Stabilization of Hydroxynitrile Lyases from Two Variants of Passion Fruit, Passiflora edulis Sims and Passiflora edulis Forma flavicarpa, by C-Terminal Truncation.

Because the synthesis of chiral compounds generally requires a broad range of substrate specificity and stable enzymes, screening for better enzymes and/or improvement of enzyme properties through molecular approaches is necessary for sustainable industrial development. Herein, the discovery of unique hydroxynitrile lyases (HNLs) from two species of passion fruits, Passiflora edulis forma flavicarpa (yellow passion fruit, PeHNL-Ny) and Passiflora edulis Sims (purple passion fruit, PeHNL-Np), isolated and purified from passion fruit leaves is reported. These are the smallest HNLs (comprising 121 amino acids). Amino acid sequences of both enzymes are 99 % identical; there is a difference of one amino acid in a consensus sequence. PeHNL-Np has an Ala residue at position 107 and is nonglycosylated at Asn105. Because it was confirmed that natural and glycosylated PeHNL-Ny showed superior thermostability, pH stability, and organic tolerance to that of PeHNL-Np, it has been speculated that protein engineering around the only glycosylation site, Asn105, located at the C-terminal region of PeHNL-Ny, might contribute to the stabilization of PeHNL. Therefore, the focus is on improved stability of the nonglycosylated PeHNL by truncating its C-terminal region. The C-terminal-truncated PeHNLΔ107 was obtained by truncating 15 amino acids from the C terminus followed by expression in Escherichia coli. PeHNLΔ107 expressed in E. coli was not glycosylated, and showed improved thermostability, solvent stability, and reusability similar to that of the wild-type glycosylated form of PeHNL expressed in Pichia pastoris. These data reveal that the lack of the high-flexibility region at the C terminus of PeHNL might be a possible reason for improving the stability of PeHNL.

Longitudinal characterization of bifidobacterial abundance and diversity profile developed in Thai healthy infants.

The early bifidobacterial colonization and development of infant gut is considered crucial for the immediate and lifelong health of human host. This study longitudinally analyzed and characterized fecal bifidobacterial profiles in association with feeding regimens observed in six infants during 5 months after birth. The dominant fecal microbiota of bifidobacteria, lactobacilli/enterococci, clostridia, bacteroides and eubacteria were specifically enumerated using fluorescent in situ hybridization (FISH) technique. Breastfeeding exhibited close association with the predomination of bifidobacteria with the highest relative abundance of 32-70% detected in both infants with exclusive breastfeeding. The nested PCR-DGGE technique revealed high diversity existing within a bifidobacterial species with multiple strain variants of B. bifidum, B. longum, B. breve and B. dentium continuously detected in feces of exclusively breast- and combination-fed infants over the period of 5 months. Contrarily, B. breve, B. adolescentis, B. dentium, B. bifidum, B. faecale, B. kashiwanohense and B. lactis detected in all exclusively formula-fed infants seem to be transient species. The persisting strains seem to derive primarily from maternal breastmilk as demonstrated by PCR-DGGE profiles of human milk and feces from three mother-infant pairs. The results suggested the pivotal role of breastfeeding regimen in supporting colonization and succession of bifidobacteria in infant gut.

Impacts of Prebiotic-Supplemented Diets and Breastmilk on Population and Diversity of Lactobacilli Established in Thai Healthy Infants.

The relative abundance and diversity of lactobacilli present in feces of infants fed with breastmilk and fructooligosaccharide-galactooligosaccharide (FOS-GOS)-, and inulin-galactooligosaccharide (inulin-GOS)-supplemented infant formulae and combination of both were compared. Fecal lactobacilli rapidly colonized and reached maximum total cell counts, which were significantly higher in the infants fed by combining breastmilk with a formula containing either FOS-GOS (C1-A infant) or inulin-GOS (C2-C infant) and the exclusively formula fed ones (F1-F and F2-H infants) than those detected in the exclusively breast-fed (B1-D and B2-E infants) (P < 0.05). The greatest relative abundance of fecal lactobacilli species was observed in all infant receiving prebiotic-containing diets, whereas bifidobacteria appeared predominantly in exclusively breast-fed infants. The species composition of lactobacilli was highly unique among individual and more variable in both groups of infants receiving breastmilk than the exclusively formula-fed infants. Breastmilk seem to be a great source of indigenous lactobacilli vertically transferred and continuously seeded infants' gut. Meanwhile, prebiotic supplementation in infant formulae enhanced and sustained the successful colonization of lactobacilli.

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

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. ß-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 ß-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 ß-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.

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.

Antibacterial metabolites secreted under glucose-limited environment of the mimicked proximal colon model by lactobacilli abundant in infant feces.

The most abundance of anti-Salmonella lactic acid bacteria (LAB) was found in feces of naturally born, exclusively breastfed Thai infants. Six strains of Lactobacillus plantarum and one strain of Lactobacillus paracasei were selected and identified. In the co-cultivation assay, L. plantarum subsp. plantarum I62 showed the strongest and broadest antibacterial activity against Escherichia coli, Shigella sonnei, Salmonella Paratyphi A, and Salmonella Typhimurium SA 2093 under the mimicked proximal colon condition, in which glucose and other nutrients were limited. According to GC-MS analysis, the major antibacterial contribution of organic acids secreted by L. plantarum I62 grown in the presence of glucose was dramatically reduced from 95.8 to 41.9 % under glucose-limited niche. The production of low-pK a acids, such as lactic, 1,2-benzenedicarboxylic, and 3-phenyllactic acids, was remarkably dropped. Surprisingly, higher-pK a acids such as 5-chlorobenzimidazole-2-carboxylic, pyroglutamic, palmitic, and oleic acids were enhanced. Moreover, cyclic dipeptides, ketones, alkanes, alcohols, and miscellaneous compounds, which were pH-independent antibacterial metabolites, became dominant. The electron microscopy strongly supported the synergistic attacks of the multiple antibacterial components targeting outer and cytoplasmic membranes leading to severe leakage and cell disruption of Salmonella Typhimurium. This strain poses to be a potential probiotic candidate for effectively controlling and treating human foodborne bacterial infection.

Histamine development and bacterial diversity in microbially-challenged tonggol (Thunnus tonggol) under temperature abuse during canning manufacture.

Histamine formation and bacteriological changes caused by temperature abuse commonly occurring in the manufacturing process of standard canned tuna was assessed in microbiologically challenged tonggol (Thunnus tonggol). The in situ challenge was performed by water-soaking at 26-28 °C for 7 h to ensure the multiplication and active phase of fish microflora. Right after pre-cooking to back-bone temperature (BBT) of 50-52 °C, histamine dropped to 5.17 ± 2.71 ppm, and slowly reached 6.84 ± 1.69 ppm at 16 h abuse. On the contrary, histamine was reduced to 2.87 ± 1.23 ppm and eventually reached 5.01 ± 1.32 ppm at 24 h abuse in the pre-cooked fish previously frozen. The numbers of total aerobic bacteria, Enterobactericeae, psychrotroph, histamine forming bacteria (HFB) and diversity of fish microflora were revealed by cultural and nested PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) techniques. Interestingly, frozen storage effectively halted histamine formation in raw fish throughout 16 h abuse despite the presence of HFB. These included the prolific strains of Morganella morganii, Proteus penneri, Proteus mirabilin, Citrobacter spp. The nested PCR-DGGE profile confirmed the presence of M. morganii and Citrobacter spp. in raw fish. These prolific strains were hardly observed in the precooked fish previously frozen. Frozen storage did not only promote even histamine distribution throughout fish muscle but also enhanced histamine loss during thawing and pre-cooking. Therefore, pre-cooking and frozen storage were proven to be the effective combined hurdles not only to reduce but also prolong histamine formation of the challenged toggol throughout 24 h of temperature abuse during canning process.

Antagonistic mechanisms of synbiosis between Lactobacillus plantarum CIF17AN2 and green banana starch in the proximal colon model challenged with Salmonella Typhimurium.

Antagonistic mechanisms of Lactobacillus plantarum CIF17AN2 (an infant isolate), saba starch, and their synbiotic combination against Salmonella Typhimurium SA2093 were evaluated. The anti-Salmonella activity was investigated under the competitive niche of fecal microbiota using the simulated proximal colon model. The alterations of the dominant fecal microbiota and beneficial bacteria were also displayed using FISH and PCR-DGGE techniques. L. plantarum CIF17AN2 exhibited anti-Salmonella mechanisms through secretion of antimicrobial compounds, adhesion ability and competitive adhesion to mucin and HT-29 cell line. However, the Salmonella inhibition was significantly reduced in the presence of human fecal microflora. The combination of saba starch with L. plantarum CIF17AN2 showed the greatest inhibition against Sal. Typhimurium SA2093 in the simulated colon model. The enhancement of anti-Salmonella activity due to the addition of saba starch corresponded to a significant decrease in pH and an increase of lactic acid and short chain fatty acids. According to PCR-DGGE analysis, L. plantarum CIF17AN2 was able to survive and effectively compete with fecal microflora. Saba starch supplement modified bifidobacterial profile but had a slight impact on the profile of lactic acid bacteria. This prebiotic approach alleviated the nutrient limitation in the proximal colon model leading to the selective stimulation of beneficial lactobacilli and bifidobacteria, hence the enhancement of anti-Salmonella activity.

Persistence of maternal milk derived Lactobacillus plantarum in the infant feces and its antagonistic activity against Escherichia coli O157:H7.

The diversity of lactic acid bacteria (LAB) in maternal milk and feces from Thai mother-infants pairs were revealed through nested PCR-DGGE. LAB species residing in maternal milk drawn from each individual demonstrated high uniqueness, yet shared similarity to her infant. Multiple strains of L. plantarum, L. fermentum, L. rhamnosus, L. mucosae, L. casei were continuously detected, suggesting direct transfer from a mother to her infant via breastfeeding. L. plantarum, the most commonly found species with many strain variants, remained persistent in infant's feces up to six months postpartum. Such success could be achieved through its ability to utilize fructooligosaccharides (FOS)/inulin together with antibacterial activity and competitive adhesion. With FOS/inulin, the prebiotic utilizing L. plantarum (M117 and M118) isolated from maternal milk effectively inhibited E. coli O157:H7 under highly microflora competitive and glucose-limited environments of colon model. The results introduce the potential trend for development of effective anti-diarrheal synbiotic infant formulae.

Effect of Lactobacillus plantarum isolated from digestive tract of wild shrimp on growth and survival of white shrimp (Litopenaeus vannamei) challenged with Vibrio harveyi.

Two hundred and two strains of lactic acid bacteria (LAB) isolated from digestive tracts of cultivated and wild adult shrimp, including Litopenaeus vannamei, Metapenaeus brevicornis and Penaeus merguiensis were selected based on their antibacterial activity against Vibrio harveyi. LAB strain of MRO3.12 exhibiting highest reduction of V. harveyi was identified as Lactobacillus plantarum MRO3.12 based on the nucleotide sequence of its 16S rDNA, which showed 99% (780/786 bp) homology to L. plantarum strain L5 (GenBank accession number DQ 239698.1). Co-cultivation of V. harveyi and L. plantarum MRO3.12 showed complete reduction of V. harveyi at 24 h under aerobic and anaerobic conditions, whereas L. plantarum increased from 5.29 to 9.47 log CFU ml(-1). After 6-week feeding trial with L. plantarum supplemented diet, white shrimp (L. vannamei) exhibited significant differences (p < 0.05) in relative growth rate (% RGR), feed conversion ratio (FCR) and survival compared to the control group fed with non-supplemented diet. LAB-fed group showed 98.89% survival, whereas only 68.89% survival was observed in the control group. LAB from the digestive tract of probiotic-fed shrimp showed higher level of 5.0 ± 0.14 log CFU/g than the non-supplemented ones (3.34 ± 0.21 log CFU/g). However, total bacterial and non-fermenting vibrios counts decreased in shrimps fed on L. plantarum. Ten days after infection with V. harveyi (5.3-5.5 log CFU ml(-1)), significant survival (p < 0.05) of 77% was observed in LAB supplemented shrimp, while only 67% survival was observed in the control.

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.

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.

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.

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.

In vitro prebiotic evaluation of exopolysaccharides produced by marine isolated lactic acid bacteria.

Lactic acid bacteria (LAB) selected based on high EPSs production yields of 14, 7.6, 4.9 and 5g/L in sucrose containing MRS broth were identified as Weissella cibaria, Weissella confusa, Lactobacillus plantarum and Pediococcus pentosaceus, respectively based on their 16S rDNA sequences. EPSs produced by these strains did not stimulate secretion of interleukin (IL)-8, and were resistant to stomach acid and human pancreatic amylase. In pure culture system, only Bifidobacterium bifidum DSM 20456 exhibited the ability to utilize these EPSs as carbon sources but not L. plantarum TISTR 875 and Lactobacillus acidophilus TISTR 1034. EPSs from W. cibaria exhibited strong bifidogenic effect in the mixed-culture of human fecal microflora using the three-stage fermentation model. In the transverse and distal colon, bifidobacteria and lactobacilli as well as acetate and propionate increased significantly. Butyrate slightly decreased in the proximal colon region after feeding EPSs, but increased in the distal region.

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.

Purification, characterization, gene cloning and nucleotide sequencing of D: -stereospecific amino acid amidase from soil bacterium: Delftia acidovorans.

The D-amino acid amidase-producing bacterium was isolated from soil samples using an enrichment culture technique in medium broth containing D-phenylalanine amide as a sole source of nitrogen. The strain exhibiting the strongest activity was identified as Delftia acidovorans strain 16. This strain produced intracellular D-amino acid amidase constitutively. The enzyme was purified about 380-fold to homogeneity and its molecular mass was estimated to be about 50 kDa, on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was active preferentially toward D-amino acid amides rather than their L-counterparts. It exhibited strong amino acid amidase activity toward aromatic amino acid amides including D-phenylalanine amide, D-tryptophan amide and D-tyrosine amide, yet it was not specifically active toward low-molecular-weight D-amino acid amides such as D-alanine amide, L-alanine amide and L-serine amide. Moreover, it was not specifically active toward oligopeptides. The enzyme showed maximum activity at 40 degrees C and pH 8.5 and appeared to be very stable, with 92.5% remaining activity after the reaction was performed at 45 degrees C for 30 min. However, it was mostly inactivated in the presence of phenylmethanesulfonyl fluoride or Cd2+, Ag+, Zn2+, Hg2+ and As3+ . The NH2 terminal and internal amino acid sequences of the enzyme were determined; and the gene was cloned and sequenced. The enzyme gene damA encodes a 466-amino-acid protein (molecular mass 49,860.46 Da); and the deduced amino acid sequence exhibits homology to the D-amino acid amidase from Variovorax paradoxus (67.9% identity), the amidotransferase A subunit from Burkholderia fungorum (50% identity) and other enantioselective amidases.