Enhanced Production of Gamma-Aminobutyric Acid (GABA) from Lactobacillus futsaii CS3 Using Agri-Food Industries By-Products Under Batch and Fed-Batch Fermentation.

Gamma-aminobutyric acid (GABA) has diverse physiological functions, but its production by lactic acid bacteria is costly due to the culture medium. This study aimed to enhance GABA production by L. futsaii CS3 using low-cost substrates and agri-food industries by-products. Optimal culture conditions were determined using response surface methodology with a central composite design (CCD). Batch and fed-batch fermentation techniques were employed. In the MRS medium with 2% (w/v) monosodium glutamate (MSG), L. futsaii CS3 produced 6.84 g/l of GABA. Further optimization revealed that 2% (w/v) cane sugar resulted in a maximum GABA production of 9.6 g/l, while cane molasses yielded 7.4 g/l. The modified MRS medium with 2% (w/v) MSG, 2% (w/v) cane sugar, 3.06% (w/v) tuna condensate, and 2.5% (w/v) surimi washing water exhibited the highest GABA concentration of 11 g/l. Surimi washing water had a lower GABA concentration of 4.12 g/l. Critical factors identified through CCD analysis were cane sugar, tuna condensate, and MSG. The optimized modified MRS medium consisted of 3.48% (w/v) cane sugar, 3.84% (w/v) tuna condensate, and 10.77% (w/v) MSG, resulting in an actual GABA concentration of 18.27 g/l. Under flask-scale and batch fermentation conditions (initial pH 5, temperature 37 °C), GABA concentrations of 20.63 g/l and 17.24 g/l were obtained after 48 h, respectively. In fed-batch fermentation, GABA concentrations reached 23.01 g/l at 72 h. The addition of cane sugar and tuna condensate effectively enhanced GABA production in L. futsaii CS3, highlighting their suitability as cost-effective substrates for industrial-scale GABA production.

Improved Survival of Freeze-Dried Lactobacillus pentosus SY130 and Applied as a Co-culture Starter with Lactobacillus plantarum KJ03 for Fermenting Stink Bean (Sataw-Dong).

Sataw-Dong, generally produced by spontaneous fermentation of mature stink beans. Monosodium glutamate effectively increases the viability of freeze-dried Lactobacillus pentosus SY130, which is able to inhibit yeast film formation in Sataw-Dong fermentation. For storage stability, the viability of freeze-dried L. pentosus SY130 and Lactobacillus plantarum KJ03, which can reduce the fermentation time of Sataw-Dong depended on storage temperature. Sataw-Dong produced by a co-culture of L. plantarum KJ03 and L. pentosus SY130 showed the highest overall acceptance score in sensory analysis. In addition, plate counts revealed lower counts of yeast and mold, and Enterobacteriaceae in inoculated Sataw-Dong, compared to the spontaneous Sataw-Dong. The main volatile organic compounds in all Sataw-Dong samples was sulfur and ketone groups. The results indicate that using a blend of the freeze-dried co-culture can be successfully used as a starter culture in Sataw-Dong fermentation, and could be further applied with other vegetables to make value-added fermented products.

Microbiota dynamics and volatilome profile during stink bean fermentation (Sataw-Dong) with Lactobacillus plantarum KJ03 as a starter culture.

Sataw-Dong is a fermented stink bean in brine, made with Parkia speciosa subjected to spontaneous fermentation. This study aimed to investigate the impact of Lactobacillus plantarum KJ03 as a starter culture during Sataw-Dong fermentation and to determine its effect on the volatilome profile. Two fermentations were performed: spontaneous and inoculated with starter. The surface of the beans and the brines were separately analyzed throughout fermentation period for 15 days. Inoculated samples clearly showed a significantly higher acidification of the brine, reaching a pH of 3.98 within 5 days of fermentation. The microbiota was investigated through 16S amplicon based sequencing and revealed the dominance of Lactobacillus plantarum and Lactobacillus sanfranciscensis throughout the fermentation in both brine and bean samples. The starter used clearly influenced volatile organic compounds (VOCs) profiles. Inoculated samples showed the lowest abundance of Brevundimonas, Corynebacterium, Enterobacteriaceae, Methylobacterium and Klebsiella, compared to the spontaneous fermentation. Correlation between OTUs and VOCs revealed that acids, aldehydes, and alcohols exhibited a positive correlation with L. plantarum and L. sanfranciscencis. Overall aldehydes were mostly produced at the beginning, while acids, alcohols, and ketones at the middle until the end of the fermentation. Sataw-Dong produced with the starter significantly perceived a positive response in the overall acceptance. As shown through microbiological changes, acidification, VOCs and sensory analysis, the successful and accelerated Sataw-Dong fermentation was achieved when using a functional starter strain.

Changes in bacterial diversity associated with bioremediation of used lubricating oil in tropical soils.

Used lubricating oil (ULO) is a widespread contaminant, particularly throughout tropical regions, and may be a candidate for bioremediation. However, little is known about the biodegradation potential or basic microbial ecology of ULO-contaminated soils. This study aims to determine the effects of used ULO on bacterial community structure and diversity. Using a combination of culture-based (agar plate counts) and molecular techniques (16S rRNA gene sequencing and DGGE), we investigated changes in soil bacterial communities from three different ULO-contaminated soils collected from motorcycle mechanical workshops (soil A, B, and C). We further explored the relationship between bacterial community structure, physiochemical soil parameters, and ULO composition in three ULO-contaminated soils. Results indicated that the three investigated soils had different community structures, which may be a result of the different ULO characteristics and physiochemical soil parameters of each site. Soil C had the highest ULO concentration and also the greatest diversity and richness of bacteria, which may be a result of higher nutrient retention, organic matter and cation exchange capacity, as well as freshness of oil compared to the other soils. In soils A and B, Proteobacteria (esp. Gammaproteobacteria) dominated the bacterial community, and in soil C, Actinobacteria and Firmicutes dominated. The genus Enterobacter, a member of the class Gammaproteobacteria, is known to include ULO-degraders, and this genus was the only one found in all three soils, suggesting that it could play a key role in the in situ degradation of ULO-contaminated tropical Thai soils. This study provides insights into our understanding of soil microbial richness, diversity, composition, and structure in tropical ULO-contaminated soils, and may be useful for the development of strategies to improve bioremediation.

Inhibition of Staphylococcus aureus in vitro by bacteriocinogenic Lactococcus lactis KTH0-1S isolated from Thai fermented shrimp (Kung-som) and safety evaluation.

Lactococcus lactis KTH0-1S isolated from Thai traditional fermented shrimp (Kung-som) is able to produce heat-stable bacteriocin and inhibits food spoilage bacteria and food-borne pathogens. The inhibitory effect of bacteriocin remained intact after treatment with different pHs and after heating, but was sensitive to some proteolytic enzymes. Addition of bacteriocin KTH0-1S to Staphylococcus aureus cultures decreased viable cell counts by 2.8 log CFU/ml, demonstrating a bactericidal mode of action. Furthermore, the growth of S. aureus decreased significantly after 12-h co-cultivation with bacteriocinogenic strain. The molecular mass of bacteriocin KTH0-1S was found to be 3.346 kDa after ammonium sulfate precipitation, reversed phase (C8 Sep-Pak), cation-exchange chromatography, RP-HPLC on C8 column and mass spectrometry (MS/MS) analysis. Bacteriocin KTH0-1S was identified as nisin Z using PCR amplification and sequencing. The majority of tested virulence factors were absent, confirming the safety. Evidenced inhibitory effect of this strain, the absence of virulence factors creates the possibility for its application as protective culture to inhibit pathogenic bacteria in the several fermented seafood products.

Enhancement of gamma-aminobutyric acid (GABA) levels using an autochthonous Lactobacillus futsaii CS3 as starter culture in Thai fermented shrimp (Kung-Som).

Gamma-aminobutyric acid (GABA) is a non-proteinogenic amino acid, which has a variety of well-characterized beneficial physiological functions. In order to improve GABA levels and the fermentation process of Thai fermented shrimp (Kung-Som), autochthonous Lactobacillus futsaii CS3 was inoculated as a starter culture into Kung-Som, and its effects on the quality of Kung-Som were studied. The optimal conditions for GABA production in Kung-Som as obtained by response surface methodology (RSM) using a central composite design (CCD) were an inoculum size of roughly 107 CFU/g (X1) of L. futsaii cells together with the addition of 0.5% (w/w) monosodium glutamate (MSG) (X2), resulting in maximum GABA levels of 10,500 mg per kg fresh product. Under these optimized conditions, the experimental GABA content of Kung-Som with an added starter culture was up to four times higher than that of the control (without starter culture) or commercial Kung-Som products (10,120 mg/kg product). Kung-Som produced by inoculation with L. futsaii CS3 but without addition of MSG showed a considerably increased GABA content of 7790 mg/kg compared to the control. Fermentation time was reduced to less than 1 week for these samples compared to the control batches, which took up to 19 days. Polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) revealed that L. futsaii CS3 remained prominently throughout the Kung-Som fermentation, and that lactic acid bacteria (LAB) rapidly dominated the total microflora because of this inoculation with L. futsaii CS3. Kung-Som samples with starter culture were accepted as well as commercial ones by 30 panelists (p > 0.05). In conclusion, L. futsaii CS3 is a good starter culture for GABA production, resulting in, improved microbiological safety as well as reduced fermentation time.

Lactobacillus futsaii CS3, a New GABA-Producing Strain Isolated from Thai Fermented Shrimp (Kung-Som).

Kung-Som is a popular traditional Thai fermented shrimp product. It is rich in glutamic acid, which is the major substrate for the biosynthesis of gamma-aminobutyric acid (GABA) by lactic acid bacteria (LAB). In the present study, LAB from Kung-Som were isolated, screened for GABA formation, and the two isolates that transform glutamic acid most efficiently into GABA were identified. Based on the API-CHL50 fermentation profile and a phylogenetic tree of 16S rDNA sequences, strain CS3 and CS5 were identified as Lactobacillus futsaii, which was for the first time shown to be a promising GABA producer. L. futsaii CS3 was the most efficient microorganism for the conversion of 25 mg/mL monosodium glutamate (MSG) to GABA, with a maximum yield of more than 99% conversion rate within 72 h. The open reading frame (ORF) of the glutamate decarboxylase (gad) gene was identified by PCR. It consists of 1410 bp encoding a polypeptide of 469 amino acids with a predicted molecular weight of 53.64 kDa and an isoelectric point (pI) of 5.56. Moreover, a good quality of the constructed model of L. futsaii CS3 was also estimated. Our results indicate that L. futsaii CS3 could be of interest for the production of GABA-enriched foods by fermentation and for other value-added products.

Utilization of palm oil decanter cake as a novel substrate for biosurfactant production from a new and promising strain of Ochrobactrum anthropi 2/3.

A biosurfactant-producing bacterium, isolate 2/3, was isolated from mangrove sediment in the south of Thailand. It was evaluated as a potential biosurfactant producer. The highest biosurfactant production (4.52 g/l) was obtained when the cells were grown on a minimal salt medium containing 25 % (v/v) palm oil decanter cake and 1 % (w/v) commercial monosodium glutamate as carbon and nitrogen sources, respectively. After microbial cultivation at 30 °C in an optimized medium for 96 h, the biosurfactant produced was found to reduce the surface tension of pure water to 25.0 mN/m with critical micelle concentrations of 8.0 mg/l. The stability of the biosurfactant at different salinities, pH and temperature and also its emulsifying activity was investigated. It is an effective surfactant at very low concentrations over a wide range of temperatures, pH and salt concentrations. The biosurfactant obtained was confirmed as a glycolipid type biosurfactant by using a biochemical test, fourier-transform infrared spectroscopy, MNR and mass spectrometry. The crude biosurfactant showed a broad spectrum of antimicrobial activity and also had the ability to emulsify oil and enhance polyaromatic hydrocarbons solubility.

An efficient biosurfactant-producing bacterium Selenomonas ruminantium CT2, isolated from mangrove sediment in south of Thailand.

Biosurfactant-producing bacteria, isolate CT2, was isolated from mangrove sediment in the south of Thailand. The sequence of the 16S rRNA gene from isolate CT2 showed 100 % similarity with Selenomonas ruminantium. The highest biosurfactant production (5.02 g/l) was obtained when the cells were grown on minimal salt medium containing 15 g/l molasses and 1 g/l commercial monosodium glutamate supplemented with 1 g/l NaCl, 0.1 g/l leucine, 5 % (v/v) inoculum size at 30 °C and 150 rpm after 54 h of cultivation. The biosurfactant obtained by extraction with ethyl acetate showed high surface tension reduction (25.5 mN/m), a small CMC value (8 mg/l), thermal and pH stability with respect to surface tension reduction and emulsification activity and a high level of salt tolerance. The biosurfactant obtained was confirmed as a lipopeptide by using a biochemical test, FT-IR, MNR and mass spectrometry. The crude biosurfactant showed a broad spectrum of antimicrobial activity and also had the ability to emulsify oil and enhance PAHs solubility.

Isolation and functional characterization of a biosurfactant produced by a new and promising strain of Oleomonas sagaranensis AT18.

Biosurfactant-producing bacteria were isolated from mangrove sediment in southern Thailand. Isolates were screened for biosurfactant production by using the surface tension test. The highest reduction of surface tension was achieved with a bacterial strain which was identified by 16S rRNA gene sequencing as Oleomonas sagaranensis AT18. It has also been investigated using different carbon and nitrogen sources. It showed that the strain was able to grow and reduce the surface tension of the culture supernatant to 25 mN/m. In all 5.30 g of biosurfactant yield was obtained after 54 h of cultivation by using molasses and NaNO3 as carbon and nitrogen sources, respectively. The biosurfactant recovery by chloroform:methanol extraction showed a small critical micelle concentration value (8 mg/l), thermal and pH stability with respect to surface tension reduction. It also showed emulsification activity and a high level of salt concentration. The biosurfactant obtained was confirmed as a glycolipid by using a biochemical test, FT-IR and mass spectra. The crude biosurfactant showed a broad spectrum of antimicrobial activity and also had the ability to emulsify oil and enhance PAHs solubility.

Lactic Acid Bacteria from Gamecock and Goat Originating from Phitsanulok, Thailand: Isolation, Identification, Technological Properties and Probiotic Potential.

From independent swab samples of the cloaca of indigenous gamecocks (CIG), anus of healthy baby goats (AHG), and vagina of goats (VG) originating from Phitsanulok, Thailand, a total of 263 isolates of lactic acid bacteria (LAB) were collected. Only three isolates, designated C707, G502, and V202, isolated from CIG, AHG, and VG, respectively, exhibited an excellent inhibitory zone diameter against foodborne pathogenic bacteria when evaluated by agar spot test. Isolates C707 and G502 were identified as Enterococcus faecium, whereas V202 was identified as Pediococcus acidilactici, based on 16S rRNA sequence analysis. When foodborne pathogenic bacteria were co-cultured with chosen LAB in mixed BHI-MRS broth at 39°C, their growth was suppressed. These LAB were found to be capable of surviving in simulated stomach conditions. Only the isolate G502 was able to survive in the conditions of simulated intestinal juice. This research suggests that selected LAB could be used as a food/feed supplement to reduce foodborne pathogenic bacteria and improve the safety of animal-based food or feed.

No distinction in the gut microbiota between diarrhea predominant-irritable bowel syndrome and healthy subjects: matched case-control study in Thailand.

BACKGROUND: Alteration in the gut microbiota has been proposed in irritable bowel syndrome (IBS) pathogenesis, especially in the diarrheal type (IBS-D). We conducted this study to evaluate the fecal microbiota in Thai IBS-D patients when compared with healthy subjects as well as to evaluate the effects of probiotics on changes in the gut microbiota correlated with symptoms. METHODS: A matched case-control study was conducted on diagnosed IBS-D patients, based on the Rome IV criteria and healthy controls. Stool samples were collected in preservation tubes. Bacterial deoxyribonucleic acid extraction was performed and amplified. Next, 16S ribosomal ribonucleic acid genes sequencing was performed to identify the microbiome in both the groups. IBS-D patients were provided with a probiotic mixture that was rich in Lactobacillus acidophillus and Bifidobacterium bifidum over 8 weeks. Changes in the symptoms, stool characteristics, and fecal microbiota were evaluated and compared with the corresponding baseline values. RESULTS: Twenty IBS-D patients with 20 age and gender-matched controls were included in this study. The baseline characteristics were not significantly different between the groups, including the mode of birth and the history of breastfeeding in infancy. No significant difference was noted in the fecal microbiota between the IBS-D patients and controls. The IBS symptom severity scales (IBS-SSS) were not statistically different after probiotic prescription; although, the bowel movements, the sense of urgency to go to the toilet and passing of mucous stool had obviously decreased. No change was noted in the fecal microbiota after receiving the experimental probiotic, except for an increase in the proportion of B. bifidum. CONCLUSION: Alteration in the gut microbiota composition was probably not the main pathogenic mechanism in the Thai IBS-D patients assessed in this study. However, modifying microbiomes with potentially protective bacteria seems to be a beneficial therapy. Thai Clinical trial registry: TCTR20191211006, Date of registration: 10 Dec 2019. Retrospectively registered, Clinical trial URL: www.clinicaltrials.in.th.

Glutamate Decarboxylase from Lactic Acid Bacteria-A Key Enzyme in GABA Synthesis.

Glutamate decarboxylase (l-glutamate-1-carboxylase, GAD; EC 4.1.1.15) is a pyridoxal-5'-phosphate-dependent enzyme that catalyzes the irreversible α-decarboxylation of l-glutamic acid to γ-aminobutyric acid (GABA) and CO2. The enzyme is widely distributed in eukaryotes as well as prokaryotes, where it-together with its reaction product GABA-fulfils very different physiological functions. The occurrence of gad genes encoding GAD has been shown for many microorganisms, and GABA-producing lactic acid bacteria (LAB) have been a focus of research during recent years. A wide range of traditional foods produced by fermentation based on LAB offer the potential of providing new functional food products enriched with GABA that may offer certain health-benefits. Different GAD enzymes and genes from several strains of LAB have been isolated and characterized recently. GABA-producing LAB, the biochemical properties of their GAD enzymes, and possible applications are reviewed here.

Application of biosurfactant from Sphingobacterium spiritivorum AS43 in the biodegradation of used lubricating oil.

This study aimed at investigating the application of biosurfactant from Sphingobacterium spiritivorum AS43 using molasses as a substrate and fertilizer to enhance the biodegradation of used lubricating oil (ULO). The cell surface hydrophobicity of bacteria, the emulsification activity, and the biodegradation efficiency of ULO were measured. The bacterial adhesion in the hydrocarbon test was used to denote the cell surface hydrophobicity of the used bacterial species. The results indicate a strong correlation between cell surface hydrophobicity, emulsification activity, and the degree of ULO biodegradation. The maximum degradation of ULO (62 %) was observed when either 1.5 % (w/v) of biosurfactant or fertilizer was added. The results also revealed that biosurfactants alone are capable of promoting biodegradation to a large extent without added fertilizer. The data indicate the potential for biosurfactant production by using low-cost substrate for application in the bioremediation of soils contaminated with petroleum hydrocarbons or oils.

Utilization of banana peel as a novel substrate for biosurfactant production by Halobacteriaceae archaeon AS65.

In this study, biosurfactant-producing bacteria was evaluated for biosurfactant production by using banana peel as a sole carbon source. From the 71 strains screened, Halobacteriaceae archaeon AS65 produced the highest biosurfactant activity. The highest biosurfactant production (5.30 g/l) was obtained when the cells were grown on a minimal salt medium containing 35 % (w/v) banana peel and 1 g/l commercial monosodium glutamate at 30 °C and 200 rpm after 54 h of cultivation. The biosurfactant obtained by extraction with ethyl acetate showed high surface tension reduction (25.5 mN/m), a small critical micelle concentration value (10 mg/l), thermal and pH stability with respect to surface tension reduction and emulsification activity, and a high level of salt tolerance. The biosurfactant obtained was confirmed as a lipopeptide by using a biochemical test FT-IR, NMR, and mass spectrometry. The crude biosurfactant showed a broad spectrum of antimicrobial activity and had the ability to emulsify oil, enhance PAHs solubility, and oil bioremediation.

Characterization and phylogenetic analysis of microbial surface active compound-producing bacteria.

Microbial surface active compounds (SACs) were isolated from various environmental sources in Thailand. Isolates were screened for SACs production in different carbon sources (crude glycerol, commercial sugar, decanter, glucose, molasses, used palm oil, and used lubricating oil) by using drop-collapsing test and emulsification activity. Molasses produced the highest number of positive results (23 of 25 isolates). Twenty-one isolate strains produced emulsions with xylene, and 15 exhibited high emulsion-stabilizing capacity, maintaining more than 50 % of the original emulsion volume for 24 h, and six isolate strains reduced the growth medium surface tension to 40 mN/m. The phylogenetic position of these 25 isolates was evaluated by 16S rRNA gene sequence analysis. The production of microbial SACs was determined for strains representative of 16 different bacterial genera, in which ten genera (Blastococcus, Erysipelothrix, Humicoccus, Methylophilus, Microlunatus, Nevskia, Pectinatus, Rubrimonas, Selenomonas, and Stenotrophomonas) were firstly reported as SAC-producing strains. Overall, the new SAC-producing strains isolated in this study display promising features for the future development and use in economically efficient industrial-scale biotechnological processes.

Molasses as a whole medium for biosurfactants production by Bacillus strains and their application.

Two types of biosurfactant (BS)-producing bacteria, Bacillus licheniformis TR7 and Bacillus subtilis SA9, were isolated from mangrove sediment in the south of Thailand. The BS production was done by using only molasses as a whole medium for growth and production. Under optimized conditions, the yields of TR7 and SA9 BS were found to be 3.30 and 3.78 g/l, respectively. It could reduce the surface tension of pure water to 28.5 and 29.5 mN/m, with the critical micelle concentrations of about 10 and 30 mg/l, respectively. Good thermal, pH, and salt stability were exhibited. Both BSs could recover oil more effectively than the two synthetic surfactants. In addition, TR7 and SA9 BS could enhance the solubility of polyaromatic hydrocarbons (PAHs). Thus, these BSs have the potential for the removal of oil and PAHs from the combined contaminated environment and facilitate its bioremediation. These studies indicate that molasses, as a renewable, relatively inexpensive and easily available resource, can be used for important biotechnological processes.

Characterization of an unexpected bioemulsifier from spent yeast obtained from Thai traditional liquor distillation.

Crude biopolymer was extracted from spent yeast, lyophilized and fractionated on Sephadex G-100 to yield two fractions coded as fraction I and II. Fraction I was composed of both carbohydrates and proteins, showing emulsifying activity whereas fraction II consisted of only proteins and possessed no activity. Hence composition and chemical characterization of the purified fraction I (bioemulsifier) was analyzed using various analytical techniques. It was found that the sample contained 96% of carbohydrates consisting mainly of glucose with minor quantities of mannose, and 4% of protein built from 17 amino acids with the highest content of serine followed by alanine. The results also indicated that the sample was protein-bound glucan with the average molecular weight of 1.93×10(5) Da. The functional groups and primary structure of the sample were revealed by FT-IR and NMR techniques. The data demonstrate that the sample comprises a mixture of (1→4)-α- and (1→3)-ß-D-glucans bound with protein. Enzymatic hydrolyses using α-amylase and endo 1,3-ß-D-glucanase confirmed the presence of both glucans. Therefore, this bioemulsifier was identified as glucan-protein complex which is different from usual mannoprotein emulsifier derived from yeasts.

Myroides pelagicus sp. nov., isolated from seawater in Thailand.

An aerobic, Gram-negative, non-motile, yellow-to-orange pigmented and rod-shaped bacterium, designated strain SM1(T), was isolated from seawater in Thailand and subjected to a polyphasic taxonomic study. Phylogenetic analyses based on 16S rRNA gene sequences revealed that the novel isolate shared 93-95 % sequence similarity with species of the genus Myroides. The DNA-DNA relatedness values of strain SM1(T) with Myroides odoratimimus JCM 7460(T) and Myroides odoratus JCM 7458(T) were below 70 %. The DNA G+C content of strain SM1(T) was 33.6 mol%, the major menaquinone was MK-6 and the major fatty acids were C(15 : 0) iso, C(17 : 1)omega9c iso and C(17 : 0) iso 3-OH. Based on its phenotypic and chemotaxonomic characteristics, strain SM1(T) was classified as representing a novel species of the genus Myroides, for which the name Myroides pelagicus sp. nov. is proposed. The type strain is SM1(T) (=IAM 15337(T)=KCTC 12661(T)).