Gamma Amino Butyric Acid (GABA) and Ferulic Acid Esterase (FAE) Producing Psychobiotic Bacteria Isolated from Cereal-Based Fermented Food.

Gut microbiota plays an important role in regulating enteric, immune and neural pathways. Many neuropsychiatric disorders such as anxiety, depression, autism and cognitive behaviour are associated with gut dysbiosis. Gamma-aminobutyric acid (GABA) and short-chain fatty acids produced by gut bacteria influence gastrointestinal and neurological functions. Ferulic acid esterase (FAE) which releases ferulic acid (FA) from feruloylated sugar ester conjugates, naturally found in grains, fruits and vegetables, is also produced by some gut bacteria and helps prevent neurodegeneration. These properties provide bacteria with the ability to maintain intestinal barrier function and prevent neuropsychiatric disorders. Therefore, this study aims to isolate GABA and FAE-producing LAB and characterize their bioactive and probiotic properties. A total of twelve cultures were isolated, of which eight bacteria positive for GABA, FAE and SCFA production were selected for further investigation. All selected bacteria were positive for bile salt hydrolase (BSH) and showed acid tolerance, resistance to bile salt, stimulated gastric and pancreatic juice, and auto- and co-aggregation properties. Furthermore, selected LAB showed mucin adhesion efficiency greater than 80% and exhibited γ-hemolytic activity. 16S rRNA sequencing identified NS0969, B1, C1, C2, M1, M2, and R2 as Limosilactobacillus fermentum and R1 as Lactiplantibacillus pentosus. This study showed that selected bacteria and/or their postbiotic preparations can be used as potential psychobiotics.

Diet-gut microbiome interaction and ferulic acid bioavailability: implications on neurodegenerative disorders.

PURPOSE OF THE REVIEW: Ferulic acid (FA), which occurs naturally as the feruloylated sugar ester in grains, fruits, and vegetables, is critical for combating oxidative stress and alleviating neurodegenerative diseases resulting from free radical-generated protein aggregates in brain cells. However, FA cannot be absorbed in conjugated form. Therefore, strategies to improve the bioavailability of FA are gaining more importance. Ferulic acid esterases (FAE) of the gut microbiota are critical enzymes that facilitate FA release from feruloylated sugar ester conjugates and influence systemic health. This review provides insight into a nutrition-based approach to preventing neurodegenerative disorders such as Alzheimer's and Parkinson's by altering the diversity of FAE-producing gut microbiota. RECENT FINDINGS: The human gut is a niche for a highly dense microbial population. Nutrient components and the quality of food shape the gut microbiota. Microbiota-diet-host interaction primarily involves an array of enzymes that hydrolyse complex polysaccharides and release covalently attached moieties, thereby increasing their bio-accessibility. Moreover, genes encoding polysaccharide degrading enzymes are substrate inducible, giving selective microorganisms a competitive advantage in scavenging nutrients. Nutraceutical therapy using specific food components holds promise as a prophylactic agent and as an adjunctive treatment strategy in neurotherapeutics, as it results in upregulation of polysaccharide utilisation loci containing fae genes in the gut microbiota, thereby increasing the release of FA and other antioxidant molecules and combat neurodegenerative processes.

Plant-Based Nutraceutical Formulation Modulates the Human Gut Microbiota and Ferulic Acid Esterase Activity During In Vitro Fermentation.

Oxidative stress is an imbalance between free reactive oxygen species and antioxidant defences leading to neurological and other chronic disorders. The interaction between food and gut microbiota and their metabolites significantly reduces oxidative stress and influences host physiology and metabolism. This process mainly involves enzymes that hydrolyse complex polysaccharides and produce metabolites. Ferulic acid esterases (FAE) one of the most important enzymes of the gut microbiome, release ferulic acid from feruloylated sugar ester conjugates, that occur naturally in grains, fruits, and vegetables. FA is crucial in combating oxidative stress resulted from free radical formation. This study investigated the effect of two plant-based nutraceutical formulations, cereal-millet-based (PC1) and fruit-vegetable-based (PC2), on gut microbiota and the production of FAE, short chain fatty acids (SCFA) and other small metabolites in in vitro fermentation using human faecal samples. After in vitro fermentation, both nutraceutical formulations increased the abundance of Bifidobacterium, Lactobacillus, Prevotella, Feacalibacteria, and Clostridium leptum. Furthermore, they induced the production of FAE, xylanase and pectinase enzymes, SCFA and other small metabolites, resulting in increased antioxidation activity of the fermentate. PC1 stimulated FAE and xylanase production more effectively. These results demonstrated a positive correlation between the feruloylated nutraceutical formulation and the production of FAE and other accessory enzymes, suggesting that PC1 and PC2 stimulate the proliferation of the FAE-producing microbial consortium of the gut microbiome and therefore, increase FA and SCFA concentration. From this study it is evident that FA-rich plant-based formulation can be used as a prophylactic nutraceutical supplement to alleviate oxidative stress by modulating the gut microbiota.

Studies on effect of multigrain use in the preparation of Litti/ Baati: A traditional food of Northern part of India (UP, Jharkhand and Bihar).

Present studies were carried out to find the effect of different multigrain viz., finger millet, foxtail millet and little millet on litti. The multigrain powder was blended in whole wheat flour. Litti composite flour was studied for nutritional, rheological, gluten, sedimentation value, falling number and compared with regular wheat flour. Flaxseed and soybean were blended with barley, besan, spices for inner composite stuffing and samples were evaluated for proximate analysis. Shelf life studies of litti were assessed for 1 month at room temperature 25 ± 2℃ and freezer at 4℃. This research work explores with the aim to have benefits of multifunctional ingredients for the improvement of litti to have a healthy product; and increase the popularity of litti all over India to make it a commercially important product because of the incorporated multifunctional ingredients. The RDA calorie for human can be met with 4-5 litti per day.

Prophylactic effect of pectic oligosaccharides against poly I: C- induced virus-like infection in BALB/c mice.

Pectin oligosaccharides (POS) are pectin-derived prebiotics that exerts anti-inflammatory effects on the host and stimulates an innate immune response. The role of POS in protective immunity against viral infections is not very obvious. Therefore, the prophylactic effect of POS in the mouse model induced by Poly I: C mimicking viral infection was examined. Mice fed POS showed a significant (p ≤ .05) increase in IgG, sIgA, IgA, IL-12, and a significant (p ≤ .05) decrease in the concentration of pro-inflammatory cytokines IL-5, IL-6, IL-13 and IL-17 in lung and blood serum after Poly I: C stimulation. However, the control group could not inhibit pro-inflammatory cytokines. POS also promoted the growth of the Lactobacillus, Prevotella, Rilenellaceae, and Lachanospiraceae groups. Therefore, this study demonstrate that POS has the potiential to protect against viral inflammation by altering gut microbiota and activating mucosal immunity. PRACTICAL APPLICATIONS: POS is 2-10 mer oligomers of pectin. The human gastrointestinal tract lacks the enzyme to break down POS. They are fermented by gut bacteria in the colon and stimulate the proliferation of specific gut bacteria that are positively correlated with the production of anti-inflammatory cytokines and SCFA. POS also stimulates the secretion of IgA, which inhibits bacterial and viral adhesion and protects the host. Therefore, POS can be used as a functional food ingredient in food to stimulate a specific group of gut bacteria and enhance preventive immunity.

Microbial ecology and functional coffee fermentation dynamics with Pichia kudriavzevii.

Specialty coffee can be developed by the application of explicit microorganisms or starters to obtain desired fermentation. In the present study, natural fermentation (NF) of Arabica coffee was carried out spontaneously, the other set was inoculated with Pichia kudriavzevii (Y) starter culture (isolated, identified and mass cultured). The effect of microbial fermentation, metagenomics, production of functional metabolites, volatiles and their sensorial aspects were studied. The bioprocess illustrated cohesive interface of coffee nutrients and microbial communities like Mycobacterium, Acinetobacter, Gordonia, etc., in NF, Lactobacillus and Leuconostoc were prevailing in Y. The Pichia and Rhodotorula dominated in both the groups. The bioactivity of bacteria and fungi induced complex changes in physicochemical features like pH (4.2-5.2), Brix° (9.5-3.0), and metabolic transition in sugar (3.0-0.7%), alcohol (1.4-2.7%), organic acids modulating flavour precursors and organoleptics in the final brew. In the roasted bean, Y exhibited higher sugar (42%), protein (25%), polyphenol (3.5%), CGA (2.5%), caffeine (17.2%), and trigonelline (2.8%) than NF. The volatile profile exhibited increased flavour molecules like furans, ketones, and pyrazines in Y, besides lactone complexes. The organoleptics in Y were highlighted with honey, malt and berry notes. P. kudriavzevii coffee fermentation could be beneficial in specialty coffee production and enhancement of distinct characteristic flavours.

Gut Dysbiosis and IL-21 Response in Patients with Severe COVID-19.

BACKGROUND: The disease severity, ranging from being asymptomatic to having acute illness, and associated inflammatory responses has suggested that alterations in the gut microbiota may play a crucial role in the development of chronic disorders due to COVID-19 infection. This study describes gut microbiota dysbiosis in COVID-19 patients and its implications relating to the disease. DESIGN: A cross sectional prospective study was performed on thirty RT-PCR-confirmed COVID-19 patients admitted to the All India Institute of Medical Sciences, Bhopal, India, between September 10 and 20, 2020. Ten healthy volunteers were recruited as the control group. IFN, TNF, and IL-21 profiling was conducted using plasma samples, and gut bacterial analysis was performed after obtaining the metagenomics data of stool samples. RESULTS: Patients with a variable COVID-19 severity showed distinct gut microflora and peripheral interleukin-21 levels. A low Firmicute/Bacteroidetes ratio, caused by the depletion of the fibre-utilizing bacteria, F. prausnitzii, B. Plebius, and Prevotella, and an increase in Bacteroidetes has associated gut microbiota dysbiosis with COVID-19 disease severity. CONCLUSIONS: The loss of the functional attributes of signature commensals in the gut, due to dysbiosis, is a predisposing factor of COVID-19 pathophysiology.

The Role of Histamine and Histamine Receptors in Mast Cell-Mediated Allergy and Inflammation: The Hunt for New Therapeutic Targets.

Histamine and its receptors (H1R-H4R) play a crucial and significant role in the development of various allergic diseases. Mast cells are multifunctional bone marrow-derived tissue-dwelling cells that are the major producer of histamine in the body. H1R are expressed in many cells, including mast cells, and are involved in Type 1 hypersensitivity reactions. H2R are involved in Th1 lymphocyte cytokine production. H3R are mainly involved in blood-brain barrier function. H4R are highly expressed on mast cells where their stimulation exacerbates histamine and cytokine generation. Both H1R and H4R have important roles in the progression and modulation of histamine-mediated allergic diseases. Antihistamines that target H1R alone are not entirely effective in the treatment of acute pruritus, atopic dermatitis, allergic asthma, and other allergic diseases. However, antagonists that target H4R have shown promising effects in preclinical and clinical studies in the treatment of several allergic diseases. In the present review, we examine the accumulating evidence suggesting novel therapeutic approaches that explore both H1R and H4R as therapeutic targets for histamine-mediated allergic diseases.

Extremozymes from metagenome: Potential applications in food processing.

The long-established use of enzymes for food processing and product formulation has resulted in an increased enzyme market compounding to 7.0% annual growth rate. Advancements in molecular biology and recognition that enzymes with specific properties have application for industrial production of infant, baby and functional foods boosted research toward sourcing the genes of microorganisms for enzymes with distinctive properties. In this regard, functional metagenomics for extremozymes has gained attention on the premise that such enzymes can catalyze specific reactions. Hence, metagenomics that can isolate functional genes of unculturable extremophilic microorganisms has expanded attention as a promising tool. Developments in this field of research in relation to food sector are reviewed.

Computational modelling and protein-ligand interaction studies of SMlipA lipase cloned from forest metagenome.

The understanding of the 3-dimensional enzyme structure is important for the point of protein engineering and applications. Computer-based molecular modelling is a vital tool for theoretical predication of enzyme activities and finding their substrates and inhibitors. SMlipA lipase was cloned from forest soil metagenome and characterized as broad spectrum enzyme with high stability in various organic solvents. In the present study, to understand the mechanism of SMlipA lipase and to identify the key residues involved in enzyme-substrate interaction, three dimensional-computational model of SMlipA has been generated and validated for stereo-chemical and amino-acid environment quality using appropriate programs, and further validation of the active-site architecture was achieved by performing docking studies with different ligand. The three dimensional structure created here provide a new understanding of the ligand preferences and their interaction with protein.

The role of IL-33 and mast cells in allergy and inflammation.

Interleukin-33 (IL-33) is a member of the interleukin-1 (IL-1) cytokine family. It is preferentially and constitutively expressed in different structural cells such as epithelial cells, endothelial cells, and smooth muscle cells. During necrosis of these cells (after tissue injury or cell damage), the IL-33 that is released may be recognized by different types of immune cells, such as eosinophils, basophils and, especially, mast cells. IL-33 needs the specific receptor ST2 (membrane-bound receptor) and Interleukin-1 receptor accessory protein heterodimer for its binding, which instigates the production of different types of cytokines and chemokines that have crucial roles in the exacerbation of allergic diseases and inflammation. IL-33 and mast cells have been influentially associated to the pathophysiology of allergic diseases and inflammation. IL-33 is a crucial regulator of mast cell functions and might be an attractive therapeutic target for the treatment of allergic and inflammatory diseases. In this review, we summarize the current knowledge regarding the roles of IL-33 and mast cells in the pathogenesis of allergies and inflammation.

Diversity of glycosyl hydrolase enzymes from metagenome and their application in food industry.

Traditional use of enzymes for food processing and production of food ingredients resulted in fast-growing enzyme industries world over. The advances in technologies gave rise to exploring newer enzymes and/or modified enzymes for specific application. Search for novel enzymes that can augment catalytic efficiency and advances in molecular biology techniques including sequencing has targeted microbial diversity through metagenomic approaches for sourcing enzymes from difficult to culture organisms. Such mining studies have received more attention in characterizing hydrolases, their prevalence, broad substrate specificities, stability, and independence of cofactors. The focus on glycosyl hydrolases from metagenome for their application in food sector is reviewed.

Cloning and molecular modelling of pectin degrading glycosyl hydrolase of family 28 from soil metagenomic library.

Western Ghats of India is recognized as one of the 12 mega diversity regions of the world and is the hot spot for unrevealed microbial diversity. To explore the diversity of polysaccharide degrading enzymes in that region, metagenomic library was constructed from forest soil of Southern Western Ghats region. Nine pectinolytic clones with the ability to degrade citrus pectin were isolated based on function based screening of the library. Sequence analysis of pg_4 clone containing revealed that it contained GH family 28 domain (pfam00295) belonging to polygalacturonase superfamily (PLN03003). Its amino acid sequence analysis showed 25-55 % identity to the other well-characterized polygalacturonases. Molecular modeling of pg_4 revealed that it comprised of three right handed-parallel ß sheets, one anti-parallel ß sheet and one α helix with three conserved catalytic residue D 2263, D 284-85 and H 312 at the C terminal end. The enzyme characterized was able to hydrolyze both apple and citrus pectin with K m values of 1.685 and 1.542 mg ml(-1) and retained more that 80 % of activity at pH 5-9 and temperature 20-60 °C.

Cloning and characterization of two functionally diverse lipases from soil metagenome.

A soil metagenomic library was constructed and two functionally diverse lipase genes, SMlipB and SMlipD, were screened by a function-driven approach and characterized. The optimal temperature for enzyme activity of SMlipB and SMlipD was 50°C and 30°C, respectively, and optimal pH was determined to be 7.0 and 9.0, respectively. Both enzymes exhibited broad substrate specificity and showed enhanced activity in the presence of SDS and Tween 20. The SMlipB enzyme was highly resistant to many organic solvents, especially isopropanol, ethanediol, DMSO, methanol and xylene, whereas SMlipD activity was inhibited in all the solvents except xylene. Sequence analysis revealed SMlipB consisted of an open reading frame of 1,212 bp and encoded for 404 amino acids. It contained the GXXGXD motifs, which are supposed to be involved in Ca(2+) binding in proteases and lipases, and an extreme C-terminal motif consisting of a negatively charged amino acid followed by four hydrophobic residues, essential for the secretion of metalloprotease, and belongs to lipase subfamily I.3. SMlipD contained 1,071 bp ORF and encoded for 357 amino acids. It contains Ca(2+) ion binding sites extending from amino acid 282 to 294 and two Cystein residues (218,308), proven necessary for forming a disulfide bridge and belongs to lipase subfamily1.2.

Potential application of pectinase in developing functional foods.

The understanding that enzymatic degradation of fruit pectin can clarify juices and improve juice yields resulted in the search for microbial pectinases and application in vegetable- and fruit-processing industries. Identified enzymes were classified on the basis of their catalytic activity to pectin or its derivatives and in terms of industrial use. Discovery of gene sequences that coded the enzymes, protein engineering, and molecular biology tools resulted in defined microbial strains that over-produced the enzymes for cost-effective technologies. Recent perspectives on the use of pectin and its derivatives as dietary fibers suggest enzymatic synthesis of the right oligomers from pectin for use in human nutrition. While summarizing the activities of pectin-degrading enzymes, their industrial applications, and gene sources, this review projects another application for pectinases, which is the use of enzymatically derived pectin moieties in functional food preparation.

Prediction of ligand binding site by insilico approach in cold resistant protein isolated from cold resistant mutant of Pseudomonas fluorescens.

Cold shock proteins perform vital functions, such as mRNA masking, coupling of transcription to translation and developmental timing and regulation, which aids in survival of microbes in cold stress. Pseudomonas fluorescens is an ecologically important bacterium which helps in plant growth promotion. Since the cold tolerant mutant of the bacterium is able to grow at the temperature ranges from 30 to 4°C, it is of interest to study the process of its survival in the extreme temperatures. Therefore, this study is focused on the three dimensional structure and molecular modeling of cold resistant protein (CRP) from P. fluorescens to predict its molecular mechanism. Investigating the structure of CRP confirmed the presence of a conserved domain characteristic of the cold shock domain (CSD) family and a single nucleotide binding domain. When 3D structure of CRP was compared with the existing cold shock proteins, major deviations were found in the loop regions connecting the ß2-ß3, ß3-ß4 and ß4-ß5 sheets. Docking studies showed that CRP forms a significant clamp like structure at the substrate binding cleft which stabilizes the ligand. Therefore, it can be concluded that CRP has a strong affinity for the poly thymidine (poly T) stretch and can be considered a candidate for transcription regulation.

Expression and purification of organic solvent stable lipase from soil metagenomic library.

Gene for organic solvent stable lipase was overexpressed from soil metagenomic library. The clone with maximum activity was selected, and enzyme was purified by gel-permeation chromatography with a molecular mass of approx. 40 kDa. The deduced aminoacid sequence indicated that the protein belongs to the lipase family I.3 and containing a C-terminal secretion signal for ABC dependent transport together with possible motifs for Ca(2+) binding sites. The enzyme expressed maximum activity at 30 °C and pH 7.0 and found to be stable in pH and temperature ranging from 6.0-9.0 and 20-60 °C, respectively. Furthermore, the enzyme was found highly resistant to many organic solvents, especially isopropanol, DMSO, methanol, xylene and hexane. The enzyme showed enhanced activity in the presence of divalent cations (Mg(2+), Mn(2+), Ca(2+), Hg(2+), Cu(2+)), whereas the presence of trivalent cation (Fe(3+)) inhibited the activity.

Comparative protein modeling, prediction of conserved residue and active sites in cold resistant protein isolated from CRPF(1), a cold tolerant mutant of Pseudomonas fluorescens.

Proteins interacting with the biological information molecules DNA and RNA play important cellular roles in all organisms. One widespread super family of proteins implicated in such function(s) is cold shock protein (CSP) that contains the cold shock domain (CSD). This work is planned to study the three-dimensional structure, conserved residues, and different active sites in the structure of cold resistant protein (CRP) from CRPF(1), cold tolerant mutant of Pseudomonas fluorescence by comparative homology modeling. Here we tried to identify crucial residues that are involved in active sites or functional sites of the protein. The study reveals that CRP represent the prototype of the CSD and share a highly similar overall fold consisting of five antiparallel beta-sheets forming a beta-barrel structure with surface exposed aromatic and basic residues that were responsible for nucleic acid binding properties of variable binding affinities and sequence selectivity and harbors the nucleic acid binding motifs RNP1 and RNP2 that is highly conserved in CSP family.

Exploration of Csp genes from temperate and glacier soils of the Indian Himalayas and in silico analysis of encoding proteins.

The metagenomic Csp library was constructed from the temperate and glacier soils of central Himalaya, India followed by polymerase chain reaction (PCR) amplification. The library was further screened for low-temperature adaptation, and the positive recombinants were sorted out by determining changes in the melting temperature (Tm). A homology search of cloned sequence showed their identity with the Csp genes of Pseudomonas fluorescens, Psychrobacter cryohalolentis K5, and Shewanella spp MR-4. Amino acid sequence analysis annotated the presence of conserved aromatic and basic amino acids as well as RNA binding motifs from the cold shock domain. Furthermore, a PROSITE scan showed a moderate identity of less than 60% with the known cold shock-inducible proteins (ribosomal proteins, rbfA, DEAD-box helicases), cold acclimation protein, and temperature-induced protein (SRP1/TIP1). This study highlighted the prevalence of Csp genes from cold Himalayan environments that can be explored for tailor-made crop constructions in future.

Characterization and localization of fluorescent Pseudomonas cold shock protein(s) by monospecific polyclonal antibodies.

Cold shock protein (CSP) from Pseudomonas fluorescens MTCC 103 and cold resistant protein (CRP) from its mutant CRPF8 of 14 and 35 kd, respectively were purified to homogeneity by HPLC. Polyclonal antibodies were raised against these proteins and the expression level was checked at different temperatures, i.e., 4, 10, 20, 30 and 37 C. Furthermore, morphological changes in P. fluorescens MTCC 103 and its mutant (CRPF8) were analyzed by transmission electron microscopy (TEM). Localization of CSP and CRP documented with immunoelectron microscopy, using colloidal gold particles conjugated with secondary antibodies being the probe were used. Nevertheless, the results of cytosolic localization of CSP and CRP were evident. Furthermore, the expression of CSP and CRP increased with decrease in temperature and the cell wall thickness of the mutant exhibited 2-fold increase, thus facilitating low temperature survival.