High-yield production of maltooligosaccharide-forming α-amylase in Bacillus subtilis.

Calcium-binding peptides derived from β-casein-depleted casein hydrolysates: optimization, structural characterization, and evaluation of osteogenic potential

Characteristics of antioxidant peptides derived from casein hydrolysis products of Lactobacillus casei 1.0351 and exploration of their pathway of action by molecular docking

Cow's milk-based infant formula aims to approximate human milk, but bovine αS1-casein remains an important allergen-related protein with a profile distinct from human milk caseins. This study controlled trypsin hydrolysis to preferentially reduce intact αS1-casein and generate candidate bioactive peptides. Under 38 °C, pH 8.0, and 5 h, the relative residual peak area of αS1-casein decreased markedly, while αS2-casein and β-casein remained detectable at 33.15% and 45.01%, respectively. Peptidomics identified 17 peptides, among which HQGLPQEVLNENLLR showed the strongest predicted binding affinity to α-amylase by molecular docking (-13.98 kcal/mol). The hydrolysate showed ABTS radical scavenging activity of 92.10% and α-amylase inhibition of 53.44%. Molecular dynamics further supported the stability of the peptide-enzyme complex. These findings provide a preliminary process window for reducing intact αS1-casein and prioritizing candidate functional peptides from casein hydrolysates.

An indigenous autochthonous bacterium, Bacillus cereus PX472023 isolated from the gut of Lates calcarifer was evaluated for its potential probiotic property. The isolated bacteria produced hydrolytic enzyme activities such as starch, esculin and casein hydrolysis along with positive result for biofilm formation. The isolate possessed desirable features like tolerance to low pH (3), high bile salt (up to 4%), pepsin (0.3%), high saline conditions (up to 3%), along with optimum hydrophobicity, auto and co aggregation activity. In addition, the isolate exhibited strong antagonistic activity against L. calcarifer pathogens, including Photobacterium damselae subsp. damselae, Lactococcus garvieae, Aeromonas veronii and Pseudomonas putida. In addition, PCR amplification revealed the presence of two probiotic marker genes (LuxS gene and E1 β-subunit of the pyruvate dehydrogenase complex gene) as well. Draft genome assembly analyses revealed genome size to be 56,16,837bp, with GC content of 34.97% and 5,605 protein coding sequences. Downstream analyses following the functional annotation of the genome demonstrated the presence of variety of bacteriocin gene clusters and presence of several genes in favour of probiotic potential of the isolate. Safety evaluation demonstrated that the isolate showed no haemolysis on sheep blood agar, was devoid of virulence-associated genes in its genome and did not induce mortality in the in vivo challenge study. The genomic investigation of antimicrobial resistance determinants revealed the absence of acquired antimicrobial resistance genes in the isolate, thereby alleviating primary concerns regarding horizontal transfer of resistance genes. The integration of in vitro and genomic analyses provided preliminary evidence supporting the probiotic potential of Bacillus cereus PX472023 in L. calcarifer and highlighted its prospective suitability for in vivo application in the same host under local environmental conditions.

Bioactive peptides identified from camel milk casein hydrolysates regulate starch digestion via α-glucosidase inhibition: integrated screening, inhibition kinetics, and peptide-enzyme interaction mechanisms.

Since the status of C. jambhiri is “rare” and C. aurantifolia is endowed with folk medicinal properties, our study aimed at producing true-to-type seedlings for further conservation by using PGRs, ferrous sulfate, and casein hydrolysate in MS medium to induce in vitro germination and “callus” formation from seeds of C. jambhiri Lush. It also focused on evaluation of suitable species and the best type of explant for organogenesis. The present study was undertaken to develop an efficient micropropagation protocol for C. jambhiri Lush. and C. aurantifolia. The frequency of callus induction increased to its maximum when 2.0 mg/L 2,4-D and 1.0 mg/L picloram were added individually. The calli derived from 2,4-D exhibited maximum regeneration potentiality. In addition, sucrose (30 g/L), dextrose (60 g/L), and coconut water (10 mL/L and 15 mL/L) also enhanced callus induction. Regarding heavy metals, 100 ppm of Fe2SO4 exhibited maximum germination percentage (84.33%) from seeds of C. jambhiri Lush. However, maximum callus induction (50.00%) was induced from the seeds of C. aurantifolia incubated in Fe2SO4 (400 ppm). The maximum number of shoots per callus was produced (5.13) with the addition of 200 mg/L casein hydrolysate in the callus-induction medium. Pearson correlation analysis revealed a positive and significant association of the number of plantlets/calluses with shoot length and regeneration percentage, respectively. It was phenotypically observed that the tissue culture traits performed better from explants derived from C. jambhiri Lush. than C. aurantifolia when subjected to varying concentrations of PGRs, carbon sources, organic adjuvants, and heavy metals, respectively.

ABSTRACTHistoric mural paintings are particularly vulnerable to biodeterioration caused by microbial colonization, which can lead to the degradation of the organic and inorganic components of wall surfaces. This study presents, to the best of our knowledge, the first identification of the Paracoccus marcusii IBR3 strain on a 19th‐century mural painting from a private Italian villa. In vitro analyses revealed that strain IBR3 exhibits significant enzymatic activities, such as casein hydrolysis and calcium carbonate dissolution. These activities implicate the strain in the degradation of proteinaceous binders and mineral substrates, which are typical of mural artwork. As part of an environmentally sustainable conservation strategy, the antimicrobial activity of Thymus serpyllum essential oil was evaluated against strain IBR3 using in vitro tests, membrane integrity assays, and ATR‐FTIR spectroscopy. The essential oil, chemically characterized by GC–MS, demonstrated promising biocidal efficacy. These findings suggest that T. serpyllum essential oil could serve as a non‐toxic alternative for preserving and maintaining culturally significant wall paintings.

Proteases from the genus Streptomyces exhibit diverse biochemical properties with significant industrial potential. This study explores the valorization of protein rich waste as a substrate for the production, purification, and characterization of a novel milk clotting protease from the endophytic isolate GZ1B10, recovered from wild Beta vulgaris tubers. Molecular identification based on 16S rDNA sequencing revealed 99.75% similarity to Streptomyces thermolineatus. The extracellular protease was purified by size exclusion chromatography followed by desalting, yielding 10.51 ± 0.8mg of homogeneous enzyme. SDS-PAGE analysis revealed a single protein band with an estimated molecular weight of 21.8kDa. Purification enhanced milk clotting activity (14.55 to 85.12 RU) and specific activity (0.21 to 8.09 RU/mg), approaching the activity profile of commercial rennet. Casein hydrolysis assays indicated selective κ-casein degradation (72%) supporting a rennet-like mode of action. Both crude and purified proteases exhibited optimal coagulation at pH 5.5, 60°C, and 0.03M CaCl₂, indicating high thermostability compared with rennet. Cheese yield measurements showed comparable results (16.25g/100 mL) with similar syneresis (34.25%) and slightly improved moisture retention. This study reports S. thermolineatus as an endophyte of wild B. vulgaris for the first time and presents, to our knowledge, the first evidence of milk clotting protease production by this species, highlighting its potential as an alternative coagulant for industrial cheese production.

A single bacterial strain, IBL03679T, from forest soil in Maryland, USA in 2009 was originally identified as belonging to the Bacillus cereus senso lato group through multi-locus sequence analysis. Here, further identification efforts with genomic and phenotypic methods demonstrate that this strain belongs to a new species. Phylogenetic analysis using whole-genome sequencing suggested the most closely related species is Bacillus proteolyticus. ANIb and dDDH values confirmed species differentiation with values below conventional 95-96% and 70% thresholds, respectively. Genomic comparisons with five other Bacillus strains with the same sequence type also group into the proposed species nov. It is a gram-stain positive, facultative anaerobe, endospore and parasporal crystal forming rod-shaped bacterium. Physiological characteristics, such as casein hydrolysis and lecithinase production, and major fatty acids identified (C16:0, iso-C15:0, and iso-C17:0), support affiliation with other Bacillus species. The polar lipid profile was primarily diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), and phosphatidylglyercol (PG); the peptidoglycan type was A1γ meso-Dpm - direct (A31); and the sole respiratory quinone was menaquinone-7. Biochemical testing revealed distinguishing test results for strain IBL03679T through inability to metabolize D-cellobiose and D-saccharose, and utilization of potassium gluconate. Collectively, the genotypic and phenotypic data demonstrate that IBL03679T belongs to a novel species. We propose the name Bacillus silvicola sp. nov. for this taxon with the type strain IBL03679T (= NRRL B-65743T, = CCUG 78293T).

IgG release and peptides production by trypsin-driven selective hydrolysis of casein in bovine colostrum to improve immunomodulatory activity

In this study, a wild-type alkaline protease-producing Bacillus strain isolated from soil was biochemically and molecularly characterized. The strain was identified as Bacillus subtilis PTK56 based on 16S rRNA analysis. Random mutagenesis using EMS (Ethyl methanesulfonate) generated multiple variants, and the mutant with the highest protease productivity was selected. Enzyme production conditions for both strains were optimized, and the partially purified enzymes were comparatively characterized. The mutant protease exhibited a 1.34-fold higher activity than the wild type. Zymogram analysis confirmed the functional impact of the mutation through the appearance of an additional activity band. Both enzymes displayed an optimal pH of 9.0 and an optimal temperature of 55°C. They retained ≥ 97% of their initial activities between 30 and 60°C and maintained ≥ 95% stability within the pH range of 6.0-11.0. In the presence of metal ions (5-10 mM) and organic solvents (10%), both enzymes preserved more than 90% activity. In non-ionic detergents (1% Triton X-100, Tween-20, and Tween-80), both enzymes exhibited high stability, retaining > 90% activity with Tween-20 and Tween-80, while the wild-type retained ~ 79% and the mutant ~ 92% with Triton X-100. The mutant enzyme showed markedly higher stability, performing 39% better than the wild type in 5% Triton X-100 and 26% better in 5% H₂O₂. Both enzymes also maintained ≥ 80% stability across 1-15% NaCl concentrations. HPLC analysis of casein hydrolysis products revealed higher amino acid release by the mutant enzyme. Overall, both proteases demonstrate strong potential for applications in detergent, photographic, and pharmaceutical industries.

Milk-clotting enzymes (MCEs) are essential biocatalysts in cheese manufacture. However, limited availability and ethical concerns associated with calf rennet have intensified the search for microbial alternatives. This study reports the purification and characterization of a milk-clotting protease from Bacillus stercoris NCCP-3139, a soil isolate from Dera Ismail Khan, Pakistan. The strain was identified as B. stercoris by standard morphological and biochemical assays and 16S rRNA gene sequencing (98.75% similarity to B. stercoris JCM 30051). The enzyme was purified by ammonium sulfate precipitation, dialysis, DEAE-cellulose ion-exchange chromatography and Sephacryl S-200 gel filtration chromatography. Milk-clotting activity was assessed in goat, sheep, cow, buffalo and camel milk. The isolate exhibited pronounced casein hydrolysis and significant milk-clotting activity (p < 0.05), consistent with the production of a rennin-like protease. Sheep and buffalo milk showed superior clotting performance compared with other species (p < 0.05). Purification increased specific activity from 1.6 to 5.0 U/mg and the milk-clotting-to-proteolytic activity (PA) ratio from 50 to 550, indicating significant catalytic selectivity. The enzyme displayed optimal activity at 55°C and pH 9.0, and was activated by Mn2+ and Sr2+ but strongly inhibited by EDTA, Hg2+ and Zn2+. SDS-PAGE revealed a single band, consistent with a monomeric enzyme. Thus isolated B. stercoris NCCP-3139 produces protease with significant specificity and selectivity indicating its potential as a microbial alternative to animal rennet in cheese production.

Tea (Camellia sinensis) is a major source of bioactive polyphenols, particularly catechins and epicatechins, which are valued for their antioxidant and health-promoting properties. The present study aimed to establish and optimize a plant cell suspension culture system for enhanced production of catechin and epicatechin using tea callus-derived cells. Callus induction and proliferation were evaluated under different combinations of plant growth regulators, followed by optimization of suspension culture conditions. Casein hydrolysate (CH) and aluminum sulfate (Al3+) were applied as elicitors to assess their synergistic effects on cell growth and secondary metabolite accumulation. The optimal suspension culture was obtained using Murashige and Skoog medium supplemented with 2 mg/L 6-benzylaminopurine and 2 mg/L naphthalene acetic acid, which supported stable cell growth without browning. HPLC analysis demonstrated that the addition of CH and AL significantly enhanced catechin and epicatechin production. The highest catechin yield (3.38 mg/L) was achieved with 25 mg/L CH, while the maximum epicatechin concentration (5.92mg/L) was obtained with a combination of 125mg/L CH and 50 mg/L AL. These findings demonstrate an efficient and scalable in vitro platform for catechin production and highlight the potential of elicitor-assisted suspension cultures as an alternative to conventional tea cultivation.

Casein hydrolysate (CMH) was proved to exert strong sleep-enhancing effects by vital amino acid metabolisms, GABAergic system, and cAMP signaling pathway in our previous study. Whether there existed a novel active peptide in CMH and its underlying synergic mechanism remain unknown. This study aims to explore novel potent sleep-enhancing peptides in CMH and to systematically investigate its underlying mechanisms by multiomics analysis. Our results showed that many potent sleep-enhancing Tyr-based peptides were released by CMH. Considering potential structural features and contents, YQEPVLGPVR (YQ10) was screened, which significantly prolonged sleep duration and recovered the corticosterone level. Moreover, ten crucial DEGs and DEMs were revealed by omics analysis. YQ10 might regulate CaMKII and neuroinflammatory pathways to communicate with GABAergic and cAMP signaling pathways, particularly regulating CaMKII, IL-1β, and IL-6, which was validated by vital factors/protein expressions and antagonist experiments. These findings could provide a theoretic basis to explore potential sleep-enhancing mechanisms of Tyr-based peptides.

The Paenibacillus srains exhibit diverse abilities to secrete hydrolases capable of degrading biomacromolecules and to act as plant growth-promoting bacteria (PGPB) through the degradation of fungal cell walls and promotion the nutrient cycling of nitrogen (N) and phosphorus (P). Despite the well-acknowledged attributes of Paenibacillus, only limited studies have identified strains of it concurrently produce multiple hydrolytic enzymes and display strong phytobeneficial characteristics. The objective of this study was to isolate and functionally characterize a novel Paenibacillus strain that combines hydrolytic capabilities with plant growth-promoting potential. Strain TH7-28T was isolated and taxonomically classified through a polyphasic identification approach based on 16S rRNA gene phylogeny, genome analysis, and physiological and biochemical characteristics. Its metabolic capabilities were characterized using CAZy and KEGG pathway annotations, supplemented by Kofam-KOALA functional profiling. Genomic relatedness to reference strains was determined using OGRIs: ANI, dDDH, and tetra-nucleotide frequency signature analyses. Biophysical and biochemical properties were evaluated through enzymatic activity assays, antimicrobial susceptibility testing, respiratory quinone system identification, fatty acid methyl ester profiling, and polar lipid composition analysis. The wheat germination test was used to verify the plant growth-promoting ability. Strain TH7-28T was Gram-stain-positive, aerobic, white and rod-shaped. Phylogenetic analysis based on the 16S rRNA gene and genomic sequence indicated that strain TH7-28T was classified within the genus Paenibacillus. The ANI values of strain TH7-28T with the closest related strains P. macerans ATCC 8244T and P. oralis KCOM 3021T were 91.00% and 92.85%, respectively. The dDDH values of strain TH7-28T with P. macerans ATCC 8244T and P. oralis KCOM 3021T were 44.60% and 52.40%, respectively. The respiratory quinone was menaquinone 7 (MK-7). The major cellular fatty acids (> 10%) comprised anteiso-C15:0, anteiso-C17:0, iso-C16:0 and C16:0. The genomic DNA G + C content was 52.1%. Phosphatidylethanolamine, two amino lipids and three phospholipids were the main polar lipids. Genomic sequencing revealed multifunctional gene clusters encoding carbohydrate-active enzymes (cellulase and amylase), proteases, and phospholipases, alongside antifungal chitinase and endo-β-1,3-glucanase. The biomolecular metabolic gene types and gene copy numbers of strain TH7-28T were significantly higher than those of other Paenibacillus genus, indicating its robust capacity for macromolecule degradation. The nitrogen-fixing potential of strain TH7-28T was confirmed via nifH/D/K genes. Phosphorus activation capacity was demonstrated through pstS/C/A/B-mediated inorganic uptake and phospholipase-driven organic solubilization. Experimental assays further demonstrated significant enzymatic activities of chitinase (8.12 ± 0.66 × 10–3 U/mL), β-1,3-glucanase (0.023 ± 0.004 U/mL), and nitrogenase (0.164 ± 0.006 U/mL), with concurrent hydrolysis of cellulose, starch, casein, chitin and lecithin. Through the wheat germination experiment, it has been confirmed that this strain significantly enhanced both the seed germination rate and stem length, thereby establishing a solid foundation for subsequent research on its practical applications. Strain TH7-28T, isolated from lake sediment, was identified to represent a novel species designated Paenibacillus lacisediminis sp. nov. The strain exhibits multifunctional hydrolase activities, antifungal enzyme synthesis, nitrogen fixation, phosphorus solubilization and plant growth promoting. The findings confirm Paenibacillus lacisediminis sp. nov. as a biotechnologically promising strain for sustainable agriculture, furthermore enriching the functional repertoire of Paenibacillus genus.

This study identified novel DPP-IV inhibitory oligopeptides from bovine casein hydrolysates for hyperglycemia regulation. Initial in vitro screenings showed that low-molecular-weight fractions (<3 kDa) exhibited superior DPP-IV inhibitory activity. From these fractions, high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) integrated with in silico, in vitro, and in situ validations identified potent oligopeptides, notably FPQYLQYL, which outperformed FVAPF and DAYPSGAWY. Kinetic analysis confirmed a competitive inhibition mode targeting the catalytic pocket. Innovatively, integrating microscale thermophoresis (MST), fluorescence quenching, molecular dynamics simulations, and atomic force microscopy (AFM) proved that the inhibitory potency was driven by high binding affinity and oligopeptide-enzyme aggregation. Crucially, structure-activity relationship analysis identified a critical substrate-mimicking N-terminal Phe-Pro motif, where the proline-driven insertion is stabilized by specific hydrophobic and aromatic interactions within the pocket. Collectively, our findings provided insights into the design and discovery of bioactive oligopeptides with hypoglycemic potential and offered a promising natural alternative to synthetic DPP-IV inhibitors.

Gluten-free diet is currently recommended for people with gluten-related diseases; however some studies document their positive effects also in other diseases. Oppositely gluten is often vilified in nutrition, but serious results about their negative effects in healthy are missing, or controversial. The objective of this study is to compare the effects of different types of diets on ovarian, testicular, and thyroid morphology in an experimental mouse model. Forty-eight (n=48) laboratory mice of the BALB/c line were included in the experiment, divided into 4 groups, and maintained on special diets for 5 weeks. The control group, (6 females, 6 males) was fed a gluten-free diet. The first (E1), second (E2) and third (E3) experimental groups, (6 females, 6 males) were fed a mixture of casein hydrolysate combined with E1: pure extracted gluten in a 30 %:70 % ratio. E2: gliadins at a ratio of 30 %:70 % and E3: avenin at a ratio of 30 %:70 %. At the end of the experiment, the mice were euthanized and ovaries, testes, and thyroid glands were sampled. The samples were fixed in a 10 % formalin solution and processed into hematoxylin-eosin-stained slides. The oocyte and follicle widths of the ovaries were measured; as well as the germinal epithelium and the width of the seminiferous tubules of the testes; as well as the follicle epithelium width and the follicle width of the thyroid gland. The results showed significant differences in the width of oocytes, follicles, testicular seminiferous tubule epithelium, testicular tubules, thyroid follicle epithelium as well as differences in the width of thyroid follicles. Concentrated gluten and gliadin-based diets showed positive results compared to concentrated avenin and gluten-free diets. On the basis of animal experiment using histological methods, it seems that gluten may not be for healthy population harmful and is not recommended to be avoided outside groups of people with gluten-related disorders. Key words Celiac disease " Gluten " Non-celiac gluten sensitivity " Cereals o Nutrition.

Optimization of tomato (Solanum lycopersicum L.) shoot regeneration by synergistic use of adenine sulfate, glutamine and casein hydrolysate

Dandelion alleviates Helicobacter pylori associated gastritis by inhibiting proteolytic activity of HtrA.