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Selection and evaluation of functional characteristics of autochthonous lactic acid bacteria isolated from traditional fermented stinky bean (Sataw-Dong)

Selection and evaluation of functional characteristics of autochthonous lactic acid bacteria... Ann Microbiol (2017) 67:25–36 DOI 10.1007/s13213-016-1233-3 ORIGINAL ARTICLE Selection and evaluation of functional characteristics of autochthonous lactic acid bacteria isolated from traditional fermented stinky bean (Sataw-Dong) 1 2 1 Krittanon Jampaphaeng & Luca Cocolin & Suppasil Maneerat Received: 19 January 2016 /Accepted: 2 September 2016 /Published online: 17 September 2016 Springer-Verlag Berlin Heidelberg and the University of Milan 2016 Abstract The aim of this study was to evaluate the techno- desirable in vitro functional properties. This strain is therefore logical and functional potential of lactic acid bacteria (LAB) a good candidate for further investigation for use in Sataw- isolated from fermented stinky bean (Sataw-Dong). Of the Dong fermentation to assess its technological performance as 114 LAB colonies isolated from spontaneously fermented a potential probiotic starter. stinky bean which showed inhibitory activity against two . . food-borne pathogens (Staphylococcus aureus DMST 4480 Keywords Lactic acid bacteria Lactobacillus fermentum . . and Escherichia coli DMST 4212), the five isolates (KJ03, Lactobacillus plantarum Probiotic Sataw-Dong KJ15, KJ17, KJ22, KJ23) exhibiting excellent antagonistic activity were subjected to further study. These five strains showed titratable acidity as lactic acid in the range of 1.47– Introduction 1.55 %, with strains KJ03 and KJ23 additionally exhibiting a high NaCl tolerance of >7 % (w/v). Using 16S rRNA gene Fermented foods are an important component of the human sequence analysis, strains KJ03 and KJ23 were identified as diet in many countries, especially at the household level. One Lactobacillus plantarum and L. fermentum, respectively, and of the advantages of fermentation is that it is an inexpensive further investigated for their functional properties in vitro. process with the potential to preserve food, improve nutrition- Both strains survived well in a simulated gastrointestinal tract al value, and enhance aroma and taste (Aloys and Angeline environment with <1 log cell decrease over 8 h (>8 log CFU/ 2009). In addition, indigenous fermented foods are strongly ml). Lactobacillus plantarum KJ03 showed the best perfor- linked to the culture and tradition of the country of their origin mance with respect to cholesterol removal (53 %), while (Guo et al. 2015). L. fermentum KJ23 showed the highest cell-surface hydropho- Foods which contain probiotics represent a group of bicity (39.5 %). Neither of the two strains showed any hemo- health-promoting, functional foods of important commer- lysis activity. Both strains hydrolyzed glycodeoxycholic and cial interest, and their share of the market is steadily in- taurodeoxycholic acids. In terms of antibiotic susceptibility, creasing (Vuyst et al. 2008). Most probiotic bacteria be- L. fermentum KJ23 was not sensitive to tetracycline. Taking long to lactic acid bacteria (LAB) groups, especially the all of the results into account, L. plantarum KJ03 possessed species of the genus Lactobacillus, which is one of the most fundamental of microbial groups. These LAB have been introduced into several of fermented food products. * Suppasil Maneerat Many studies have reported that dairy products are the suppasil.m@psu.ac.th most commonly used food vehicles for the delivery of probiotics (Rubio et al. 2014). However, there has been a Biotechnology for Bioresource Utilization Laboratory, Department growing interest in developing non-dairy probiotics, and it of Industrial Biotechnology, Faculty of Agro-Industry, Prince of is known that some traditional fermented foods may con- Songkla University, Hat Yai, Songkla 90112, Thailand 2 stitute a suitable base for the development of probiotic- Department of Agricultural, Forest and Food Science of the type functional foods (Ruiz-Moyano et al. 2009). The po- University of Torino (DISAFA), University of Turin, Grugliasco, Turin 10095, Italy tential to develop non-dairy probiotic products has 26 Ann Microbiol (2017) 67:25–36 attracted the interest of individuals with lactose intolerance which were covered with lids and allowed to ferment sponta- and consumers with cholesterol-restricted diets (Granato neously at room temperature (27–32 °C) for 10 days. Samples et al. 2010). Moreover, traditional fermented foods are a obtained at 0, 1, 2, 4, 6, 8, and 10 days of fermentation were plentiful source of microorganisms, and some of them used to isolate LAB. show probiotic properties (Rivera-Espinoza and Gallardo- Navorra 2010). Thus, non-dairy products made from fruits, Isolation of LAB showing antagonistic activity vegetables, and cereals would appear to have a promising against foodborne pathogens future (Martins et al. 2013). Stink bean (Parkia speciose), a Southeast Asian plant of Samples (25 g) of Sataw-Dong were added to 0.1 % (w/v) the genus Parkia in the family Fabaceae, grows wild in trop- peptone water (225 ml) and placed in a stomacher for 2 min. ical forests and is often cultivated in Southern Thailand. The Ten-fold serial dilutions were made in peptone water, and the beans are flattened and elliptical in shape with a nutty and firm appropriate dilutions were spread onto MRS agar (Lab M texture. They are believed to contain medicinal compounds Ltd., Heywoord, UK) supplemented with bromocresol purple which exhibit potential biological activity, such as antibacte- (0.02 %, w/v), and NaCl (3 %, w/v), and the plates were rial (Sakunpak and Panichayupakaranant 2012), incubated at 37 °C for 24 h for the isolation of presumptive antiangiogenic (Aisha et al. 2012), anticancer (Ali et al. LAB. After 24 h, the plates of LAB were individually overlaid 2006), antioxidant (Aisha et al. 2012) and hypoglycemic ac- with Tryptone Soy Agar (TSA; Hi Media Laboratories, tivities (Jamaluddin and Mohamad 1993). Stink beans pre- Mumbai, India) (0.75 % agar, w/v) seeded with served in brine for several days undergo fermentation, with Staphylococcus aureus DMST 8840 and Escherichia coli the production of lactic acid; the fermentation product is called DMST 4212 [approx. 10 colony-forming units (CFU)/ml]. fermented stink bean or Sataw-Dong (in the Thai language) The plates were then incubated at 37 °C for 24 h. Inhibition and consumed as a raw pickle. While there are many similar zones were detected by a clear zone around the tested strains LA fermentation products, to date cucumber, cabbage and (Schillinger and Lücke 1989). olives are the only vegetables that are fermented in large vol- Bacterial colonies exhibiting an inhibition zone were indi- umes for human consumption (Montet et al. 2006). vidually picked and streaked on MRS agar two to three times However, all natural fermentation processes have a com- to purify the isolates. All Gram-positive, catalase-negative mon problem in that they are relatively uncontrollable, isolates were defined as LAB (Hwanhlem et al. 2011), and resulting in variations in product stability and quality attri- these isolates were maintained in MRS broth containing glyc- butes (Parkouda et al. 2010). It has been reported that these erol (60 %) at −20 °C. For routine analysis, strains were problems can be overcome by using a starter culture, control- subcultured twice in MRS broth at 37 °C for 24 h. ling the microflora, accelerating the ripening time, inhibiting the growth of pathogenic and spoilage bacteria and alleviating Confirmation of the antagonistic activity variations in organoleptic quality problems in fermented foods, thereby improving the overall quality of fermented veg- Antagonistic activity against bacterial indicators was investi- etable products (Font de Valdez et al. 1990; Caplice and gated using the agar spot test and agar well diffusion assay as Fitzgerald 1999). described by Jones et al. (2008) and Schillinger and Lücke The aim of the study reported here was to: (1) characterize (1989), respectively. The agar spot test experiment was con- LAB isolated from traditionally fermented stink bean; (2) se- ducted by spotting each overnight LAB culture (5 μl) onto the lect the most suitable strains according to their technological surface of a MRS agar plate and incubating the plate at 37 °C characteristics, including antagonistic activity against for 24 h. These plates were then overlaid with 10 ml of TSA foodborne pathogens; (3) investigate a number of the func- (0.75 % agar, w/v) seeded with 100 μl of each tested indicator tional properties the isolates may have. strain. After an overnight incubation at 37 °C, the plates were examined for zones of inhibition. The agar well diffusion assay was performed to character- Materials and Methods ize the type of antimicrobial compounds. Cell-free superna- tants were collected by centrifugation (8000 g, 10 min, 4 °C) Sataw-Dong preparation from overnight cultures. Two samples of supernatant taken from each strain were used in trials as follows: (1) the pH Sataw-Dong was prepared according to the traditional recipe. was adjusted to 6.5 and the supernatant heated at 90 °C for Whole pods were blanched and added to a brine prepared by 10 min, and (2) the supernatant without adjustment was used boiling clean water containing 3–4 % NaCl, 5 % sugars, and as control. Sterile culture plates containing 20 ml TSB and 1.0 0.5 % Malabar tamarind (Garcinia cambogia). The beans to- % (w/v) agar were then prepared and subsequently seeded gether with the brine were transferred to 12 glass jars (100 g), with each bacterial indicator. Wells (diameter 7 mm) were Ann Microbiol (2017) 67:25–36 27 punched into the agar layer, and cell-free supernatants (50 μl) harvested by centrifugation (8000 g, 10 min) and resuspended were placed into each well; the plates were then incubated at in 2 ml of phosphate buffer saline (PBS) in the presence of 37 °C for 24 h. The inhibition activity of each supernatant was lysozyme (100 mg/L) (Sigma-Aldrich, St. Louis, MO). assessed based on the diameter of the inhibition zone. Bacterial suspensions without lysozyme were used as con- trols. Samples were incubated at 37 °C, and viable cell counts Determination of pH value and total titratable acidity after 10 and 20 min were enumerated on MRS agar by the drop plate method. Survival rates were calculated as a percent- All LAB cultures were diluted to an absorbance of 0.5 age of growth. (10 CFU/ml). Each LAB suspension (1 %) was inoculated into MRS broth and the cultures incubated at 37 °C for 24 h, Survival of LAB under simulated gastric and intestinal following which aliquots (20 ml) were taken for the measure- juices Cells of the LAB isolates were collected by centrifuga- ments. The pH was directly measured using a pH meter tion (8000 g, 10 min) and washed twice with PBS before (OHAUS, Shanghai, China). A cell-free supernatant was used being resuspended in PBS solution at pH 2.5 containing pep- to determine the total titratable activity by titration against sin (3 mg/ml; Sigma-Aldrich) as simulated gastric juice 0.1 N NaOH using phenolphthalein (0.1 % w/v in 95 % eth- (Maragkoudakis et al. 2006). Viable counts were carried out anol) as an indicator. after incubation at 37 °C for 2 h. After this step, the cultures were centrifuged and washed twice with PBS solution before Determination of NaCl tolerance of LAB being transferred to PBS solution, pH 8.0, containing pancre- atin (1 mg/ml) and ox bile salts (3 mg/ml) (Sigma-Aldrich) as The NaCl concentration in MRS broth was adjusted to 0, 3, 5, simulated intestinal fluid. The samples were incubated at 7, 9, and 12 % (w/v), following which 1 % (v/v) of active LAB 37 °C in for 6 h. Survival of LAB was enumerated after 3 h was inoculated into MRS broth containing the different con- and 6 h of incubation. centrations of NaCl and cultivated at 37 °C for 24 h. The survival of LAB was determined by the drop plate method Effect of micro-aerobic and anaerobic conditions on on MRS agar and reported as colony-forming units per growth of LAB LAB strains (1 %, v/v) were inoculated into milliliter. MRS broth (micro-aerobic condition) and MRS broth supple- mented with L-cysteine (0.5 mg/ml) covered with liquid par- Molecular identification and sequencing of 16S rRNA affin (anaerobic condition) (Talwalkar et al. 2001). The sam- gene of selected LAB ples were incubated at 37 °C for 24 h. For micro-aerobic condition, cells were enumerated on MRS agar and incubated DNA of the selected LAB was extracted using an extraction at 37 °C for 24 h. On the other hand, cells were dropped on kit (QIAgen, Hilden, Germany) according to the manufac- MRS agar supplemented with L-cysteine and overlaid with turer’s protocol and stored at −20 °C. 16S rRNA gene ampli- agar, followedbyincubationat37°Cfor 24h inananaerobic fication was performed in a thermocycler (Techne, Abingdon, jar. UK) with the following universal primers: 27 F (5′-AGAG TTTGATCCTGG CTCAG-3′) and 1492R (5′-GGTT Cell-surface hydrophobicity assay Cell-surface hydropho- ACCTTGTTACGACTT-3′) according to the protocol de- bicity was determined by a microbial adhesion to hydrocarbon scribed by Cai et al. (1999). The PCR products were purified test as described by Otero et al. (2004). LAB were grown in using a PCR purification kit (QIAgen) and sequenced (Ward MRS broth at 37 °C for 24 h and harvested by centrifugation Medic Ltd., Selangor, Malaysia). The basic local alignment (8,000 g, 10 min). The pellets were washed twice with PBS search tool (BLAST) was used to compare the obtained se- (pH 7.4) and resuspended in the same buffer to an absorbance quences against the public data library of the National Center of 0.8–1.0 at 600 nm (OD ). Equal volumes of cell suspen- for Biotechnology Information (http://www.ncbi.nlm.nih. sion and n-hexadecane were mixed in duplicate and vortexed gov). The nucleotide sequences of all the bacterial isolates thoroughly for 2 min. The tubes were allowed to separate into were deposited in the GenBank database (accession numbers two phases for 30 min. The aqueous phase was then measured LC016617 and LC037354). in a spectrophotometer at 600 nm. The decrease in the absor- bance of the aqueous phase was taken as a measure of the cell Determination of functional properties in vitro surface hydrophobicity (% H). Resistance to lysozyme Lysozyme resistance to assess the Blood hemolytic activity Fresh overnight LAB cultures were in vitro ability of the strains to survive transit through the oral streaked in triplicate on TSA plates supplemented with 5 % cavity was performed as described by Turchi et al. (2013), (v/v) human blood (obtained from Songklanagarind Hospital, with slight modifications. LAB grown overnight were Songkhla, Thailand) and incubated at 37 °C for 48 h. 28 Ann Microbiol (2017) 67:25–36 Hemolytic activities of the bacterial culture were examined for and evaporated. The residue was dissolved in 2 ml of O- signs of β-hemolysis (clear zones around colonies), α- phthalaldehyde reagent. After mixing, 1 ml of concentrated hemolysis (green zones around colonies), or γ-haemolysis sulfuric acid was added and the mixture vortexed for 1 min. (no clear zones around colonies) (Hargrove and Alford 1978). Absorbance was read at 540 nm. All experiments were per- formed in duplicate. Bile salt hydrolase activity Qualitative bile salt hydrolase (BSH) activity was evaluated as described by Wang et al. Determination of antibiotic susceptibility The LAB strains (2012). The MRS agar plate was prepared by adding 0.5 % were tested for resistance to antibiotics by the broth (w/v) of bile salt (Sigma-Aldrich) as follows: glycocholic ac- microdilution method. The minimum inhibitory concentration id, taurocholic acid, glycodeoxycholic acid (GDC) and (MIC) values were determined in the LAB susceptibility test taurodeoxycholic acids (TDC). Overnight cultures of each medium (LSM) broth formulation described by Federici et al. LAB strain (10 μl) were spotted onto the agar plates and (2014) using ampicillin, vancomycin, chloramphenicol, eryth- incubated anaerobically at 37 °C for 24–72 h. The presence romycin, kanamycin, streptomycin, tetracycline, clindamycin, of precipitated bile acid around the colonies (opaque halo) was and ciprofloxacin at concentrations of 0.065–1024 mg/L. The considered to be a positive result. MRS agar plate without individual inoculum was adjusted to an absorbance of 0.2–0.3 supplemented bile acid was used as control. (600 nm), equivalent to 10 CFU/ml. In brief, 100 μlof bac- terial suspension was mixed with the antibiotic solution (100 μl) to be tested in 96-well plates and incubated overnight Cholesterol-lowering property The cholesterol-lowering at 37 °C. Growth was determined visually after incubation. property was determined according to Wang et al. (2014)with Susceptible and resistant strains were distinguished according some modification. The MRS broth was supplemented with to the breakpoints (cutoff values) reported by European Food 0.3 % (w/v) Ox gall (Hi-Media). Water-soluble cholesterol Safety Authority (2012). Accordingly, strains showing MICs (polyoxyethylene cholesteryl sebacate; Sigma-Aldrich) was higher than the respective cutoff values were considered to be filter-sterilized and added into the broth at a final concentra- resistant. tion of 100 μg/ml, inoculated with each LAB strain, and in- cubated at 37 °C for 24 h. After the incubation, cells were removed by centrifugation, and residual cholesterol concen- Detection of the presence of virulence genes To evaluate the tration in the broth was determined using a modified colori- safety of the selected LAB for their application in fermenta- metric method. Briefly, 1 ml of the broth was added to 1 ml of tion processes, we performed PCR assays to detect the pres- KOH (33 %, w/v) and 2 ml of absolute ethanol, mixed for ence of genes encoding potential virulence genes using geno- 1 min, then heated at 70 °C for 30 min. After cooling, 5 ml of mic DNA obtained as described in section Molecular identifi- hexane was added to the tube and the tube vortexed for 1 min. cation and sequencing of 16S rRNA gene of selected LAB. The hexane layer (4 ml) was transferred to a clean glass tube The specific primers are described in Table 1 (Vankerckhoven Table 1 PCR primers and products for the detection of virulence determinants Target genes Sequence (5′ to 3′)T (°C) Product size (bp) References ace 5′-GAATTGAGCAAAAGTTCAATCG-3′ 56 1008 Ben Omar et al. (2004) 5′-GTCTGTCTTTTCACTTGTTTC-3′ asa15′-GCACGCTATTACGAACTATGA-3′ 56 375 Vankerckhoven et al. (2004) 5′-TAAGAAAGAACATCACCACGA-3′ cylA5′-ACTCGGGGATTGATAGGC-3′ 58 688 Vankerckhoven et al. (2004) 5′-GCTGCTAAAGCTGCGCTT-3′ clyB5′-AAGTACACTAGTAGAACTAAGGGA-3′ 52 843 Semedo et al. (2003) 5′-ACAGTGAACGATATAACTCGCTATT-3′ efaAfs 5′- GACAGACCCTCACGAATA-3′ 54 705 Eaton and Gasson (2001) 5′- AGTTCATCATGCTGTAGTA −3′ esp 5′-AGATTTCATCTTTGATTCTTGG-3′ 56 510 Vankerckhoven et al. (2004) 5′-AATTGATTCTTTAGCATCTGG-3′ gelE5′-ACCCCGTATCATTGGTTT-3′ 52 419 Eaton and Gasson (2001) 5′-ACGCATTGCTTTTCCATC-3′ T , Melting temperature asa1, Aggregation substance gene; CylA/B, cytolysin A/B genes; efaAfs, cell-wall adhesion gene; esp, enterococcal surface protein gene; gelE, gelatinase gene Ann Microbiol (2017) 67:25–36 29 et al. 2004). The target genes were ace (adhesin of collagen tested for antagonistic activity against Staphylococcus aureus protein), asa1 (aggregation substance), CylA/B (cytolysins), DMST 8840 and Escherichia coli DMST 4212 according to efaAfs (cell-wall adhesion), esp (enterococcal surface protein) the safety criteria of Thai Community Product Standard (317/ and gelE (gelatinase). Of the strains tested, Enterococcus 2004). However, only five strains (KJ03, KJ15, KJ17, KJ22, feacalis Van B tested positive for the ace, asa1, CylA/B, KJ23) showed excellent inhibition zones (diameter > 15 mm), efaAfs,and gelE genes and Enterococcus feacalis 13–5tested with strain KJ03 having the highest inhibition zone (Table 2). positive for the esp gene. Those five LAB strains were subsequently analyzed for their production of antibacterial compounds by the agar well Statistical analysis diffusion method. Supernatants obtained from the five strains did not exhibit the inhibition zones after pH adjustment to 6.5 Statistical analysis was conducted using one-way analysis of (data not shown), suggesting that the antimicrobial activity of variance with Duncan’s post hoc test. Significance was set at these five LAB strains may come from organic acids. In fact, p < 0.05. All data are expressed as the mean ± standard the drop in pH arising from the production of lactic acid can be deviation. sufficient to inhibit certain bacteria as the non-dissociated form of lactic acid triggers a lowering of the internal pH of the cell which causes a collapse in the electrochemical proton gradient in sensitive bacteria, thereby resulting in a bacterio- Results and Discussion static or bactericidal effect (González et al. 2007). The anti- bacterial activity of LAB strains selected from vegetables has Isolation of LAB with primary antagonistic activity been confirmed in several previous studies; i.e., from fermented cucumber, organic leafy vegetables (Ponce et al. Lactic acid bacteria originally isolated from vegetables are 2008) and from fermented Himalayan vegetables (Dewan probably the most suitable candidates for improving the mi- and Tamang 2007). crobiological safety of fermented vegetable products because they are well-adapted to the conditions of the matrices used in the fermentation process and should therefore be more com- Lactic acid production and pH reduction ability petitive than LAB obtained from other sources (Ponce et al. 2008). A total of 114 colonies which showed clear zones Lactic acid produced by LAB is an essential compound for against S. aureus DMST 8840 and E. coli DMST 4212. All food preservation because it maintains the acidity condi- of those isolates were identified as LAB based on two criteria: tions of the fermented foods and it is antagonistic against Gram-positive and catalase-negative (data not shown). food spoilage and poisoning bacteria (Hwanhlem et al. 2011). LAB ferment sugars via a number of different path- Screening for antagonistic activity ways, resulting in homo-, or mixed acid fermentation. Homofermentation produces lactic acid as the sole end Antagonistic activity of LAB strains may contribute to the product of glucose metabolism when the Embden– improvement in the quality of fermented foods, which is Meyerhof–Parnas pathway is used (Hofvendahl and achieved through the control of spoilage and pathogenic bac- Hahn–Hägerdal 2000). teria, thus extending shelf-life and improving sensory quality We therefore measured lactic acid production and pH re- (Begonović et al. 2014). All 114 LAB strains identified were duction in these five LAB isolates (KJ03, KJ15, KJ17, KJ22, Table 2 Inhibition zone diameter LAB isolate no. Inhibition zone (mm) of lactic acid bacteria isolated from Sataw-Dong on agar in the Staphylococcus aureus DMST8840 Escherichia coli DMST4212 presence of indicator bacterial species Staphylococcus aureus KJ03 29.7 ± 0.6 23.0 ± 1.0 DMST8840 and Escherichia coli DMST4212 KJ15 26.7 ± 0.6 17.7 ± 1.2 KJ17 27.7 ± 0.5 18.3 ± 0.6 KJ22 25.3 ± 0.7 19.0 ± 1.0 KJ23 29.3 ± 1.2 21.7 ± 0.6 LAB, Lactic acid bacteria Data are presented as the mean ±standard deviation (SD) from triplicate determinations 30 Ann Microbiol (2017) 67:25–36 KJ23) exhibiting excellent antagonistic activity against Identification of LAB isolates S. aureus DMST 8840 and E. coli DMST 4212. After 24 h of incubation, strain KJ23 was the highest producer of lactic The 16S rRNA gene sequences of strains KJ03 and KJ23 were acid at 1.55 % (in % equivalents lactic acid), and strain KJ15 compared with those other bacterial strains in GenBank data- was the lowest lactic acid producer at 1.47 %. The remaining base and identified and subsequently confirmed as three strains showed no significant lactic acid production, with Lactobacillus plantarum (99 % similarity) and L. fermentum the exception of strain KJ15. The pH values were correlated (99 % similarity), respectively. The 16S rRNA gene se- with these results in the range of 3.86–4.00 (Table 3). quences of strains KJ03 and KJ23 were deposited in the DDBJ/EMBL/Genbank with accession numbers LC016617 and LC037354, respectively. These strains were found to be Effect of NaCl on growth of isolates Gram-positive, non-spore-forming rods; they were catalase- negative and form off-white cream colonies (Table 4). The The cell viability of the five LAB isolates after incubation in fermentation of vegetables is mainly carried out by MRS broth supplemented with NaCl is shown in Fig. 1 re- L. plantarum and L. fermentum strains for improving their ports. NaCl supplemented at a concentration of 3–5% did not nutritional and sensory features, and these strains are also significantly affect the growth of these LAB strains as they considered to be the best candidates for starters with probiotic grew over 9 log CFU/ml. In addition, the growth of three properties (Swain et al. 2014). isolates (KJ03, KJ22, KJ23) appeared to be slightly stimulated with the addition of 3 % NaCl to the incubation broth. As Functional properties of selected LAB in vitro predicted, increasing NaCl concentration had a negative effect on the growth of all isolates, with a subsequent decrease in cell An important step towards the selection of potential probiotic viability (Fig. 1). In particular, LAB viability was strongly LAB is to evaluate their resistance and survival through the affected by 7 % NaCl. Isolates KJ03 and KJ23 best tolerated human gastrointestinal tract (GIT). Lactobacillus plantarum the highest NaCl concentrations (>8 log CFU/ml), and we subsp. plantarum strain JCM1149, originally isolated from therefore selected these two strains for further investigations. pickled cabbage, is a commercial type strain with probiotic In MRS broth supplemented with 9–12 % NaCl, these two properties (Zago et al. 2011; Nazzaro et al. 2012) and was also LAB strains were not able to grow throughout the 24-h incu- used in this study to compare properties among our isolates. bation period, although cell counts remained constant (approx. The results of our study on the functional properties of our two 5–6 log CFU/ml) for the entire incubation time. The tolerance isolated Lactobacillus strains (KJ03 and KJ23) are shown in to salt concentrations of 2–10 % is a limiting factor affecting Table 5. the persistence, competitiveness, and metabolism of the starter culture over the entire fermentation due to its water binding and ionic characteristics (Ammor and Mayo 2007). The Resistance to lysozyme The resistance of L. plantarum KJ03 growth of LAB is sometimes enhanced in the presence of and L. fermentum KJ23 to lysozyme, expressed as percentage low concentration of NaCl (1–2%,w/v)(Leroyandde of survival, ranged from a minimum mean value of 99.73 % to Vuyst 1999). Consequently, a salt tolerance of at least 7 % is a maximum mean value of 100 %. These isolates revealed a considered to be necessary for potential starter cultures in high lysozyme resistance after both 10 and 20 min incubation, Sataw-Dong fermentation because the presence of 5–7% with a survival rate >98 % (Table 5). Lysozyme present in NaCl is normally found in many recipes of Sataw-Dong. human saliva is the first challenge to survival. Resistance to Table 3 pH values and titratable Isolated no. pH Titratable acidity (%) acidity of cell-free supernatants of the five lactic acid bacterial strains 3h 6h 12 h 24 h 3 h 6 h 12 h 24h isolated from Sataw-Dong after 3, 6, 12, and 24 h of incubation a a a a KJ03 6.93 4.91 3.89 3.86 0.07 ± 0.01 0.44 ± 0.01 1.35 ± 0.01 1.53 ± 0.01 a a b a KJ15 6.96 4.91 3.94 3.88 0.05 ± 0.01 0.44 ± 0.01 1.29 ± 0.01 1.52 ± 0.01 a b a b KJ17 6.93 5.05 3.97 3.90 0.07 ± 0.03 0.40 ± 0.03 1.36 ± 0.01 1.47 ± 0.02 a c a a KJ22 6.90 5.35 4.01 4.00 0.05 ± 0.01 0.31 ± 0.02 1.35 ± 0.00 1.53 ± 0.02 a a b a KJ23 6.94 5.14 3.99 3.97 0.06 ± 0.01 0.46 ± 0.01 1.27 ± 0.01 1.55 ± 0.01 Data on titratable acidity are presented as the mean ± SD from triplicate determinations. Mean values followed by different superscript lowercase letter in the same column are significantly different at p ≤ 0.05 Ann Microbiol (2017) 67:25–36 31 Fig. 1 Viability of lactic acid MRS (control) MRS + 3% NaCl MRS + 5% NaCl bacteria isolates KJ03, KJ15, MRS + 7% NaCl MRS + 9% NaCl MRS + 12% NaCl KJ17, KJ22, and KJ23 after cultivation in MRS broth supplemented with various concentrations of NaCl (0–12 %) at 37 °C for up to 24 h. Lb. Lactobacillus KJ03 KJ15 KJ17 KJ22 KJ23 Lb. plantarum JCM1149 lysozyme has been attributed to the peptidoglycan structure in plantarum KJ03 and L. fermentum KJ23 survived more than 9 the cell wall, the physiological state of the cell, and lysozyme log CFU/ml (>95 %) after their cell suspensions were exposed structure in the medium (Cunningham et al. 1991). This result to the simulated gastric juice. The surviving cells were further confirmed the high resistance of the two Lactobacillus strains tested for bile salt tolerance. Lactobacillus plantarum KJ03 to 100 mg/l of lysozyme under conditions stimulating the exhibited better survival, at >9 log CFU/ml (92.55 %), than in vivo dilution by saliva (Zago et al. 2011). L. fermentum KJ23 (81.32 %) (Table 5). The ability to survive the acidic and bile challenges in the GIT is advantageous for probiotics. Acid tolerance of Survival of LAB under simulated gastric and intestinal juices The behavior of L. plantarum KJ03 and L. fermentum Lactobacillus strains was also reported in previous studies. Srinu et al. (2013) revealed that all of the L. plantarum and KJ23 under simulated GIT conditions enables strains to be selected which are likely to survive these conditions, a neces- L. casei strains they tested showed good survival in the acidic sary prerequisite for probiotic cultures. Stresses to microor- pH range (1.5–3.5) tested. Jamaly et al. (2011)also reported ganisms begin in the mouth, with lysozyme-containing saliva, that L. plantarum was able to tolerate 3 h of acid exposure and continue in the stomach (pH 1.5–3.0) and in the upper (pH. 2.0 and 3.0). Good probiotic bacteria should survive well in a pancreatin solution at pH 8.0 in the presence of bile salts intestine which contains bile (Zago et al. 2011). Lactobacillus Table 4 Physiological properties of the five selected lactic acid bacterial strains isolated from Sataw-Dong Property Lactobacillus plantarum KJ03 Lactobacillus fementum KJ23 Lactobacillus plantarum JCM1149 Shape Rod Short rod Rod Gram stain Positive Positive Positive Catalase test –– – Growth at 15/45 °C +/+ +/+ +/+ Growth at pH 2.0 + + + 3.0 ++ + ++ 4.0 +++ +++ +++ 5.0 +++ +++ +++ Lactobacillus plantarum JCM1149 was used as a positive control for the two probiotic strains isolated in this study (KJ03 and KJ23) +, Positive reaction; −, negative reaction; +, ++, +++: Low, moderate, and high turbidity of growth, respectively Log CFU/ml 32 Ann Microbiol (2017) 67:25–36 Table 5 In vitro probiotic properties of the selected isolates of lactic acid bacteria Properties Lactobacillus plantarum KJ03 Lactobacillus fementum KJ23 Lactobacillus plantarum JCM1149 Survival (log CFU/ml) a a a Control (0 h) 9.584 ± 0.193 9.498 ± 0.147 9.315 ± 0.229 a a a Simulated lysozyme, 10 min 9.550 ± 0.146 9.475 ± 0.285 9.280 ± 0.167 a a a Simulated lysozyme, 20 min 9.445 ± 0.140 9.392 ± 0.121 9.225 ± 0.181 a b b Simulated gastric juice (pH 2.5), 2 h 9.211 ± 0.061 9.011 ± 0.008 9.004 ± 0.083 a b b Simulated intestinal juice (pH 8.0), 3 h 9.020 ± 0.010 8.542 ± 0.022 8.788 ± 0.103 a c b Simulated intestinal juice (pH 8.0), 6 h 8.735 ± 0.015 7.621 ± 0.035 8.312 ± 0.045 Viable cell count (log CFU/ml) a,A a,A b,A Micro-aerobic condition 9.541 ± 0.112 9.522 ± 0.075 9.122 ± 0.063 a,A a,A a,A Anaerobic condition 9.531 ± 0.065 9.538 ± 0.082 9.250 ± 0.008 b a c Cell surface hydrophobicity (%) 34.82 ± 1.57 39.53 ± 1.34 19.90 ± 1.33 Bile salt hydrolase activity Glycocholic acid –– – Taurocholic acid –– – Glycodeoxycholic acid + + + Taurodeoxycholic acid –– – Blood hemolysis γγ γ Cholesterol removal (%) 53.0 ± 0.6 49.2 ± 1.4 38.0 ± 1.1 Where applicable, data are presented as the mean ± SD. Means followed by different superscript lowercase letters in the same row are significantly different at p ≤ 0.05. Means followed by different superscript uppercase letters in the same row are significantly different at p ≤ 0.05 +, precipitated bile salt acid around colonies (0.3 %, w/v), simulating the near neutral small intestine envi- their use in commercial products could ensure high cell sur- ronment. Many studies have reported that the majority of the vival during storage (Talwalkar et al. 2001). strains survived well under such conditions, suggesting a po- tential recuperation of the initial levels during the passage of the small intestine (Maragkoudakis et al. 2006). Zielińska Cell surface of hydrophobicity of LAB isolates The analysis et al. (2015)reported that L. plantarum strains K1 and O23 of the adhesion ability of the food bacteria with probiotic isolated from pickled vegetables showed good resistance un- potential has been conducted in many species and strains. In der the bile salt stress condition. fact, it is commonly accepted that adhesion properties and mechanisms in Lactobacillus are strain- and matrix- dependent (Federici et al. 2014). In this study, L. fermentum Effect of micro-aerobic and anaerobic conditions on LAB KJ23 gave the highest affinities value of 39.5 %, followed by isolates growth There was no significant difference in the 34.8 % and 19.9 % in L. plantarum KJ03 and L. plantarum growth of L. plantarum KJ03 or L. fermentum KJ23 when JCM 1149, respectively, toward n-hexadecane (Table 5). tested under micro-aerobic and anaerobic conditions Nevertheless, both strains showed moderate affinity to n- (Table 5). Moreover, both strains grew >log 9 CFU/ml in all hexadecane, which were in range of 30–40 % cell-surface conditions after a 24-h incubation. These results are in accor- hydrophobicity. The determination of microbial adhesion to dance with those reported by Smetankova et al. (2012)who hexadecane as a method to estimate the ability of a strain to observed that wild strains of L. plantarum produced lactic acid adhere to epithelial cells is a valid qualitative phenomenolog- equally well under aerobic and anaerobic conditions. ical approach (Kiely and Olson 2000). Adhesion is believed to L. fermentum was able to grow and show probiotic properties be a requirement for the realization of probiotic effect, as it is in the absence of oxygen (Lingani et al. 2008). Probiotic bac- required for colonization of the GIT; it is also an important teria generally grow and colonize the small intestine, which is prerequisite for competitive exclusion of enteropathogens and a strictly anaerobic environment, and have been reported to immunomodulation of the host (Begonović et al. 2014). Our survive under these conditions (Talwalkar and Kailasapathy results indicate that the LAB strains isolated from our food 2003). Oxygen toxicity is a major problem in the survival of source have the potential to adhere to and colonize the gut epithelial cells of the human intestine. probiotics. Screening probiotics for oxygen tolerance before Ann Microbiol (2017) 67:25–36 33 Blood hemolytic activity and bile salt hydrolase All strains displayed no haemolysis (γ -hemolysis) when tested with hu- man blood agar (Table 5). Absence of hemolytic activity is considered to be a safety prerequisite for the selection of a probiotic strain (Ruiz-Moyano et al. 2009). Results from re- cent studies strongly suggest that BSH activity is a relevant property of a candidate probiotic. BSH activity is associated with the protection of bacteria from toxicity through the de- toxification of bile salts, thereby increasing the intestinal sur- vival and persistence of the BSH-producing strains (Guo et al. 2010). Du Toit et al. (1998) reported a beneficial effect of BSH-positive Lactobacillus strains in vivo. In our study, L. plantarum KJ03 and L. fermentum KJ23 both showed similar BSH activity in culture, as they both hydrolyzed GDC and TCD. In populations with a high incidence of colorectal cancer, fecal concentrations of bile acids are increased, suggesting that increased exposure of the colonic lumen to high levels of bile acids plays a role in the natural course of development of colon cancer. Bayerdörffer et al. (1995) also reported a positive association between deoxycholic acid (DOC) in the serum and colorectal adeno- mas, the precursors of colorectal cancer, further indicating a pathogenic role of DOC in colonic carcinogenesis. DOC also appears to be the most significant bile acid with respect to human colorectal cancer (Hill 1990). Cholesterol-lowering property A high level of serum cho- lesterol in humans is generally considered to be a risk factor for coronary heart disease (Klaver and van der Meer 1993). Among the isolates tested for the removal of cholesterol, L. plantarum KJ03 showed the highest cholesterol reduction (52.27 %), followed by L. fermentum KJ23 (49.33 %); the lowest value was found for L. plantarum JCM 1149 (36.73 %). Studies have indicated that many Lactobacillus spp. have cholesterol-reducing effects in vitro or in vivo (Kumar et al. 2012). It was hypothesized that deconjugated bile salts may contribute to lower cholesterol levels as free bile salts may be more readily excreted from the GIT than conju- gated bile salts (Fukushima et al. 1999). However, the exact mechanism of serum cholesterol reduction by probiotic bac- teria is not completely understood (Choi and Chang 2015). Klaver and van der Meer (1993) suggested that in vitro cho- lesterol reduction by some Lactobacillus spp. results from their coprecipitation with deconjugated bile salts. Interestingly, Mann and Spoerry (1974) studied cholesteremia in a tribe of Maasai and found that serum cholesterol levels of Maasai men decreased after consumption of large amounts of milk fermented with a wild Lactobacillus strain. Determination of antibiotic susceptibility The selected strains were tested for their susceptibility to nine antibiotics by the broth microdilution assay. Lactobacillus plantarum KJ03 was found to be susceptible to six antibiotics Table 6 Minimum inhibitory concentrations for selected strains of lactic acid bacteria a −1 Strain Minimum inhibitory concentration (μgmL ) Ampicillin Vancomycin Ciprofloxacin Kanamycin Streptomycin Erythromycin Clindamycin Tetracycline Chloramphenicol Lactobacillus plantarum KJ03 2 >64 16 16 8 <0.5 <0.125 32 8 Break point for Lactobacillus plantarum 2 Not required – 64 Not required 1 2 32 8 Lactobacillus fermentum KJ23 1 >64 32 16 8 <0.5 <1.25 32 4 Breakpoint for Lactobacillus fermentum 2 Not required – 32 64 1 1 8 4 Strains with a minimum inhibitory concentration lower than or equal to the breakpoints are considered here to be susceptible Breakpoints are according to the guidelines of the European Food Safety Authority (2012) 34 Ann Microbiol (2017) 67:25–36 (erythromycin, chloramphenicol, ampicillin, kanamycin, tet- collagen protein (ace), aggregation substances (agg and asa), racycline, clindamycin) but not to streptomycin and vancomy- enterococcal surface protein (esp) and gelatinase (gelE)were cin. Lactobacillus fermentum KJ23 was also susceptible to six absent, whereas the expected PCR products were observed for antibiotics (erythromycin, chloramphenicol, ampicillin, kana- Enterococcus faecalis 13–5and E. faecalis VanB as positive mycin, streptomycin, clindamycin) but not to tetracycline ac- control (Table 7). The positive control also exhibited negative cording to the breakpoint levels suggested by European Food results for cell wall-adhesion (Cyl) due to the low levels or Safety Authority (2012). All strains were resistant to cipro- downregulation of gene expression or an inactive gene floxacin in accordance with the Scientific Committee for product. Animal Nutrition (2002). However, they were susceptible to It is well known that virulence of microorganisms is regu- the breakpoints values proposed by Danielsen and Wind lated by virulence coding genes present on the genome in (2003) who reported relevant microbiological breakpoints special regions. Several putative virulence factors have been for a wider range of Lactobacillus species (Table 6). Strains described which cause serious disease in humans, such as KJ03 and KJ23 showed resistance to vancomycin at the aggregation substance protein, gelatinase, cytolysin, entero- highest amount tested (64 μg). Since vancomycin is an anti- coccal surface proteins, accessory colonization factors and biotic belonging to glycopeptide antibiotics, it inhibits the endocarditis antigens (Moraes et al. 2012). The verification synthesis of peptidoglycan which is an important structural of virulence factors by molecular procedures is important component of the bacterial cell wall. Therefore, Gram- due to the risk of genetic transfer because these genes are positive bacteria, including LAB, are especially vulnerable usually located in conjugative plasmids (Eaton and Gasson to vancomycin treatment (Reynolds 1989). Moreover, resis- 2001). tance to vancomycin has been reported as a natural or an intrinsic property of many LAB (Gotcheva et al. 2002). The Lactobacillus spp. exhibited resistance against inhibi- Conclusion tion of nucleic acid synthesis (ciprofloxacin), resulting in its natural resistance, which may be inherent to a bacterial genus The results of our study demonstrate that L. plantarum KJ03 or species, but may also be acquired through an exchange of isolated from fermented stinky bean possesses desirable genetic material, mutations and the incorporation of new in vitro functional properties that are similar or superior to genes (Ammor et al. 2007). Moreover, a drawback to antibi- those of the reference probiotic strain, L. plantarum subsp. otic resistance is that a transfer of antibiotic resistance genes is plantarum JCM 1149. Strain KJ03 was able to survive and possible because antibiotic resistance genes are generally car- establish in an environment similar to the human GIT, inhibit ried on plasmids, which can be transferred to other bacteria by potential pathogenic bacteria under the safety of Thai means of conjugation (Cebeci and Gurakan 2003). This may Community Product Standard (317/2004), and considered to result in pathogenic bacteria possessing a high level of antibi- be safe to be used with regard to their antibiotic resistance otic resistance. Owing to tetracycline resistance of pattern. Therefore, L. plantarum KJ03 can be considered as L. fermentum KJ23, we selected L. plantarum KJ03 as a po- a starter culture to improve the quality of Sataw-Dong.A tential LAB for further starter culture applications. probiotic potential is expected to greatly enhance the already important nutritional value of stink bean, which is regarded as Detection of virulence genes Virulence factors can be crucial a source of organic acids, vitamins, and minerals. for strain pathogenicity depending on their type. The presence Development of a functional product may indeed convey a of virulence genes in both LAB strains KJ03 and KJ23 was favorable impact in rural economy, especially knowing that investigated using PCR technology. The genes for adhesion such product originates from less well-developed regions. 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Selection and evaluation of functional characteristics of autochthonous lactic acid bacteria isolated from traditional fermented stinky bean (Sataw-Dong)

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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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Ann Microbiol (2017) 67:25–36 DOI 10.1007/s13213-016-1233-3 ORIGINAL ARTICLE Selection and evaluation of functional characteristics of autochthonous lactic acid bacteria isolated from traditional fermented stinky bean (Sataw-Dong) 1 2 1 Krittanon Jampaphaeng & Luca Cocolin & Suppasil Maneerat Received: 19 January 2016 /Accepted: 2 September 2016 /Published online: 17 September 2016 Springer-Verlag Berlin Heidelberg and the University of Milan 2016 Abstract The aim of this study was to evaluate the techno- desirable in vitro functional properties. This strain is therefore logical and functional potential of lactic acid bacteria (LAB) a good candidate for further investigation for use in Sataw- isolated from fermented stinky bean (Sataw-Dong). Of the Dong fermentation to assess its technological performance as 114 LAB colonies isolated from spontaneously fermented a potential probiotic starter. stinky bean which showed inhibitory activity against two . . food-borne pathogens (Staphylococcus aureus DMST 4480 Keywords Lactic acid bacteria Lactobacillus fermentum . . and Escherichia coli DMST 4212), the five isolates (KJ03, Lactobacillus plantarum Probiotic Sataw-Dong KJ15, KJ17, KJ22, KJ23) exhibiting excellent antagonistic activity were subjected to further study. These five strains showed titratable acidity as lactic acid in the range of 1.47– Introduction 1.55 %, with strains KJ03 and KJ23 additionally exhibiting a high NaCl tolerance of >7 % (w/v). Using 16S rRNA gene Fermented foods are an important component of the human sequence analysis, strains KJ03 and KJ23 were identified as diet in many countries, especially at the household level. One Lactobacillus plantarum and L. fermentum, respectively, and of the advantages of fermentation is that it is an inexpensive further investigated for their functional properties in vitro. process with the potential to preserve food, improve nutrition- Both strains survived well in a simulated gastrointestinal tract al value, and enhance aroma and taste (Aloys and Angeline environment with <1 log cell decrease over 8 h (>8 log CFU/ 2009). In addition, indigenous fermented foods are strongly ml). Lactobacillus plantarum KJ03 showed the best perfor- linked to the culture and tradition of the country of their origin mance with respect to cholesterol removal (53 %), while (Guo et al. 2015). L. fermentum KJ23 showed the highest cell-surface hydropho- Foods which contain probiotics represent a group of bicity (39.5 %). Neither of the two strains showed any hemo- health-promoting, functional foods of important commer- lysis activity. Both strains hydrolyzed glycodeoxycholic and cial interest, and their share of the market is steadily in- taurodeoxycholic acids. In terms of antibiotic susceptibility, creasing (Vuyst et al. 2008). Most probiotic bacteria be- L. fermentum KJ23 was not sensitive to tetracycline. Taking long to lactic acid bacteria (LAB) groups, especially the all of the results into account, L. plantarum KJ03 possessed species of the genus Lactobacillus, which is one of the most fundamental of microbial groups. These LAB have been introduced into several of fermented food products. * Suppasil Maneerat Many studies have reported that dairy products are the suppasil.m@psu.ac.th most commonly used food vehicles for the delivery of probiotics (Rubio et al. 2014). However, there has been a Biotechnology for Bioresource Utilization Laboratory, Department growing interest in developing non-dairy probiotics, and it of Industrial Biotechnology, Faculty of Agro-Industry, Prince of is known that some traditional fermented foods may con- Songkla University, Hat Yai, Songkla 90112, Thailand 2 stitute a suitable base for the development of probiotic- Department of Agricultural, Forest and Food Science of the type functional foods (Ruiz-Moyano et al. 2009). The po- University of Torino (DISAFA), University of Turin, Grugliasco, Turin 10095, Italy tential to develop non-dairy probiotic products has 26 Ann Microbiol (2017) 67:25–36 attracted the interest of individuals with lactose intolerance which were covered with lids and allowed to ferment sponta- and consumers with cholesterol-restricted diets (Granato neously at room temperature (27–32 °C) for 10 days. Samples et al. 2010). Moreover, traditional fermented foods are a obtained at 0, 1, 2, 4, 6, 8, and 10 days of fermentation were plentiful source of microorganisms, and some of them used to isolate LAB. show probiotic properties (Rivera-Espinoza and Gallardo- Navorra 2010). Thus, non-dairy products made from fruits, Isolation of LAB showing antagonistic activity vegetables, and cereals would appear to have a promising against foodborne pathogens future (Martins et al. 2013). Stink bean (Parkia speciose), a Southeast Asian plant of Samples (25 g) of Sataw-Dong were added to 0.1 % (w/v) the genus Parkia in the family Fabaceae, grows wild in trop- peptone water (225 ml) and placed in a stomacher for 2 min. ical forests and is often cultivated in Southern Thailand. The Ten-fold serial dilutions were made in peptone water, and the beans are flattened and elliptical in shape with a nutty and firm appropriate dilutions were spread onto MRS agar (Lab M texture. They are believed to contain medicinal compounds Ltd., Heywoord, UK) supplemented with bromocresol purple which exhibit potential biological activity, such as antibacte- (0.02 %, w/v), and NaCl (3 %, w/v), and the plates were rial (Sakunpak and Panichayupakaranant 2012), incubated at 37 °C for 24 h for the isolation of presumptive antiangiogenic (Aisha et al. 2012), anticancer (Ali et al. LAB. After 24 h, the plates of LAB were individually overlaid 2006), antioxidant (Aisha et al. 2012) and hypoglycemic ac- with Tryptone Soy Agar (TSA; Hi Media Laboratories, tivities (Jamaluddin and Mohamad 1993). Stink beans pre- Mumbai, India) (0.75 % agar, w/v) seeded with served in brine for several days undergo fermentation, with Staphylococcus aureus DMST 8840 and Escherichia coli the production of lactic acid; the fermentation product is called DMST 4212 [approx. 10 colony-forming units (CFU)/ml]. fermented stink bean or Sataw-Dong (in the Thai language) The plates were then incubated at 37 °C for 24 h. Inhibition and consumed as a raw pickle. While there are many similar zones were detected by a clear zone around the tested strains LA fermentation products, to date cucumber, cabbage and (Schillinger and Lücke 1989). olives are the only vegetables that are fermented in large vol- Bacterial colonies exhibiting an inhibition zone were indi- umes for human consumption (Montet et al. 2006). vidually picked and streaked on MRS agar two to three times However, all natural fermentation processes have a com- to purify the isolates. All Gram-positive, catalase-negative mon problem in that they are relatively uncontrollable, isolates were defined as LAB (Hwanhlem et al. 2011), and resulting in variations in product stability and quality attri- these isolates were maintained in MRS broth containing glyc- butes (Parkouda et al. 2010). It has been reported that these erol (60 %) at −20 °C. For routine analysis, strains were problems can be overcome by using a starter culture, control- subcultured twice in MRS broth at 37 °C for 24 h. ling the microflora, accelerating the ripening time, inhibiting the growth of pathogenic and spoilage bacteria and alleviating Confirmation of the antagonistic activity variations in organoleptic quality problems in fermented foods, thereby improving the overall quality of fermented veg- Antagonistic activity against bacterial indicators was investi- etable products (Font de Valdez et al. 1990; Caplice and gated using the agar spot test and agar well diffusion assay as Fitzgerald 1999). described by Jones et al. (2008) and Schillinger and Lücke The aim of the study reported here was to: (1) characterize (1989), respectively. The agar spot test experiment was con- LAB isolated from traditionally fermented stink bean; (2) se- ducted by spotting each overnight LAB culture (5 μl) onto the lect the most suitable strains according to their technological surface of a MRS agar plate and incubating the plate at 37 °C characteristics, including antagonistic activity against for 24 h. These plates were then overlaid with 10 ml of TSA foodborne pathogens; (3) investigate a number of the func- (0.75 % agar, w/v) seeded with 100 μl of each tested indicator tional properties the isolates may have. strain. After an overnight incubation at 37 °C, the plates were examined for zones of inhibition. The agar well diffusion assay was performed to character- Materials and Methods ize the type of antimicrobial compounds. Cell-free superna- tants were collected by centrifugation (8000 g, 10 min, 4 °C) Sataw-Dong preparation from overnight cultures. Two samples of supernatant taken from each strain were used in trials as follows: (1) the pH Sataw-Dong was prepared according to the traditional recipe. was adjusted to 6.5 and the supernatant heated at 90 °C for Whole pods were blanched and added to a brine prepared by 10 min, and (2) the supernatant without adjustment was used boiling clean water containing 3–4 % NaCl, 5 % sugars, and as control. Sterile culture plates containing 20 ml TSB and 1.0 0.5 % Malabar tamarind (Garcinia cambogia). The beans to- % (w/v) agar were then prepared and subsequently seeded gether with the brine were transferred to 12 glass jars (100 g), with each bacterial indicator. Wells (diameter 7 mm) were Ann Microbiol (2017) 67:25–36 27 punched into the agar layer, and cell-free supernatants (50 μl) harvested by centrifugation (8000 g, 10 min) and resuspended were placed into each well; the plates were then incubated at in 2 ml of phosphate buffer saline (PBS) in the presence of 37 °C for 24 h. The inhibition activity of each supernatant was lysozyme (100 mg/L) (Sigma-Aldrich, St. Louis, MO). assessed based on the diameter of the inhibition zone. Bacterial suspensions without lysozyme were used as con- trols. Samples were incubated at 37 °C, and viable cell counts Determination of pH value and total titratable acidity after 10 and 20 min were enumerated on MRS agar by the drop plate method. Survival rates were calculated as a percent- All LAB cultures were diluted to an absorbance of 0.5 age of growth. (10 CFU/ml). Each LAB suspension (1 %) was inoculated into MRS broth and the cultures incubated at 37 °C for 24 h, Survival of LAB under simulated gastric and intestinal following which aliquots (20 ml) were taken for the measure- juices Cells of the LAB isolates were collected by centrifuga- ments. The pH was directly measured using a pH meter tion (8000 g, 10 min) and washed twice with PBS before (OHAUS, Shanghai, China). A cell-free supernatant was used being resuspended in PBS solution at pH 2.5 containing pep- to determine the total titratable activity by titration against sin (3 mg/ml; Sigma-Aldrich) as simulated gastric juice 0.1 N NaOH using phenolphthalein (0.1 % w/v in 95 % eth- (Maragkoudakis et al. 2006). Viable counts were carried out anol) as an indicator. after incubation at 37 °C for 2 h. After this step, the cultures were centrifuged and washed twice with PBS solution before Determination of NaCl tolerance of LAB being transferred to PBS solution, pH 8.0, containing pancre- atin (1 mg/ml) and ox bile salts (3 mg/ml) (Sigma-Aldrich) as The NaCl concentration in MRS broth was adjusted to 0, 3, 5, simulated intestinal fluid. The samples were incubated at 7, 9, and 12 % (w/v), following which 1 % (v/v) of active LAB 37 °C in for 6 h. Survival of LAB was enumerated after 3 h was inoculated into MRS broth containing the different con- and 6 h of incubation. centrations of NaCl and cultivated at 37 °C for 24 h. The survival of LAB was determined by the drop plate method Effect of micro-aerobic and anaerobic conditions on on MRS agar and reported as colony-forming units per growth of LAB LAB strains (1 %, v/v) were inoculated into milliliter. MRS broth (micro-aerobic condition) and MRS broth supple- mented with L-cysteine (0.5 mg/ml) covered with liquid par- Molecular identification and sequencing of 16S rRNA affin (anaerobic condition) (Talwalkar et al. 2001). The sam- gene of selected LAB ples were incubated at 37 °C for 24 h. For micro-aerobic condition, cells were enumerated on MRS agar and incubated DNA of the selected LAB was extracted using an extraction at 37 °C for 24 h. On the other hand, cells were dropped on kit (QIAgen, Hilden, Germany) according to the manufac- MRS agar supplemented with L-cysteine and overlaid with turer’s protocol and stored at −20 °C. 16S rRNA gene ampli- agar, followedbyincubationat37°Cfor 24h inananaerobic fication was performed in a thermocycler (Techne, Abingdon, jar. UK) with the following universal primers: 27 F (5′-AGAG TTTGATCCTGG CTCAG-3′) and 1492R (5′-GGTT Cell-surface hydrophobicity assay Cell-surface hydropho- ACCTTGTTACGACTT-3′) according to the protocol de- bicity was determined by a microbial adhesion to hydrocarbon scribed by Cai et al. (1999). The PCR products were purified test as described by Otero et al. (2004). LAB were grown in using a PCR purification kit (QIAgen) and sequenced (Ward MRS broth at 37 °C for 24 h and harvested by centrifugation Medic Ltd., Selangor, Malaysia). The basic local alignment (8,000 g, 10 min). The pellets were washed twice with PBS search tool (BLAST) was used to compare the obtained se- (pH 7.4) and resuspended in the same buffer to an absorbance quences against the public data library of the National Center of 0.8–1.0 at 600 nm (OD ). Equal volumes of cell suspen- for Biotechnology Information (http://www.ncbi.nlm.nih. sion and n-hexadecane were mixed in duplicate and vortexed gov). The nucleotide sequences of all the bacterial isolates thoroughly for 2 min. The tubes were allowed to separate into were deposited in the GenBank database (accession numbers two phases for 30 min. The aqueous phase was then measured LC016617 and LC037354). in a spectrophotometer at 600 nm. The decrease in the absor- bance of the aqueous phase was taken as a measure of the cell Determination of functional properties in vitro surface hydrophobicity (% H). Resistance to lysozyme Lysozyme resistance to assess the Blood hemolytic activity Fresh overnight LAB cultures were in vitro ability of the strains to survive transit through the oral streaked in triplicate on TSA plates supplemented with 5 % cavity was performed as described by Turchi et al. (2013), (v/v) human blood (obtained from Songklanagarind Hospital, with slight modifications. LAB grown overnight were Songkhla, Thailand) and incubated at 37 °C for 48 h. 28 Ann Microbiol (2017) 67:25–36 Hemolytic activities of the bacterial culture were examined for and evaporated. The residue was dissolved in 2 ml of O- signs of β-hemolysis (clear zones around colonies), α- phthalaldehyde reagent. After mixing, 1 ml of concentrated hemolysis (green zones around colonies), or γ-haemolysis sulfuric acid was added and the mixture vortexed for 1 min. (no clear zones around colonies) (Hargrove and Alford 1978). Absorbance was read at 540 nm. All experiments were per- formed in duplicate. Bile salt hydrolase activity Qualitative bile salt hydrolase (BSH) activity was evaluated as described by Wang et al. Determination of antibiotic susceptibility The LAB strains (2012). The MRS agar plate was prepared by adding 0.5 % were tested for resistance to antibiotics by the broth (w/v) of bile salt (Sigma-Aldrich) as follows: glycocholic ac- microdilution method. The minimum inhibitory concentration id, taurocholic acid, glycodeoxycholic acid (GDC) and (MIC) values were determined in the LAB susceptibility test taurodeoxycholic acids (TDC). Overnight cultures of each medium (LSM) broth formulation described by Federici et al. LAB strain (10 μl) were spotted onto the agar plates and (2014) using ampicillin, vancomycin, chloramphenicol, eryth- incubated anaerobically at 37 °C for 24–72 h. The presence romycin, kanamycin, streptomycin, tetracycline, clindamycin, of precipitated bile acid around the colonies (opaque halo) was and ciprofloxacin at concentrations of 0.065–1024 mg/L. The considered to be a positive result. MRS agar plate without individual inoculum was adjusted to an absorbance of 0.2–0.3 supplemented bile acid was used as control. (600 nm), equivalent to 10 CFU/ml. In brief, 100 μlof bac- terial suspension was mixed with the antibiotic solution (100 μl) to be tested in 96-well plates and incubated overnight Cholesterol-lowering property The cholesterol-lowering at 37 °C. Growth was determined visually after incubation. property was determined according to Wang et al. (2014)with Susceptible and resistant strains were distinguished according some modification. The MRS broth was supplemented with to the breakpoints (cutoff values) reported by European Food 0.3 % (w/v) Ox gall (Hi-Media). Water-soluble cholesterol Safety Authority (2012). Accordingly, strains showing MICs (polyoxyethylene cholesteryl sebacate; Sigma-Aldrich) was higher than the respective cutoff values were considered to be filter-sterilized and added into the broth at a final concentra- resistant. tion of 100 μg/ml, inoculated with each LAB strain, and in- cubated at 37 °C for 24 h. After the incubation, cells were removed by centrifugation, and residual cholesterol concen- Detection of the presence of virulence genes To evaluate the tration in the broth was determined using a modified colori- safety of the selected LAB for their application in fermenta- metric method. Briefly, 1 ml of the broth was added to 1 ml of tion processes, we performed PCR assays to detect the pres- KOH (33 %, w/v) and 2 ml of absolute ethanol, mixed for ence of genes encoding potential virulence genes using geno- 1 min, then heated at 70 °C for 30 min. After cooling, 5 ml of mic DNA obtained as described in section Molecular identifi- hexane was added to the tube and the tube vortexed for 1 min. cation and sequencing of 16S rRNA gene of selected LAB. The hexane layer (4 ml) was transferred to a clean glass tube The specific primers are described in Table 1 (Vankerckhoven Table 1 PCR primers and products for the detection of virulence determinants Target genes Sequence (5′ to 3′)T (°C) Product size (bp) References ace 5′-GAATTGAGCAAAAGTTCAATCG-3′ 56 1008 Ben Omar et al. (2004) 5′-GTCTGTCTTTTCACTTGTTTC-3′ asa15′-GCACGCTATTACGAACTATGA-3′ 56 375 Vankerckhoven et al. (2004) 5′-TAAGAAAGAACATCACCACGA-3′ cylA5′-ACTCGGGGATTGATAGGC-3′ 58 688 Vankerckhoven et al. (2004) 5′-GCTGCTAAAGCTGCGCTT-3′ clyB5′-AAGTACACTAGTAGAACTAAGGGA-3′ 52 843 Semedo et al. (2003) 5′-ACAGTGAACGATATAACTCGCTATT-3′ efaAfs 5′- GACAGACCCTCACGAATA-3′ 54 705 Eaton and Gasson (2001) 5′- AGTTCATCATGCTGTAGTA −3′ esp 5′-AGATTTCATCTTTGATTCTTGG-3′ 56 510 Vankerckhoven et al. (2004) 5′-AATTGATTCTTTAGCATCTGG-3′ gelE5′-ACCCCGTATCATTGGTTT-3′ 52 419 Eaton and Gasson (2001) 5′-ACGCATTGCTTTTCCATC-3′ T , Melting temperature asa1, Aggregation substance gene; CylA/B, cytolysin A/B genes; efaAfs, cell-wall adhesion gene; esp, enterococcal surface protein gene; gelE, gelatinase gene Ann Microbiol (2017) 67:25–36 29 et al. 2004). The target genes were ace (adhesin of collagen tested for antagonistic activity against Staphylococcus aureus protein), asa1 (aggregation substance), CylA/B (cytolysins), DMST 8840 and Escherichia coli DMST 4212 according to efaAfs (cell-wall adhesion), esp (enterococcal surface protein) the safety criteria of Thai Community Product Standard (317/ and gelE (gelatinase). Of the strains tested, Enterococcus 2004). However, only five strains (KJ03, KJ15, KJ17, KJ22, feacalis Van B tested positive for the ace, asa1, CylA/B, KJ23) showed excellent inhibition zones (diameter > 15 mm), efaAfs,and gelE genes and Enterococcus feacalis 13–5tested with strain KJ03 having the highest inhibition zone (Table 2). positive for the esp gene. Those five LAB strains were subsequently analyzed for their production of antibacterial compounds by the agar well Statistical analysis diffusion method. Supernatants obtained from the five strains did not exhibit the inhibition zones after pH adjustment to 6.5 Statistical analysis was conducted using one-way analysis of (data not shown), suggesting that the antimicrobial activity of variance with Duncan’s post hoc test. Significance was set at these five LAB strains may come from organic acids. In fact, p < 0.05. All data are expressed as the mean ± standard the drop in pH arising from the production of lactic acid can be deviation. sufficient to inhibit certain bacteria as the non-dissociated form of lactic acid triggers a lowering of the internal pH of the cell which causes a collapse in the electrochemical proton gradient in sensitive bacteria, thereby resulting in a bacterio- Results and Discussion static or bactericidal effect (González et al. 2007). The anti- bacterial activity of LAB strains selected from vegetables has Isolation of LAB with primary antagonistic activity been confirmed in several previous studies; i.e., from fermented cucumber, organic leafy vegetables (Ponce et al. Lactic acid bacteria originally isolated from vegetables are 2008) and from fermented Himalayan vegetables (Dewan probably the most suitable candidates for improving the mi- and Tamang 2007). crobiological safety of fermented vegetable products because they are well-adapted to the conditions of the matrices used in the fermentation process and should therefore be more com- Lactic acid production and pH reduction ability petitive than LAB obtained from other sources (Ponce et al. 2008). A total of 114 colonies which showed clear zones Lactic acid produced by LAB is an essential compound for against S. aureus DMST 8840 and E. coli DMST 4212. All food preservation because it maintains the acidity condi- of those isolates were identified as LAB based on two criteria: tions of the fermented foods and it is antagonistic against Gram-positive and catalase-negative (data not shown). food spoilage and poisoning bacteria (Hwanhlem et al. 2011). LAB ferment sugars via a number of different path- Screening for antagonistic activity ways, resulting in homo-, or mixed acid fermentation. Homofermentation produces lactic acid as the sole end Antagonistic activity of LAB strains may contribute to the product of glucose metabolism when the Embden– improvement in the quality of fermented foods, which is Meyerhof–Parnas pathway is used (Hofvendahl and achieved through the control of spoilage and pathogenic bac- Hahn–Hägerdal 2000). teria, thus extending shelf-life and improving sensory quality We therefore measured lactic acid production and pH re- (Begonović et al. 2014). All 114 LAB strains identified were duction in these five LAB isolates (KJ03, KJ15, KJ17, KJ22, Table 2 Inhibition zone diameter LAB isolate no. Inhibition zone (mm) of lactic acid bacteria isolated from Sataw-Dong on agar in the Staphylococcus aureus DMST8840 Escherichia coli DMST4212 presence of indicator bacterial species Staphylococcus aureus KJ03 29.7 ± 0.6 23.0 ± 1.0 DMST8840 and Escherichia coli DMST4212 KJ15 26.7 ± 0.6 17.7 ± 1.2 KJ17 27.7 ± 0.5 18.3 ± 0.6 KJ22 25.3 ± 0.7 19.0 ± 1.0 KJ23 29.3 ± 1.2 21.7 ± 0.6 LAB, Lactic acid bacteria Data are presented as the mean ±standard deviation (SD) from triplicate determinations 30 Ann Microbiol (2017) 67:25–36 KJ23) exhibiting excellent antagonistic activity against Identification of LAB isolates S. aureus DMST 8840 and E. coli DMST 4212. After 24 h of incubation, strain KJ23 was the highest producer of lactic The 16S rRNA gene sequences of strains KJ03 and KJ23 were acid at 1.55 % (in % equivalents lactic acid), and strain KJ15 compared with those other bacterial strains in GenBank data- was the lowest lactic acid producer at 1.47 %. The remaining base and identified and subsequently confirmed as three strains showed no significant lactic acid production, with Lactobacillus plantarum (99 % similarity) and L. fermentum the exception of strain KJ15. The pH values were correlated (99 % similarity), respectively. The 16S rRNA gene se- with these results in the range of 3.86–4.00 (Table 3). quences of strains KJ03 and KJ23 were deposited in the DDBJ/EMBL/Genbank with accession numbers LC016617 and LC037354, respectively. These strains were found to be Effect of NaCl on growth of isolates Gram-positive, non-spore-forming rods; they were catalase- negative and form off-white cream colonies (Table 4). The The cell viability of the five LAB isolates after incubation in fermentation of vegetables is mainly carried out by MRS broth supplemented with NaCl is shown in Fig. 1 re- L. plantarum and L. fermentum strains for improving their ports. NaCl supplemented at a concentration of 3–5% did not nutritional and sensory features, and these strains are also significantly affect the growth of these LAB strains as they considered to be the best candidates for starters with probiotic grew over 9 log CFU/ml. In addition, the growth of three properties (Swain et al. 2014). isolates (KJ03, KJ22, KJ23) appeared to be slightly stimulated with the addition of 3 % NaCl to the incubation broth. As Functional properties of selected LAB in vitro predicted, increasing NaCl concentration had a negative effect on the growth of all isolates, with a subsequent decrease in cell An important step towards the selection of potential probiotic viability (Fig. 1). In particular, LAB viability was strongly LAB is to evaluate their resistance and survival through the affected by 7 % NaCl. Isolates KJ03 and KJ23 best tolerated human gastrointestinal tract (GIT). Lactobacillus plantarum the highest NaCl concentrations (>8 log CFU/ml), and we subsp. plantarum strain JCM1149, originally isolated from therefore selected these two strains for further investigations. pickled cabbage, is a commercial type strain with probiotic In MRS broth supplemented with 9–12 % NaCl, these two properties (Zago et al. 2011; Nazzaro et al. 2012) and was also LAB strains were not able to grow throughout the 24-h incu- used in this study to compare properties among our isolates. bation period, although cell counts remained constant (approx. The results of our study on the functional properties of our two 5–6 log CFU/ml) for the entire incubation time. The tolerance isolated Lactobacillus strains (KJ03 and KJ23) are shown in to salt concentrations of 2–10 % is a limiting factor affecting Table 5. the persistence, competitiveness, and metabolism of the starter culture over the entire fermentation due to its water binding and ionic characteristics (Ammor and Mayo 2007). The Resistance to lysozyme The resistance of L. plantarum KJ03 growth of LAB is sometimes enhanced in the presence of and L. fermentum KJ23 to lysozyme, expressed as percentage low concentration of NaCl (1–2%,w/v)(Leroyandde of survival, ranged from a minimum mean value of 99.73 % to Vuyst 1999). Consequently, a salt tolerance of at least 7 % is a maximum mean value of 100 %. These isolates revealed a considered to be necessary for potential starter cultures in high lysozyme resistance after both 10 and 20 min incubation, Sataw-Dong fermentation because the presence of 5–7% with a survival rate >98 % (Table 5). Lysozyme present in NaCl is normally found in many recipes of Sataw-Dong. human saliva is the first challenge to survival. Resistance to Table 3 pH values and titratable Isolated no. pH Titratable acidity (%) acidity of cell-free supernatants of the five lactic acid bacterial strains 3h 6h 12 h 24 h 3 h 6 h 12 h 24h isolated from Sataw-Dong after 3, 6, 12, and 24 h of incubation a a a a KJ03 6.93 4.91 3.89 3.86 0.07 ± 0.01 0.44 ± 0.01 1.35 ± 0.01 1.53 ± 0.01 a a b a KJ15 6.96 4.91 3.94 3.88 0.05 ± 0.01 0.44 ± 0.01 1.29 ± 0.01 1.52 ± 0.01 a b a b KJ17 6.93 5.05 3.97 3.90 0.07 ± 0.03 0.40 ± 0.03 1.36 ± 0.01 1.47 ± 0.02 a c a a KJ22 6.90 5.35 4.01 4.00 0.05 ± 0.01 0.31 ± 0.02 1.35 ± 0.00 1.53 ± 0.02 a a b a KJ23 6.94 5.14 3.99 3.97 0.06 ± 0.01 0.46 ± 0.01 1.27 ± 0.01 1.55 ± 0.01 Data on titratable acidity are presented as the mean ± SD from triplicate determinations. Mean values followed by different superscript lowercase letter in the same column are significantly different at p ≤ 0.05 Ann Microbiol (2017) 67:25–36 31 Fig. 1 Viability of lactic acid MRS (control) MRS + 3% NaCl MRS + 5% NaCl bacteria isolates KJ03, KJ15, MRS + 7% NaCl MRS + 9% NaCl MRS + 12% NaCl KJ17, KJ22, and KJ23 after cultivation in MRS broth supplemented with various concentrations of NaCl (0–12 %) at 37 °C for up to 24 h. Lb. Lactobacillus KJ03 KJ15 KJ17 KJ22 KJ23 Lb. plantarum JCM1149 lysozyme has been attributed to the peptidoglycan structure in plantarum KJ03 and L. fermentum KJ23 survived more than 9 the cell wall, the physiological state of the cell, and lysozyme log CFU/ml (>95 %) after their cell suspensions were exposed structure in the medium (Cunningham et al. 1991). This result to the simulated gastric juice. The surviving cells were further confirmed the high resistance of the two Lactobacillus strains tested for bile salt tolerance. Lactobacillus plantarum KJ03 to 100 mg/l of lysozyme under conditions stimulating the exhibited better survival, at >9 log CFU/ml (92.55 %), than in vivo dilution by saliva (Zago et al. 2011). L. fermentum KJ23 (81.32 %) (Table 5). The ability to survive the acidic and bile challenges in the GIT is advantageous for probiotics. Acid tolerance of Survival of LAB under simulated gastric and intestinal juices The behavior of L. plantarum KJ03 and L. fermentum Lactobacillus strains was also reported in previous studies. Srinu et al. (2013) revealed that all of the L. plantarum and KJ23 under simulated GIT conditions enables strains to be selected which are likely to survive these conditions, a neces- L. casei strains they tested showed good survival in the acidic sary prerequisite for probiotic cultures. Stresses to microor- pH range (1.5–3.5) tested. Jamaly et al. (2011)also reported ganisms begin in the mouth, with lysozyme-containing saliva, that L. plantarum was able to tolerate 3 h of acid exposure and continue in the stomach (pH 1.5–3.0) and in the upper (pH. 2.0 and 3.0). Good probiotic bacteria should survive well in a pancreatin solution at pH 8.0 in the presence of bile salts intestine which contains bile (Zago et al. 2011). Lactobacillus Table 4 Physiological properties of the five selected lactic acid bacterial strains isolated from Sataw-Dong Property Lactobacillus plantarum KJ03 Lactobacillus fementum KJ23 Lactobacillus plantarum JCM1149 Shape Rod Short rod Rod Gram stain Positive Positive Positive Catalase test –– – Growth at 15/45 °C +/+ +/+ +/+ Growth at pH 2.0 + + + 3.0 ++ + ++ 4.0 +++ +++ +++ 5.0 +++ +++ +++ Lactobacillus plantarum JCM1149 was used as a positive control for the two probiotic strains isolated in this study (KJ03 and KJ23) +, Positive reaction; −, negative reaction; +, ++, +++: Low, moderate, and high turbidity of growth, respectively Log CFU/ml 32 Ann Microbiol (2017) 67:25–36 Table 5 In vitro probiotic properties of the selected isolates of lactic acid bacteria Properties Lactobacillus plantarum KJ03 Lactobacillus fementum KJ23 Lactobacillus plantarum JCM1149 Survival (log CFU/ml) a a a Control (0 h) 9.584 ± 0.193 9.498 ± 0.147 9.315 ± 0.229 a a a Simulated lysozyme, 10 min 9.550 ± 0.146 9.475 ± 0.285 9.280 ± 0.167 a a a Simulated lysozyme, 20 min 9.445 ± 0.140 9.392 ± 0.121 9.225 ± 0.181 a b b Simulated gastric juice (pH 2.5), 2 h 9.211 ± 0.061 9.011 ± 0.008 9.004 ± 0.083 a b b Simulated intestinal juice (pH 8.0), 3 h 9.020 ± 0.010 8.542 ± 0.022 8.788 ± 0.103 a c b Simulated intestinal juice (pH 8.0), 6 h 8.735 ± 0.015 7.621 ± 0.035 8.312 ± 0.045 Viable cell count (log CFU/ml) a,A a,A b,A Micro-aerobic condition 9.541 ± 0.112 9.522 ± 0.075 9.122 ± 0.063 a,A a,A a,A Anaerobic condition 9.531 ± 0.065 9.538 ± 0.082 9.250 ± 0.008 b a c Cell surface hydrophobicity (%) 34.82 ± 1.57 39.53 ± 1.34 19.90 ± 1.33 Bile salt hydrolase activity Glycocholic acid –– – Taurocholic acid –– – Glycodeoxycholic acid + + + Taurodeoxycholic acid –– – Blood hemolysis γγ γ Cholesterol removal (%) 53.0 ± 0.6 49.2 ± 1.4 38.0 ± 1.1 Where applicable, data are presented as the mean ± SD. Means followed by different superscript lowercase letters in the same row are significantly different at p ≤ 0.05. Means followed by different superscript uppercase letters in the same row are significantly different at p ≤ 0.05 +, precipitated bile salt acid around colonies (0.3 %, w/v), simulating the near neutral small intestine envi- their use in commercial products could ensure high cell sur- ronment. Many studies have reported that the majority of the vival during storage (Talwalkar et al. 2001). strains survived well under such conditions, suggesting a po- tential recuperation of the initial levels during the passage of the small intestine (Maragkoudakis et al. 2006). Zielińska Cell surface of hydrophobicity of LAB isolates The analysis et al. (2015)reported that L. plantarum strains K1 and O23 of the adhesion ability of the food bacteria with probiotic isolated from pickled vegetables showed good resistance un- potential has been conducted in many species and strains. In der the bile salt stress condition. fact, it is commonly accepted that adhesion properties and mechanisms in Lactobacillus are strain- and matrix- dependent (Federici et al. 2014). In this study, L. fermentum Effect of micro-aerobic and anaerobic conditions on LAB KJ23 gave the highest affinities value of 39.5 %, followed by isolates growth There was no significant difference in the 34.8 % and 19.9 % in L. plantarum KJ03 and L. plantarum growth of L. plantarum KJ03 or L. fermentum KJ23 when JCM 1149, respectively, toward n-hexadecane (Table 5). tested under micro-aerobic and anaerobic conditions Nevertheless, both strains showed moderate affinity to n- (Table 5). Moreover, both strains grew >log 9 CFU/ml in all hexadecane, which were in range of 30–40 % cell-surface conditions after a 24-h incubation. These results are in accor- hydrophobicity. The determination of microbial adhesion to dance with those reported by Smetankova et al. (2012)who hexadecane as a method to estimate the ability of a strain to observed that wild strains of L. plantarum produced lactic acid adhere to epithelial cells is a valid qualitative phenomenolog- equally well under aerobic and anaerobic conditions. ical approach (Kiely and Olson 2000). Adhesion is believed to L. fermentum was able to grow and show probiotic properties be a requirement for the realization of probiotic effect, as it is in the absence of oxygen (Lingani et al. 2008). Probiotic bac- required for colonization of the GIT; it is also an important teria generally grow and colonize the small intestine, which is prerequisite for competitive exclusion of enteropathogens and a strictly anaerobic environment, and have been reported to immunomodulation of the host (Begonović et al. 2014). Our survive under these conditions (Talwalkar and Kailasapathy results indicate that the LAB strains isolated from our food 2003). Oxygen toxicity is a major problem in the survival of source have the potential to adhere to and colonize the gut epithelial cells of the human intestine. probiotics. Screening probiotics for oxygen tolerance before Ann Microbiol (2017) 67:25–36 33 Blood hemolytic activity and bile salt hydrolase All strains displayed no haemolysis (γ -hemolysis) when tested with hu- man blood agar (Table 5). Absence of hemolytic activity is considered to be a safety prerequisite for the selection of a probiotic strain (Ruiz-Moyano et al. 2009). Results from re- cent studies strongly suggest that BSH activity is a relevant property of a candidate probiotic. BSH activity is associated with the protection of bacteria from toxicity through the de- toxification of bile salts, thereby increasing the intestinal sur- vival and persistence of the BSH-producing strains (Guo et al. 2010). Du Toit et al. (1998) reported a beneficial effect of BSH-positive Lactobacillus strains in vivo. In our study, L. plantarum KJ03 and L. fermentum KJ23 both showed similar BSH activity in culture, as they both hydrolyzed GDC and TCD. In populations with a high incidence of colorectal cancer, fecal concentrations of bile acids are increased, suggesting that increased exposure of the colonic lumen to high levels of bile acids plays a role in the natural course of development of colon cancer. Bayerdörffer et al. (1995) also reported a positive association between deoxycholic acid (DOC) in the serum and colorectal adeno- mas, the precursors of colorectal cancer, further indicating a pathogenic role of DOC in colonic carcinogenesis. DOC also appears to be the most significant bile acid with respect to human colorectal cancer (Hill 1990). Cholesterol-lowering property A high level of serum cho- lesterol in humans is generally considered to be a risk factor for coronary heart disease (Klaver and van der Meer 1993). Among the isolates tested for the removal of cholesterol, L. plantarum KJ03 showed the highest cholesterol reduction (52.27 %), followed by L. fermentum KJ23 (49.33 %); the lowest value was found for L. plantarum JCM 1149 (36.73 %). Studies have indicated that many Lactobacillus spp. have cholesterol-reducing effects in vitro or in vivo (Kumar et al. 2012). It was hypothesized that deconjugated bile salts may contribute to lower cholesterol levels as free bile salts may be more readily excreted from the GIT than conju- gated bile salts (Fukushima et al. 1999). However, the exact mechanism of serum cholesterol reduction by probiotic bac- teria is not completely understood (Choi and Chang 2015). Klaver and van der Meer (1993) suggested that in vitro cho- lesterol reduction by some Lactobacillus spp. results from their coprecipitation with deconjugated bile salts. Interestingly, Mann and Spoerry (1974) studied cholesteremia in a tribe of Maasai and found that serum cholesterol levels of Maasai men decreased after consumption of large amounts of milk fermented with a wild Lactobacillus strain. Determination of antibiotic susceptibility The selected strains were tested for their susceptibility to nine antibiotics by the broth microdilution assay. Lactobacillus plantarum KJ03 was found to be susceptible to six antibiotics Table 6 Minimum inhibitory concentrations for selected strains of lactic acid bacteria a −1 Strain Minimum inhibitory concentration (μgmL ) Ampicillin Vancomycin Ciprofloxacin Kanamycin Streptomycin Erythromycin Clindamycin Tetracycline Chloramphenicol Lactobacillus plantarum KJ03 2 >64 16 16 8 <0.5 <0.125 32 8 Break point for Lactobacillus plantarum 2 Not required – 64 Not required 1 2 32 8 Lactobacillus fermentum KJ23 1 >64 32 16 8 <0.5 <1.25 32 4 Breakpoint for Lactobacillus fermentum 2 Not required – 32 64 1 1 8 4 Strains with a minimum inhibitory concentration lower than or equal to the breakpoints are considered here to be susceptible Breakpoints are according to the guidelines of the European Food Safety Authority (2012) 34 Ann Microbiol (2017) 67:25–36 (erythromycin, chloramphenicol, ampicillin, kanamycin, tet- collagen protein (ace), aggregation substances (agg and asa), racycline, clindamycin) but not to streptomycin and vancomy- enterococcal surface protein (esp) and gelatinase (gelE)were cin. Lactobacillus fermentum KJ23 was also susceptible to six absent, whereas the expected PCR products were observed for antibiotics (erythromycin, chloramphenicol, ampicillin, kana- Enterococcus faecalis 13–5and E. faecalis VanB as positive mycin, streptomycin, clindamycin) but not to tetracycline ac- control (Table 7). The positive control also exhibited negative cording to the breakpoint levels suggested by European Food results for cell wall-adhesion (Cyl) due to the low levels or Safety Authority (2012). All strains were resistant to cipro- downregulation of gene expression or an inactive gene floxacin in accordance with the Scientific Committee for product. Animal Nutrition (2002). However, they were susceptible to It is well known that virulence of microorganisms is regu- the breakpoints values proposed by Danielsen and Wind lated by virulence coding genes present on the genome in (2003) who reported relevant microbiological breakpoints special regions. Several putative virulence factors have been for a wider range of Lactobacillus species (Table 6). Strains described which cause serious disease in humans, such as KJ03 and KJ23 showed resistance to vancomycin at the aggregation substance protein, gelatinase, cytolysin, entero- highest amount tested (64 μg). Since vancomycin is an anti- coccal surface proteins, accessory colonization factors and biotic belonging to glycopeptide antibiotics, it inhibits the endocarditis antigens (Moraes et al. 2012). The verification synthesis of peptidoglycan which is an important structural of virulence factors by molecular procedures is important component of the bacterial cell wall. Therefore, Gram- due to the risk of genetic transfer because these genes are positive bacteria, including LAB, are especially vulnerable usually located in conjugative plasmids (Eaton and Gasson to vancomycin treatment (Reynolds 1989). Moreover, resis- 2001). tance to vancomycin has been reported as a natural or an intrinsic property of many LAB (Gotcheva et al. 2002). The Lactobacillus spp. exhibited resistance against inhibi- Conclusion tion of nucleic acid synthesis (ciprofloxacin), resulting in its natural resistance, which may be inherent to a bacterial genus The results of our study demonstrate that L. plantarum KJ03 or species, but may also be acquired through an exchange of isolated from fermented stinky bean possesses desirable genetic material, mutations and the incorporation of new in vitro functional properties that are similar or superior to genes (Ammor et al. 2007). Moreover, a drawback to antibi- those of the reference probiotic strain, L. plantarum subsp. otic resistance is that a transfer of antibiotic resistance genes is plantarum JCM 1149. Strain KJ03 was able to survive and possible because antibiotic resistance genes are generally car- establish in an environment similar to the human GIT, inhibit ried on plasmids, which can be transferred to other bacteria by potential pathogenic bacteria under the safety of Thai means of conjugation (Cebeci and Gurakan 2003). This may Community Product Standard (317/2004), and considered to result in pathogenic bacteria possessing a high level of antibi- be safe to be used with regard to their antibiotic resistance otic resistance. Owing to tetracycline resistance of pattern. Therefore, L. plantarum KJ03 can be considered as L. fermentum KJ23, we selected L. plantarum KJ03 as a po- a starter culture to improve the quality of Sataw-Dong.A tential LAB for further starter culture applications. probiotic potential is expected to greatly enhance the already important nutritional value of stink bean, which is regarded as Detection of virulence genes Virulence factors can be crucial a source of organic acids, vitamins, and minerals. for strain pathogenicity depending on their type. The presence Development of a functional product may indeed convey a of virulence genes in both LAB strains KJ03 and KJ23 was favorable impact in rural economy, especially knowing that investigated using PCR technology. The genes for adhesion such product originates from less well-developed regions. Table 7 The evaluation of Isolates Virulence genes virulence genes in strains of lactic acid bacteria esp ace asa1 gelE efaAfs CylA CylB Lactobacillus plantarum KJ03 −− − − − − − Lactobacillus fermentum KJ23 −− − − − − − Enterococcus faecalis VanB − ++ + + −− Enterococcus faecalis 13-5 + ND ND ND ND ND ND The results are presented as: +, Positive; −, negative; ND, not detected See Table 1 fordefinitionofgenes Ann Microbiol (2017) 67:25–36 35 Acknowledgments This work has been financially supported by the Font de Valdez G, de Giori GS, Garro M, Mozzi F, Oliver G (1990) Lactic Thailand Research Fund through the Royal Golden Jubilee Ph.D. acid bacteria from naturally fermented vegetables. Microbiol Alim Program (Grant No. PHD/0049/2554) and the Graduate School of Nutr 8:175–179 Prince of Songkla University. Fukushima M, Fujii S, Yoshimura Y et al (1999) Effect of rice bran on intraintestinal fermentation and cholesterol metabolism in cecectomized rats. 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Annals of Microbiology – Springer Journals

Published: Sep 17, 2016

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