Acid Protease Research Articles

Revealing the proteolytic characteristics of lactobacillus, lacticaseibacillus, and lactiplantibacillus isolates by in vitro and in situ perspectives

This study examined in vitro and in situ proteolytic characteristics of 8 lactobacilli strains isolated from Turkish Tulum cheese, consisting of L. plantarum, L. helveticus, L. rhamnosus, and L. paracasei, whose beneficial properties and safety concerns previously evaluated. The studied isolates presented versatile proteolytic activity with acidic, neutral, and alkaline proteases. The proteolytic activity of isolates against azocasein substrate was determined between 10.30 and 19.90 U/mg, and L. plantarum isolates had the highest proteolytic activity at varying pH values. Isolates 15, 449, 480 and 675 harboured proteinase genes in the catalytic region of prtR and prtP, and in the prtP/prtM intergenic region. All isolates were also evaluated in the production of fermented milk. As a result of SDS-PAGE analysis of fermented milk samples, L. plantarum isolates 449, 480 and 675 showed more proteolytic activity on milk caseins. Besides, isolate 480 was also effective in hydrolysis of beta-lactoglobulin. L. plantarum isolates resulted in an increase in amino acid (AA) concentrations especially in arginine, alanine, histidine, and proline during the 24-h fermentation of milk. AA production abilities varied depending on the strain. Generally, isolate 675 offered superior AA production ability. The proteolytic properties of cultures are effective on both human health and technological properties of the product in terms of reducing the allergenicity of milk proteins, forming functional peptides, and improving the sensory properties of the product. Based on the results of this study, L. plantarum isolates 449, 480 and 675 could potentially be used as starter cultures or co-cultures for these purposes.

Reserve mobilization and secondary metabolites during seed germination and seedling establishment of the tree Erythrina velutina (Fabaceae)

Introduction: The lack of knowledge on seed germination and seedling establishment is a main constraint for the restoration of degraded areas, including the tropical dry forest known as Caatinga. Objective: To assess reserve and secondary metabolite mobilization during seed germination and seedling establishment in Erythina velutina. Methods: We scarified, disinfected, imbibed, sown between towel paper, and incubated seeds under controlled conditions. We hydroponically cultivated seedlings in a greenhouse. We harvested cotyledons at seed imbibition, radicle protrusion, hypocotyl emergence, apical hook formation and expansion of cordiform leaves, first trifoliate leaf, and second trifoliate leaf. Results: Seeds contained approximately 20 % starch, 14.5 storage proteins, 11.6 neutral lipids, and 5.7 % non-reducing sugars on a dry weight basis. Soluble sugars were mainly consumed from hypocotyl emergence to apical hook formation, while major reserves were mobilized from apical hook formation to expansion of first trifoliate leaf. Enzymatic activity increased from mid to late seedling establishment, causing the mobilization of starch, oils, and proteins. Terpenoid-derivatives, flavonoids, phenolic acids, and alkaloids were detected. Flavonoids and phenolic acids were present at almost all stages and terpenoid-derivatives disappeared at expansion of cordiform leaves. Conclusion: Soluble sugars support early seedling growth, while starch, oils and proteins are simultaneously mobilized from mid to late establishment by amylases, lipases, and acid proteases. The cotyledons contain secondary metabolites, which may act in seedling defense. High content of reserves and presence of secondary metabolites in the cotyledons could enable E. velutina seedlings endure stress, validating their use in the restoration of degraded areas.

Revitalizing allicin for cancer therapy: advances in formulation strategies to enhance bioavailability, stability, and clinical efficacy.

The main objective of this review is to highlight the therapeutic potential of allicin, a defense molecule in garlic known for its diverse health benefits, and address the key challenges of its bioavailability and stability. The research further aims to evaluate various formulation strategies and nanotechnology-based delivery systems that can resolve these issues and improve allicin's clinical efficacy, especially in cancer therapy. We conducted a comprehensive review of the available literature and previous studies, focusing on the therapeutic properties of allicin, its bioavailability, stability issues, and novel formulation strategies. We assessed the mechanism of action of allicin in cancer, including its effects on signaling pathways, cell cycle, apoptosis, autophagy, and tumor development. We also evaluated the outcomes of both in vitro and in vivo studies on different types of cancers, such as breast, cervical, colon, lung, and gastric cancer. Despite allicin's significant therapeutic benefits, including cardiovascular, antihypertensive, cholesterol-lowering, antimicrobial, antifungal, anticancer, and immune-modulatory activity, its clinical utility is limited due to poor stability and unpredictable bioavailability. Allicin's bioavailability in the gastrointestinal tract is dependent on the activity of the enzyme alliinase, and its stability can be affected by various conditions like gastric acid and intestinal enzyme proteases. Recent advances in formulation strategies and nanotechnology-based drug delivery systems show promise in addressing these challenges, potentially improving allicin's solubility, stability, and bioavailability. Allicin offers substantial potential for cancer therapy, yet its application is hindered by its instability and poor bioavailability. Novel formulation strategies and nanotechnology-based delivery systems can significantly overcome these limitations, enhancing the therapeutic efficacy of allicin. Future research should focus on refining these formulation strategies and delivery systems, ensuring the safety and efficacy of these new allicin formulations. Clinical trials and long-term studies should be carried out to determine the optimal dosage, assess potential side effects, and evaluate their real-world applicability. The comparative analysis of different drug delivery approaches and the development of targeted delivery systems can also provide further insight into enhancing the therapeutic potential of allicin.

Molecular Characterization and Expression of Calpains and Cathepsins in Tilapia Muscle in Response to Starvation

Calpains are a class of calcium-dependent, non-lysosomal proteins. Two main calpain isoforms are expressed in skeletal muscle, m-calpain, and mu-calpain, which exert critical proteolytic activities related to muscle functions, muscle atrophy, and myogenesis. Cathepsins are lysosomal acid proteases. The main cathepsin isoforms implicated in muscle aging and atrophy are cathepsins B, L, and H. This study characterized calpain1, calpain2, cathepsin L, cathepsin B, and cathepsin H genes in the tilapia genome. In addition, the expressions of these genes were examined in the muscle tissues of starved versus fed; and refed versus control tilapia, Oreochromis niloticus. qPCR expression data showed that Calpain1 and 2 catalytic subunits and the regulatory subunit were significantly higher in starved compared to the fed tilapia. Similarly, cathepsin L, B, and H showed significant upregulation in the starved compared to the fed fish. Besides, calpain and cathepsin L enzymatic catalytic activity increased in the starved fish relative to the fed fish. The results indicate that the studied genes are involved in atrophying muscle and are considered to have a potential role as markers of tilapia muscle accretion.

Enhancement of Monacolin K Production by Intergeneric Hybridization between Monascus purpureus (Arg-) and Monascus ruber (Thi-, Met-) Auxotrophs

This study aims to reconcile industrially suitable strains of higher red pigment producing Monascus purpureus MPA5 and higher Monacolin K producing Monascus ruber MRA7, were tried to fuse for development of increased efficacy of targeted secondary metabolites production. The optimum concentration of protoplast mixture was standardized, before the fusion experiment. The effective fusion was observed with the solution of 30 % PEG 6000, CaCl2.2H2O (0.01 M) and glycine (0.05 M). Colonies of the fusants were segregated using complete medium. The fusants were irradiated under UV- light in LD50 dose. On the basis of non-segregation of colony appearance after repeated subculturing in complete medium, 8 colonies were selected. Finally, one of the stable fusant MF11 grew more rapidly than one of the parental strain M. purpureus MPA5, but not as fast as M. ruber MRA7, were selected and grown on low grade rice as solid substrate for production of metabolites. Production of red pigments, alpha-amylase and acid protease were at intermediate stage between two parental strains but enhancement of Monacolin K was observed. High performance liquid chromatography (HPLC) analysis was revealed that Monacolin K content was increased as much as two and half times than the parents. No fluorescent yellow band was detected in TLC plate, indicating of citrinin negativity of the fusant MF11. It is the first report of enhanced Monacolin K production by a stable fusant of Monascus sp.

Changes in digestive enzymes and nutritional ontogeny reserves in newly hatched Pacific pygmy octopus, Paroctopus digueti

In this study, changes in digestive physiology and nutritional reserve content during the early stages of the Pacific pygmy octopus, Paroctopus digueti, were examined. Body growth, biochemical composition (soluble protein and glycogen concentrations) in the digestive gland and arm muscle, hepatosomatic index, as well as lipases and proteases (acid and alkaline) activities were determined every two days up until 14 days after hatching. Newly hatched octopuses were obtained in the laboratory, from the eggs of a female that spawned in the wild and were kept in individual chambers and fed fresh crab meat. A significant decrease in soluble protein was observed in both, the digestive gland (8 days after hatching) and the arm muscle (2–4 days after hatching). Thereafter, soluble protein gradually increases in digestive gland whereas, in the arm muscle, afterward a significant increase at 6 days after hatching, a decreasing pattern until 14 days after hatching was noticed, possibly due to the start of exogenous feeding. In arm muscle, 2 days after hatching the glycogen showed a peak with subsequent marked fluctuations up to 14 days after hatching, indicative of the complete use of internal nutritional reserves and dependence on exogenous feeding at this time. Higher lipase activity was observed 2 days after hatching, with a significant decrease occurring between 6 and 14 days after hatching. Acid protease activity increased between 6 and 14 days after hatching, while alkaline protease activity remained stable during the developmental period, with a peak occurring 8 days after hatching. These results contribute to the further understanding of this critical period of transition from endogenous to exogenous feeding that takes place between 6 and 8 days after hatching and may be useful in developing effective feeding schemes employing natural or formulated foods for this species.

Impact of fertilization and grazing on soil N and enzyme activities in a karst pasture ecosystem

The biggest obstacle to economic development in karst areas comes from the ecological crisis, so healthy soil and adequate food are important. Soil nitrogen (N) is an important factor in maintaining soil quality. Understanding the changes in soil N fractions and enzyme activities under different management practices is important to maintain sustainable pastureland in karst areas. In this study, we investigated how grazing and fertilization affect different soil nitrogen fractions and enzyme activities of karst pastureland. The four treatments were unused land (UL, undisturbed utilization), abandoned-grazing pastureland (APL, grazing for three years followed by abandonment for six years), grazing pastureland (GPL, controlled grazing without fertilizer) and fertilized-grazing pastureland (FGPL, controlled grazing + fertilizer), with the aim of determining the management practices that is most beneficial to soil N in the area. Our results indicated that FGPL is the most beneficial in enhancing soil N fractions. This is reflected in greater total nitrogen (TN), alkaline driven nitrogen (NOH), inorganic nitrogen (TON), anaerobic mineralized nitrogen (The average increase in each soil layer over UL was 1.43 times; TMN), microbial biomass nitrogen (The average increase in each soil layer over UL was 3.51 times; MBN), amino acid nitrogen (AAN), amino sugar nitrogen (ASN), acidolyzed unknown nitrogen (TUN) and unacidified nitrogen (AIN) in FGPL soils. Likewise, greater enzyme activities were also found in nitrogen conversion-related enzymes (soil urease (S-UE), soil N-acetyl-β-D-aminoglucosidase (S-NAG), nitrate reductase (S-NR), and soil acid protease (S-ACPT)). In addition, the combination of grazing and balanced nutrient inputs slowed soil acidification and maintained high levels of total phosphorus (TP) and total potassium (TK). In contrast, the soil N fractions responded significantly to APL (low TN and plant effective nitrogen), while insufficient conversion capacity (enzyme activity, MBN) was the main reason for the compromised soil N availability at GPL. Government policy makers and livestock producers should encourage controlled grazing with proper fertilizer supplementation, and avoid prolonged abandonment of grazing lands.

Inhibition of protein degradation increases the Bt protein concentration in Bt cotton

Bacillus thuringiensis (Bt) cotton production is challenged by two main problems, i.e., the low concentration of Bt protein at the boll setting stage and the lowest insect resistance in bolls among all the cotton plant’s organs. Therefore, increasing the Bt protein concentration at the boll stage, especially in bolls, has become the main goal for increasing insect resistance in cotton. In this study, two protein degradation inhibitors (ethylene diamine tetra acetic acid (EDTA) and leupeptin) were sprayed on the bolls, subtending leaves, and whole cotton plants at the peak flowering stage of two Bt cultivars (medium maturation Sikang 1 (SK1) and early maturation Zhongmian 425 (ZM425) in 2019 and 2020. The Bt protein content and protein degradation metabolism were assessed. The results showed that the Bt protein concentrations were enhanced by 21.3 to 38.8% and 25.0 to 38.6% in the treated bolls of SK1 and ZM425 respectively, while they were decreased in the subtending leaves of these treated bolls. In the treated leaves, the Bt protein concentrations increased by 7.6 to 23.5% and 11.2 to 14.9% in SK1 and ZM425, respectively. The combined application of EDTA and leupeptin to the whole cotton plant increased the Bt protein concentrations in both bolls and subtending leaves. The Bt protein concentrations in bolls were higher, increasing by 22.5 to 31.0% and 19.6 to 32.5% for SK1 and ZM425, respectively. The organs treated with EDTA or/and leupeptin showed reduced free amino acid contents, protease and peptidase activities and significant enhancements in soluble protein contents. These results indicated that inhibiting protein degradation could improve the protein content, thus increasing the Bt protein concentrations in the bolls or/and leaves of cotton plants. Therefore, the increase in the Bt protein concentration without yield reduction suggested that these two protein degradation inhibitors may be applicable for improving insect resistance in cotton production.

Untargeted metabolomic profiling of Aspergillus sojae 3.495 and Aspergillus oryzae 3.042 fermented soy sauce koji and effect on moromi fermentation flavor

Aspergillus sojae 3.495 and Aspergillus oryzae 3.042 are essential microbial starters in fermented foods due to their high hydrolytic enzyme activities. However, their metabolic characteristics and differences during soy sauce fermentation are still unclear. This study identified 42 non-volatile differential metabolites based on UHPLC-MS/MS, among which 10 amino acids and derivatives and 4 non-volatile acids were high in A. sojae 3.495 koji, while 3 sugar alcohols, 5 flavonoids, and 4 fatty acids were prominent in A. oryzae 3.042. Further analysis revealed that higher acidic protease (1887.43 U/g) in A. sojae 3.495 and higher glucoamylase (2413.36 U/g) and cellulase (890.78 U/g) in A. oryzae 3.042 dominated the formation of differential metabolites. Metabolic pathway analysis showed that shikimate and phenylalanine metabolic pathways in A. sojae 3.495 were more active and enhanced 3-methyl-butanal and 2-phenylethanal in moromi fermentation. The more prominent pathways of sugar alcohol metabolism and flavonoid biosynthesis in A. oryzae 3.042 koji enhanced 2-methyl-butanoic acid and ethyl 3-methylbutyrate in moromi and significantly elevated reducing sugars (1.56 g/100 mL). Overall, comparative metabolomics elucidated the metabolic framework and flavor formation mechanisms of different inocula in soy sauce koji, providing ideas for scientific control of soy sauce fermentation and multi-strain koji-making for flavor enhancement.

Caspase-8 and Tyrosine Kinases: A Dangerous Liaison in Cancer

Caspase-8 is a cysteine-aspartic acid protease that has been identified as an initiator caspase that plays an essential role in the extrinsic apoptotic pathway. Evasion of apoptosis is a hallmark of cancer and Caspase-8 expression is silenced in some tumors, consistent with its central role in apoptosis. However, in the past years, several studies reported an increased expression of Caspase-8 levels in many tumors and consistently identified novel “non-canonical” non-apoptotic functions of Caspase-8 that overall promote cancer progression and sustain therapy resistance. These reports point to the ability of cancer cells to rewire Caspase-8 function in cancer and raise the question of which are the signaling pathways aberrantly activated in cancer that may contribute to the hijack of Caspase-8 activity. In this regard, tyrosine kinases are among the first oncogenes ever identified and genomic, transcriptomic and proteomic studies indeed show that they represent a class of signaling molecules constitutively activated in most of the tumors. Here, we aim to review and discuss the role of Caspase-8 in cancer and its interplay with Src and other tyrosine kinases.

Biosynthesis of the acid protease produced by Lacticaseibacillus casei LBC 237 and Limosilactobacillus fermentum LBF 433 and their potential application in the bovine milk clotting

This study aimed to produce and evaluate proteases from Lacticaseibacillus casei (LBC 237) and Limosilactobacillus fermentum (LBF 433) for milk coagulation. Proteases were assessed for acid protease activity (PA) and stability within temperature range of 30–75 °C and pH levels of 5.0, 5.5, and 6.8. Substrate concentration (0.025 %–2.5 %) and storage conditions (up to 120 days) were also investigated. Milk-clotting activity (MCA) and the effect of NaCl (1–4 %) on coagulation were evaluated, with subsequent characterization of resulting clots in terms of molecular size, structure, and flavor profile. Chemical changes in fermentation, proteases, and clots were determined. The proteases showed better PA at 60 °C and a pH of 5.5, with no change over 120 days. LBC-P demonstrated a higher affinity for the substrate. The proteases generated bands at ∼35 kDa, similar to bovine chymosin bands, and LBC-P exhibited MCA (480 SU/mL). Clots formed using LBC-P and bovine chymosin displayed similar bands (50 kDa, 35 kDa, and 15 kDa), while the LBC-P clot exhibited a more porous structure. LBC-P was inactivated by NaCl concentrations (≥1 %), and the clots showed changes in amides I. The bitterness intensity was lower for the clot obtained with LBC-P. While both LBC-P and LBF-P showed potential for protein hydrolysis, only LBC-P demonstrated potential for milk coagulation for cheese production. This study suggests acid proteases with high hydrolytic activity. In conclusion, L. casei (LBC 237) derived proteases show potential for milk coagulation, offering a promising alternative to bovine chymosin in yellow cheese production.

Mechanisms of biochar-mediated promotion of acidogenic fermentation in waste activated sludge and propionic acid production pathways

The conversion of organic matter in waste activated sludge (WAS) into high value-added chemicals of volatile fatty acids (VFAs) has become a focus of attention worldwide. However, the conversion of WAS to a certain acid among VFAs is difficult. Herein, for the first time, biochar was used to promote sludge solubilization, hydrolysis and acidogenic fermentation for the production of propionic acid. The results illustrated that biochar (prepared at 450 °C, containing environmental persistent free radicals (EPFRs) of 15.91 × 1015 spins/g) stimulated sludge solubilization, hydrolysis and VFAs production, enhancing sludge cell lysis rate, VFAs production, propionic acid ratio, protease, α-glucosidase and nicotinamide adenine dinucleotide hydrogen (NADH)/nicotinamide adenine dinucleotide (NAD+) ratio by 10.53 %, 107.42 %, 17.52 %, 68 %, 32 % and 76.10 % respectively, compared with those observed in the control group without biochar addition. And once the EPFRs were shielded, they decreased. The signal of EPFRs decreased from 15.91 × 1015 spins/g to 15.20 × 1015 spins/g on day 2 and 8.50 × 1015 spins/g on day 4. The EPFRs contained in the biochar played an important role in the intermediary function and interfered with the bacterial metabolic pathways. Metagenomics sequencing also revealed that biochar EPFRs promoted the sludge acidogenic fermentation to produce propionic acid ([EC:7.1.1.2]) and partly inhibited acetic acid production ([EC:1.2.1.3]). This study reveals that biochar could promote sludge solubilization, hydrolysis and promote propionic acid production, which could be used as a new strategy for acid production in the future.

Analysis on driving factors of microbial community succession in Jiuyao of Shaoxing Huangjiu (Chinese yellow rice wine)

The microbial ecosystem of fermented food is greatly disturbed by human activities.Jiuyao is important saccharification starter for brewing huangjiu. The interaction between environmental factors and microorganisms significantly affected the microbial community structure at different stages of Jiuyao manufacturing. This study combined environmental factor analysis and high-throughput sequencing technology to comprehensively analyze the specific changes of microbial community and environmental factors in each fermentation stage of Jiuyao production and their correlation. The results showed that the activities of liquefaction enzyme, glycosylation enzyme and acid protease reached the highest value on the 8 th day (192 h) after the beginning of fermentation, and the cellulase activity reached the highest value at the end of fermentation. Pediococcus（37.5 %-58.2 %）, Weissella（9.2 %-27.0 %） and Pelomonas（0.1 %-12.1 %） were the main microbial genera in the genus bacteria, and Saccharomycopsis（37.1 %-52.0 %）, Rhizopus（12.5 %-31.0 %） and Saccharomyces（4.0 %-20.5 %） were the main microbial genera in the genus fungi. The results of correlation analysis showed that the microbial communities in Jiuyao were closely related to environmental factors. Most microbial communities were positively correlated with temperature, but negatively correlated with ambient humidity, CO2 concentration, acidity and water content of Jiuyao. In addition, the transcription levels of enzymes related to microbial glucose metabolism in Jiuyao were higher in the late stage of Jiuyao fermentation. Interestingly, these enzymes had high transcription levels in fungi such as Saccharomycopsis, Rhizopus and Saccharomyces, as well as in bacteria such as Pediococcus and Lactobacillus. This study provides a reference for revealing the succession rule of microbial community structure caused by environmental factors during the preparation of Jiuyao in Shaoxing Huangjiu.

Effects of Co-Modification by Extrusion and Enzymatic Hydrolysis on Physicochemical Properties of Black Wheat Bran and Its Prebiotic Potential.

Black wheat bran (BWB) is an important source of dietary fiber (DF) and phenolic compounds and has stronger nutritional advantages than ordinary WB. However, the low content of soluble dietary fiber (SDF) negatively influences its physicochemical properties and nutritive functions. To obtain a higher content of SDF in BWB, we evaluated the impact of co-modification by extrusion and enzymes (cellulase, xylanase, high-temperature α-amylase, and acid protease) on water extractable arabinoxylan (WEAX) in BWB. An optimized co-modification method was obtained through single-factor and orthogonal experiments. The prebiotic potential of co-modified BWB was also evaluated using pooled fecal microbiota from young, healthy volunteers. The commonly investigated inulin served as a positive control. After co-modification, WEAX content was dramatically increased from 0.31 g/100 g to 3.03 g/100 g (p < 0.05). The water holding capacity, oil holding capacity, and cholesterol adsorption capacity (pH = 2.0 and pH = 7.0) of BWB were increased by 100%, 71%, 131%, and 133%, respectively (p < 0.05). Scanning electron microscopy demonstrated a looser and more porous microstructure for co-modified BWB granules. Through in vitro anerobic fermentation, co-modified BWB achieved a higher content of Bifidobacterium and Lactobacillus than inulin fermentation. In addition, co-modified BWB induced the highest butyric acid production, indicating high potential as prebiotics. The results may contribute to improving technologies for developing high-fiber-content cereal products.

The prebiotic effect of agavin inclusion levels in low fishmeal diets for Totoaba macdonaldi juveniles

The prebiotic effects of dietary agavin on growth performance, feed efficiency and digestibility, digestive enzyme activity, and distal intestine integrity in low fishmeal diets for Totoaba macdonaldi juveniles were evaluated in the present study. For this purpose, totoaba juveniles with a mean initial weight of 29 ± 1 g were fed for 56 days with four experiential diets formulated to replace fishmeal (FM) and poultry by-product meal (PBM) with soybean meal (SBM). The reference diet (RD) contained FM and PBM solely as the primary protein source. The following three diets contained SBM included at 240 g kg−1 with graded levels of agavin; 10, 20, and 30 g kg−1, in order to determine if a lower or higher quantity of the prebiotic in the diet would maximize growth parameters and fish intestinal integrity and reduce putative adverse effects of SBM in the diet. The inclusion of agavin in the diet, independently of the inclusion level, resulted in higher growth performance without significant differences in feed utilization and nutrient digestibility compared with the RD. Acid proteases (i.e., pepsin-like), trypsin, and total alkaline proteases activities resulted in significant differences among treatments but were not correlated with growth improvements. In terms of the intestinal health, significant differences in the number of mucosal folds and brush border height were found among dietary treatments and the light microscopy images and our semi-quantitative scoring revealed that agavin prevented the typical alterations in the distal intestine morphology associated with SBM induced enteritis in totoaba juveniles after 56 days of feeding. Thus, based on the results from the present study a 1 % agavin is recommended as the minimum dietary inclusion level in a low fishmeal diet (18 % FM) containing 24 % SBM to prevent typical symptoms of enteritis in totoaba juveniles under culture conditions.

Tyrosinase Inhibitory Peptides from Enzyme Hydrolyzed Royal Jelly: Production, Separation, Identification and Docking Analysis.

Tyrosinase is inextricably related to the development of Alzheimer's disease. The effects of natural tyrosinase inhibitors on human health have attracted widespread attention. This study aimed to isolate and analyze the tyrosinase (TYR) inhibitory peptides in the enzymatic digestion products of royal jelly. We first analyzed optimal process conditions for the enzymatic digestion of royal jelly by single-factor and orthogonal experiments and then used gel filtration chromatography to obtain five fractions (D1~D5) with molecular weights ranging from 600 to 1100 Da. LC-MS/MS was applied to identify the fractions with the highest activity, and the obtained peptides were screened and molecularly docked using AutoDock Vina. The results showed that the optimal enzymatic conditions for tyrosinase inhibition rate were acid protease, enzyme addition 10,000 U/g, initial pH 4, feed-to-liquid ratio 1:4, enzymatic temperature 55 °C, and enzymatic time 4 h. The D4 fraction had the most significant TYR inhibitory activity. The IC50 values of the three new peptides with the strongest TYR inhibitory activity, TIPPPT, IIPFIF, and ILFTLL, were obtained as 7.59 mg/mL, 6.16 mg/mL, and 9.25 mg/mL, respectively. The molecular docking results showed that aromatic and hydrophobic amino acids were more favorable to occupy the catalytic center of TYR. In conclusion, the new peptide extracted from royal jelly has the potential to be used as a natural TYR inhibitory peptide in food products with health-promoting properties.

Application of Acid Protease for Eco-friendly Pre-treatment of Goat Skin to Improve Antimicrobial Finish Using Herbal Natural Extracts

Due to its moisture retention capacity and huge surface area, leather is highly prone to microbial proliferation and biodeterioration; hence, leather products desired have an antimicrobial finish. In this study, acid protease enzyme pre-treatment of goat skin was utilized as an eco-friendly substitute for conventional wet-chemical processing. The treatment can impart the desired surface properties to improve the antimicrobial finish with natural extracts obtained from leaves of Azadirachta indica (Neem Tree), Ocimum sanctum (Holy Basil, Tulsi), and Camellia sinensis (Green Tea). The procedure was optimized for different process parameters, including enzyme concentration, pH, material to liquor ratio (MLR), treatment time, and temperature. The effect of the treatment on bulk and surface properties of the skin was characterized by weight loss analysis, X-Ray Diffraction (XRD), Thermal Gravimetric Analysis (TGA), and Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR), X-Ray Photoelectron Spectroscopy (XPS), Water contact angle measurement, Scanning Electron Microscopy (SEM) respectively. The effect of the enzymatic treatment on organoleptic properties and the mechanical strength of the skin was also studied. The enzymatic treatment resulted in weight loss, and removal of non-collagen components, thus opening the fibrous collagen matrix of the skin. Hence, the skin treated with acid protease enzyme provides better affinity and accessibility for phytoactive compounds from the natural extracts and better attachment by electrostatic attachment due to an increase in surface functional groups after the enzymatic treatment compared to untreated skin. The effectiveness of the antimicrobial finish was measured as a zone of inhibition and with a modified Hohenstein test against test microorganisms E. coli and S. aureus.Azadirachta indica (Neem Tree) extract showed the highest inhibitory activity (97%) against E. coli, while the Ocimum sanctum (Holy Basil, Tulsi) extract exhibited the highest inhibitory activity (95%) against S. aureus.

Effects of Different Proteases on Protein Digestion In Vitro and In Vivo and Growth Performance of Broilers Fed Corn-Soybean Meal Diets.

This study was conducted to investigate the effects of different proteases alone or in combination on protein digestibility of broilers. In vitro, the properties of four proteases in broilers, including acidic protease (AcP), alkaline protease (AlP), neutral protease (NeP) and keratinase (Ker), on endogenous protease activity and their effects on protein digestibility of common ingredients in broiler diets were investigated using a gut-mimicking model. In vivo, 640 1-day-old male broilers were randomly divided into 8 groups of 10 with 8 replicates of 10 birds per replicate cage. Eight dietary treatments included a corn-soybean meal basal diet (control), and the basal diet with 1.6 U AcP/g, 0.8 U NeP/g, 0.8 U AlP/g, 0.4 U Ker/g, 1.6 U AcP/g + 0.8 U NeP/g, 1.6 U AcP/g + 0.8 U AlP/g, or 1.6 U AcP/g + 0.4 U Ker/g added. The experiment lasted for 31 days. The results showed that the optimum pH values of AcP, NeP, AlP and Ker were 3.0, 9.0, 11.0 and 11.0 in vitro, respectively. Ker recovery proportion was 37.68% at pH 3.3-6.2. AcP alone or in combination with NeP, AlP or Ker increased in vitro crude protein digestibility (IVCPD) and decreased ileal apparent digestibility of crude protein in 31-day-old broilers (p < 0.05). All protease supplementation reduced the ileal apparent digestibility of amino acids compared to the control (p < 0.05). Acidic protease had a positive effect on trypsin and chymotrypsin activities, while AlP and Ker showed a negative effect. In vivo, average daily gain and average daily feed intake were significantly (p < 0.05) increased in broiler diets supplemented with AcP compared to the control group. When adding exogenous proteases to broiler diets, their sensitivity to digestive pH and their negative effects on endogenous protease activity, dosage and combination effects should be taken into account. In addition, the properties and dosage of proteases and the protein level in the feed should be considered.

Characteristics of the soy sauce taste and koji enzyme profiles as affected by soybean traits

Soybean is the main raw material for soy sauce fermentation and has a significant impact on the quality of soy sauce. Understanding the impact of soybean traits on the soy sauce quality is important for the development of the industry. The present study compared the traits of 12 soybeans and the quality of their fermented kojis and soy sauces, revealing the differences in total sugar and protein content may be the key factors responsible for differences in soy sauce characteristics. On this basis, 6 representative soybeans including 3 high protein and low total sugar content soybeans (HPLS) and 3 low protein and high total sugar content soybeans (LPHS) were selected for further study. Results of the changes in enzyme activities during koji-making fermentation showed that neutral protease and beta-glucosidase activities were higher in HPLS kojis, while acid protease and xylanase activities were higher in LPHS kojis after 24 h of fermentation. Differences in enzyme activity further caused variations in the soy sauce quality and formed some correlations with soy sauce tastes, such as xylanase and umami (r = 0.885), kokumi (r = 0.916). The intensities of umami and kokumi were higher in LPHS soy sauces, which had a higher dipeptides content of X-Glu, X-Thr, X-Ser and Gly-X.

Acid protease functionalized novel silver nanoparticles (APTs-AgNPs): A new approach towards photocatalytic and biological applications

Herein, we described for the first time, an efficient biogenic synthesis of APTs-AgNPs using acid protease from Melilotus indicus leaf extract. The acid protease (APTs) has an essential role in the stabilization, reduction, and capping of APTs-AgNPs. The crystalline nature, size, and surface morphology of APTs-AgNPs were examined using different techniques such as XRD, UV, FTIR, SEM, EDS, HRTEM, and DLS analysis. The generated APTs-AgNPs demonstrated notable performance as dual functionality (photocatalyst and antibacterial disinfection). By destroying 91 % of methylene blue (MB) in <90 min of exposure, APTs-AgNPs demonstrated remarkable photocatalytic activity. APTs-AgNPs also showed remarkable stability as a photocatalyst after five test cycles. Furthermore, the APTs-AgNPs was found to be a potent antibacterial agent with inhibition zones of 30(±0.5 mm), 27(±0.4 mm), 16(±0.1 mm), and 19(±0.7 mm) against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria, respectively, under both light and dark conditions. Furthermore, APTs-AgNPs effectively scavenged 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, demonstrating their potent antioxidant activity. The outcomes of this study thus demonstrates the dual functionality of APTs-AgNPs produced using the biogenic approach method as a photocatalyst and an antibacterial agent for effective microbial and environmental control.