Probiotic and genomic evaluation of Lactiplantibacillus plantarum T31 isolated from tempoyak in mung bean substrate

Comparison between two techniques for endometrial swab culture and between biopsy and culture in barren mares

Abstract. Harahap NIP, Munir E, Hutahaean S. 2023. Immunomodulatory effects of probiotics isolated from traditional fermented foods and beverages of Sumatra (Indonesia) and synbiotics in mice. Biodiversitas 24: 1157-1162. Traditional Sumatran foods and beverages such as sugar palm sap, dadih, and tempoyak have a higher nutritional value than the original ingredients. Lactic Acid Bacteria (LAB), a group of gut bacteria, is the starter that commonly appeared abundantly during fermentation of these natural products. Probiotics are microorganisms that, when consumed in sufficient amounts, provide health benefits to the host, one of which is immunomodulation. When probiotics are coupled with prebiotics to create a unified product known as synbiotics, their function as immunomodulators improves. The goal of this study is to identify new probiotic candidate LAB strains from traditional Sumatran fermented products in combination with inulin as a prebiotic, that can act as immunomodulators in mice through phagocytosis activity. Eight bacterial isolates encoded as NI01, NI02, NI04, DA01, DA02, TE01, TE02 and TE03 were isolated as LAB strains. Four isolates i.e. NI02, NI04, DA01, and TE02 were identified molecularly as Lactiplantibacillus plantarum, Leuconostoc mesenteroides, Lacticaseibacillus paracasei and Lactiplantibacillus plantarum, respectively which were antagonists to human pathogenic bacteria, Escherichia coli and Staphylococcus aureus. The highest phagocytic activity of macrophages was observed through the administration of synbiotics from DA01 + Inulin at 98%, followed by TE02 (96%), NI04 (95%), and NI02 (92%). The findings of this study demonstrated the potential of LAB strains in traditional fermented Sumatran products as probiotic agents and immunomodulators for future application in the health sector.

Indonesia as an archipelagic country is rich in ethnicity and culture. This condition causes Indonesia to have a lot of uniqueness, one of which is traditional food. Traditional foods are varied from traditional non-fermented foods to traditional fermented foods. Based on the essential ingredients, traditional fermented foods from Indonesia can be grouped into 2 types, plant-based, and animal-based fermented foods. Information about plant-based fermented foods has been widely discussed, one of which is tempeh. However, a comprehensive discussion of traditional animal-based fermented food products, especially meat and fish, is rarely found. Therefore, the discussion on this topic can be a new thing to provide information to the public about Indonesia's treasures from a culinary perspective. This review discusses several traditional fermented foods from Indonesia, such as shrimp paste (terasi), urutan, budik, bekasam, naniura, buntilan/bontot, and ikan peda. In most cases, these foods utilize lactic acid bacteria (LAB) for their manufacture. This article is expected to be able to complete information about traditional fermented food products from Indonesia, as well as to promote the treasures of culinary origins from Indonesia to the societies.

This study aimed to isolate Lactiplantibacillus plantarum strains with potent anti-Listeria activity, desirable probiotic properties, and safety from traditional Chinese fermented foods. Initial screening of 102 lactic acid bacteria (LAB) isolates yielded 43 strains inhibitory to Listeria monocytogenes. Eight L. plantarum strains exhibiting strong inhibition zones (>15 mm) were selected for probiotic characterization and safety assessment. Among these, strain Z-5 demonstrated remarkable cell surface properties and a favorable safety profile. Further analysis revealed that strain Z-5 demonstrated significant tolerance to simulated gastrointestinal conditions. Whole-genome sequencing revealed a 3.38 Mb chromosome and identified a complete plantaricin biosynthesis gene cluster encoding Pln A, Pln E, and Pln F. The crude bacteriocin extract exhibited high stability and significantly reduced L. monocytogenes counts in milk stored at 4 °C and 25 °C in a concentration-dependent manner. Our integrated phenotypic and genomic analyses confirm L. plantarum Z-5 as a promising multifunctional probiotic candidate and natural biopreservative for controlling L. monocytogenes in foods.

This study aimed to evaluate and characterize lactic acid bacteria for their probiotic attributes from traditional fermented food, Finger millet batter, and to explore the ability of probiotic lactic acid bacteria to inhibit food borne pathogens. Among all 40 isolates, five bioactive isolates Lactiplantibacillus plantarum MYSRD57, Lacticaseibacillus paracasei MYSRD68, Lactiplantibacillus plantarum MYSDS3, Lactiplantibacillus plantarum MYSDS4 and Lactiplantibacillus plantarum MYSDVN3 with best probiotic attributes were selected and presented in this study. The antimicrobial efficacy of Lactobacillus and their cell free supernatant were effective in controlling pathogens all the foodborne pathogens in vitro. Furthermore, these strains exhibited important probiotic characteristics, such as antibiotics susceptibility, anti-hemolytic activity, tolerance to acidic pH, phenol, sodium chloride, 0.3% bile and survival ability at different temperatures. All the strains were also exhibited high auto-aggregation and cell surface hydrophobicity. Therefore, our findings demonstrated important probiotic features of lactic acid bacteria , while inhibiting multiple pathogens, suggesting possible applications of these potential probiotics strains.

Indonesia has abundant traditional fermented food with various lactic acid bacteria (LAB), which can be developed into probiotics for pharmaceutical and functional food and feed products. This research is aimed at (1) obtaining and identifying LAB isolates and (2) studying the microbiome (bacterial diversity and abundance) of spontaneously-fermented traditional foods of Kalimantan Island, Cincalok, Tempoyak, and Mandai. To obtain LAB isolates, food samples were serially diluted and inoculated on MRS agar that contained 1% CaCO3 (MRSA). Isolates forming clear zones were purified and identified by DNA barcoding. The microbiome was studied using genomic-sequencing techniques and analysed for taxonomic composition. Seven pure isolates were obtained from Cincalok, two Tempoyak, and one Mandai. DNA barcoding revealed that the Cincalok seven isolates were Staphylococcus carnosus (strain HSP-S16), Tetragenococcus halophilus (FSB201), Corynebacterium phoceense, Vagococcus vulneris (SS1995), Enterococcus faecalis (S11-6), Pisciglobus halotolerans (C01), and Priestia filamentosa (P3.1); two from Tempoyak, Levilactobacillus brevis (E1D3BL1) and Lactiplantibacillus plantarum (UMCC-2996); and one from Mandai, Staphylococcus cohnii (XAAS.x13; non-LAB). The T. halophilus, E. faecalis, P. halotolerans, L. brevis, and L. plantarum belong to LAB. The P. halotolerans from Cincalok and non-LAB in these three fermented foods were the first documented report. The microbiome revealed the dominance of Firmicutes phyla in the fermented foods, with 93% in Cincalok, 89.94% in Tempoyak, and 60.32% in Mandai. On the genus level, Cincalok was dominated by Tetragenococcus 40.33%, Anaerococcus 23.29%, Vagococcus 9.27%, and Lactobacillus 6.84%. Meanwhile, Tempoyak was dominated only by Lactobacillus 89.94%. Mandai were dominated by Lactobacillus 31.97%, Proteus 17.14%, Aerococcus 16.85%, Mangrovibacter 15.15%, and Vagococcus 6.2%. However, Mandai's microbiome LAB was not culturable/isolated on MRSA. The plausibility is that those unculturable LAB require coculturing with other bacteria and additional media components to grow on MRSA. This study is the first report regarding the microbiome of Cincalok, Tempoyak, and Mandai, along with their culturable LAB isolates.

This study aims to determine differences in the viability of Lactic Acid Bacteria (LAB) isolates on MRS medium supplemented with various concentrations molasses. Concentration variation molasis used as controls were 0%, 1%, 2% and 3%. Variation of time incubation on each of the media that is 0 hours, 24 hours, 48 hours and 72 hours. The viability of each isolate was viewed with the population of Log (cells/ml) which grow on the medium and the number of generations and time of generations. The results showed that the concentration molasses added to MRS affect the viability of LAB. Concentration of 1% makes LAB better viability than controls. Total population continues to increase at the end of the incubation with a yield of 1.17 generations in time 20.46 hours/generation. In contrast to controls, reduced population size at the end of the incubation so as not to produce a generation. LAB viability in addition molasses 2% and 3% live on 48 hours of incubation. This is indicated by the number of population increased at 48 hours of incubation, and then decreased at the end of incubation. The addition of concentration variation in the types of bacteria showed different viability. At B1 adding molasses 2% and 3% live on 48 hours of incubation and control can only live on a 24 hours incubation, whereas the B3, the control can remain alive at 48 hours of incubation. In contrast to the controls on B4 can survive on 48 hours of incubation, whereas the addition of 3% molasses can only live on a 24 hours incubation. Key words : Lactic Acid Bacteria/LAB, Molasses, Viability

In this study, lactic acid bacteria were isolated from traditional fermented foods in Guizhou. The fermentation supernatant and cell disruption extract were used to screen lactic acid bacteria with α-glucosidase inhibitory activity, and the active lactic acid bacteria strains with potential hypoglycemic effect were screened. The results showed that 10 strains were screened from 27 strains of lactic acid bacteria with α-glucosidase inhibitory activity, of which 5 strains had good acid and bile salt tolerance. The best acid resistance was XG01, and the survival rate was as high as 98.5%. ST01 had the strongest tolerance to bile salt, up to 77.3%. The lactic acid bacteria cell extracts of HLG01, YR01, ST01 and XG01 could better inhibit the activity of α-glucosidase, and the inhibition rates were 39.27, 38.6, 38.53, and 34.4%, respectively. The fermentation supernatant of JR01 could better inhibit the activity of α-glucosidase, and the inhibition rate reached 33.8%. Through molecular biological identification, HLG01, YR01, and ST01 were were Lactiplantibacillus plantarum, XG01 was Pediococcus pentosaceus, and JR01 was Weissella cibaria. Weissella cibaria JR01 is the first reported lactic acid bacterium with α-glucosidase inhibitory activity. Five strains of lactic acid bacteria with good tolerance were screened out, which provided a strain basis for the subsequent study of the hypoglycemic effect of lactic acid bacteria and the development of various functional foods.

Evaluation of new candidate probiotic lactobacillus strains isolated from a traditional fermented food- multigrain-millet dosa batter

The eastern part of Hainan, China, has a flat terrain and a suitable climate with abundant sunshine and rain. This unique environment makes the tropical microbial resources of natural fermented food unique and rich. Therefore, we combined Shotgun metagenomic sequencing, 16S rRNA sequencing and pure culture technology to analyze the microbial diversity, microbiota composition, species differences and correlation of 30 traditional fermented food samples collected from Wenchang, Qionghai, Wanning and Lingshui in the eastern part of Hainan province, and isolated, identified and preserved the microorganisms in them. The results showed that the microbial community structure differs significantly between samples from different regions and between different substrates. The alpha diversity of microorganisms in traditional fermented foods in the Wanning area was higher than those of the other three areas. The beta diversity indicated that the microbiota structural difference between Wanning and Qionghai was smaller. This is consistent with the fact that the precipitation in the Wanning area is the highest and similar to that of Qionghai. The alpha diversity of microorganisms was the highest in fermented vegetables, followed by fermented grains, and the lowest in fermented seafood. Beta diversity showed that the microbiota composition of fermented grains and fermented vegetables is very similar, but that of fermented seafood is significantly different. The results of microbiota structural analysis showed that firmicutes and proteobacteria are the dominant bacterial phyla, and Lactobacillus plantarum and Lactobacillus fermentum are the dominant species in traditional fermented foods in eastern Hainan. Lactic acid bacteria are the dominant species in traditional fermented foods from the eastern Hainan region of China, regardless of the substrate used in fermentation. According to the microbial functional characteristics, the microbial metabolism and biosynthesis pathways in traditional fermented foods in Hainan tend to be active. In addition, combined with pure culture technology, we isolated, identified and preserved 342 lactic acid bacteria strains from traditional fermented food in eastern Hainan province. This study helped us understand the different characteristics of microbial communities in tropical southern China and supplement the Lactobacillus species resource pool in tropical southern China. Moreover, it provided new insights and directions for the development and utilization of fermented foods.

In this study, lactic acid bacteria (LAB) isolation from fermented foods and molecular identification using magnetic bead technology were performed. And then exopolysaccharide (EPS) production possibility was tested in agar medium, and the positive ones were selected for the next step. The bacteria that could produce higher carbohydrate level were grown in MRS medium fortified with whey and pumpkin waste. In our study, 19 different LAB species were identified from fermented products collected from different places in Hatay(Türkiye) province. In molecular identification, universal primer pairs, p806R/p8FPL, and PEU7/DG74 were used for PCR amplification. After that, PCR products purified using paramagnetic bead technology were sequenced by the Sanger sequencing method. The dominant species, 23.8% of the isolates, were identified as Lactiplantibacillus plantarum. As a technological property of LAB, exopolysaccharide production capability of forty-two LAB isolate was tested in agar medium, and after eleven isolates were selected as positive. Two LAB (Latilactobacillus curvatus SHA2-3B and Loigolactobacillus coryniformis SHA6-3B) had higher EPS production capability when they were grown in MRS broth fortified with pumpkin waste and whey. The highest EPS content (1750mg/L glucose equivalent) was determined in Loigolactobacillus coryniformis SHA6-3B grown in MRS broth fortified with 10% pumpkin waste. Besides the produced EPS samples were validated with FTIR and SEM methods.

Lactiplantibacillus plantarum Dad-13, Lactiplantibacillus plantarum FNCC-0250 and Lacticaseibacillus paracasei GMRMP-001 are probiotics strain isolated from Indonesian local and traditional fermented foods. This study aims to evaluate the administration of multi-strain probiotics at dose of 109 CFU/mL/day for 14 days against the diarrhea-causing bacteria Escherichia coli FNCC-0091 at a dose of 1011 CFU/mL/day. Thirty rats were randomized and the divided into 5 groups, including control group, skim milk group, single-strain group, multi-strain group and E. coli group. Antibacterial activity was analyzed in vitro, feed intake, body weight were analyzed, microbial analysis was carried out on feces and cecum, short chain fatty acids were analyzed and intestinal morphology was measured. The result showed that the single-strain and multi-strain inhibition zone in in vitro test produces a very strong inhibitory potential (> 9 mm). The number of L. plantarum and L. paracasei increased and the number of E. coli decreased significantly in the feces and cecum of Rat indicating the survival rate of the probiotic bacteria in the digestive tract and their ability to fight pathogenic bacteria. The concentration of acetic acid, propionic acid and butyric acid compounds also increased, indicating that the digestive tract is at a low pH so that it can inhibit the growth of pathogenic bacteria in rats. In addition, consumption of multi-strain probiotics does not have a negative effect on intestinal morphology. The conclusion is that multi-strain probiotics are able to inhibit pathogenic bacteria in vivo and in vitro. HIGHLIGHTS Multi-strain probiotics isolated from traditional Indonesian fermented foods can reduce the potential for diarrhea. Single-strain and multi-strain have the same effectiveness in inhibiting pathogenic bacteria as seen from the growth of the bacteria. Digestive conditions were damaged in rat that were not given probiotic intervention, but when they received probiotic intervention, digestive conditions improved. GRAPHICAL ABSTRACT

Fermented foods are important parts of traditional food culture with a long history worldwide. Abundant nutritional materials and open fermentation contribute to the diversity of microorganisms, resulting in unique product quality and flavor. Lactic acid bacteria (LAB), as important part of traditional fermented foods, play a decisive role in the quality and safety of fermented foods. Reproduction and metabolic of microorganisms drive the food fermentation, and microbial interaction plays a major role in the fermentation process. Nowadays, LAB have attracted considerable interest due to their potentialities to add functional properties to certain foods or as supplements along with the research of gut microbiome. This review focuses on the characteristics of diversity and variability of LAB in traditional fermented foods, and describes the principal mechanisms involved in the flavor formation dominated by LAB. Moreover, microbial interactions and their mechanisms in fermented foods are presented. They provide a theoretical basis for exploiting LAB in fermented foods and improving the quality of traditional fermented foods. The traditional fermented food industry should face the challenge of equipment automation, green manufacturing, and quality control and safety in the production.

Traditional Bulgarian fermented foods are prominent for their uniqueness of local ingredients, production methods, and endemic microbial species. The present research investigated the diversity and beneficial biological potential of lactic acid bacteria (LAB) isolated from various types of unique Bulgarian fermented foods. Species identification was performed via 16S rDNA sequencing. Biological activity was evaluated by determining antibacterial activity (via agar well diffusion assay), H2O2 production, spectrophotometrically determined auto- and co-aggregation, microbial adhesion to hydrocarbon, and biofilm formation. The biosafety of the isolated lactic acid bacteria was established based on hemolytic activity and phenotypic and genotypic antibiotic susceptibility. Forty-five strains were isolated from fermented foods (sauerkraut, fermented green tomatoes, fermented cucumbers, kefir, white cheese, and Izvara (curdled milk)). Five species were detected: Lactiplantibacillus plantarum, Levilactobacillus koreensis, Levilactobacillus brevis, Lactobacillus helveticus, and Levilactobacillus yonginensis. The most prominent species was L. plantarum, at 47%. For the first time, L. koreensis and L. yonginensis, isolated from unique Bulgarian fermented foods, are reported in this study. The antibacterial effect of the cell-free supernatants was evaluated. An antagonistic effect was observed against Escherichia coli (57%) and Salmonella enterica subsp. enterica serotype Enteritidis (19%) for several L. plantarum strains. Only one L. brevis (Sauerkraut, S15) strain showed activity against E. coli. The best autoaggregation ability at hour 4 was observed for L. koreensis (fermented cucumbers, FC4) (48%) and L. brevis S2 (44%). The highest percentage of co-aggregation with Candida albicans, at hou 4 in the experiments, was observed for strains L. koreensis (fermented green tomato, FGT1) (70%), L. plantarum strains S2 (54%), S13 (51%), and S6 (50%), while at hour 24 for strains L. koreensis FGT1 (95%), L. brevis (Kefir, K7) (89%), L. plantarum S2 (72%), and L. koreensis FC2 (70%). Seven of the isolated LAB strains showed hydrophobicity above 40%. Our results showed that the ability of biofilm formation is strain-dependent. No hemolytic activity was detected. The antibiotic resistance to 10 antibiotics from different groups was tested phenotypically and genotypically. No amplification products were observed in any strains, confirming that the isolates did not carry antibiotic-resistance genes. Traditional fermented Bulgarian foods can be considered functional foods and beneficial LAB sources.

In this study, Lactic acid bacteria were isolated from traditional fermented foods in Guizhou. The fermentation supernatant and cell disruption extract were used to screen Lactic acid bacteria with α-glucosidase inhibitory activity, and the active Lactic acid bacteria strains with potential hypoglycemic effect were screened. The results showed that 10 strains were screened from 27 strains of Lactic acid bacteria with α-glucosidase inhibitory activity, of which 5 strains had good acid and bile salt tolerance. The best acid resistance was XG01, and the survival rate was as high as 98.5%. ST01 had the strongest tolerance to bile salt, up to 77.3%. Through molecular biological identification, HLG01, YR01, and ST01 were Lactiplantibacillus plantarum, XG01 was Pediococcus pentosaceus, and JR01 was Weissella cibaria. Five strains of Lactic acid bacteria with good tolerance were screened out, which provided a strain basis for the subsequent study of the hypoglycemic effect of Lactic acid bacteria and the development of various functional foods.