015) that Pediococcus pentosaceus, L. plantarum and Weissella confusa isolated from fermented meat were able to inhibit the growth of colon cancer cells in a dose-dependent manner and, more interestingly, cell-free filtrate of L. plantarum also showed stronger effects compared with the other two strains. Our findings are also in accordance with a previous study by Haghshenas et al. (2015) which demonstrated that secreted metabolites of L. plantarum 17C isolated from ewe colostrum exhibited antiproliferative effect on HT-29 cell line. Antiproliferative activity of probiotic strains on colon cancer cells could be due to the presence of EPS (Sadeghi-Aliabadi, Mohammadi, Fazeli, & Mirlohi, 2014). According to Kim et al. (2006), cell-bound EPS (cb-EPS) and released EPS (r-EPS) of L. rhamnosus inhibited the growth of HT-29 and PANC-1 pancreatic cancer cells. When comparing the two types of EPS, r-EPS exhibited stronger cytotoxic effects than cb-EPS. The ability of probiotic bacteria to exert cytotoxicity effects on colon cancer cells thus suggests its potential to be developed as chemopreventive for colon cancer.
Adhesion of lactobacilli has been deemed essential for exertion of beneficial probiotic effects in the large intestine (Maragkoudakis et al., 2006). In our study, adhesion of L. plantarum on HT-29 cells was quantified ( Figure 4) and showed an average of 159 ± 10 cells adhered to 100 HT-29 epithelial cells (Table 4) immune system efficiently (Gopal et al., 2001;Wagner & Johnson, 2017). Previous studies by Oguntoyinbo and Narbad (2015) and Lee, Kim, Han, Eom, and Paik (2014) also showed that L. plantarum isolated from fermented cereal food and kimchi possessed strong adhesive properties on HT-29 cells. The bacterial adhesion is most probably due to cell-surface adhesive factors such as lectin/adhesion proteins of S-layers, secreted lectin-like bacteriocins, and lectin-like complexes on lactobacilli and bifido BC walls (Lakhtin et al., 2006).
According to Pretzer et al. (2005), in vitro adhesion of L. plantarum was also mediated by mannose components on the bacterial surface that bind to mannose receptor on the epithelial surface of human intestinal cells.

| CON CLUS ION
Lactobacillus plantarum isolated from tempoyak showed reasonably good probiotic properties including acid and bile salts tolerance, antioxidative, antiproliferative effects, and remarkable adhesion on HT-29 cells. This was the first study to demonstrate probiotic potentials of lactic acid bacterium isolated from tempoyak and thus particularly useful as a reference for future developments of L. plantarum AN6 as a potential probiotic bacterium in functional food industry. In order to better apply the isolate as probiotic strain, in vivo experiments are needed to better understand the mechanisms of actions involved in its probiotic properties.

E TH I C A L R E V I E W
This study does not involve any human or animal testing.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest. In vitro adhesion of L. plantarum was monitored after 2 hr of incubation. The data are presented as mean ± standard deviation of bacteria adhered to cell line (n = 3).