Tipparat Hongpattarakere

Selection of lipase producing yeasts for methanol-tolerant biocatalyst as whole cell application for palm-oil transesterification.

Methanol-tolerant lipase producing yeast was successfully isolated and selected thorough ecological screening using palm oil-rhodamine B agar as one step-approach. All 49 lipase-producing yeasts exhibited the ability to catalyze esterification reaction of oleic acid and methanol at 3 molar equivalents. However, only 16 isolates catalyzed transesterification reaction of refined palm oil and methanol. Rhodotorula mucilagenosa P11I89 isolated from oil contaminated soil showed the strongest hydrolytic lipase activity of 1.2U/ml against palm oil. The production of oleic methyl ester and fatty acid methyl ester (FAME) of 64.123 and 51.260% was obtained from esterification and transesterification reaction catalyzed by whole cell of R. mucilagenosa P11I89 in the presence of methanol at 3 molar equivalents against the substrates, respectively. FAME content increased dramatically to 83.29% when 6 molar equivalents of methanol were added. Application of the methanol-tolerant-lipase producing yeast as a whole cell biocatalyst was effectively resolved major technical obstacles in term of enzyme stability and high cost of lipase, leading to the feasibility of green biodiesel industrialization.

Bifidogenic characteristic and protective effect of saba starch on survival of Lactobacillus plantarum CIF17AN2 during vacuum-drying and storage

Resistant starch (RS) from unripe saba banana (Musa sapientum (Linn)) (Kluai Hin) exhibited high resistance to gastric acid and intestinal amylases. Its bifidogenic effect under competition of human fecal microflora was determined in the simulated proximal region of human colon. In addition, saba RS effectively protected Lactobacillus plantarum CIF17AN2 during drying process. The maximum survival of 85.81% was achieved under vacuum drying operated at 37 °C when saba RS was added. The addition of saba RS to formulate a synbiotic product was able to retain high viability of the vacuum-dried L. plantarum during 8-week storage at ambient temperature. This is because saba RS can stabilize the moisture content of the synbiotic product. In contrast, the dramatic increase of moisture content in the vacuum-dried L. plantarum without saba RS led to significant decrease in cell survival. Moreover, saba RS could potentially protect the vacuum-dried L. plantarum from gastric acid and bile exposures.

An Inducible Operon Is Involved in Inulin Utilization in Lactobacillus plantarum Strains, as Revealed by Comparative Proteogenomics and Metabolic Profiling

ABSTRACT The draft genomes of Lactobacillus plantarum strains isolated from Asian fermented foods, infant feces, and shrimp intestines were sequenced and compared to those of well-studied strains. Among 28 strains of L. plantarum, variations in the genomic features involved in ecological adaptation were elucidated. The genome sizes ranged from approximately 3.1 to 3.5 Mb, of which about 2,932 to 3,345 protein-coding sequences (CDS) were predicted. The food-derived isolates contained a higher number of carbohydrate metabolism-associated genes than those from infant feces. This observation correlated to their phenotypic carbohydrate metabolic profile, indicating their ability to metabolize the largest range of sugars. Surprisingly, two strains (P14 and P76) isolated from fermented fish utilized inulin. β-Fructosidase, the inulin-degrading enzyme, was detected in the supernatants and cell wall extracts of both strains. No activity was observed in the cytoplasmic fraction, indicating that this key enzyme was either membrane-bound or extracellularly secreted. From genomic mining analysis, a predicted inulin operon of fosRABCDXE, which encodes β-fructosidase and many fructose transporting proteins, was found within the genomes of strains P14 and P76. Moreover, pts1BCA genes, encoding sucrose-specific IIBCA components involved in sucrose transport, were also identified. The proteomic analysis revealed the mechanism and functional characteristic of the fosRABCDXE operon involved in the inulin utilization of L. plantarum. The expression levels of the fos operon and pst genes were upregulated at mid-log phase. FosE and the LPXTG-motif cell wall anchored β-fructosidase were induced to a high abundance when inulin was present as a carbon source. IMPORTANCE Inulin is a long-chain carbohydrate that may act as a prebiotic, which provides many health benefits to the host by selectively stimulating the growth and activity of beneficial bacteria in the colon. While certain lactobacilli can catabolize inulin, this has not yet been described for Lactobacillus plantarum, and an associated putative inulin operon has not been reported in this species. By using comparative and functional genomics, we showed that two L. plantarum strains utilized inulin and identified functional inulin operons in their genomes. The proteogenomic data revealed that inulin degradation and uptake routes, which related to the fosRABCDXE operon and pstBCA genes, were widely expressed among L. plantarum strains. The present work provides a novel understanding of gene regulation and mechanisms of inulin utilization in probiotic L. plantarum generating opportunities for synbiotic product development.

Antagonistic Characteristics Against Food-borne Pathogenic Bacteria of Lactic Acid Bacteria and Bifidobacteria Isolated from Feces of Healthy Thai Infants.

Background: Food-borne pathogens are among the most significant problems in maintaining the health of people. Many probiotics have been widely reported to alleviate and protect against gastrointestinal infections through antibacterial secretion. However, the majority of them cannot always play antagonistic roles under gut conditions. Probiotic bacteria of human origin must possess other protective mechanisms to survive, out-compete intestinal flora and to successfully establish in their new host at a significant level. Objectives: Probiotic characteristics of Lactic Acid Bacteria (LAB) and bifidobacteria isolated from the feces of Thai infants were primarily investigated in terms of gastric acid and bile resistances, antibacterial activity and mucin adhesion ability. Antagonistic interaction through secretion of antibacterial compounds and competitive exclusion against food-borne pathogens were also evaluated. Materials and Methods: Culturable LAB and bifidobacteria were isolated from feces of Thai infants. Their ability to withstand gastric acid and bile were then evaluated. Acid and bile salt tolerant LAB and bifidobacteria were identified. They were then further assessed according to their antagonistic interactions through antibacterial secretion, mucin adhesion and competitive mucin adhesion against various food-borne pathogenic bacteria. Results: Gastric acid and bile tolerant LAB and bifidobacteria isolated from healthy infant feces were identified and selected according to their antagonistic interaction against various food-borne pathogenic bacteria. These antagonistic probiotics included four strains of Lactobacillus rhamnosus, two strains of L. casei, five strains of L. plantarum, two strains of Bifidobacterium longum subsp. longum and three strains of B. bifidum. All strains of the selected LAB inhibited all pathogenic bacteria tested through antibacterial secretion, while bifidobacteria showed high level of competitive exclusion against the pathogenic bacteria. Conclusions: These human-derived LAB and bifidobacteria exhibited different mechanisms involved in pathogenic inhibition. Therefore a combination of these probiotic strains could be a great promise and possibility for the development of probiotic products to effectively prevent and control food-borne infection in humans.

Variation of mucin adhesion, cell surface characteristics, and molecular mechanisms among Lactobacillus plantarum isolated from different habitats

The adhesion ability to mucin varied greatly among 18 Lactobacillus plantarum isolates depending on their isolation habitats. Such ability remained at high level even though they were sequentially exposed to the gastrointestinal (GI) stresses. The majority of L. plantarum isolated from shrimp intestine and about half of food isolates exhibited adhesion ability (51.06–55.04%) about the same as the well-known adhesive L. plantarum 299v. Interestingly, five infant isolates of CIF17A2, CIF17A4, CIF17A5, CIF17AN2, and CIF17AN8 exhibited extremely high adhesion ranging from 62.69 to 72.06%. Such highly adhesive property correlating to distinctively high cell surface hydrophobicity was significantly weaken after pretreatment with LiCl and guanidine-HCl confirming the entailment of protein moiety. Regarding the draft genome information, all molecular structures of major cell wall-anchored proteins involved in the adhesion based on L. plantarum WCSF1, including lp_0964, lp_1643, lp_3114, lp_2486, lp_3127, and lp_3059 orthologues were detected in all isolates. Exceptionally, the gene-trait matching between yeast agglutination assay and the relevant mannose-specific adhesin (lp_1229) encoding gene confirmed the Msa absence in five infant isolates expressed distinctively high adhesion. Interestingly, the predicted flagellin encoding genes (fliC) firstly revealed in lp_1643, lp_2486, and lp_3114 orthologues may potentially contribute to such highly adhesive property of these isolates.

Prebiotic efficacy and mechanism of inulin combined with inulin-degrading Lactobacillus paracasei I321 in competition with Salmonella

Inulin efficacy was proven for raising anti-Salmonella activity of the inulin-degrading Lactobacillus paracasei I321 (infant isolate) through the mechanisms of antibacterial secretion and competitive adhesion. Inulin significantly promoted competitive adhesion of L. paracasei I321 against many Gram-negative foodborne pathogenic bacteria. Salmonella Typhimurium SA2093 was completely suppressed under highly competitive niche of fecal microflora when inulin, L. paracasei I321, and their synbiotic combination were applied. Inulin supplementation obviously alleviated nutrient limitation in the mimicked proximal colon leading to complete destruction of Salmonella. This was achieved through the manipulation of fecal bifidobacteria which subsequently altered antibacterial metabolites. Acetate was the major metabolite correlating to high number of fecal bifidobacteria in the presence of inulin, whereas lactate became dominant when L. paracasei I321 was coupled. This inulin-degrading strain was able to persist and dominate in the competitive environment of fecal microflora and functioned synergistically with inulin to completely destroy Salmonella.

Identification of saturated and unsaturated 1-methoxyalkanes from the Thai millipede Orthomorpha communis as potential “Raincoat Compounds”

Mixtures of saturated and unsaturated 1-methoxyalkanes (alkyl methyl ethers, representing more than 45.4% of the millipede hexane extracts) were newly identified from the Thai polydesmid millipede, Orthomorpha communis, in addition to well-known polydesmid defense allomones (benzaldehyde, benzoyl cyanide, benzoic acid, mandelonitrile, and mandelonitrile benzoate) and phenolics (phenol, o- and p-cresol, 2-methoxyphenol, 2-methoxy-5-methylphenol and 3-methoxy-4-methylphenol). The major compound was 1-methoxy-n-hexadecane (32.9%), and the mixture might function as “raincoat compounds” for the species to keep off water penetration and also to prevent desiccation.