Targeted Genome Mining Reveals the Psychrophilic Clostridium estertheticum Complex as a Potential Source for Novel Bacteriocins, Including Cesin A and Estercticin A

Antimicrobial resistance in pathogenic bacteria is considered a major public health issue necessitating the discovery of alternative antimicrobial compounds. In this regard, targeted genome mining in bacteria occupying under-explored ecological niches has the potential to reveal such compounds, including bacteriocins. In this study, we determined the bacteriocin biosynthetic potential of the psychrophilic Clostridium estertheticum complex (CEC) through a combination of genome mining and phenotypic screening assays. The genome mining was performed in 40 CEC genomes using antiSMASH. The production of bacteriocin-like compounds was phenotypically validated through agar well (primary screening) and disk diffusion (secondary screening) assays using cell free supernatants (CFS) and partially purified extracts, respectively. Stability of four selected CFS against proteolytic enzymes, temperature and pH was determined while one CFS was analyzed by HRMS and MS/MS to identify potential bacteriocins. Twenty novel bacteriocin biosynthetic gene clusters (BBGC), which were classified into eight (six lantibiotics and two sactipeptides) distinct groups, were discovered in 18 genomes belonging to C. estertheticum (n = 12), C. tagluense (n = 3) and genomospecies2 (n = 3). Primary screening linked six BBGC with narrow antimicrobial activity against closely related clostridia species. All four preselected CFS retained activity after exposure to different proteolytic, temperature and pH conditions. Secondary screening linked BBGC1 and BBGC7 encoding a lantibiotic and sactipeptide, respectively, with activity against Bacillus cereus while lantibiotic-encoding BBGC2 and BBGC3 were linked with activity against B. cereus, Staphylococcus aureus (methicillin-resistant), Escherichia coli and Pseudomonas aeruginosa. MS/MS analysis revealed that C. estertheticum CF004 produces cesin A, a short natural variant of nisin, and HRMS indicated the production of a novel sactipeptide named estercticin A. Therefore, we have shown the CEC, in particular C. estertheticum, is a source of novel and stable bacteriocins that have activities against clinically relevant pathogens.

The Feasibility of Using Agarophyte Processing Waste

When agarophytes are processed, 16-89% algal waste (AW) is formed. Due to the high content of minerals in this waste and the presence of proteins that are resistant to the action of proteolytic enzymes, such AW is mainly used in the production of feed for farm animals. The significant content of polysaccharides in dry AW indicates that it could be used as a raw material for the production of dietary fiber (DF). Due to the difference in the chemical composition of AW depending on the type of red algae, different approaches for its deproteinization have been used. Thus, a 3% alkali solution should be used for the deproteinization of algal waste from the red algae Gracilaria, a 0.5% alkali solution for GelidiumAW, and a 1% sodium carbonate solution for A. plicataAW.The duration of the deproteinization process is 30 min at a temperature of 97±2 ∘C for all types of AW. In this study, functional and technological solutions of DF from AW were developed. The results showed that their water binding capacity was 6-22 g of water per 1 g of the preparation, the fat binding capacity was 1.6-3.3 g of fat per 1 g of the preparation, and the swelling capacity was 46-312% depending on the type of red algae.The obtained DF was used in the production of minced fish food products of the ‘fish sticks’ type, based on minced fish and consisting of cod and pink salmon. The study of the chemical composition and calorie content of the developed food product showed the possibility of its use in dietary nutrition. Keywords: red algae, algal waste, deproteinization, dietary fiber

Antibiotics Alter Pocillopora Coral-Symbiodiniaceae-Bacteria Interactions and Cause Microbial Dysbiosis During Heat Stress

Symbioses between eukaryotes and their associated microbial communities are fundamental processes that affect organisms’ ecology and evolution. A unique example of this is reef-building corals that maintain symbiotic associations with dinoflagellate algae (Symbiodiniaceae) and bacteria that affect coral health through various mechanisms. However, little is understood about how coral-associated bacteria communities affect holobiont heat tolerance. In this study, we investigated these interactions in four Pocillopora coral colonies belonging to three cryptic species by subjecting fragments to treatments with antibiotics intended to suppress the normal bacteria community, followed by acute heat stress. Separate treatments with only antibiotics or heat stress were conducted to compare the effects of individual stressors on holobiont transcriptome responses and microbiome shifts. Across all Pocillopora species examined, combined antibiotics and heat stress treatment significantly altered coral-associated bacteria communities and caused major changes in both coral and Cladocopium algal symbiont gene expression. Individually, heat stress impaired Pocillopora protein translation and activated DNA repair processes, while antibiotics treatments caused downregulation of Pocillopora amino acid and inorganic ion transport and metabolism genes and Cladocopium photosynthesis genes. Combined antibiotics-heat stress treatments caused synergistic effects on Pocillopora and Cladocopium gene expression including enhanced expression of oxidative stress response genes, programed cell death pathways and proteolytic enzymes that indicate an exacerbated response to heat stress following bacteria community suppression. Collectively, these results provide further evidence that corals and their Symbiodiniaceae and bacteria communities engage in highly coordinated metabolic interactions that are crucial for coral holobiont health, homeostasis, and heat tolerance.

Veni, Vidi, Vici: Immobilized Peptide-Based Conjugates as Tools for Capture, Analysis, and Transformation

Analysis of peptide biomarkers of pathological states of the organism is often a serious challenge, due to a very complex composition of the cell and insufficient sensitivity of the current analytical methods (including mass spectrometry). One of the possible ways to overcome this problem is sample enrichment by capturing the selected components using a specific solid support. Another option is increasing the detectability of the desired compound by its selective tagging. Appropriately modified and immobilized peptides can be used for these purposes. In addition, they find application in studying the specificity and activity of proteolytic enzymes. Immobilized heterocyclic peptide conjugates may serve as metal ligands, to form complexes used as catalysts or analytical markers. In this review, we describe various applications of immobilized peptides, including selective capturing of cysteine-containing peptides, tagging of the carbonyl compounds to increase the sensitivity of their detection, enrichment of biological samples in deoxyfructosylated peptides, and fishing out of tyrosine–containing peptides by the formation of azo bond. Moreover, the use of the one-bead-one-compound peptide library for the analysis of substrate specificity and activity of caspases is described. Furthermore, the evolution of immobilization from the solid support used in peptide synthesis to nanocarriers is presented. Taken together, the examples presented here demonstrate immobilized peptides as a multifunctional tool, which can be successfully used to solve multiple analytical problems.

The role of cysteine in the formation of domain structures of papain and legumin in peas, involved in limited proteolysis

The limited use of plant proteins for food is explained by their low bioavailability and poor digestibility by enzymes of the gastrointestinal tract. Partially reproduced enzymatic processes of limited proteolysis that occur during seed germination are used to modify and improve the edibility characteristics of seed proteins. The present work discusses the possibility of reducing the duration of seed germination processes by optimising the conditions and parameters of limited proteolysis. To optimise manufacturing high-quality final product, enzymes (additional to the natural enzymes in the seed) and proteolysis conditions (in this case, temperature), as well as added substances (hydrolysis activators), were selected. The influence of cysteine on the formation of domain structures of proteins (enzymes and globulins) was evaluated. The proposed expressions can be used to determine those fragments of protein molecules that form stable domains and become unstructured when exposed to enzymes. Optimal conditions for limited proteolysis were identified based on the physical mechanism of action of papain-like proteolytic enzymes on pea legumin LegA (3KSC, CAA10722). It is shown that the decomposition of protein secondary structures takes 6–8 times longer, since the formed hydrogen bonds limit the access of enzymes to the corresponding amino-acid residues. It is also demonstrated that the decomposition of hydrogen bonds, e.g. by preliminary heat treatment of proteins, will broaden the prospects for limited proteolysis.

Enzymatic Hydrolysis and Fermentation of Pea Protein Isolate and Its Effects on Antigenic Proteins, Functional Properties, and Sensory Profile

Combinations of enzymatic hydrolysis using different proteolytic enzymes (papain, Esperase®, trypsin) and lactic fermentation with Lactobacillus plantarum were used to alter potential pea allergens, the functional properties and sensory profile of pea protein isolate (PPI). The order in which the treatments were performed had a major impact on the changes in the properties of the pea protein isolate; the highest changes were seen with the combination of fermentation followed by enzymatic hydrolysis. SDS-PAGE, gel filtration, and ELISA results showed changes in the protein molecular weight and a reduced immunogenicity of treated samples. Treated samples showed significantly increased protein solubility at pH 4.5 (31.19–66.55%) and at pH 7.0 (47.37–74.95%), compared to the untreated PPI (6.98% and 40.26%, respectively). The foaming capacity was significantly increased (1190–2575%) compared to the untreated PPI (840%). The treated PPI showed reduced pea characteristic off-flavors, where only the treatment with Esperase® significantly increased the bitterness. The results from this study suggest that the combination of enzymatic hydrolysis and lactic fermentation is a promising method to be used in the food industry to produce pea protein ingredients with higher functionality and a highly neutral taste. A reduced detection signal of polyclonal rabbit anti-pea-antibodies against the processed protein preparations in ELISA furthermore might indicate a decreased immunological reaction after consumption.

Roles of Epithelial and Mesenchymal TRP Channels in Mediating Inflammatory Fibrosis

The maintenance of normal vision is dependent on preserving corneal transparency. For this to occur, this tissue must remain avascular and its stromal architecture needs to be retained. Epithelial transparency is maintained provided the uppermost stratified layers of this tissue are composed of terminally differentiated non-keratinizing cells. In addition, it is essential that the underlying stromal connective tissue remains avascular and scar-free. Keratocytes are the source of fibroblasts that are interspersed within the collagenous framework and the extracellular matrix. In addition, there are sensory nerve fibers whose lineage is possibly either neural crest or mesenchymal. Corneal wound healing studies have been undertaken to delineate the underlying pathogenic responses that result in the development of opacification following chemical injury. An alkali burn is one type of injury that can result in severe and long- lasting losses in ocular transparency. During the subsequent wound healing process, numerous different proinflammatory cytokines and proteolytic enzymes undergo upregulation. Such increases in their expression levels induce maladaptive expression of sustained stromal inflammatory fibrosis, neovascularization, and losses in the smooth optical properties of the corneal outer surface. It is becoming apparent that different transient receptor potential channel (TRP) isoforms are important players in mediating these different events underlying the wound healing process since injury upregulates both their expression levels and functional involvement. In this review, we focus on the involvement of TRPV1, TRPA1 and TRPV4 in mediating some of the responses that underlie the control of anterior ocular tissue homeostasis under normal and pathological conditions. They are expressed on both different cell types throughout this tissue and also on corneal sensory nerve endings. Their roles have been extensively studied as sensors and transducers of environmental stimuli resulting from exposure to intrinsic modulators and extrinsic ligands. These triggers include alteration of the ambient temperature and mechanical stress, etc., that can induce pathophysiological responses underlying losses in tissue transparency activated by wound healing in mice losses in tissue transparency. In this article, experimental findings are reviewed about the role of injury-induced TRP channel activation in mediating inflammatory fibrotic responses during wound healing in mice.

Conjugation with gold nanoparticles improves the stability of the KT2 peptide and maintains its anticancer properties

One of the major weaknesses of therapeutic peptides is their sensitivity to degradation by proteolytic enzymes in vivo.

Effect of Proteolytic Enzymes (Bromelain and Papain) on Sensory and Chemical Quality of Sukuti (an Indigenous Dried Meat Product of Nepal)

The present work was undertaken to compare the effect of bromelain and papain on the sensory quality of sukuti (Nepalese indigenous dried buffalo meat). Buffalo lean meat (round cut) was purchased from the local market of Dharan and used for the preparation of sukuti after injection of 10% m/m enzyme (bromelain and papain) solution at the concentration (0-100 mg/L) and resting for 4h followed by drying at 65°C up to the moisture of 5%. The optimized concentration of each enzyme was selected by sensory evaluation based on color, flavor, texture, and overall acceptability. The proximate composition, collagen content, and collagen solubility were studied for two optimized samples with enzyme treatment and control. The best tenderizing effect of papain and bromelain enzyme was found at 40 mg/L and 10 mg/L respectively from the sensory analysis and the sensory attributes for these concentrations were significantly (p<0.05) higher than other samples. The soluble collagen content increased from 0.44 mg/g tissue in untreated meat to 0.52 for bromelain treated and to 0.98 mg/g for papain treated samples. The collagen solubility for the untreated sample was 4.74% which increases to 7.80% for bromelain and 13.82% for papain-treated samples. The protein content of optimized papain and bromelain treated samples was significantly decreased (p<0.05) from 82.44 to 80.25% and 81.43% respectively for papain and bromelain treated samples. There were no significant changes in fat, ash, and moisture on enzyme treatment.

Development of a recipe for a multicomponent mixture "Solodok +" to improve the consumer properties of bakery products

The object of research is the technology of bakery products enriched with a mixture of germinated grains of wheat, corn, barley and oats. Investigated problem: The problem of using a mixture of germinated grains is the formation of a closure sticky crumb in baked goods with a mixture. The reason for this is the high activity in the mixture of amylolytic and proteolytic enzymes. The solution to the problem consists in the developed multicomponent mixture (MM) to improve the consumer properties of bakery products, the formulation of which includes 15 % of the flour mass of the germinated grain mixture. Main scientific results: On the basis of experimental studies, the formulation of the "Solodok+" multicomponent mixture has been developed. The mixture contains: chicory inulin, dry milk whey enriched with Mg and Mn, apple pectin, phosphatide concentrate, enzyme preparation Deltamalt FN-A 50 and ascorbic acid. The optimal dose of the "Solodok+" MM for bakery products is 2.5 % by weight of flour. The area of practical use of the research results: "Solodok+" MM is recommended to be used in the production of bakery products enriched with sprouted grains at enterprises of the bakery industry of various capacities. An innovative technological product: "Solodok+" MM helps not only to reduce the stickiness of the crumb, improve its porosity, increase the volume of products, but also lengthen the freshness of unpackaged products. Scope of application of the innovative technological product: Bakery products with a mixture of sprouted grains and "Solodok+" MM have increased nutritional value, high consumer properties and are intended for a wide range of consumers.