Characterization, molecular identification and antimicrobial activity of lactic acid bacteria with potentials as halal probiotics isolated from Rinuak fish (Psilopsis sp.) in Lake Maninjau, West Sumatra, Indonesia

Abstract

Background and Objective: The exploration of lactic acid bacteria in integration of specific halal certification is one of the major research topics in the fields of health, food, animal husbandry and agriculture. This study aimed to investigate antimicrobial potentials of probiotic lactic acid bacteria isolated from Rinuak fish (Psilopsis sp.) from Lake Maninjau, Indonesia. Material and Methods: Totally, 15 lactic acid bacteria were isolated from four samples of Rinuak fish (Psilopsis sp.) and investigated for their characteristics as probiotic candidates using conventional laboratory assessments and 16S rRNA sequencing methods. Results and Conclusion: Five isolates were identified as probiotic candidates, including IR2.2, IR2.4, IR4.1, IR4.3 and IR4.5 due to their good resistance of gastric pH ranging 84.24–88.01% and their survival ability against bile salts (resistance of 50.37–57.35%). The IR4.3 was identified to generate the greatest antimicrobial activity against Escherichia coli ATCC 0157, Staphylococcus aureus ATCC 25923 and Salmonella enteridis ATCC 13076 with their diameter of inhibition zone of 22.46, 19.34 and 9.41 mm, respectively. The 16S RRNA sequencing method verified that the lactic acid bacteria isolated from rinuak fish (Psilopsis sp.) included 97.69% similarity to Lactobacillus fermentum strain 4901. This strain promised as an antidiarrheal and antityphoid agent and a natural food preservative appropriate for incorporation into HALAL-compliant foods and pharmaceutical products. Keywords: Food preservative, Endemic Fish, Microbial fermentation, Molecular characterization, Lactobacillus fermentum strain 4901، Indigenous probiotics،Food microbiology،Gastrointestinal health Introduction   Global challenges in various aspects of life are becoming larger. This can be seen from the increasing human population and hence demands for food products are increasing as well; one of which, is fishery products. This fishery product is a basic requirement to meet the needs of protein sources. The protein must be ensured that is halal due to the tauths of the Islamic religion, because Islam emphasizes that maintaining a healthy body by consuming halal foods and drinks is an obligation for every muslim [1]. For halal food development, it is essential to ensure that all ingredients and processes comply with Islamic dietary laws. Lactic acid bacteria (LAB) sourced from permissible animals such as fish and processed without contamination from non-halal substances can be addressed as halal. However, clear certification is warranted for commercial uses. Fermented fish is one of the products from fisheries, which is rich in proteins and LAB [2]. In the fermentation process, microbes and enzymes can stimulate specific flavors, increase the digestibility of food ingredients, decrease the content of anti-nutrients and other undesirable ingredients and produce derivative products and compounds that are beneficial for human life [3]. In general, LAB are food-grade microorganisms. These bacteria can provide flavors to foods, inhibit spoilage bacteria in foods and inhibit pathogenic bacteria. The LAB can be isolated from various sources, especially from fermented foods [4]. In addition, LAB can create an acidic atmosphere, which can decrease the number of pathogen colonies [5]. The existence of selected strains of LAB has demonstrated beneficial effects, as probiotics for humans [6]. The LAB isolation is possible from plant and animal-based products; for example fish, fruits and milk [7].  Rinuak fish (Psilopsis sp.) of Lake Maninjau, Indonesia, is one of those products. Rinuak fish is an domestic fish of Lake Maninjau that includes animal proteins, which is potentially developed due to its high nutritional compounds. The flesh of rinuak fish (Psilopsis sp.) contains proteins of 14.52%, magnesium of 0.21% (in fresh rinuak), phosphorus of 2.4% (in fresh rinuak), water content of 78.62% and ash content of 6.4% (in fresh rinuak). Rinuak fish also contains calcium [8]. The mineral composition especially magnesium and calcium are able to stimulate the bacterial growth. Magnesium (especially in gluconate form) improves probiotic survival, texture, acidity and viability (> 10⁶ CFU g-1) during storage [9]. Research by [10] showed that addition of calcium (calcium carbonate) to fermented feed helped boost growth of Lactiplantibacillus plantarum, Lacticaseibacillus rhamnosus and Bacillus subtilis. Therefore, these mineral nutrients of rinuak fish support the potential of rinuak fish for LAB growth. However, there is a lack of studies that investigate its antimicrobial activity and potential characteristics as probiotics. One of the most important characteristics of LAB is that they can produce the antimicrobial compounds of bacteriocins [11]. Bacteriocins are secondary metabolite products of LAB that include similar actions to antibiotics, being able to inhibit certain bacteria from growing [12]. Previous studies were carried out worldwide to investigate the LAB content in fish. For example, studies on swamp fish fermented with the addition of pure coconut oil resulted in the discovery of LAB with antimicrobial activity [13]. Potential antimicrobials can be achieved from LAB produced by fermentation of rinuak fish (Psilopsis sp.) in Lake Maninjau by isolating and characterizing DNA from LAB through polymerase chain reaction (PCR) analysis of 16s rRNA gene and genome sequencing. Based on the results of this molecular analysis, the phylogenetics of LAB from the fermented rinuak fish (Psilopsis sp.) of Lake Maninjau was investigated. Numerous previous studies were carried out on the nutritient of rinuak and LAB contents of rinuaks. However, study on the antimicrobial potential of fermented rinuak fish and its probiotic characteristics has not been investigated. Therefore, this study aimed to characterize the isolated LAB morphologically and biochemically and carry out theit molecular identification using 16s RNA sequence method. The probiotics effectiveness in this study was assessed on a strain spesific basis. Materials and Methods Research Equipment and Materials Equipment of this study included incubator (Infors HT Ecotorn, USA), anaerobic jars, refrigerator, autoclave (ALP KT40S, Japan), hot plate (Sybron nouveau II, USA), vortex (Labmart 3000, China), analytical balance (Kern ABT 320-4M, Germany), bunsen, microtubes, hockey sticks, inoculation needles, glass slides, pipettes and micropippette tips (DragonLab, BIO-RAD, USA), light microscope (Irmeco, USA), UV-vis spectrophotometer, oven (Memmert UF 100 Plus, Germany), centrifuge (Eppendorf 5417 R, Germany), thin layer chromatography (TLC) chamber and paper chromatograph, capilary tubes, 96-well microplates, microplate reader (PR 4100, BIO-RAD, USA), pH meter, shaker waterbath (IC DK-540b, Japan), PCR equipment (Techne TC-312, UK), spinner down (BIO-RAD, USA) electrophoresis equipment (BIO-RAD, USA), gel documantation imager (BIO-RAD, USA) and labobartory standard glasswares. Materials of this study included rinuak fish sampels collected from Lake Maninjaus, Indonesia, de Mann-Rogosa-Sharpe (MRS) broth and agar (Merck, Germany), distilled water (DW), 70 and 90% ethanol, peptone water, MRS agar (Merck, Germany), 37% HCl (Merck, Germany), ox-gall, nutrient agar (NA), penicillin, kanamycin, ampicillin, NaOH (Merck, Germany), genomic DNA mini kit (Invitrogen Pure-Link, USA) and lysozyme (Invitrogen PureLink, USA) Fermentation of Rinuak Fish (Psilopsis sp.) from Lake Maninjau The process of making rinuak fish fermentation (Psilopsis sp.) was as follows. The ingredients were fish, salt and rice. First, fish was washed and then salt and rice were add to nourish the taste. Samples were transferred into a jar and the lid was close tightly. Jars were stored at room temperature (RT) for 4 d and then dried for 3–5 d [14]. The flowchart of the fermentation process for rinuak fish (Psilopsis sp.) from Lake Maninjau can be seen in Figure 1. Sampling Locations for Rinuak Fish (Psilopsis sp.) Samples of rinuak Fish (Psilopsis sp.) were collected from fishermen in Lake Maninjau at various locations based on the easy access to collection locations, residential areas in the lake area, upstream rivers around the lake, depth of the lake and food sources for rinuak Fish. Sample 1 was collected in a dense settlement and included a traditional market in the Lake Maninjau area. Sample 2 was collected from the middle of the lake with a depth of nearly ±150 M. Sample 3 was collected in a tourist attraction area at Lake Maninjau and Sample 4 was collected in the upstream area of the river, the Batang Sri Antokan River; where, there was the Maninjau Hydroelectric Power Plant. Fish was immediately transferred into a cold box and carried to the Microbiology Laboratory of Stikes Syedza Saintika. Then, isolation and molecular characterization of DNA of LAB were carried out at the Biotechnology laboratory of Andalas University, Indonesia. The location of each sample is illustrated in Table 1 and a map showing the locations for rinuak fish in Lake Maninjau, West Sumatra, Indonesia, is demonstrated in Figure 2. Isolation of Lactic Acid Bacteria from Rinuak Fish (Psilopsis sp) Lake Maninjau, West Sumatra, Indonesia The LAB isolation process began with an enrichment process; 1 g of rinuak fish sample was added into 9 ml of MRS broth and then homogenized to achieve a 10-1 dilution. This was incubated at 37 oC for 24 h. After incubation, 100 µl of the enrichment or 10-1 dilution were added into a microtube with 900 µl of peptone water and then homogenized using vortex to achieve a 10-2 dilution. This procedure was repeated until 10-8 dilutions were achieved. Then, plating process was carried out and the culture from the 10-8 dilution was inoculated on MRS agar and incubated anaerobically at 37 oC for 48 h. Single LAB colonies that grew on the surface of the media were re-purified by inoculating the colony onto MRS agar, incubating anaerobically at 37 oC for 24 h [15]. Characterization of Lactic Acid Bacteria from Rinuak Fish (Psilopsis sp.) in Lake Maninjau, West Sumatra, Indonesia Identification of lactic acid bacteria morphology     Morphological identification was carried out macroscopically on LAB cultures inoculated on MRS agar to identify the shape, color and diameter of LAB isolates. Then, Gram staining was carried out to investigate LAB morphology microscopically by verifying the color and the shape of the cells. Meanwhile, biochemical characterization was carried out using fermentation-type and catalase assays [15]. Selection of Lactic Acid Bacteria from Rinuak Fish (Psilopsis sp) from Lake Maninjau, West Sumatra, Indonesia, as Probiotic Candidates 1) Resistance of Rinuak Fish (Psilopsis sp.) from Lake Maninjau, West Sumatra, Indonesia, to Gastric pH The resistance assessment for gastric pH was carried out based on Kim et al. [16] method with modifications. This assay used two types of media, including MRS broth with addition of 37% HCl to achieve a pH of 2.5 and MRS broth without addition of HCl to maintain a pH of 6.8 (as a control). The medium was sterilized at 121 °C for 15 min using autoclave. Moreover, 5 ml of MRS broth-HCl were added to 0.5 ml of the bacterial isolate and incubated at 37 oC for 3 and 6 h. Then, absorbance was read at 600 nm. This was carried out three times. Resistance was expressed as a percentage. According to [17], percentage of the resistance of LAB isolates can be calculated using the following formula:     2) Resistance of Rinuak Fish (Psilopsis sp.) from Lake Maninjau, West Sumatra, Indonesia, to Bile Salts The resistance assessment of bile salt was carried out based on a method from Gotcheva V et al. [18] with modifications. Various concentrations of bile salt of 0, 0.3 and 0.5% were added to the MRS broth media. The media were autoclaved at 121 °C for 15 min. The bacterial isolate (0.5 ml) was transferred into 5 ml of MRS broth added with 0, 0.3 and 0.5% ox-gall and incubated at 37 oC for 5 h. The MRS broth containing no bile salt was set as the control and compared with the treatments. The LAB growth was assessed using UV spectrophotometry based on the absorbance at 600 nm. All assessments were carried out in three replications. Isolate resistance was expressed as a percentage. According to [19], percentage of the resistance of LAB isolates can be calculated using the following formula:       Screening of Lactic Acid Bacteria from Rinuak Fish (Psilopsis sp.) in Lake Maninjau for their Antimicrobial Potentials The lactic acid bacteria antimicrobial activity assay Three pathogens of Salmonella typhi, Staphylococcus aureus and Escherichia coli were assessed for antimicrobial activity of LAB using modified paper diffusion method [20]. Briefly, 1 ml of LAB culture enriched for 48 h was collected using micropipette and transferred into a sterile microtube. This was centrifuged at 10,000 rpm for 5 min and the supernatant was collected for antimicrobial resistance assessment. Then, 0.4 g of NA media was homogenized by heating using hot plate and sterilizing using autoclave. Then, 40 µl of the bacterial isolate enriched for 24 h were poured into a Petri dish containing 20 ml of NA media and cooled down until the media hardened. The paper disk was soaked in LAB isolate suspension for approximately 10 min. After the agar solidified, the paper disk was set on NA media, which contained isolates of pathogenic bacteria. Then, positive control was set by dropping LAB supernatant (20 µl) onto sterile test papers, including 10 g of penicillin, 30 g of kanamycin and 10 g of ampicillin. This was incubated at 37 °C for 24 h anaerobically. After 24 h, diameter of the inhibition zone was reported using caliper [21]. Antimicrobial Assay of Crude Bacteriocin Supernatant Briefly, 1 ml was cultured in 9 ml of MRS solution at 37 oC and incubated for 2 d. This was centrifuged at 14,000 rpm for 5 min. Then, 0.22-µl membrane filter was used to filter the supernatant. To eliminate the barrier effect because of the presence of organic acids, 1 N NaOH solution was added to the cell-free supernatant to maintain pH 6.5 [21]. The bacterial pathogens were grown aerobically at 37 °C for 24 h. Then, 0.2% pathogenic bacteria were transferred onto 20 ml of MHA solution at 50 °C. After the gelatin was solid, a 6-mm well was made in the media using cork borer. Furthermore, 50 µl of supernatant were transferred into the wells and set 10–15 min. The incubation was carried out at 37 °C for 24 h under aerobic conditions. Antimicrobial activity of bacteriocins in the supernatant was verified by varying the time intervals of 15, 30 and 60 min at 100 ºC. The supernatant of LAB was investigated for its ability in inhibiting bacteria, including E. coli O157, S. aureus ATCC 25923 and Salmonella enteritidis ATCC 13076 via similar methods. The inhibition zone was associated with existence of bacteriocin compounds and its dimension was recorded using caliper [22]. Isolation and Characterization of 16S rRNA Genome isolation was carried out using genomic DNA mini kit. Lysozyme was used at a concentration of 20 mg ml-1 to lyse the bacterial cell walls. The 16S rRNA gene was amplified using selected bacterial genomic DNA kit. Amplification was carried out using reverse primer 1387R (5'-GGGCGGGGTGTACAAGGC-3') and forward primer 63F (5'-CAGGCCTAACACATGCAAGTC-3'). Reactions were carried out in a total volume of 50 μl. The PCR mixture contained 25 μl of DreamTaq Green DNA polymerase (Thermo Fisher Scientific, USA), 22 μl of milli Q water (MQ), 1 μl of the template and 1 μl of each forward or reverse primers (10 μM each, IDT synthesis). Amplification conditions included preheating at 95 °C for 5 min and then denaturation at 95 °C for 30 s, primer annealing at 58 °C for 30 s, 1 min extension step at 72 °C for 35 cycles and post cycling extension for 5 min at 72 °C. The reaction was carried out in a thermal cycler (Biometra T-Personal Thermal Cycler, USA). The amplified DNA was added to GelRed nucleic acid gel stain and electrophoresed on agarose gels [1.5% (w/v) in TBE buffer] at 100 V for 60 min. The amplicons could be visualized using gel documentation imager. The PCR amplification product was purified using absolute ethanol Na-acetate method and then sequenced [23]. The BLAST and Phylogenetic Analysis Phylogenetic analysis method was carried out based on former studies [24]. Sequence data were collected using BioEdit software and then converted to FASTA format. The BLAST was used to carry out sequence analysis (http://www.ncbi.nl-m.nih.gov/blast/cgi). Moreover, DNA sequences were imported into Clustal W2 (http://www.ebi.ac.uk/Tools/clustalw2/) to carry out phylogenetic analysis. A phylogenetic tree was created using BLAST MEGA v.6.0 (http://www.megasoftware.net) and neighbor-joining (NJ) method. Data Analysis Data were present as mean ±SD (standard deviation). Statistical significance was reported using one-way analysis of variance (ANOVA) and SPSS software v.26 (IBM, USA). Tukey post-hoc test was used to assess significant differences between group means, with a p-value of < 0.05 as statistically significant. Results and Discussion Isolation of Lactic Acid Bacteria in Rinuak Fish (Psilopsis sp.) from Lake Maninjau, West Sumatra, Indonesia The LAB selective media (MRS broth and agar), providing appropriate nutrients and pH for LAB growth, were used in the serial dilution-agar plate method. This allowed LAB to grow and reproduce effectively. Serial dilution was necessary to decrease bacterial density, ensuring individual colonies grow separately instead of clustering together. The MRS broth and agar as the sources of nutrition and appropriate pH for LAB growth were used in serial dilution-agar plates to isolate LAB. Serial dilution was needed to decrease density of the inoculated LAB to enable the LAB to develop in colonies independently of one another, rather than in piles [19] (Figure 3). Single colonies that were round, convex, yellowish-white and shiny grew separately with various diameter sizes. These were re-inoculated on MRS agar using streak method to achieve pure isolates of LAB from rinuak fish. These findings were similar to those of Purwati study, which showed colonies of LAB on MRS agar as yellowish-white colonies. Details on the isolation and purification of 15 LAB isolates were as follow: three isolates of IR1 (IR1.1, IR1.2 and IR1.3), five isolates of IR2 (IR2.1, IR2.2, IR2.3, IR2.4 and IR2.5), one isolate of IR3 (IR3) and six isolates of IR4 (IR4.1, IR4.2, IR4.3, IR4.4, IR4.5 and IR4.6). A higher number of LAB isolates (n = 15) was achieved in this study, compared to previous reports, which achieved 9–12 isolates in Tilapia fish [25] and 10–13 isolates in Bilih fish [24]. The observed differences in the diversity of LAB types within the samples might indeed be affected by the environmental conditions at the respective sampling locations. For example, Sample IR.1 was collected from Maninjau Village, an area with high population density and a traditional market, while IR.3 originated from Duo Koto Village, a well-known tourist destination. Such anthropogenic activities in these areas might contribute to pollution of the close lake ecosystem, including household and market waste directly discharged into Lake Maninjau. In contrast, IR.2 and IR.4 were collected from relatively undisturbed mid-lake regions—Tanjung Sani Village and Koto Malintang Village respectively—where the water depth exceeded 100 m and minimal human activity was observed, potentially preserving a further appropriate aquatic environment. However, it is acknowledged that geographic origin alone might not fully responsible for the variations in LAB diversity. Several other ecological and biological factors such as the natural diet of rinuak fish, differences in water temperature, seasonal fluctuations and post-capture handling practices (e.g. time to processing or storage conditions) could play significant roles in shaping the microbiota. These variables might affect selective pressures and survival conditions for specific LAB strains. Further studies incorporating water physicochemical analyses, fish diet profiling and standardized handling procedures are recommended to strengthen the interpretation of LAB diversity patterns. A further holistic approach is necessary to elucidate the multifactorial nature of LAB colonization and persistence in rinuak fish across various environments. Survival of microorganisms depends greatly on environmental conditions and is affected by their food sources [26]. Characterization of Lactic Acid Bacteria in Rinuak Fish (Psilopsis sp.) from Lake Maninjau, West Sumatra, Indonesia                 The morphological characteristics of all isolates were carried out microscopically through Gram staining (Table 2). The LAB is classified as Gram-positive bacteria that can bind crystal violet-iodine complexes and preserve the purple color of their cells [27]. The stages in Gram staining are crystal violet as the primary dye, Lugol solution (KI-I2) as the mordant, 96% alcohol as the decolorizing agent and safranin solution as the counterstain. Crystal violet dissociates in solution into CV+ and Cl-. These ions then penetrate the bacterial membranes and cell walls. Moreover, CV+ ions react with negatively charged bacterial cell components and make bacterial cells (-) react with CV+ purple. Addition of iodine solution (I- or I3) forms a crystal violet-iodine complex (CV-I) in the inner and outer layers of the cells; thereby, strengthening purple color of the bacterial cells [28]. The cell walls of Gram-positive bacteria are in the form of thick fibers consisting of 50–90% peptidoglycan. T