Enzymes Of Lactic Acid Bacteria Research Articles

Assaying D-Amino Acid in Japanese Sake Using L-Amino Acid Derivatizing Agent.

The D-amino acids of D-alanine, D-glutamic acid, and D-aspartic acid increase tasting evaluation scores of Sake, a Japanese traditional alcohol beverage. Sake is brewed using seed mash for growth of brewing yeast without growth of contaminating microorganisms. Kimoto is brewed using lactic acid bacteria growth to decrease pH. Sake brewed using the Kimoto method also has a rich taste and a higher tasting evaluation score than Sake brewed using the Sokujyo Syubo (Moto) method, which adds lactic acid instead of using lactic acid bacteria growth. D-alanine, D-glutamic acid, and D-aspartic acid in Sake have the function of increasing tasting evaluation scores. They are converted by enzymes in lactic acid bacteria respectively as alanine racemase (EC 5.1.1.1), glutamate racemase (EC 5.1.1.3), and aspartate racemase (EC 5.1.1.13) in Kimoto Mash. Herein, simultaneous assay methods for D-alanine, D-glutamic acid, and D-aspartic acid are explained. Sample solutions adjusted to alkalinity are derivatized by an L-FDLA solution only for L-amino acid. Results demonstrate that, for D-alanine, D-glutamic acid, and D-aspartic acid, this method can assay them easily using no expensive or specialized equipment.

The Problem of Bitter Peptides Formed in the Process of Cheese Ripening

Bitter peptides are formed by the breakdown of proteins and high-molecular peptides during proteolysis. Their formation in cheeses is related to the proteolytic activity of rennet in balance with the peptidase activity of microbial enzymes of lactic acid bacteria. The bitter taste then arises when there is a disproportion between the formation and degradation of bitter peptides by increasing their concentration above the perception threshold. The extent to which bitter peptides affect the overall taste of cheese depends on the balance between their formation and breakdown to non-bitter lower peptides and amino acids. Only a perfectly balanced proteolysis process enables the creation of quality matured cheese with a characteristic taste and aroma. Therefore, a complete clarification of the mechanism is desirable, and thus also the possibility of control and regulation of this process.

The use of the multicomplex sports nutrition "MDX" in martial arts athletes

The composition of the diet is very significant for the development of various physical qualities in humans. The use of balanced supplements has a positive effect on the body’s condition and increases the success of trainees in many sports. One of the options for optimizing nutrition for athletes can be the use of the sports nutrition multicomplex MDX. This product is obtained by microbiological processing of whey followed by low-temperature thickening. It contains oligopeptides and free amino acids, glucose, galactose, nucleic acids, a number of vitamins and microelements, betacarotene, ergosterol, folic acid, endosomal enzymes of lactic acid bacteria. In addition, the product contains a live culture of lactic acid bacteria: L. Lactis, L. Thermophyilus, L. Bulgaricus. Hand-to-hand fighters, judokas and Greco-Roman wrestlers took part in the work. Athletes (70 males: adolescents and young men) regularly took this product according to the following scheme: five days 1g/kg body weight and two days off. The daily dose was divided into two parts. Combat athletes took one part 30 minutes before the start of training, the second they took 30 minutes after the end of training. It was found that among the sports specializations taken into account, judoists who received the multicomplex MDX sports nutrition had the highest speed. They were faster than hand-to-hand fighters and fighters.

Expression profiles of milk proteolysis-related genes in Lactobacillus paracasei EG9, a non-starter lactic acid bacterial strain, during Gouda-type cheese ripening

We investigated the expression profiles of proteolysis-related genes in Lactobacillus paracasei EG9, a non-starter lactic acid bacterial strain used as an adjunct starter in Gouda-type cheese production. The amino acid sequences of previously reported proteolytic enzymes in lactic acid bacteria were matched against the EG9 genome by translated BLAST search, and 31 candidate genes, including those coding for peptidases, oligopeptide transporters, and cell-envelope proteinases, were listed. The relative expression levels of these genes were evaluated at 0 and 30 days of cheese ripening. On day 0, 27 genes showed significantly up-regulated expression compared with the genes in the stationary phase of the reference cultured cells. On day 30, 25 genes were found to be significantly down-regulated. Our findings suggest that the proteolysis-related genes in EG9 play an important role in the proteolytic process from casein degradation through peptide uptake to free amino acid production during cheesemaking.

Characterization of a 4,6‑α‑glucanotransferase from Lactobacillus reuteri E81 and production of malto-oligosaccharides with immune-modulatory roles

A wide number of Lactic Acid Bacteria (LAB) species produce α-glucans with their ability to synthesize glucansucrases (GS) which use sucrose as substrate for the glucan production. Recently another group of enzymes in LAB gained special interest for their ability to produce α-glucans targeting the substrates containing α1-4-linkages and synthesizing new (α1-6) or (α1-3)–linkages as α‑glucanotransferases. In this study, a putative 4,6‑α‑glucanotransferase (GTFB) from sourdough isolate Lactobacillus reuteri E81 was identified and expressed in Escherichia coli. The biochemical characterization of the GTFB-E81 confirmed its function as it cleaved the α1-4-linkages in different substrates and produced new gluco-oligomers/polymers containing α1-6 linkages together with the α1-4-linkages detected by NMR analysis. GTFB-E81 produced malto-oligosaccharides targeting maltose and maltoheptaose as substrates with up to DP 8 detected by TLC and ESI-MS/MS analysis. The functional roles of these malto-oligosaccharides were determined by testing their immune-modulatory functions in HT29 cells and they triggered the production of anti-inflammatory 1L-4 and pro-inflammatory IL-12 cytokines.

Modification of IgE binding to αS1-casein by proteolytic activity of Enterococcus faecium isolated from Iranian camel milk samples

Milk is a perfect source of nutrients for neonates. When breast feeding cannot be done, an infant’s alimentation is usually initiated to cow’s milk, among the primary foods. It has been reported that about 2.5% of juveniles under the age of 3 years manifest allergic reactions to cow’s milk proteins. Among the cow’s milk proteins, casein fractions are considered as the strongest allergenic proteins. The proteolytic enzymes of lactic acid bacteria (LAB), during fermentation of dairy products, can break down milk proteins especially caseins and subsequently reduce the immune reactivity of allergenic proteins. In this research, raw bovine and camel milk samples were screened for cocci LAB strains and after isolation, their proteolytic activity against bovine milk caseins were evaluated by SDS-PAGE and RP-HPLC. The potential of cocci LAB strains on αS1-casein degradation and their potential to break down the principle allergenic epitopes of this protein was detected using indirect competitive ELISA. Molecular identification of the best proteolytic strain was fulfilled by 16S rDNA fragment sequencing with universal primers. The obtained results demonstrated that Enterococcus faecium isolated from raw camel milk samples was the most efficient isolate in hydrolyzing Na-caseinate and αS1-casein. Hydrolysated αS1-casein by Enterococcus faecium was also less recognized by IgE of bovine milk allergic patients’ sera in comparison with native αS1-casein. It has been proposed that Enterococcus faecium could be an efficient strain in allergenicity reduction of cow’s milk proteins. So it could be an excellent candidate to be potentially used in dairy industries.

Synthesis of New Hyperbranched α-Glucans from Sucrose by Lactobacillus reuteri 180 Glucansucrase Mutants.

α-Glucans produced by glucansucrase enzymes of lactic acid bacteria attract strong attention as novel ingredients and functional biopolymers in the food industry. In the present study, α-helix 4 amino acid residues D1085, R1088, and N1089 of glucansucrase GTF180 of Lactobacillus reuteri 180 were targeted for mutagenesis both jointly and separately. Analysis of the mutational effects on enzyme function revealed that all D1085 and R1088 mutants catalyzed the synthesis of hyperbranched α-glucans with 15-22% branching (α1→3,6) linkages, compared to 13% in the wild-type GTF180. In addition, besides native (α1→6) and (α1→3) linkages, all of the mutations introduced a small amount of (α1→4) linkages (5% at most) in the polysaccharides produced. We conclude that α-helix 4 residues, especially D1085 and R1088, constituting part of the +2 acceptor binding subsite, are important determinants for the linkage specificity. The new hyperbranched α-glucans provide very interesting structural diversities and may find applications in the food industry.

Fermented Dairy Products in the Nutrition of Infants in the Russian Federation: Past and Present

Fermented dairy products have a high nutritional and biological value and functional properties beneficial to human health; they are very diverse and have a long history. Fermentation of milk is a complex technological, physical and biochemical process that occurs under the influence of two enzymes of lactic acid bacteria — -galactosidase and lactate dehydrogenase. Requirements for biological properties of starter microorganisms and fermentation technology are strictly regulated. Based on the starter cultures used, we can single out fermented dairy products of lactic acid and mixed (lactic acid and alcohol) fermentation. There are adapted, partially adapted and non-adapted cultured milk products for children, some of which are enriched with pro- and prebiotics to enhance functional properties. The article provides information about one of the first Russian non-adapted fermented milk products for infants enriched with inulin, fruit and cereals.

Inventory of the GH70 enzymes encoded by Leuconostoc citreum NRRL B‐1299 – identification of three novel α‐transglucosylases

Leuconostoc citreum NRRL B-1299 has long been known to produce α-glucans containing both α-(1→6) and α-(1→2) linkages, which are synthesized by α-transglucosylases of the GH70 family. We sequenced the genome of Leuconostoc citreum NRRL B-1299 to identify the full inventory of GH70 enzymes in this strain. Three new genes (brsA, dsrM and dsrDP) putatively encoding GH70 enzymes were identified. The corresponding recombinant enzymes were characterized. Branching sucrase A (BRS-A) grafts linear α-(1→6) dextran with α-(1→2)-linked glucosyl units, and is probably involved in the α-(1→2) branching of L. citreum NRRL B-1299 dextran. This is the first report of a naturally occurring α-(1→2) branching sucrase. DSR-M and DSR-DP are dextransucrases that are specific for α-(1→6) linkage synthesis and mainly produce oligomers or short dextrans with molar masses between 580 and 27 000 g·mol(-1) . In addition, DSR-DP contains sequences that diverge from the consensus sequences that are typically present in enzymes that synthesize linear dextran. Comparison of the genome with five other L. citreum genomes further revealed that dsrDP is unique to L. citreum NRRL B-1299. The presence of this gene in a prophage represents the first evidence of phage-mediated horizontal transfer of genes encoding such enzymes in lactic acid bacteria. Finally, brsA and dsrM are located in a chromosomal region in which genes encoding strain-specific GH70 enzymes are consistently located. This region may be a good target on which to focus in order to rapidly evaluate the diversity of GH70 enzymes in L. citreum strains.

Starch‐modifying enzymes of lactic acid bacteria – structures, properties, and applications

AbstractIn spite that lactic acid bacteria (LAB) are used for production of fermented foods and drinks for millennia, their ability to grow using starch as a sole carbon source was noticed by the scientists in the last 30 years. A number of amylolytic LAB (ALAB) strains were isolated and several detailed investigations of biochemical and genetic basis of starch hydrolysis were performed. The purpose of this review is to summarize for the first time the available data about the starch‐modifying enzymes in ALAB. The most important amylolytic representatives of the genera Lactobacillus, Lactococcus, Streptococcus, Pediococcus, Carnobacterium, and Weissella are described. Amino acid sequences, corresponding to ALAB amylase enzymes are compared and some features of the gene expression are analyzed. The possible application of ALAB strains for direct production of lactic acid from starch, as well as their participation in food manufacturing is discussed.

Screening of lactic acid bacteria from Indonesia reveals glucansucrase and fructansucrase genes in two differentWeissella confusastrains from soya

Homopolysaccharide (glucan and fructan) synthesis from sucrose by sucrase enzymes in lactic acid bacteria (LAB) has been well studied in the genera Leuconostoc, Streptococcus and Lactobacillus. This study aimed to identify and characterize genes encoding glucansucrase/glucosyltransferase (GTF) and fructansucrases/fructosyltransferase (FTF) enzymes from genomic DNA of 'rare' Indonesian exopolysaccharide-producing LAB. From a total of 63 exopolysaccharide-producing LAB isolates obtained from foods, beverages and environmental samples, 18 isolates showing the most slimy and mucoid colony morphologies on sucrose were chosen for further study. By comparing bacterial growth on De Man, Rogosa and Sharpe (MRS)-sucrose with that on MRS-raffinose, and using the results of a previous PCR screening study with degenerate primer pairs targeting the conserved catalytic domain of GTFs, various strains were identified as producers of fructan (13), of glucan only (five) or as potential producers of both glucan and fructan (nine). Here, we report the characteristics of three gtf genes and one ftf gene obtained from Weissella confusa strains MBF8-1 and MBF8-2. Strain MBF8-1 harbored two putative gtf genes with high sequence similarity to GTFB of Lactobacillus reuteri 121 and GTF180 of L. reuteri 180, respectively. Strain MBF8-2 possessed single gtf and ftf genes with high sequence similarity to GTFKg3 of Lactobacillus fermentum Kg3 and DSRWC of Weissella cibaria, and FTF levansucrase of L. reuteri 121, respectively.

Fructansucrase enzymes and sucrose analogues: A new approach for the synthesis of unique fructo-oligosaccharides

Fructansucrase enzymes of lactic acid bacteria use the cheap compound sucrose (Glc–Fru) to synthesize a variety of poly- and oligosaccharide products. Recently, it has been shown that a variety of sucrose analogues (e.g. Gal–Fru, Man–Fru) can be efficiently synthesized. This has exciting potential for the development of novel (fructo) oligosaccharide derivatives.

Rational Transformation of Lactobacillus reuteri 121 Reuteransucrase into a Dextransucrase

Glucansucrase or glucosyltransferase (GTF) enzymes of lactic acid bacteria display high sequence similarity but catalyze synthesis of different alpha-glucans (e.g., dextran, mutan, alternan, and reuteran) from sucrose. The variations in glucosidic linkage specificity observed in products of different glucansucrase enzymes appear to be based on relatively small differences in amino acid sequences in their sugar-binding acceptor subsites. This notion was derived from mutagenesis of amino acids of GTFA (reuteransucrase) from Lactobacillus reuteri strain 121 putatively involved in acceptor substrate binding. A triple amino acid mutation (N1134S:N1135E:S1136V) in a region immediately next to the catalytic Asp1133 (putative transition state stabilizing residue) converted GTFA from a mainly alpha-(1-->4) ( approximately 45%, reuteran) to a mainly alpha-(1-->6) ( approximately 80%, dextran) synthesizing enzyme. The subsequent introduction of mutation P1026V:I1029V, involving two residues located in a region next to the catalytic Asp1024 (nucleophile), resulted in synthesis of an alpha-glucan containing only a very small percentage of alpha-(1-->4) glucosidic linkages ( approximately 5%) and a further increased percentage of alpha-(1-->6) glucosidic linkages ( approximately 85%). This changed glucosidic linkage specificity was also observed in the oligosaccharide products synthesized by the different mutant GTFA enzymes from (iso)maltose and sucrose. Amino acids crucial for glucosidic linkage type specificity of reuteransucrase have been identified in this report. The data show that a combination of mutations in different regions of GTF enzymes influences the nature of both the glucan and oligosaccharide products. The amino acids involved most likely contribute to sugar-binding acceptor subsites in glucansucrase enzymes.

Bioactive peptides in dairy products: synthesis and interaction with proteolytic enzymes

Food proteins are sources of peptides with various biological activities, such as opioids, mineral carriers, antihypertensives, immuno-stimulants, antithrombotics and antigastrics. These bioactive peptides are generated in vivo, in vitro and during food processing. Bioactive peptides are largely found in milk, fermented milks and cheeses. Proteolytic enzymes naturally occurring in milk, and enzymes from lactic acid bacteria or from exogenous sources contribute to the generation of bioactive peptides. Dairy processing conditions such as cheese ripening are also relevant. Once produced, bioactive peptides play a significant role in cheesemaking by selectively inhibiting proteolytic enzymes of lactic acid bacteria and subsequently affecting cheese quality. Sensitivity to inhibition is highly specific: enzymes of the same biochemical class and strains of the same species differ. Bioactive peptides also inhibit dairy spoilage enzymes.

Peptides from several italian cheeses inhibitory to proteolytic enzymes of lactic acid bacteria, pseudomonas fluorescens ATCC 948 and to the angiotensin I-converting enzyme

Water-soluble peptides from Mozzarella, Italico, Crescenza, and Gorgonzola cheeses were fractionated by reverse-phase fast protein liquid chromatography. Peptide fractions with inhibitory activity to amino- and endo-peptidases from Lactobacillus delbrueckii ssp. bulgaricus B397, Streptococcus thermophilus 305, and Lactococcus lactis ssp. cremoris Wg2 were found. Enzymes from Lactobacillus casei ssp. casei 2752 were less sensitive. Endopeptidase from Lactobacillus casei ssp. casei 2752 also had a different response to the effect of some inhibitors. It probably showed limited differences in catalysis and substrate positioning. Most of these inhibitory peptides were also effective in reducing the activity of the Pseudomonas fluorescens ATCC 948 endopeptidase and the angiotensin I-converting enzyme. Inhibitory peptide fractions from Mozzarella, Italico, and Crescenza cheeses had a certain degree of hydrophobicity while the peptide fraction from Gorgonzola cheese eluted in the initial part of the acetonitrile gradient. One of the inhibitory peptides contained in the water-soluble extract of Crescenza cheese was further purified and sequenced. It corresponded to the β-casein fragment 58-72.

Proteolytic enzymes of lactic acid bacteria

The proteolytic system of lactic acid bacteria is essential for their growth in milk and contributes significantly to flavour development in fermented milk products where these microorganisms are used as starter cultures. The proteolytic system is composed of proteinases which initially cleave the milk protein to peptides, peptidases which cleave the peptides thus formed into smaller peptides and amino acids and transport systems which are involved in the cellular uptake of small peptides and amino acids. An overview of the literature on the research to date performed on the proteolytic enzymes of lactic acid bacteria is presented. The review highlights the different types of lactococcal and non-lactococcal proteinases as well as the approach to molecular cloning of the lactococcal proteinase genes and the molecular control of the regulation of proteinase production. A wide spectrum of peptidases have been identified in lactic acid bacteria. The research on the characterisation, substrate specificity and localisation of endopeptidases, aminopeptidases, dipeptidases, tripeptidases and proline specific peptidases is presented in addition to the illuminating research which has been performed on the transport systems for peptides and amino acids in lactic acid bacteria.

乳酸菌の酵素に関する研究(第11報)

乳酸菌の酵素に関する研究(第11報)

乳酸菌の酵素に関する研究(第13報)

乳酸菌の酵素に関する研究(第13報)

乳酸菌の酵素に関する研究(第10報)

乳酸菌の酵素に関する研究(第10報)

乳酸菌の酵素に關する研究

(1) Lactobacillus plantarum No. 11から得たcell-free racemiaseの溶液を透析して, racemiaseには透析性の助酵素のあることを認めた. (2) このCoracemiase溶液は殆んどその力を減衰することなく減圧濃縮し又乾燥粉末とすることが出来る. (3) Coracemiase溶液をアセトンで分別沈澱すると,50%と90%の間で沈澱する区分に活力の殆んど総てが移行する. (4) 90%アセトンで沈澱させた粗製coracemiase区分の収量は乾菌1gに対し70～80mgで黄色琥珀状を呈し,有機性P2O56.34%を含有し, 260mμの紫外線部に顕著な吸収帯を示す. OHLMEYER法による還元を行つても吸収帯には全く変化が見られない. (5) ビタミンB複合体,無機イオン, ATP, DPN等通常考え得る助酵素類及びその混合物を以つてしてもcoracemiaseの作用を代替し得ない. (6) Racemiaseの作用機構がピルビン酸を介する水素授受でないという自説を更に強く主張した. 終りに臨み本実験に種々の側面的事実を提供された福井農学士に心から謝意を表し度い.尚本研究の経費の一部は文部省科学研究費に仰いで行ることを附記する.