Genome sequences and functional analysis of Levilactobacillus brevis LSF9-1 and Pediococcus acidilactici LSF1-1 from fermented fish cake (Som-fak) with gamma-aminobutyric acid (GABA) production.

Gamma-aminobutyric acid (GABA) is a crucial inhibitory neurotransmitter in the sympathetic nervous system that exerts regulatory effects on the blood, immune, and nervous systems. GABA production in som-fak, a traditional fermented fish of Thailand, has been attributed to the activity of lactic acid bacteria (LAB). The present study aims to characterize the LAB isolates and compare the genomes and GABA synthesis genes of selected isolates capable of GABA production. Thirteen isolates demonstrating GABA synthesis capability were identified based on their phenotypic and genotypic characteristics. Seven isolates (group I: LSF3-3, LSF8-3, LSF9-1, LSF9-3, LSF9-6, LSF9-7, and LSF10-14) were identified as Levilactobacillus brevis with 99.78-100% similarity. LSF2-1, LSF3-2, LSF5-4, and LSF6-5 (group II) were identified as Lactiplantibacillus pentosus with 99.86-100% similarity. Strain LSF1-1 (group III) was identified as Pediococcus acidilactici (99.47%), and LSF10-4 (group IV) was identified as Pediococcus pentosaceus with 99.93% similarity. The GABA production of isolates ranged from 0.087 to 16.935 g/L. The maximum production of 16.935 g/L from 3% monosodium glutamate was obtained from strain LSF9-1. Gene and genome analysis revealed that L. brevis LSF9-1 has multiple gad genes in the genome, such as gadB1, gadB2, gadC1, and gadC2, making it the potential strain for GABA production. Additionally, the genome analysis of P. acidilactici LSF1-1 consists of gadA, gadB, and gadC, which respond to controlling GABA production and export. Furthermore, strain LSF1-1 was considered safe, containing no virulence factors. Thus, Levilactobacillus brevis LSF9-1 and Pediococcus acidilactici LSF1-1 have the potential for GABA production and probiotic use in future studies.

Terrilactibacillus tamarindi sp. nov., isolated from bark of Tamarindus indica.

A Gram-stain-positive, catalase-positive, facultatively anaerobic, terminal-spore-forming rod, designated strain BCM23-1T, was isolated from bark of Tamarindus indica collected from Chiang Mai Province, Thailand. This strain produced d-lactic acid from glucose. It grew at 20-45 °C (optimum, 30 °C), pH 3.5-9 (optimum, pH 7.0) and in the presence of 1-4 % (w/v) NaCl. The cell-wall peptidoglycan contained meso-diaminopimelic acid (A1γ). The major isoprenoid quinone was menaquinone 7 (MK-7). Polar lipids analysis revealed the presence of diphosphatidylglycerol, phosphatidylglycerol, an unidentified aminophospholipid, an unidentified phospholipid and an unidentified lipid. The predominant cellular fatty acids were anteiso-C17 : 0, anteiso-C15:0, and iso-C16 : 0 when cultivated on GYP agar plates. The 16S rRNA gene sequence similarity between strain BCM23-1T and Terrilactibacillus laevilacticus NK26-11T was 98.3 %. The draft genome of BCM23-1T was 3.24 Mb in size and contained 3088 coding sequences with an in silico DNA G+C content of 37.1 mol%. The values of ANIb, ANIm and digital DNA-DNA hybridization between strain BCM23-1T and T. laevilacticus NK26-11T were 89.9, 90.8 and 40.4 %, respectively. The results of phenotypic and chemotaxonomic, 16S rRNA gene sequence similarity, and whole genome analyses support strain BCM23-1T as representing a novel species of Terrilactibacillus for which the name Terrilactibacillus tamarindi sp. nov. is proposed. The type strain is BCM23-1T (=LMG 31662T=JCM 33748T=TISTR 2841T).

Anaerobic flora, Selenomonas ruminis sp. nov., and the bacteriocinogenic Ligilactobacillus salivarius strain MP3 from crossbred-lactating goats.

This study aimed to examine the distribution of anaerobic bacteria in the rumen fluid of Thai crossbred goats and to screen potential probiotic strains capable of producing antimicrobial compounds and inhibiting bacteria that cause milk fat depression. Thirty-four strains of bacteria from the rumen fluid were divided into 13 groups within 12 genera based on 16S rRNA gene sequences. The RF1-5 and RF5-12 were identified as Streptococcus luteliensis and Bacillus licheniformis, respectively, and demonstrated non-ropy exopolysaccharide. Furthermore, mPRGC5T was closely related to Selenomonas caprae JCM 33725 T (97.8% similarity) based on 16S rRNA gene sequences. It exhibited low average nucleotide identity, digital DNA-DNA hybridization, and average amino acid identity values with related type strains ranging from 84.9 to 86.0%, 21.3 to 21.8%, and 73.8 to 76.1%, respectively. The genotypic and phenotypic characteristics of mPRGC5T strongly support this strain as a new species of the genus Selenomonas for which the name Selenomonas ruminis mPRGC5T was proposed. The type strain is mPRGC5T (= JCM 33724 T = KCTC 25177 T). Ligilactobacillus salivarius MP3 showed antibacterial activity against Cutibacterium acnes subsp. acnes DSM 1897 T and Kocuria rhizophila MIII. The enterolysin A cluster gene was identified in its genome. The auto-aggregation of L. salivarius MP3 was 93.6 ± 0.2%. Additionally, co-aggregation of L. salivarius MP3 with C. acnes DSM 1897 T and K. rhizophila MIII had 92.2 ± 3.4% and 87.3 ± 4.5%, respectively. The adhesion capacity of strain MP3 was 76.11 ± 2.2%. Probiogenomic analysis revealed that L. salivarius MP3 was nonhazardous to animal supplementation and included acid- and bile-tolerant ability. However, strain MP3 contained three antibiotic resistance genes. Thus, the supplementation of L. salivarius MP3 could increase the milk fat content by suppressing C. acnes DSM 1897 T with antibiotic resistance gene horizontal transfer awareness.

Genomic Assessment of Potential Probiotic Lactiplantibacillus plantarum CRM56-2 Isolated from Fermented Tea Leaves.

Lactiplantibacillus plantarum is a widely studied species known for its probiotic properties that can help alleviate serum cholesterol levels. Whole-genome sequencing provides genetic information on probiotic attributes, metabolic activities and safety assessment. This study investigates the probiotic properties of strain CRM56-2, isolated from Thai fermented tea leaves, using Whole-Genome Sequencing (WGS) to evaluate the safety, health-promoting genes and functional analysis. Strain CRM56-2 showed bile salt hydrolase (BSH) activity, assimilated cholesterol at a rate of 75.94%, tolerated acidic and bile environments and attached to Caco-2 cells. Based on ANIb (98.9%), ANIm (99.2%), and digital DNA-DNA hybridisation (98.3%), strain CRM56-2 was identified as L. plantarum. In silico analysis revealed that it was not pathogenic and contained no antibiotic-resistance genes or plasmids. L. plantarum CRM56-2 possessed genes linked to several probiotic properties and beneficial impacts. The genome of strain CRM56-2 suggested that L. plantarum CRM56-2 is non-hazardous, with potential probiotic characteristics and beneficial impacts, which could enhance its probiotic application. Consequently, L. plantarum CRM56-2 demonstrated excellent cholesterol-lowering activity and probiotic properties.

Comparative genomic analysis and optimization of astaxanthin production of Rhodotorula paludigena TL35-5 and Rhodotorula sampaioana PL61-2.

Astaxanthin is a powerful antioxidant known to enhance skin, cardiovascular, eye, and brain health. In this study, the genome insights and astaxanthin production of two newly isolated astaxanthin-producing yeasts (TL35-5 and PL61-2) were evaluated and compared. Based on their phenotypic and genotypic characteristics, TL35-5 and PL61-2 were identified as basidiomycetous yeasts belonging to Rhodotorula paludigena and Rhodotorula sampaioana, respectively. To optimize astaxanthin production, the effects of cultural medium composition and cultivation conditions were examined. The optimal conditions for astaxanthin production in R. paludigena TL35-5 involved cultivation in AP medium containing 10 g/L glucose as the sole carbon source, supplemented with 1.92 g/L potassium nitrate, pH 6.5, and incubation at 20°C for 3 days with shaking at 200 rpm. For R. sampaioana PL61-2, the optimal medium composition for astaxanthin production consisted of AP medium with 40 g/L glucose, supplemented with 0.67 g/L urea, pH 7.5, and the fermentation was carried out at 20°C for 3 days with agitating at 200 rpm. Under their optimal conditions, R. paludigena TL35-5 and R. sampaioana PL61-2 gave the highest astaxanthin yields of 3.689 ± 0.031 and 4.680 ± 0.019 mg/L, respectively. The genome of TL35-5 was 20,982,417 bp in length, with a GC content of 64.20%. A total of 6,789 protein-encoding genes were predicted. Similarly, the genome of PL61-2 was 21,374,169 bp long, with a GC content of 64.88%. It contained 6,802 predicted protein-encoding genes. Furthermore, all essential genes involved in astaxanthin biosynthesis, including CrtE, CrtYB, CrtI, CrtS, and CrtR, were identified in both R. paludigena TL35-5 and R. sampaioana PL61-2, providing evidence for their ability to produce astaxanthin.

Probiogenomic Analysis of Lactiplantibacillus sp. LM14-2 from Fermented Mussel (Hoi-dong), and Evaluation of its Cholesterol-lowering and Immunomodulation Effects.

Lactiplantibacillus sp. LM14-2, isolated from Thai-fermented mussel (Hoi-dong), showed attractive probiotic properties. This strain was identified as Lactiplantibacillus plantarum based on its phenotypic, chemotaxonomic, and genetic characteristics including whole-genome sequencing (WGS). The draft genome sequence was analyzed and annotated for the molecular mechanisms involved in the safety assessment, the adaptation and adhesion of L. plantarum LM14-2 to the gastrointestinal tract (GIT), and the beneficial genes involved in bacteria-host interactions. The L. plantarum LM14-2 exhibited bile salt hydrolase (BSH) activity, assimilated cholesterol at 86.07 ± 5.03%, stimulated the secretion of interleukin-12, interferon-gamma, and human beta defensin-2, and induced nitric oxide production. In addition, L. plantarum LM14-2 showed excellent gastrointestinal tolerance and adhesion ability to Caco-2 cells. Furthermore, the in silico analysis showed that L. plantarum LM14-2 was a non-human pathogen and did not contain antibiotic resistance genes or plasmids. L. plantarum LM14-2 also contained potential genes associated with various probiotic characteristics and health-promoting effects. Consequently, this study suggested that L. plantarum LM14-2 could be considered safe, with potential probiotic properties and health-promoting impacts, which could facilitate its probiotic application.

Characterization of Lactic Acid Bacteria from Fermented Fish (pla-paeng-daeng) and Their Cholesterol-lowering and Immunomodulatory Effects.

The cholesterol-lowering and immunomodulatory effects and probiotic properties of 25 lactic acid bacteria (LAB) isolated from fermented fish (pla-paeng-daeng) in Thailand were examined in the present study. Based on their phenotypic and genetic characteristics, LAB were identified as Lactiplantibacillus pentosus (Group I, 6 isolates), Lactiplantibacillus argentoratensis (Group II, 1 isolate), Limosilactobacillus fermentum (Group III, 2 isolates), Companilactobacillus pabuli (Group IV, 4 isolates), Companilactobacillus farciminis (Group V, 5 isolates), Companilactobacillus futsaii (Group VI, 6 isolates), and Enterococcus lactis (Group VII, 1 isolate). Lactiplantibacillus pentosus PD3-1 and PD9-2 and Enterococcus lactis PD3-2 exhibited bile salt hydrolase (BSH) activities. The percentage of cholesterol assimilated by all isolates ranged between 21.40 and 54.07%. Bile salt hydrolase-producing isolates tolerated acidic and bile conditions and possessed adhesion properties. They also exerted immunomodulatory effects that affected the production of interleukin-12 (IL-12), interferon-γ (IFN-γ), human ß-defensin-2 (hBD-2), and nitric oxide (NO). These isolates meet standard probiotic requirements and exert beneficial effects.

Diversity, astaxanthin production, and genomic analysis of Rhodotorula paludigena SP9-15.

Astaxanthin is a carotenoid known for its powerful antioxidant properties. This study focused on isolating yeast strains capable of producing astaxanthin from flower and fruit samples collected in Thailand. Out of 115 isolates, 11 strains were identified that produced astaxanthin. Molecular identification techniques revealed that these isolates belonged to two species: Rhodotorula paludigena (5 isolates) and Rhodosporidiobolus ruineniae (6 isolates). Whole-genome analysis of one representative strain, R. paludigena SP9-15, identified putative candidate astaxanthin synthesis-associated genes, such as CrtE, CrtYB, CrtI, CrtS, CrtR, CrtW, CrtO, and CrtZ. High-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) confirmed astaxanthin production. Further optimization of astaxanthin production was carried out by investigating the effects of various factors on the growth rate and astaxanthin production. The optimal conditions were 40 g/L glucose as a carbon source, pH 7.5, and cultivation at 25 °C with 200 rpm for 3 days. Under these conditions, R. paludigena SP9-15 synthesized biomass of 11.771 ± 0.003 g/L, resulting in astaxanthin with a content of 0.558 ± 0.018 mg/g DCW (dry cell weight), an astaxanthin yield of 6.565 ± 0.238 mg/L, and astaxanthin productivity of 2.188 ± 0.069 g/L/day. These findings provide insights into astaxanthin production using red yeast strains from Thailand and highlight the potential of R. paludigena SP9-15 for further application.

Functional genome analysis and anti-Helicobacter pylori activity of a novel bacteriocinogenic Lactococcus sp. NH2-7C from Thai fermented pork (Nham).

Helicobacter pylori, linked to gastric diseases, is targeted for probiotic treatment through bacteriocin production. Bacteriocins have gained recognition for their non-toxic effects on host cells and their ability to combat a wide range of pathogens. This study aimed to taxonomically characterize and evaluate the safety and probiotic properties of the novel species of Lactococcus sp. NH2-7C isolated from fermented pork, as well as its bacteriocin NH2-7C, both in vitro and in silico. Comparative genotypic analysis revealed an average nucleotide identity of 94.96%, an average amino acid identity of 94.29%, and a digital DNA-DNA hybridization value of 63.80% when compared to Lactococcus lactis subsp. lactis JCM 5805T. These findings suggest that strain NH2-7C represents a novel species within the genus Lactococcus. In silico assessments confirmed the non-pathogenic nature of strain NH2-7C and the absence of genes associated with virulence and biogenic amine formation. Whole-genome analysis revealed the presence of the nisA gene responsible for nisin A production, indicating its potential as a beneficial compound with anti-Helicobacter pylori activity and non-toxic characteristics. Probiotic assessments indicated bile salt hydrolase and cholesterol assimilation activities, along with the modulation of interleukin-6 and tumour necrosis factor-α secretion. Strain NH2-7C demonstrated gastrointestinal tolerance and the ability to adhere to Caco-2 cells, affirming its safety and probiotic potential. Additionally, its ability to produce bacteriocins supports its suitability as a functional probiotic strain with therapeutic potential. However, further in vitro and in vivo investigations are crucial to ensure its safety and explore potential applications for Lactococcus sp. NH2-7C as a probiotic agent.

Distribution, cholesterol-lowering and immunomodulation effects of lactic acid bacteria from fermented mussel (Hoi-dong).

Forty-eight lactic acid bacteria (LAB) isolated from fermented mussels in Thailand were evaluated for their probiotic properties, bile salt hydrolase (BSH), cholesterol assimilation and immunomodulatory effects. They were identified as Companilactobacillus formosensis (Group I, 10 isolates), Lentilactobacillus buchneri (Group II, 8 isolates), Lactiplantibacillus plantarum subsp. plantarum (Group III, 16 isolates), Lacticaseibacillus rhamnosus (Group IV, 1 isolate), Pediococcus pentosaceus (Group V, 5 isolates) and P. acidilactici (Group V, 1 isolate), Enterococcus thailandicus (Group VI, 2 isolates), En. hirae (Group VII, 1 isolate), En. durans (Group VI, 1 isolate), Lactococcus lactis subsp. lactis (Group VII, 1 isolate), Lc. lactis subsp. hordinae (Group VII, 1 isolate), and Leuconostoc lactis (Group VIII, 1 isolate), based on their phenotypic and genetic characteristics. Seven isolates, L. plantarum subsp. plantarum LM6-1, LM6-2, LM7-2-2B, LM12-1, LM14-1, LM15-1P and LM15-2 expressed bile salt hydrolase activity. All isolates assimilated cholesterol ranging from 20.73 to 79.40%. BSH-producing isolates were tolerant to acidic and bile conditions and showed the adhesion ability to Caco-2 cells. The BSH-producing and selected isolates showed the immunomodulatory effects to stimulate interleukin-12 (IL-12), interferon-gamma (IFN-γ), human beta defensin-2 (hBD-2) and nitric oxide (NO) production at various levels. Therefore, these results indicated that the isolates meet the standard probiotic criteria and beneficial effects.

Draft genome sequencing data of the moderately halophilic bacterium, Allobacillus halotolerans SKP2-8 from shrimp paste (ka-pi).

A moderately halophilic, Gram-stain-positive, spore-forming rod-shaped bacterium, designated SKP2-8 was isolated from a traditional fermented shrimp paste (Ka-pi) collected from the market in Samut Sakhon province, Thailand. This isolate SKP2-8 was closely related to Allobacillus halotolerans LMG 24826T with 99.56% similarity based on 16S rRNA gene sequence. The draft genome of SKP2-8 was 2.53 Mb with 2,515 coding sequences with an average G+C content of 39.5 mol%. The ANIb, ANIm, AAI and the digital DNA-DNA hybridization values of isolate SKP2-8 were 97.22%, 97.64%, 97.75% and 78.0%, respectively, compared with A. halotolerans LMG 24826T. Based on the phenotypic characteristics, DNA-DNA relatedness and phylogenomic analysis, it was identified as Allobacillus halotolerans. The genome sequence data of this isolate provide information for further analysis of the potential biotechnological use of this microorganism and guide the characterization. The draft genome was deposited at DDBJ/ EMBL/GenBank (DNA Databank of Japan/European Molecular Biology Laboratory/Genbank) (VMHF00000000).