Cell Envelope Proteinase Research Articles

Acetate kinase and peptidases are associated with the proteolytic activity of Lactobacillus helveticus isolated from fermented food

Lactobacillus (L.) helveticus is widely used in food industry due to its high proteolytic activity. However, such activity varies greatly between isolates, and the determining factors regulating the strength of proteolytic activity in L. helveticus are unclear. This study sequenced the genomes of 60 fermented food-originated L. helveticus and systemically examined the proteolytic activity-determining factors. Our analyses found that the strength of proteolytic activity in L. helveticus was independent of the isolation source, geographic location, phylogenetic closeness between isolates, and distribution of cell envelope proteinases (CEPs). Genome-wide association study (GWAS) identified two genes, the acetate kinase (ackA) and a hypothetical protein, and 15 single nucleotide polymorphisms (SNPs) that were associated with the strength of the proteolytic activity. Further investigating the functions of these gene components revealed that ackA and two cysteine peptidases coding genes (pepC and srtA) rather than the highly heterogeneous and intraspecific CEPs were linked to the level of proteolytic activity. Moreover, the sequence type (ST) defined by SNP analysis revealed a total of ten STs, and significantly weaker proteolytic activity was observed among isolates of ST2. This study provides practical information for future selection of L. helveticus of strong proteolytic activity.

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.

A Systems-Wide Analysis of Proteolytic and Lipolytic Pathways Uncovers The Flavor-Forming Potential of The Gram-Positive Bacterium Macrococcus caseolyticus subsp. caseolyticus.

Macrococcus caseolyticus subsp. caseolyticus is a Gram-positive, commensal organism documented to be present as a component of the secondary microflora in fermented foods such as Ragusano and Fontina cheeses and Cantonese sausage. In these products, the organism appears to play a role in ripening and the development of the final organoleptic qualities. However, the role of this organism in flavor generation is not well understood. Therefore, the objective of this study was to investigate the role of M. caseolyticus subsp. caseolyticus in flavor compound formation through an examination of enzymatic, metabolomic and genomic data. A bank of M. caseolyticus subsp. caseolyticus strains derived from a variety of niches were examined. Enzyme activities analyzed comprised those of the proteolytic and lipolytic cascades including cell-envelope proteinase (CEP), peptidases, esterases, lipases, aminotransferases and glutamate dehydrogenase (GDH). Strain to strain variation was observed, often associated with niche. All strains, except those isolated from non-dairy sources, demonstrated high CEP activity. Such high CEP activity associated with dairy strains implies the importance of this characteristic in the adaptation of these strains to a dairy-specific niche. However, limited downstream peptidolytic activity, in addition to a limited ability to generate free amino acids (FAA) was observed across all strains, indicating weak ability of this organism to generate amino-acid derived flavor compounds. Interestingly, the strains with high CEP activity also demonstrated high esterase activity and gas chromatography-mass spectrometry (GC-MS) analysis of the volatile compounds produced when these strains were grown in lactose-free milk demonstrated differences in the range and types of volatiles produced. In contrast to this metabolic versatility, comparative genome analysis revealed the distribution of components of the proteolytic and lipolytic system in these strains to be conserved. Overall, this study demonstrates the potential of M. caseolyticus subsp. caseolyticus to generate diverse volatile flavor compounds. Additionally, the identification of the highly active strain-specific cell wall bound caseolytic proteases deriving extensive casein hydrolysis, serves as a promising avenue which can be potentially harnessed in the future to produce greater and more diverse flavor compounds.

Screening of the main nutrients for cell envelope proteinases production by Lactobacillus plantarum LP69 using Plackett-Burman design

In order to select the most important nutrients that influence the production of cell-envelope proteinase (CEP), Plackett–Burman design was employed to assess the effects of glucose, maltose, peptone, casein peptone, inulin, isomaltooligosaccharide, Na2HPO4, CH3COONa, leucine, and serine on the activity of CEP, protein content, specific activity, optical density (OD600) value and pH in MRS broth fermented by Lactobacillus plantarum LP69. The results revealed that Na2HPO4, inulin, casein peptone and leucine significantly affect (P<0.05) the production of CEP. Additionally, Na2HPO4, inulin and leucine were negatively correlated with the enzyme activity and the specific activity, while casein peptone was positively correlated with the enzyme activity and the specific activity.

Development of an Enzyme-Mediated, Site-Specific Method to Conjugate Toll-Like Receptor 2 Agonists onto Protein Antigens: Toward a Broadly Protective, Four Component, Group A Streptococcal Self-Adjuvanting Lipoprotein-Fusion Combination Vaccine.

Subunit vaccines composed of protein antigens covalently attached to Toll-like receptor (TLR) agonists elicit superior immune responses compared to mixtures of antigens and TLR agonists. Among different conjugation approaches, enzyme-mediated ligation is one of the few that provides an opportunity for the generation of homogeneous, molecularly defined products in which protein antigens are maintained with native structures, which is most critical to elicit protective immune responses upon vaccination. Four highly conserved protein antigens from Group A Streptococcus (GAS) have the potential to be safe and efficacious vaccine candidates. After a TLR2 agonist fibroblast-stimulating lipopeptide-1 (FSL-1) was successfully attached onto each antigen using sortase A and techniques for their purification were developed, a combination vaccine containing interleukin 8 (IL-8) protease (Streptococcus pyogenes cell envelope proteinase [SpyCEP]), Group A Streptococcal C5a peptidase (SCPA), anchorless virulence factor arginine deiminase (ADI), and trigger factor (TF)-TLR2 conjugates was produced. This combination was assessed for immunity in mice and compared with mixtures of the four antigens with FSL-1 or alum. High titer antigen-specific IgG antibodies were detected from all vaccine groups, with antibodies elicited from FSL-1 conjugates around 10-fold higher compared to the FSL-1 mixture group. Furthermore, the FSL-1 conjugates afforded a more balanced TH1/TH2 immune response than the alum-adjuvanted group, suggesting that this combination vaccine represents a promising candidate for the prevention of GAS diseases. Thus, we established a conjugation platform that allows for the production of defined, site-specific antigen-adjuvant conjugates, which maintain the native three-dimensional structure of antigens and can be potentially applied to a variety of protein antigens.

Matching starter phenotype to functionality for low salt Cheddar cheese production based on viability, permeability, autolysis, enzyme accessibility and release in model systems

Lactococcal starters were evaluated for suitability in low salt Cheddar cheese manufacture using model systems containing 0%, 3% or 5% (w/v) salt. Strains underwent the Pearce test and storage at 14 days and were monitored for viability, permeabilisation, autolysis, PepX activity and cell envelope proteinase activities. Salt exerted a species and strain-dependent effect on viability throughout storage. Flow cytometry indicated live, permeabilised or dead cells varied in a strain and salt-dependent manner. Salt affected the dynamics of live and permeabilised cells during storage; cooking temperature influenced permeability and accessible intracellular PepX activity. Higher permeabilisation, autolysis, released and accessible intracellular PepX activity with lower cell envelope proteinase activities, indicated AM2, SK11 and Z8 strains may perform better in reduced salt environments. Strains HP, R1 and 303 had low autolysis and permeabilisation but higher cell envelope proteinase activities at reduced salt levels and may not be suitable for low salt cheese production.

Structure, dynamics and immunogenicity of a catalytically inactive CXC chemokine-degrading protease SpyCEP from Streptococcus pyogenes

Over 18 million disease cases and half a million deaths worldwide are estimated to be caused annually by Group A Streptococcus. A vaccine to prevent GAS disease is urgently needed. SpyCEP (Streptococcus pyogenes Cell-Envelope Proteinase) is a surface-exposed serine protease that inactivates chemokines, impairing neutrophil recruitment and bacterial clearance, and has shown promising immunogenicity in preclinical models. Although SpyCEP structure has been partially characterized, a more complete and higher resolution understanding of its antigenic features would be desirable prior to large scale manufacturing. To address these gaps and facilitate development of this globally important vaccine, we performed immunogenicity studies with a safety-engineered SpyCEP mutant, and comprehensively characterized its structure by combining X-ray crystallography, NMR spectroscopy and molecular dynamics simulations. We found that the catalytically-inactive SpyCEP antigen conferred protection similar to wild-type SpyCEP in a mouse infection model. Further, a new higher-resolution crystal structure of the inactive SpyCEP mutant provided new insights into this large chemokine protease comprising nine domains derived from two non-covalently linked fragments. NMR spectroscopy and molecular simulation analyses revealed conformational flexibility that is likely important for optimal substrate recognition and overall function. These combined immunogenicity and structural data demonstrate that the full-length SpyCEP inactive mutant is a strong candidate human vaccine antigen. These findings show how a multi-disciplinary study was used to overcome obstacles in the development of a GAS vaccine, an approach applicable to other future vaccine programs. Moreover, the information provided may also facilitate the structure-based discovery of small-molecule therapeutics targeting SpyCEP protease inhibition.

Effect of prebiotics, inorganic salts and amino acids for cell envelope proteinase production from Lactobacillus plantarum LP69.

Cell-envelope proteinases (CEPs) can improve the texture and organoleptic characteristics of dairy products, and may also cause the release of bioactive peptides, which contribute to improving the host&rsquo;s health. Thus, the CEPs with high activity produced by L. plantarum has great potential in the development of functional dairy products. A single factor experiment was used to investigate the effects of prebiotics (inulin, stachyose, isomaltooligosaccharide, xylooligosaccharides, galactooligosaccharides and fructooligosaccharides), inorganic salts (Na2HPO4, NaH2PO4, K2HPO4, KH2PO4, CH3COONa and C6H14N2O7) and amino acids (arginine, leucine, serine, lysine, alanine and glutamic acid) on the activity of cell envelope proteinases (CEPs), specific activity, protein content, OD600 value and pH in MRS broth fermented by Lactobacillus plantarum LP69. The optimum concentration of inulin (0.7%), isomaltooligosaccharide (0.5%), Na2HPO4 (0.4%), CH3COONa (0.4%), leucine (20 mg/L), serine (20 mg/L) for L. plantarum LP69 was estimated with the activity of CEP in the range 17.36&ndash;21.47 U/mL, protein content in the range 19.18&ndash;22.53 mg/mL, specific activity in the range 0.77&ndash;1.12 U/mg. Inulin, isomaltooligosaccharide, Na2HPO4, CH3COONa, leucine and serine are superior to other selected substances, and have a significant influence on the CEP activity and specific activity of L. plantarum LP69. This would provide a reference for further optimization of CEP-producing media of L. plantarum LP69.

Effect of prebiotics, inorganic salts and amino acids for cell envelope proteinase production from Lactobacillus plantarum LP69 [pdf

Effect of prebiotics, inorganic salts and amino acids for cell envelope proteinase production from Lactobacillus plantarum LP69 [pdf

Effects of Carbon and Nitrogen Sources on Activity of Cell Envelope Proteinase Produced by Lactobacillus plantarum LP69

Abstract In present study, the effects of carbon sources (glucose, lactose, sucrose, galactose, maltose and soluble starch) and nitrogen sources (casein peptone, whey protein, soy peptone, yeast, tryptone, beef extract and peptone) on activity of cell envelope proteinases (CEP), specific activity, protein content, OD600 value and pH in MRS broth fermented by Lactobacillus plantarum LP69 were investigated by individual factor experiment. The results indicated that carbon and nitrogen sources have significant influence on the activity of CEP and specific activitys of L. plantarum LP69, glucose, maltose, casein peptone and peptone are superior to other selected carbon and nitrogen sources. The optimum concentrations of glucose, maltose, casein peptone and peptone for L. plantarum LP69 are 2%, 2%, 1% and 1%; the activity of CEP are 19.52U/mL, 21.13U/mL, 13.49U/mL and 20.61U/mL, respectively.

Transcription activity of lactic acid bacterial proteolysis-related genes during cheese maturation

The catabolism of milk protein in cheese is one way how the microorganisms influence the sensorial characteristics of the final product. In this investigation, we paid attention to four genes [prtP (cell-envelope proteinase gene), pepX (X-prolyl dipeptidyl aminopeptidase gene), pepN (aminopeptidase gene) and bcaT (branched chain aminotransferase gene)] responsible for the production of volatile aroma-active compounds from milk proteins by lactic acid bacteria (LAB). We studied the dynamics of these genes and their corresponding LAB host, during the maturation of a raw ewes’ milk-based cheese, using metagenomics and metatranscriptomics approaches. The transcriptome-oriented experiments included the analysis of total RNA (at three stages of cheese maturation) and also the construction of specific cDNA sub-libraries of the abovementioned genes. The proteolytic transcriptome analysis was supported by following the transcription activity of 16S rRNA gene and by metagenomic investigation. The combination of the described methods permitted to screen the dynamics of targeted genes throughout the cheese production. Lactococci were the major players in the LAB group, but the analysis provided also information on the role and properties of members of the genus Lactobacillus, such as Lb. rhamnosus, Lb. helveticus, Lb. pentosus, Lb. curvatus, Lb. parabuchneri, Lb. plantarum, Lb. brevis, Lb. delbrueckii, Lb. paracasei, Lb. fermentum and Lb. heilongjiangensis, proteolysis-related genes of which were active during cheese ripening.

Identification and partial characterization of proteolytic activity of Enterococcus faecalis relevant to their application in dairy industry.

Bacteria of the genus Enterococcus are lactic acid bacteria (LAB), which occur ubiquitous in many traditional fermented foods, especially artisanal cheeses, playing positive role in the development of cheese flavor. Moreover, several enterococci are successfully used as a pharmaceutical probiotic and some of them are able to produce bacteriocin and bioactive peptides, thanks to which the possibilities of application of enterococci in dairy technology and biotechnology are increased. The aims of the study were to investigate the proteolytic potential and identify the key enzymes of proteolytic system of Enterococcus faecalis isolated from artisan Polish cheeses. An extracellular - secreted (E) and a cell envelope proteinase (CEP) were isolated and enzyme activity depending on bacterial growth phase was evaluated. CEP showed a higher protease activity than E and this fraction has been purified 70-fold by a method including precipitation, diafiltration and gel filtration chromatography. The molecular mass of the enzyme has been estimated to be ~25 kDa by SDS-PAGE. Maximum enzyme activity of the proteinase has been observed at pH 6,9 and 37 ºC. The enzyme was able to hydrolyze: casein, bovine serum albumin, α-lactalbumin, β-lactoglobulin, but not Leu-pNa. The results of zymography, SDS- PAGE and LC-MS-MS/MS data allowed us to identify the key enzymes of proteolytic system of E. faecalis as coccolysin and glutamylendopeptidase. To asses microbiological safety of the tested strain, the evaluation of the presence of virulence factors and antibiotic susceptibility was also conducted.

Study of Extraction and Enzymatic Properties of Cell-Envelope Proteinases from a Novel Wild Lactobacillus plantarum LP69

Lactobacilli cell-envelope proteinases (CEPs) have been widely used in the development of new streams of blockbuster nutraceuticals because of numerous biopharmaceutical potentials; thus, the development of viable methods for CEP extraction and the improvement of extraction efficiency will promote their full-scale application. In this study, CEP from a novel wild Lactobacillus plantarum LP69 was released from cells by incubating in calcium-free buffer. The extraction conditions of CEP were optimized by response surface methodology with the enzyme activity and specific activity as the detective marker. The optimal extraction conditions were: time of 80 min, temperature of 39 °C and buffer pH of 6.5. Under these conditions, enzyme activity and specific activity were (23.94 ± 0.86) U/mL and (1.37 ± 0.03) U/mg, respectively, which were well matched with the predicted values (22.12 U/mL and 1.36 U/mg). Optimal activity of the crude CEP occurred at pH 8.0 and 40 °C. It is a metallopeptidase, activated by Ca2+, inhibited by Zn2+ and ethylene-diamine-tetra-acetic acid, and a serine proteinase which is inhibited by phenylmethylsulfonyl fluoride. Kinetic studies showed that CEP from LP69 could hydrolyze whey protein, lactoglobulin and casein. Our study improves the extraction efficiency of CEPs from LP69, providing the reference for their industrial development.

Distribution of Cell Envelope Proteinases Genes among Polish Strains of Lactobacillus helveticus.

Most of the lactic acid bacteria (LAB) are able to grow in milk mainly due to the activity of a complex and well-developed proteolytic system. Cell envelope-associated proteinases (CEPs) begin casein hydrolysis and allow for releasing the peptides, enclosed in the structure of native milk proteins that are essential for growth of Lactobacillus helveticus. The biodiversity of genes encoding CEPs among L. helveticus strains can have an effect on some technological parameters such as acid production, bacterial growth rate in milk as well as liberation of biologically active peptides. The study reveals significant differences in the presence of various variants of CEPs encoding genes among ten novel Polish strains and indicates the intraspecific diversity exhibited by L. helveticus. In terms of distribution of CEPs genes, four different genetic profiles were found among the microorganisms analyzed. Furthermore, the strains exhibited also various levels of proteolytic activity. Molecular analysis revealed that prtH3 is the most abundant CEPs-encoding gene among the strains investigated. The results indicate also that ecological niche and environmental conditions might affect proteolytic properties of L. helveticus strains. The greatest variety in terms of quantity of the detected CEP encoding genes was noticed in L. helveticus 141, T105 and T104 strains. In these strains, the combination of three nucleotide gene sequences (prtH/prtH2/prtH3) was identified. Interestingly, T104 and T105 exhibited the highest proteolytic activity and also the fastest dynamic of milk acidification among the tested strains of L. helveticus.

Peptidomic study of casein proteolysis in bovine milk by Lactobacillus casei PRA205 and Lactobacillus rhamnosus PRA331

Lactobacilli contain different cell envelope proteinases (CEPs) responsible for the hydrolysis of caseins and the release of various bioactive peptides. In this work, we explored the CEP activity of Lactobacillus casei PRA205 and Lactobacillus rhamnosus PRA331 whole cells towards β-, αS1-, κ- and αS2-caseins in bovine milk. Mass spectrometry analysis of fermented milk hydrolysates identified a total of 331 peptides, which were mainly derived from β-caseins (59.0 and 60.1% for PRA205 and PRA331, respectively). Analysis of αS1-casein (f1–23) cleavage site specificity congruently supports that Lb. casei PRA205 and Lb. rhamnosus PRA331 exhibited a mixed-type CEPI/III activity. PRA205 and PRA331 CEPs also showed cleavage site specificity toward β-casein. These CEPs cleaved the peptide bond preferentially when hydrophobic or negatively charged amino acids were present. 13.5% and 13.7% of peptides released by Lb. casei PRA205 and Lb. rhamnosus PRA331 CEPs were found to have 100% homology with previously identified bioactive peptides.

Characterization of Goat Milk Hydrolyzed by Cell Envelope Proteinases from Lactobacillus plantarum LP69: Proteolytic System Optimization, Bioactivity, and Storage Stability Evaluation.

Despite the widespread application of lactic acid bacterium in dairy production through its contribution to acidification, development of sensorial properties, and health-promoting effects, relatively little information is available on the cell envelope proteinases (CEPs) of Lactobacillus plantarum, especially on the proteolytic system and the production of bioactivity peptides. In this study, CEPs from a novel L. plantarum LP69 were involved in goat milk hydrolysis and generated a product with high activity that showed a degree of hydrolysis of 15.68 ± 0.74%, Angiotensin I-Converting Enzyme (ACE)-inhibitory rate of 83.25 ± 1.05%, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging rate of 64.91 ± 1.27%, and hydroxyl radical scavenging rate of 89.17 ± 1.13%. The optimized hydrolysis conditions were time of 4.5 h, temperature of 41 °C, initial pH of 8.5, and enzyme to substrate ratio (E/S) of 12% (w/w) by orthogonal experiments. Application of a stabilizer greatly promoted milk stability. A well-designed stabilizer consists of 0.05% carrageenan, 0.15% gellan gum, and 0.15% sucrose esters, which significantly raised the milk stability coefficient, R, from 70.67% to 98.57%. The storage stability of milk was evaluated during 84 days at room temperature or 4 °C. Our study depicts the contribution of CEPs from L. plantarum LP69 in goat milk, exploring a new way for the development of a functional milk product.

Invited review: Bioactive compounds produced during cheese ripening and health effects associated with aged cheese consumption

Traditionally, cheese is manufactured by converting fluid milk to a semisolid mass through the use of a coagulating agent, such as rennet, acid, heat plus acid, or a combination thereof. Cheese can vary widely in its characteristics, including color, aroma, texture, flavor, and firmness, which can generally be attributed to the production technology, source of the milk, moisture content, and length of aging, in addition to the presence of specific molds, yeast, and bacteria. Among the most important bacteria, lactic acid bacteria (LAB) play a critical role during the cheese-making process. In general, LAB contain cell-envelope proteinases that contribute to the proteolysis of cheese proteins, breaking them down into oligopeptides that can be subsequently taken up by cells via specific peptide transport systems or further degraded into shorter peptides and amino acids through the collaborative action of various intracellular peptidases. Such peptides, amino acids, and their derivatives contribute to the development of texture and flavor in the final cheese. In vitro and in vivo assays have demonstrated that specific sequences of released peptides exhibit biological properties including antioxidant, antimicrobial, anti-inflammatory, immunomodulatory, and analgesic/opioid activity, in addition to angiotensin-converting enzyme inhibition and antiproliferative activity. Some LAB also produce functional lipids (e.g., conjugated linoleic acid) with anti-inflammatory and anticarcinogenic activity, synthesize vitamins and antimicrobial peptides (bacteriocins), or release γ-aminobutyric acid, a nonprotein amino acid that participates in physiological functions, such as neurotransmission and hypotension induction, with diuretic effects. This review provides an overview of the main bioactive components present or released during the ripening process of different types of cheese.

Comparative Genomics of Completely Sequenced Lactobacillus helveticus Genomes Provides Insights into Strain-Specific Genes and Resolves Metagenomics Data Down to the Strain Level.

Although complete genome sequences hold particular value for an accurate description of core genomes, the identification of strain-specific genes, and as the optimal basis for functional genomics studies, they are still largely underrepresented in public repositories. Based on an assessment of the genome assembly complexity for all lactobacilli, we used Pacific Biosciences' long read technology to sequence and de novo assemble the genomes of three Lactobacillus helveticus starter strains, raising the number of completely sequenced strains to 12. The first comparative genomics study for L. helveticus—to our knowledge—identified a core genome of 988 genes and sets of unique, strain-specific genes ranging from about 30 to more than 200 genes. Importantly, the comparison of MiSeq- and PacBio-based assemblies uncovered that not only accessory but also core genes can be missed in incomplete genome assemblies based on short reads. Analysis of the three genomes revealed that a large number of pseudogenes were enriched for functional Gene Ontology categories such as amino acid transmembrane transport and carbohydrate metabolism, which is in line with a reductive genome evolution in the rich natural habitat of L. helveticus. Notably, the functional Clusters of Orthologous Groups of proteins categories “cell wall/membrane biogenesis” and “defense mechanisms” were found to be enriched among the strain-specific genes. A genome mining effort uncovered examples where an experimentally observed phenotype could be linked to the underlying genotype, such as for cell envelope proteinase PrtH3 of strain FAM8627. Another possible link identified for peptidoglycan hydrolases will require further experiments. Of note, strain FAM22155 did not harbor a CRISPR/Cas system; its loss was also observed in other L. helveticus strains and lactobacillus species, thus questioning the value of the CRISPR/Cas system for diagnostic purposes. Importantly, the complete genome sequences proved to be very useful for the analysis of natural whey starter cultures with metagenomics, as a larger percentage of the sequenced reads of these complex mixtures could be unambiguously assigned down to the strain level.

High γ-aminobutyric acid production from lactic acid bacteria: Emphasis on Lactobacillus brevis as a functional dairy starter

ABSTRACTγ-Aminobutyric acid (GABA) and GABA-rich foods have shown anti-hypertensive and anti-depressant activities as the major functions in humans and animals. Hence, high GABA-producing lactic acid bacteria (LAB) could be used as functional starters for manufacturing novel fermented dairy foods. Glutamic acid decarboxylases (GADs) from LAB are highly conserved at the species level based on the phylogenetic tree of GADs from LAB. Moreover, two functionally distinct GADs and one intact gad operon were observed in all the completely sequenced Lactobacillus brevis strains suggesting its common capability to synthesize GABA. Difficulties and strategies for the manufacture of GABA-rich fermented dairy foods have been discussed and proposed, respectively. In addition, a genetic survey on the sequenced LAB strains demonstrated the absence of cell envelope proteinases in the majority of LAB including Lb. brevis, which diminishes their cell viabilities in milk environments due to their non-proteolytic nature. Thus, several strategies have been proposed to overcome the non-proteolytic nature of Lb. brevis in order to produce GABA-rich dairy foods.

Transcriptomic And Proteomic Analysis To Delineate The Mechanism Of Antibiofilm Activity Of 3‐Furancarboxaldehyde On Group A Streptococcus

Group A Streptococcus (GAS, Streptococcus pyogenes), an uncompromising human exclusive pathogen is responsible for various invasive and non‐invasive diseases. The biofilm forming ability of the multi‐virulent pathogen is considered as an important factor for its growth and persistence in the human host. The current study demonstrates the antibiofilm activity of 3Furancarboxaldehyde (3FCA), a compound present in floral honey, against GAS biofilm, and analyses its effect on multiple aspects of GAS (morphology, virulence, aggregation, gene expression and proteomic analysis) at its minimal biofilm inhibitory concentration (132μg/ml). 3FCA was found to modify the growth pattern of GAS in solid and liquid media and enhance the rate of auto‐aggregation. Real Time gene expression analysis showed downregulation of covR gene, which is speculated to be the prime target for the antibiofilm activity. The gel based proteomic and Matrix‐assisted laser desorption/ionization (MALDI) analysis of GAS upon treatment with 3FCA revealed upregulation of proteins such as ABC transporter (ATP‐binding protein) MtsB SPy_0454, putative cell envelope proteinase PrtS SPy_0416, which are speculated to be involved in increased hyaluronic acid capsule production and increased virulence of GAS respectively. Few other proteins which are involved in TCA cycle were also found to be differentially regulated. This study reveals the mechanism of antibiofilm activity of 3FCA at both genomic and proteomic levels.