Effects of temperature and pH on the structure of a metalloprotease from Lactobacillus fermentum R6 isolated from Harbin dry sausages and molecular docking between protease and meat protein.

BACKGROUND: Microbial protease can interact with meat protein in fermented meat products at a certain pH and temperature. To investigate the effects of various pH values and temperatures on the structural characteristics of Lactobacillus fermentum R6 protease, which was isolated from Harbin dry sausages, spectroscopy techniques and molecular dynamics were utilized to evaluate structural changes. RESULTS: The protease exhibited a stable spatial structure at pH 7 and 40 °C, and the extension of the protease structure was also promoted. Although the structure of the protease could be changed or destroyed by pH 8 and 70 °C, it was mainly determined by the changes of secondary and tertiary structures such as α-helix, ß-sheet, ß-turn and random coil. In addition, carbonyl vibration, -NH vibration, C-H stretching vibration and disulphide bonds were present in L. fermentum R6 protease under various pH and temperature conditions. Molecular docking showed that the protease can interact with myosin light chain, myosin heavy chain, actin and myoglobin. CONCLUSION: The protease can maintain stable structure and interact with meat protein, which reflected certain application prospects in the fermentation of Harbin dry sausages. © 2021 Society of Chemical Industry.

Novel Feruloyl Esterase from Lactobacillus fermentum NRRL B-1932 and Analysis of the Recombinant Enzyme Produced in Escherichia coli.

UNLABELLED: A total of 33 Lactobacillus strains were screened for feruloyl esterase (FE) activity using agar plates containing ethyl ferulate as the sole carbon source, and Lactobacillus fermentum NRRL B-1932 demonstrated the strongest FE activity among a dozen species showing a clearing zone on the opaque plate containing ethyl ferulate. FE activities were monitored using high-performance liquid chromatography with an acetonitrile-trifluoroacetic acid gradient. To produce sufficient purified FE from L. fermentum strain NRRL B-1932 (LfFE), the cDNA encoding LfFE (Lffae) was amplified and cloned by using available closely related genome sequences and overexpressed in Escherichia coli A 29.6-kDa LfFE protein was detected from the protein extract of E. coli BL21(pLysS) carrying pET28bLffae upon IPTG (isopropyl-ß-d-thiogalactopyranoside) induction. The recombinant LfFE containing a polyhistidine tag was purified by nickel-nitrilotriacetic acid affinity resin. The purified LfFE showed strong activities against several artificial substrates, including p-nitrophenyl acetate and 4-methylumbelliferyl p-trimethylammoniocinnamate chloride. The optimum pH and temperature of the recombinant LfFE were around 6.5 and 37°C, respectively, as determined using either crude or purified recombinant LfFE. This study will be essential for the production of the LfFE in E. coli on a larger scale that could not be readily achieved by L. fermentum fermentation. IMPORTANCE: The production of feruloyl esterase (FE) from Lactobacillus fermentum NRRL B-1932 reported in this study will have immense potential commercial applications not only in biofuel production but also in pharmaceutical, polymer, oleo chemical, cosmetic additive, and detergent industries, as well as human health-related applications, including food flavoring, functional foods, probiotic agents, preventive medicine, and animal feed. Given the essential role FE plays in the production of hydroxycinnamic acids and ferulic acid, plus the generally regarded as safe status of lactobacilli, which therefore have less regulatory concerns, LfFE from the probiotic L. fermentum reported in this work can be directly used for increased production of high-value hydroxycinnamates and ferulic acid from natural or synthetic carbon sources.

Superoxide dismutase recombinant Lactobacillus fermentum ameliorates intestinal oxidative stress through inhibiting NF-κB activation in a trinitrobenzene sulphonic acid-induced colitis mouse model.

AIMS: Superoxide dismutase (SOD) can prevent and cure inflammatory bowel diseases by decreasing the amount of reactive oxygen species. Unfortunately, short half-life of SOD in the gastrointestinal tract limited its application in the intestinal tract. This study aimed to investigate the treatment effects of recombinant SOD Lactobacillus fermentum in a colitis mouse model. METHODS AND RESULTS: In this study, we expressed the sodA gene in Lact. fermentum I5007 to obtain the SOD recombinant strain. Then, we determined the therapeutic effects of this SOD recombinant strain in a trinitrobenzene sulphonic acid (TNBS)-induced colitis mouse model. We found that SOD activity in the recombinant Lact. fermentum was increased by almost eightfold compared with that in the wild type. Additionally, both the wild type and the recombinant Lact. fermentum increased the numbers of lactobacilli in the colon of mice (P < 0·05). Colitis mice treated with recombinant Lact. fermentum showed a higher survival rate and lower disease activity index (P < 0·05). Recombinant Lact. fermentum significantly decreased colonic mucosa histological scoring for infiltration of inflammatory cells, lipid peroxidation, the expression of pro-inflammatory cytokines and myeloperoxidase (P < 0·05) and inhibited NF-κB activity in colitis mice (P < 0·05). CONCLUSIONS: SOD recombinant Lact. fermentum significantly reduced oxidative stress and inflammation through inhibiting NF-κB activation in the TNBS-induced colitis model. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides insights into the anti-inflammatory effects of SOD recombinant Lact. fermentum, indicating the potential therapeutic effects in preventing and curing intestinal bowel diseases.

Function of aspartic acid residues in optimum pH control of L-arabinose isomerase from Lactobacillus fermentum.

L-Arabinose isomerase (L-AI) catalyzes the isomerization of L-arabinose to L-ribulose and D-galactose to D-tagatose. Most reported L-AIs exhibit neutral or alkaline optimum pH, which is less beneficial than acidophilic ones in industrial D-tagatose production. Lactobacillus fermentum L-AI (LFAI) is a thermostable enzyme that can achieve a high conversion rate for D-galactose isomerization. However, its biocatalytic activity at acidic conditions can still be further improved. In this study, we report the single- and multiple-site mutagenesis on LFAI targeting three aspartic acid residues (D268, D269, and D299). Some of the lysine mutants, especially D268K/D269K/D299K, exhibited significant optimum pH shifts (from 6.5 to 5.0) and enhancement of pH stability (half-life time increased from 30 to 62 h at pH 6.0), which are more favorable for industrial applications. With the addition of borate, D-galactose was isomerized into D-tagatose by D268K/D269K/D299K at pH 5.0, resulting in a high conversion rate of 62 %. Based on the obtained 3.2-Å crystal structure of LFAI, the three aspartic acid residues were found to be distant from the active site and possibly did not participate in substrate catalysis. However, they were proven to possess similar optimum pH control ability in other L-AI, such as that derived from Escherichia coli. This study sheds light on the essential residues of L-AIs that can be modified for desired optimum pH and better pH stability, which are useful in D-tagatose bioproduction.

L-arabinose isomerase from Lactobacillus fermentum C6: Enzymatic characteristics and its recombinant Bacillus subtilis whole cells achieving a significantly increased production of D-tagatose.

L-arabinose isomerase (L-AI) is a functional enzyme for the isomerizing of D-galactose to produce D-tagatose. In this study, L-AI-C6-encoding gene from the probiotic Lactobacillus fermentum C6 was cloned and expressed in Bacillus subtilis WB600 for investigating enzymatic characteristics and bioconverting D-tagatose by means of whole-cell catalysis. Results showed that the engineered B. subtilis WB600-pMA5-LAI achieved a maximum specific activity of L-AI-C6 (232.65 ± 15.54 U/mg protein) under cultivation in LB medium at 28 °C for 40 h. The recombinant L-AI-C6 was purified, and enzymatic characteristics test showed its optimum reaction temperature and pH at 60 °C and 8.0, respectively. In addition, L-AI-C6 exhibited good stability within the pH range of 5.5-9.0. By using B. subtilis WB600-pMA5-LAI cells as whole-cell catalyst, the highest D-tagatose yield reached 42.91 ± 0.28 % with D-galactose as substrate, which was 2.41 times that of L. fermentum C6 (17.79 ± 0.11 %). This suggested that the cloning and heterologous expression of L-AI-C6 was an effective strategy for improving D-tagatose conversion by whole-cell catalysis. In brief, the present study demonstrated that the reaction temperature, pH, and stability of L-AI-C6 from L. fermentum C6 meet the demands of industrial application, and the constructed B. subtilis WB600-pMA5-LAI shows promising potential for the whole-cell biotransformation of D-tagatose.

Acceptor Subsite Mutants of Limosilactobacillus fermentum NCC 2970 GtfB 4,3-α-Glucanotransferase Regulate the Ratio of (α1 → 3)/(α1 → 6) Linkages in Biosynthesized α-Glucans.

Limosilactobacillus fermentum NCC 2970 GtfB (Lf2970 GtfB) is the only characterized 4,3-α-glucanotransferase (4,3-α-GTase) in the glycoside hydrolase (GH) 70 family belonging to the GtfB subfamily. However, the mechanism for its (α1 → 3) linkage formation remains unclear, and the structural determinants of its linkage specificity remain to be explored. Here, sequence alignment and structural comparison were conducted to identify key amino acids that may be critical for linkage specificity. Five residues of Lf2970 GtfB (D991, G1028, A1398, T1400, and E1405), located at donor and acceptor subsites, were selected for mutation. Product structure analysis revealed that D991 and G1028, located near the acceptor binding subsites, played crucial roles in linkage formation. Besides native (α1 → 4) and (α1 → 3) linkages, mutants G1028R and D991N showed 8 and 10% (α1 → 6) linkage increases compared to 1% for wild-type in products. Additionally, molecular docking studies demonstrated that the orientation of acceptor binding in G1028R and D991N mutants was favorable for (α1 → 6) linkage synthesis. However, the mutation at positions A1398, T1400, and E1405 indicated that the donor subsites contribute less to the linkage specificity. These results shed light on the structural determinants of linkage specificity of 4,3-α-GTase Lf2970 GtfB and provided insights into the structure-function relationship of family GH70.

Molecular cloning, characterization and heterologous expression of bile salt hydrolase (Bsh) from Lactobacillus fermentum NCDO394.

Bile salt hydrolase (Bsh) active probiotic strains hydrolyze bile acid amino conjugates in vivo, which triggers cholesterol consumption in liver to synthesize new bile leading to consequential cholesterol lowering. Hence, bile salt hydrolyzing potential was the criterion to select L. fermentum NCDO394 for this study and its gene encoding Bsh was identified and cloned. The resulting nucleotide sequence of bsh gene contained an open reading frame (ORF) of 978 nucleotides encoding a predicted protein of 325 amino acids with a theoretical pI of 6.39. Moreover, deduced Bsh protein had high similarity with the Bshs of L. fermentum only and also exhibited significant similarity to the Pencillin V amidases of other Lactobacillus spp. Five catalytically important amino acids were highly conserved in L. fermentum Bsh while four amino acid motifs around these active sites, were not as consistent as in other Bsh proteins. Furthermore, L. fermentum bsh gene was sub-cloned into pET-28b(+) vector, and its expression was induced with 0.05 mM isopropylthiogalactopyranoside (IPTG) in Escherichia coli BL21(DE3). The recombinant Bsh (rBsh) was purified with homogeneity using Ni+2-NTA column and characterized for substrate specificity, pH and temperature. The rBsh hydrolyzed six major human bile salts with a slight preference towards glycine-conjugated bile salts. The optimum pH of rBsh was six, and its enzymatic activity declined below pH 5 and above pH 7. The enzyme was stable and functional even at 65 °C while showed its maximum activity at 37 °C. In conclusion, L. fermentum NCDO394 may be a promising candidate probiotic which may affect cholesterol metabolism in vivo.

Expression of catalase in Lactobacillus fermentum and evaluation of its anti-oxidative properties in a dextran sodium sulfate induced mouse colitis model.

Lactic acid bacteria are generally sensitive to hydrogen peroxide (H2O2). Lactobacillus plantarum ATCC14431 is one of the few lactic acid bacteria able to degrade H2O2 through the action of a manganese-dependent catalase (containing the katA gene). However, it is not a natural inhabitant of the intestinal tract and its bio-efficacy and survival in the gastrointestinal tract have never been tested. In this study, we successfully expressed the katA gene from L. plantarum ATCC14431 in L. fermentum I5007 and the recombinant L. fermentum exhibited almost 20-fold higher catalase activity than the empty vector control. The anti-oxidative properties of this catalase-producing L. fermentum were evaluated using a dextran sodium sulphate (DSS) induced colitis mice model. Compared with the control, mice receiving DSS alone had increased diarrhea and mucosa histological scores (P < 0.05), as well as lipid peroxidation (P < 0.05), myeloperoxidase (P < 0.05), and active NF-κB in colonic tissue (P < 0.05). Similar to vitamin E, treatment with recombinant L. fermentum mitigate these effects accompanied by a improvement in mucosa histological scores in the proximal colon (P < 0.05) and decreased lipid peroxidation (P < 0.05), myeloperoxidase (P < 0.05) and active NF-κB in colonic tissue (P < 0.05). In conclusion, the expression of catalase in L. fermentum increased its ability to survive when exposed to aerated environment in vitro and conferred the anti-oxidative and anti-inflammatory effects in the DSS induced colitis model.

Surface-associated MUC5B mucins promote protease activity in Lactobacillus fermentum biofilms.

BACKGROUND: Mucosal surfaces are coated with layers of mucus gel that protect the underlying tissues and promote colonization by members of the commensal microflora. Lactobacillus fermentum is a common inhabitant of the oral cavity, gastrointestinal and reproductive tracts and is one of the most important lactic acid bacteria contributing to the formation of a healthy intestinal microflora. We have investigated the proteolytic activity in L. fermentum in response to interactions with the MUC5B mucin, which is a major component of mucus gels at sites colonized by this micro-organism. METHODS: Biofilms of Lactobacillus fermentum were established in mini-flow cells in the presence or absence of human salivary MUC5B. The proteolytic activity of biofilm cells was examined in a confocal scanning laser microscope with a fluorescent protease substrate. Degradation of MUC5B by L. fermentum was analysed using SDS-PAGE followed by Western blotting with antisera raised against the MUC5B peptide. Cell surface proteins differentialy expressed in a MUC5B-rich environment were identified with the aid of comparative two-dimensional electrophoresis followed by LC-MS/MS. RESULTS: Lactobacillus fermentum adhered well to surfaces coated with MUC5B mucin and in biofilms of L. fermentum formed in a MUC5B environment, the proportion of proteolytically-active cells (47 ± 0.6% of the population), as shown by cleavage of a fluorescent casein substrate, was significantly greater (p < 0.01) than that in biofilms formed in nutrient broth (0.4 ± 0.04% of the population). Thus, the presence of MUC5B mucins enhanced bacterial protease activity. This effect was mainly attributable to contact with surface-associated mucins rather than those present in the fluid phase. Biofilms of L. fermentum were capable of degrading MUC5B mucins suggesting that this complex glycoprotein can be exploited as a nutrient source by the bacteria.Comparison of the surface proteomes of biofilm cells of L. fermentum in a MUC5B environment with those in nutrient broth using two-dimensional electrophoresis and mass spectroscopy, showed that the enhanced proteolytic activity was associated with increased expression of a glycoprotease; O-sialoglycoprotein endopeptidase, as well as chaperone proteins such as DnaK and trigger factor. CONCLUSIONS: Adhesion to mucin-coated surfaces leads to a shift towards a more protease-active phenotype within L. fermentum biofilms and proteases produced within the biofilms can degrade MUC5B mucins. The enhanced proteolytic activity was associated with an increase in O-sialoglycoprotein endopeptidase on the cell surface. We propose that the upregulation of chaperone proteins in the mucin environment may contribute to the protease-active phenotype through activation of the glycopeptidase. This would represent one way for commensal lactobacilli e.g. L. fermentum to exploit complex substrates in their local environment in order to survive on mucosal surfaces.

ß-Galactosidase with transgalactosylation activity from Lactobacillus fermentum K4.

The LacLM ß-galactosidase of Lactobacillus fermentum K4 is encoded by 2 consecutive genes, lacL (large subunit) and lacM (small subunit), that share 17 overlapping nucleotides. Phylogenetic analysis revealed that this enzyme was closely related to other Lactobacillus ß-galactosidases and provided significant insight into its common and distinct characteristics. We cloned both the lacL and lacM genes of L. fermentum K4 and heterologously expressed each in Escherichia coli, although the recombinant enzyme was only functional when both were expressed on the same plasmid. We evaluated the enzymatic properties of this species-specific LacLM ß-galactosidase and discovered that it acts as both a hydrolase, bioconverting lactose into glucose and galactose, and a transgalactosylase, generating prebiotic galacto-oligosaccharides (GOS). The recombinant ß-galactosidase showed a broad pH optimum and stability around neutral pH. The optimal temperature and Michaelis constant (K(m)) for the substrates o-nitrophenyl-ß-D-galactopyranoside and lactose were, respectively, 40°C and 45 to 50°C and 1.31 mM and 27 mM. The enzyme activity was stimulated by some cations such as Na⁺, K⁺, and Mg²âº. In addition, activity was also enhanced by ethanol (15%, wt/vol). The transgalactosylation activity of L. fermentum K4 ß-galactosidase effectively and rapidly generated GOS, up to 37% of the total sugars from the reaction. Collectively, our results suggested that the ß-galactosidase from L. fermentum K4 could be exploited for the formation of GOS.

[Characterization and identification of Lactobacillus fermentum 4,6-α-glucosyltransferase and its products].

4,6-α-glucosyltransferases (4,6-α-GTs), which converts amylose into α(1-6) bonds-containing α-glucan, possesses great application potential in enzymatic synthesis of dietary fiber. Primers were designed according to the conserved motifs existing in the amino acid sequence of 4,6-α-GTs, and used to amplify a putative GTFB-Like 4,6-α-GTs gene (named as gtf16) from the genomic DNA of Lactobacillus. The gtf16 gene was cloned into the plasmid pET15b, expressed in Escherichia coli BL21(DE3), followed by purification and characterization. The optimum pH and the optimum temperature of the purified enzyme were 5.0 and 40 °C, respectively. The biotransformation product of this enzyme was systematically characterized by thin-layer chromatography, NMR spectroscopy, and hydrolysis reaction. The Gtf16-catalyzed product shows a similar structure to that of the isomalto/malto-polysaccharide (IMMP), which is the amylose-derived product catalyzed by GtfB from Lactobacillus reuteri 121. Moreover, The Gtf16-catalyzed product contains up to 75% of α(1-6) bonds and has an average molecular weight of 23 793 Da. Furthermore, the content of the anti-digestive components was 88.22% upon hydrolysis with digestive enzymes.

Nitrosylmyoglobin formation in meat by Lactobacillus fermentum AS1.1880 is due to its nitric oxide synthase activity.

The objective of this study was to clarify whether formation of nitrosylmyoglobin (MbFeIINO) by Lactobacillus fermentum AS1.1880 in meat is due to nitric oxide synthase (NOS) activity. Confocal laser scanning microscopy exhibited strong green fluorescence in the L. fermentum sample treated with a nitric oxide (NO)-specific probe, directly indicating that NO was produced. Furthermore, determination of NOS activity based on the presence of NO metabolites indicated the existence of NOS in L.fermentum. A NOS inhibitor, NG-nitro-L-arginine methyl ester, significantly inhibited the activity of NOS in L.fermentum (P < 0.05). Futhermore, NOS protein was detected in L.fermentum by Western blot analysis. L-arginine addition largely increased the NOS activity of L.fermentum (P < 0.05). In meat batters, the redness of a sample inoculated with L.fermentum was higher than that of the control and colour was significantly improved with the addition of L-arginine (P < 0.05), indicating that more MbFeIINO was formed.

Purification, characterization and molecular cloning of a dicaffeoylquinic acid-hydrolyzing esterase from human-derived Lactobacillus fermentum LF-12.

Dicaffeoylquinic acids (DiCQAs), the main components of kudingcha made from the leaves of Ilex kudingcha, could be transformed by gut microbiota. However, the information about the related microorganisms and enzymes involved in the biotransformation of DiCQAs in the human gut is limited. Therefore, a strain of bacteria that could hydrolyze DiCQAs, belonging to Lactobacillus fermentum named L. fermentum LF-12, was isolated from human feces in the present study. Furthermore, an esterase for the hydrolysis of DiCQAs was purified from L. fermentum LF-12 and heterogeneously expressed in Escherichia coli. The esterase could be induced to exert superior hydrolytic activity in the presence of lactose as the carbon source. The molecular weight of the purified esterase was determined to be 31.9 kDa, and the isoelectric point, optimal pH and temperature for the esterase were 4.71, 6.5 and 45 °C, respectively. The enzyme activity was improved by Mg2+ and Ca2+, and reduced by Co2+, Cu2+, EDTA and some kinds of organic solvents. The present results provide new insights into the metabolism of DiCQAs by the human gut.

Expression and characterisation of feruloyl esterases from Lactobacillus fermentum JN248 and release of ferulic acid from wheat bran.

Genes encoding six feruloyl esterases (FAEs; lbff0997, lbff0272, lbff1432, lbff1695, lbff1849, lbff0153) from Lactobacillus fermentum JN248 were cloned, overexpressed and characterised. Maximum enzyme activity was observed at 35 °C for recombinant FAEs LFFae0997, LFFae0272 and LFFae0153, at 30 °C for LFFae1695, and at 40 °C for LFFae1432and LFFae1849. For five of the enzymes, optimal activity was observed at pH 7.0 or pH 8.0, and high thermostability was measured up to 55 °C. By contrast, LFFae1432 lost less than 10.0% activity after incubation at 40 °C for 2 h, and pH stability was highest between pH 7.0 and pH 9.0. In addition, LFFae1432 was the most robust esterase, with a higher affinity and hydrolytic activity against synthetic esters. The enzymes released ferulic acids (FAs) from de-starched wheat bran (DSWB), and 60.7% of the total alkali-extractable FAs were released when LFFae1432 was added alone, compared with less than 10% for the other enzymes. The amount of FAs released by FAEs increased when combined with xylanase. These FAEs could serve as promising biocatalysts for biodegradation, and LFFae1432 may hold promise for potential industrial applications.

Microencapsulated feruloyl esterase-producing lactobacilli ameliorate lipid profile and glycaemia in high fat diet-induced obese mice.

The effect of oral administration of spray-dried microcapsules of feruloyl esterase (FE) producing Lactobacillus fermentum CRL1446 (Lf) and Lactobacillus johnsonii CRL1231 (Lj) on high fat diet-induced obese mice was investigated to evaluate whether these strains could be used as a biotherapeutic for obesity. Swiss albino mice were divided into a normal diet fed group receiving empty microcapsules (control), a high fat diet plus empty microcapsules (HFD group), HFD plus microcapsules with Lf (HFD-Lf group) and HDF plus microcapsules with Lj (HFD-Lj group). Microcapsules containing Lf or Lj at a dose of ~107 cells/day/mouse were given orally for 7 weeks. Body weight gain, adiposity index, plasma leptin, lipid profiles, glycaemia, insulinemia, oral glucose tolerance, intestinal FE, glutathione peroxidase and glutathione reductase (GR) activities were determined. Administration of lactobacilli (HFD-Lf and HFD-Lj groups) improved metabolic parameters (triglyceride, total cholesterol, low-density lipoprotein cholesterol levels) and cardiovascular risk indicators (37-46% decrease of atherogenic index), and reduced body weight gain (29-38%), adiposity index (42-62%), plasma leptin levels, liver weight and fat deposition in liver. Intestinal FE activities significantly increased in HFD-Lf (62%) and HFD-Lj group (48%), thus improving hepatic GR activity (42% increment) compared to HFD group. Moreover, L. johnsonii increased HDL-cholesterol and L. fermentum reduced blood glucose to levels similar to the control. These FE-producing lactobacilli have the potential to improve biomarkers involved in obesity by increasing intestinal FE activity.

Preparation and in vitro analysis of microencapsulated live Lactobacillus fermentum 11976 for augmentation of feruloyl esterase in the gastrointestinal tract.

FA (ferulic acid) is a well-known phenolic phytochemical present in plant cell walls. Various studies have indicated that FA has many physiological functions in the prevention of chronic disease. It has been shown to play an important chemoprotective role in degenerative diseases. FA also shows strong antioxidant and nitrite-scavenging potential and anticarcinogenic and antiinflammatory properties. The in vivo physiological importance of FA depends on its availability for absorption. Dietary fibre-bound FA is partially released by gut micro-organisms; however, the concentration of the released FA is too low to act as a chemopreventive agent. Therefore it is important to augment the bioavailability of FA to appreciate more fully its real physiological effect. This paper evaluates the suitability of the alginate-poly(L-lysine)-alginate microcapsules for oral delivery of live feruloyl esterase-producing Lactobacillus fermentum 11976 cells, in vitro, by using a dynamic simulated human GI (gastrointestinal) model. The present study shows that microencapsulated L. fermentum 11976 cells can efficiently break down a FA-containing substrate, and establishes the biotechnological basis for their use in supplementing the bioavailability of dietary FA in the intestine.

Purification and characterization of a novel α-D-glucosidase from Lactobacillus fermentum with unique substrate specificity towards resistant starch.

Resistant starch is not digestible in the small intestine and is fermented by lactic acid bacteria in the large intestine into short chain fatty acids, such as acetate, propionate and butyrate, which result in several health benefits in analogy with dietary fibre components. The mode and mechanism of resistant starch degradation by lactic acid bacteria is still not understood. In the present study, we have purified α-D-glucosidase from Lactobacillus fermentum NCDC 156 by employing three sequential steps i.e. ultra filtration, DEAE-cellulose and Sephadex G-100 chromatographies. It was found to be a monomeric protein (~50 kDa). The optimum pH and temperature of this enzyme were found to be 5.5 and 37°C, respectively. Under optimised conditions with p-nitrophenyl-D-glucopyranoside as the substrate, the enzyme exhibited a Km of 0.97 mM. Its activity was inhibited by Hg2+ and oxalic acid. N-terminal blocked purified enzyme was subjected to lysyl endopeptidase digestion and the resultant peptides were subjected to BLAST analysis to understand their homology with other α-D-glucosidases from lactobacillus species.

Site-specific hydrolysis of chlorogenic acids by selected Lactobacillus species.

Hydroxycinnamic acids are a major group of phenolic compounds widely distributed in plants. Among them, chlorogenic acids and caffeic acid have been in the focus of interest due to their impact on food quality and their putative health benefits. Numerous microorganisms like lactic acid bacteria are able to hydrolyze chlorogenic acids by cinnamoyl esterase enzymes. Data on the specificity of theses enzymes regarding the cleavage of distinct isomers of mono- or dichlorogenic acids is lacking. Lactobacillus reuteri, Lactobacillus helveticus, and Lactobacillus fermentum were screened for their ability to hydrolyze chlorogenic acid isomers in culture medium. Concentrations of chlorogenic acids and the released caffeic acid were determined by UHPLC-ESI-MS. The highest hydrolysis rate (100%) was observed for the hydrolysis of 5-CQA by Lactobacillus helveticus. A so far unknown metabolic pathway for the cleavage of 4-CQA is proposed including isomerization to 5-CQA and 3-CQA followed by hydrolysis.

Modeling of urea degradation in white and rosé wines by acid urease.

UNLABELLED: The specific activity of a whole cell acid urease preparation was tested in five wines manufactured in the Apulia region of Italy in the 2003 vintage at both short and long treatment times, thus confirming the validity of the pseudo-first-order kinetic model to describe urea removal in real wines. The ratio between the experimental pseudo-first-order kinetic rate constant (kIe) for any real wine tested and that (kI) referred to a model wine solution having the same composition and pH reduced from about 0.21 to 0.02 as the overall content of phenolic compounds (P) increased from 109 to 853 g m-3 of gallic acid equivalent (GAE). The specific inhibitory effect of such compounds was explained by accounting for the equilibrium constant (KP) of the reaction of polyphenols with acid urease, which was found to be about 21 g of GAE m-3 for the real wines tested, whereas it ranged from about 16 to 45 g of GAE m-3 when the model wine solution was enriched with tannins extracted from grape seeds or skins, respectively. A sequential experimental procedure consisting of accelerated acid urease tests at high doses of enzyme followed by accelerated ethyl carbamate tests on the resulting acid urease treated wine was recommended to assess preliminarily the technoeconomic feasibility of the acid urease hydrolytic process for the wine of concern. KEYWORDS: Acid urease; real and model wines; phenolics; pseudo-first-order kinetic rate constant; inhibitory effect; urea degradation kinetics.

A Zymogram technique for preliminary screening and characterization of feruloyl esterases.

We report here, for the first time, a zymogram technique designed for rapid screening of feruloyl esterase using ethyl ferulate as enzyme substrate and casein precipitation as enzyme activity indicator.