Cathepsin G Research Articles

Effect of Peony (Paeonia ostii) Seed Meal Supplement on Enzyme Activities and Flavor Compounds of Chinese Traditional Soybean Paste during Fermentation.

Peony seed meal (PSM) is the by-product obtained from peony seeds after oil extraction. In this study, PSM was incorporated into traditional koji-making, and its impacts on koji enzyme activities and flavor compounds in final products were investigated. In the process of koji fermentation, the optimal addition ratio of PSM to soybean was determined as 7:3. Under this ratio, the maximum enzyme activities of neutral protease, amylase, and glucoamylase were 1177.85, 686.58, and 1564.36 U/g, respectively, and the koji obtained was subjected to maturation. During post-fermentation, changes in the fermentation characteristics of the paste samples were monitored, and it was found that compared to the soybean paste without PSM, the enzyme activities maintained at a relatively good level. The PSM soybean paste contained a total of 80 flavor compounds and 11 key flavor compounds (OAV ≥ 1), including ethyl isovalerate, isovaleric acid, hexanal, phenylacetaldehyde, 3-Methyl-1-butanol 4-heptanone, 2-pentylfuran, methanethiol ester caproate, isoamyl acetate, 3-methyl-4-heptanone, and isovaleraldehyde. These findings could be used to improve the quality of traditional fermented paste, enrich its flavor, and simultaneously promote PSM as a valuable resource for fermented foods.

Nemopilema nomurai jellyfish venom attenuates phenotypic modulation of PDGF BB-induced vascular smooth muscle cells and κ-carrageenan-induced rat tail thrombosis

Jellyfish venoms have long been recognized as a potentially rich source of natural bioactive compounds with pharmacological potential for the creation of innovative drugs. Our previous study demonstrated that Nemopilema nomurai jellyfish venom (NnV) has a chymotrypsin-like serine protease with fibrinolytic activity in vitro. Therefore, the present study aims to investigate the potential effect of NnV on cell migration, proliferation, and differentiation of vascular smooth muscle cells (VSMC; A7r5 cells) involved in the probable mechanism pathways. We also determined its anti-thrombotic effect through κ-carrageenan-induced Sprague–Dawley (SD) rat tail thrombus model. NnV inhibits on Platelet-derived growth factor (PDGF)-BB-stimulated A7r5 cells migration and proliferation by decreasing matrix metalloproteinase 2 (MMP-2) level and phosphorylation of ERK and Akt in a dose-dependent manner, but not p38. Furthermore, NnV regulates the phenotype transition of differentiation in PDGF-BB-stimulated A7r5 cells via ɑ-SMA and calponin in a dose-dependent manner. In an in vivo study, NnV treatment demonstrated clear anti-thrombotic activity in a dose-dependent manner, which was associated with decreased thrombus formation and length in κ-carrageenan-induced SD rat tail. These findings suggested that NnV has a novel fibrinolytic enzyme that can be used to prevent and/or treat thrombosis-related cardiovascular disorders.

Substrate specificity of human chymotrypsin-like protease (CTRL) characterized by phage display-selected small-protein inhibitors

Chymotrypsin-like protease (CTRL) is one of the four chymotrypsin isoforms expressed in the human exocrine pancreas. Human genetic and experimental evidence indicate that chymotrypsins B1, B2, and C (CTRB1, CTRB2 and CTRC) are important not only for protein digestion but also for protecting the pancreas against pancreatitis by degrading potentially harmful trypsinogen. CTRL has not been reported to play a similar role, possibly due to its low abundance and/or different substrate specificity. To address this problem, we investigated the specificity of the substrate-binding groove of CTRL by evolving the substrate-like canonical loop of the Schistocerca gregaria proteinase inhibitor 2 (SGPI-2), a small-protein reversible chymotrypsin inhibitor to bind CTRL. We found that phage-associated SGPI-2 variants with strong affinity to CTRL were similar to those evolved previously against CTRB1, CTRB2 or bovine chymotrypsin A (bCTRA), indicating comparable substrate specificity. When tested as recombinant proteins, SGPI-2 variants inhibited CTRL with similar or slightly weaker affinity than bCTRA, confirming that CTRL is a typical chymotrypsin. Interestingly, an SGPI-2 variant selected with a Thr29His mutation in its reactive loop was found to inhibit CTRL strongly, but it was digested rapidly by bCTRA. Finally, CTRL was shown to degrade human anionic trypsinogen, however, at a much slower rate than CTRB2, suggesting that CTRL may not have a significant role in the pancreatic defense mechanisms against inappropriate trypsinogen activation and pancreatitis.

Conversion of Baijiu distillers’ grains to functional peptides: Process optimization and antioxidant activity evaluation

Distiller’s grains resourcing is a crucial problem for the sustainable development of the Chinese Baijiu industry. In order to improve the protein utilization of distiller’s grains. In this study, distiller’s grains pretreated by wet crushing combined with amylase/cellulase were further hydrolyzed with neutral protease combined with papain at 50 °C for 3 h. The hydrolysates were decolorized by 1% coconut shell activated carbon for 15 min, and separated and purified by macroporous resin and Sephadex gel to yield peptide fractions (F1, F2, F3 and F4). The F3 fraction with the highest radical scavenging activity was analyzed by nano-LC-MS/MS and peptidomics to identify peptides. Three peptides (especially newly discovered AAHVLAAAFL) displayed strong alcohol solubility and antioxidant activity and were stably bound to key regulatory pathway receptors (Keap1-Nrf2) and myeloperoxidase (MPO), further elucidating molecular mechanism of anti-oxidation. These findings provided knowledge for potential functional peptide candidates and facilitates the high-value commercial development of distiller’s grains.

Comparative Study of the Preparation of High-Molecular-Weight Fibroin by Degumming Silk with Several Neutral Proteases.

Removing sericin from the periphery of silk without damage to silk fibroin (SF) to obtain high-molecular-weight SF is a major challenge in the field of SF-based biomaterials. In this study, four neutral proteases, subtilisin, trypsin, bromelain and papain, were used to degum silk, and the degumming efficiency of the proteases and their influence on the molecular weight (MW) of regenerated silk fibroin were studied. The results indicated that all four neutral proteases could remove sericin from silk almost completely, and they caused less damage to SF fibers than Na2CO3 degumming did. The degumming efficiency of trypsin and papain was strong, but they caused relatively high damage to SF, whereas bromelain caused the least damage. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel permeation chromatography and shear viscosity showed that the MWs of regenerated SF derived from neutral protease degumming were significantly higher than that of SF derived from Na2CO3 degumming. The MW of regenerated SF derived from bromelain degumming was the highest, while the MWs of regenerated SF derived from papain and trypsin degumming were relatively low. This study provides an efficient and environmentally friendly biological degumming method for obtaining high-molecular-weight silk fibroin.

A Novel Mechanically Robust and Biodegradable Egg White Hydrogel Membrane by Combined Unidirectional Nanopore Dehydration and Annealing.

A homogeneous egg white obtained by high-speed shearing and centrifugation was dehydrated into a fragile and water-soluble egg white glass (EWG) by unidirectional nanopore dehydration (UND). After EWG annealing, it can become an egg white hydrogel membrane (EWHM) that is water-insoluble, flexible, biocompatible, and mechanically robust. Its tensile strength, elongation at break, and the swelling ratio are about 5.84 MPa, 50-110%, and 60-130%, respectively. Protein structure analysis showed that UND caused the rearrangement of the protein molecules to form EWG with random coil and α-helix structures. The thermal decomposition temperature of the EWG was 309.25 °C. After EWG annealing at over 100 or 110 °C for 1.0 h or 45 min, the porous network EWHM was mainly composed of β-sheet structures, and the thermal decomposition temperature increased to 317.25-318.43 °C. Their 12-day residues in five proteases ranged from 1% to 99%, and the order was pepsin > neutral protease > papain > trypsin > alkaline protease. Mouse fibroblast L929 cells can adhere, grow, and proliferate well on these EWHMs. Therefore, the combined technology of UND and annealing for green and novel processing of EWHM has potential applications in the field of biomimetic and biomedical materials.

Design and synthesis of isatin derivatives as effective SARS-CoV-2 3CL protease inhibitors.

SARS-CoV-2 chymotrypsin-like cysteine protease (3CLpro ) is one of the most widely developed drug targets for COVID-19. This study aimed to design and synthesize isatin derivatives to target SARS-CoV-2 3CLpro in a covalent binding manner. Through the process, a potent 3CLpro inhibitor (5g) was discovered with an IC50 value of 0.43 ± 0.17 μM. To understand the binding affinity and specificity of 5g as a candidate inhibitor of SARS-CoV-2 3CLpro , several assays were conducted, including FRET enzyme activity assays, thermodynamic-based and kinetic-based validation of inhibitor-target interactions, and cell-based FlipGFP assays. The interaction mechanism between 3CLpro -5g was characterized by docking. Overall, these findings suggest that 5g is a new potent SARS-CoV-2 3CLpro inhibitor for the treatment of COVID-19.

Identification of lysosome-related genes in connection with prognosis and immune cell infiltration for drug candidates in head and neck cancer.

Lysosome dysfunction has been shown to play an important role in cancer progression. However, few research studies have reported the role of lysosomes in head and neck squamous cell carcinoma (HNSCC) progression. Lysosome-related genes (LRGs) were collected from the Molecular Signatures Database. Differentially expressed lysosome-related genes (DELRGs) were identified from the TCGA-HNSCC dataset. The least absolute shrinkage and selection operator and multivariate Cox regression analysis were used to identify the prognostic genes. The prognostic values and expression of hub DELRGs were further validated by GEO datasets. Estimation of STromal and Immune cells in MAlignant Tumors using Expression data and the single-sample gene set enrichment analysis were applied to evaluate the correlation between cathepsin G (CTSG) and immune infiltrates. Twenty-two DELRGs were identified. Among them, CTSG was an independent prognostic biomarker for HNSCC patients. Gene set enrichment analysis indicated that the potential mechanism of CTSG in regulating HNSCC was associated with the immune- and inflammation-related pathways. CTSG expression was highly correlated with immune cell infiltration. Finally, two potential compounds (CH and MAN) targeting CTSG protein were identified, and their reliability was validated through molecular docking analysis. CTSG was associated with immune infiltration and had prognostic value in HNSCC patients, which may be a potential biomarker for predicting the outcome of immunotherapy.

Exploring the characteristics of microbial community and enzyme activity in the aged refuse landfill environment of Taiyuan, China

Municipal solid waste landfill is the main disposal option for domestic garbage, in which microbial activities play an important role. However, despite the widespread practice of landfilling, the metagenomic microbial profiles of landfill sites remain poorly characterized. In this study, we used a combination of physicochemical analysis, ultraviolet-visible spectrophotometry, and high-throughput Illumina shotgun sequencing to systematically investigate the changes in soil enzyme activities, microbial community structure, and functional attributes in aged refuse collected from the Xingou Municipal Solid Waste Landfill in Taiyuan, China, with ordinary topsoil from an area within 5 km of the landfill as control soil. Except for neutral phosphatase (P = 0.065), the activities of urease, laccase, dehydrogenase, sucrase, neutral protease, and β-glucosidase were all significantly reduced (P < 0.05) in the aged refuse compared with the control soil. Contrastingly, catalase activity was found to be significantly elevated in the aged refuse. Compared with the control soil, aged refuse was characterized by higher richness and diversity of microbial communities, as reflected by the higher values of community richness estimators (Chao 1 and ACE) and diversity indices (Shannon and Simpson). In total, 186 phyla, 4 354 genera, and 34 459 species were identified, with 132 phyla, 1 914 genera, and 7 369 species showing significantly different abundances between the aged refuse and the control soil. Actinobacteria and Acidobacteria were identified as the dominant phyla in the control soil, whereas Proteobacteria, Euryarchaeota (archaea), and Firmicutes were found to predominate in the aged refuse. Notably, Euryarchaeota and Methanoculleus were the major taxa detected in the aged refuse, but were almost completely absent in the control soil. Xenobiotic biodegradation and bacterial chemotaxis were the main functions of the microflora in the aged refuse, whereas the carbohydrate, amino acid, energy, and lipid metabolism pathways were significantly enriched in the control soil. Moreover, the aged refuse contained a high abundance of genes involved in quorum sensing. Our findings in this study revealed close associations between enzyme activities and variations in the microbial community structure and genes that were actively involved in biodegradation activities at landfill sites. It was found that the landfill environment was characterized by a more complex spectrum of microbial activities than expected. Further investigations are needed to gain a more comprehensive understanding of the microbial community structure and functional attributes as well as their potential influencing factors in the landfill environment.

Improving protein utilization and fermentation quality of soy sauce by adding protease

Soy sauce is the most popular traditional fermented condiment in Asia. However, some proteins are unutilized during fermentation, causing resource waste. Therefore, this study aimed to improve protein utilization and fermentation quality by adding protease in the moromi. Firstly, Aspergillus oryzae (A. oryzae) endogenous proteases were found to play an important role in nitrogen element utilization, including increasing total nitrogen (TN), amino acid nitrogen (AAN), and relative protein expression. Secondly, among six exogenous proteases, neutral protease (NP) performed better both alone and synergistically with A. oryzae crude protease powder (CPP), because of the enhancement of small peptides and enzymatic effect. Importantly, adding NP increased AAN and TN by 7.45 % and 2.21 % respectively, as well as increasing peptides below 500 Da, Maillard peptides and the proportion of lactic to acetic acid, hence improving the umami and richness of soy sauce. Overall, adding NP could increase soy sauce protein utilization and fermentation quality.

Effects of UV-B Radiation on the Chemical Composition of Azolla and Its Decomposition after Returning to the Field and Nitrogen Transformation in Soil

In the present research, the effects of UV-B radiation (5.00 kJ·m−2) on the chemical composition of Azolla were investigated, and the decomposition of Azolla residues after UV-B radiation, the nitrogen form, enzyme activity, and bacterial community in paddy soil were analyzed. Compared to the natural light treatment, the total nitrogen content of Azolla was significantly increased by 17.0% under UV-B radiation treatment. Compared to returned Azolla grown under natural light, the decomposition rate of cellulose, lignin, and total nitrogen of returned Azolla grown under UV-B radiation significantly increased, which led to an increase in the activities of nitrogen transformation enzymes, including neutral protease, ammonia monooxygenase, nitrogenase, nitrate reductase, and nitrite reductase, and the contents of different nitrogen forms (NH4+-N, NO3−-N, soluble organic nitrogen, and microbial biomass nitrogen) in paddy soil, while N2O emission flux was significantly reduced by 20–30%. The dominant bacteria in soil supplied with Azolla grown under natural light were Firmicutes, Clostridia, Clostridiales, and Lachnospiraceae. However, returning Azolla grown under UV-B radiation to the soil significantly changed the bacterial community structure in soil, resulting in a decrease in the number of ammonifying bacteria, nitrifying bacteria, and nitrogen-fixing bacteria and an increase in the number of denitrifying bacteria, inducing changes in the dominant bacteria to Methanomicrobiales, Methanoregulaceae, and Methanoregula. According to the structural equation model, returning Azolla to the field would reduce N2O emissions by increasing Azolla lignin decomposition and ammonia monooxygenase activity, reducing the number of nitrifying bacteria and reducing nitrite reductase activity in soil. Thus, UV-B radiation can directly change the phytochemical components and their decomposition in soil, thus indirectly affecting the bacterial community structure, enzyme activity, and nitrogen transformation, which play important ecological roles in regulating the nutrient transformation of terrestrial ecosystems.

Characterization of Protease from Endophytic Fungi Fusarium sp. JE-DP4a

Protease is a catalytic enzyme that catalyzes a proteolysis process and breaks down a protein into smaller polypeptides or amino acids. Endophytic fungi can produce bioactive compounds, one of which is protease enzymes. The papaya leaf endophytic fungi used in this study was Fusarium sp. strain JE-DP4a. This study was conducted to determine the characterization of protease from endophytic fungi Fusarium sp. strain JE-DP4a with variations in incubation temperature of 30, 37, and 44oC and pH variation of 5.5-8. Measurement of protease enzyme activity using a spectrophotometer UV-Vis at 280 nm. Based on the research results, the highest protease activity produced at a temperature of 44oC is 53.78 U/mL, and specific activity is 17.31 U/mg protein. Protease activity at pH 7 shows the highest was 46.72 U/mL.Thus it is ably concluded that the protease produced by Fusarium sp. strain JE-DP4a belongs to the neutral protease group.

The serine/threonine protein kinase VSKM1 promotes cold stress resistance of the postharvest Volvariella volvacea by enhancing mitoribosome activity

The edible straw mushroom Volvariella volvacea has a desirable taste and diverse effects on health; however, it undergoes rapid autolysis at 4 °C. Spray treatment with the β-glucosidase inhibitor DGlucono-1,5-lactone (DGase) at 100 µmol/L maintained the quality of V. volvacea during storage at 4 °C for 72 h. Enhanced alkaline protease and neutral protease activities can act as indicators of cold stress resistance (CSR) in V. volvacea, as well as the stable ATP level. The total sugar and amino acid content is negatively correlated with CSR in V. volvacea. DGase treatment enhanced the CSR of V. volvacea by inducing the overexpression of genes in the mitochondrial ribosome (mitoribosome). Overexpression of a serine/threonine-protein kinase gene in V. volvacea (VSKM1), which promotes mitoribosome activity in yeast, enhanced freezing tolerance. Our findings indicated that overexpression of VSKM1 promoted CSR by enhancing mitoribosome activity for the quality retention of postharvest V. volvacea.

A Rapid Method for Testing Filtration Performance of Malt and the Optimization of the Method

A rapid filtration test method that predicts malt filtration efficiency has been developed in this research. This rapid filtration test method has an advantage over existing beer filtration tests, as it can be easily operated and there is no need to brew. Six kinds of malts with distinct filtration performance were selected, and the filtration performance was determined by measuring the turbidity of their clarified wort through simple simulation of mashing. The results indicate the filtering performance ranking as given: Copeland &gt; Pearl malt 1 &gt; Pearl malt 2 &gt; Synergy &gt; Planet &gt; Wheat malt. When a sample is purchased for six times of parallelization, RSD is less than 10%, and the experimental results are significantly correlated with viscosity and β-glucan content. Therefore, this method can effectively predict the filtration performance of malt. The response surface design 3 × 3 factor experiment was used to optimize the fast detection method, which increased the sample differentiation (F-value) and made it easier to judge the experimental results. Response surface experiment results show the optimal reaction conditions for enhancing differentiation, i.e., an enzyme ratio of 0.5 (α-amylase, β-glucanase, neutral protease = 1:2:1), a holding period of 38 min, and a reaction temperature of 44 °C. The F-value is 147.00.

Boophilin D1, a Kunitz type protease inhibitor, as a source of inhibitors for the ZIKA virus NS2B-NS3 protease

Arboviruses are a global concern for a multitude of reasons, including their increased incidence and human mortality. Vectors associated with arboviruses include the mosquito Aedes sp., which is responsible for transmitting the Zika virus. Flaviviruses, like the Zika virus, present only one chymotrypsin-like serine protease (NS3) in their genome. Together with host enzymes, the NS2B co-factor NS3 protease complex are essential for the viral replication cycle by virus polyprotein processing. To search for Zika virus NS2B-NS3 protease (ZIKVPro) inhibitors, a phage display library was constructed using the Boophilin domain 1 (BoophD1), a thrombin inhibitor from the Kunitz family. A BoophilinD1 library mutated at positions P1–P4′ was constructed, presenting a titer of 2.9x106 (cfu), and screened utilizing purified ZIKVPro. The results demonstrated at the P1–P4’ positions the occurrence of 47% RALHA sequence (mut 12) and 11.8% RASWA sequence (mut14), SMRPT, or KALIP (wt) sequence. BoophD1-wt and mutants 12 and 14 were expressed and purified. The purified BoophD1 wt, mut 12 and 14, presented Ki values for ZIKVPro of 0.103, 0.116, and 0.101 μM, respectively. The BoophD1 mutant inhibitors inhibit the Dengue virus 2 protease (DENV2) with Ki values of 0.298, 0.271, and 0.379 μM, respectively. In conclusion, BoophD1 mut 12 and 14 selected for ZIKVPro demonstrated inhibitory activity like BoophD1-wt, suggesting that these are the strongest Zika inhibitors present in the BoophD1 mutated phage display library. Furthermore, BoophD1 mutants selected for ZIKVPro inhibit both Zika and Dengue 2 proteases making them potential pan-flavivirus inhibitors.

Insilico generation of novel ligands for the inhibition of SARS-CoV-2 main protease (3CLpro) using deep learning.

The recent emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing the coronavirus disease (COVID-19) has become a global public health crisis, and a crucial need exists for rapid identification and development of novel therapeutic interventions. In this study, a recurrent neural network (RNN) is trained and optimized to produce novel ligands that could serve as potential inhibitors to the SARS-CoV-2 viral protease: 3 chymotrypsin-like protease (3CLpro). Structure-based virtual screening was performed through molecular docking, ADMET profiling, and predictions of various molecular properties were done to evaluate the toxicity and drug-likeness of the generated novel ligands. The properties of the generated ligands were also compared with current drugs under various phases of clinical trials to assess the efficacy of the novel ligands. Twenty novel ligands were selected that exhibited good drug-likeness properties, with most ligands conforming to Lipinski's rule of 5, high binding affinity (highest binding affinity: -9.4 kcal/mol), and promising ADMET profile. Additionally, the generated ligands complexed with 3CLpro were found to be stable based on the results of molecular dynamics simulation studies conducted over a 100 ns period. Overall, the findings offer a promising avenue for the rapid identification and development of effective therapeutic interventions to treat COVID-19.

The determination of epidermal growth factor in Edible bird's nest by enzyme-linked immunosorbent assay

Edible bird's nest (EBN) is a traditional food which was nourishing and functional. Particularly, there is the epidermal growth factor (EGF) in EBN, which is thought to play an important role in promoting skin repair. However, the type and content of EGF in EBN were not determined yet. In this study, the type of EGF in EBN was identified as bird EGF by enzyme-linked immunosorbent assay and this method was validated to be accurate and precise. Moreover, it was found that the content of EGF in raw-unclean EBN, raw-clean EBN and stewed EBN was 3000 pg/g–4000 pg/g and there were no significant differences, which suggested that the batches, origins, forms, stewing temperatures and stewing times of EBN had no effect on the content of EGF in EBN. However, it was due to that enzyme destroyed the primary structure of EGF, the EGF content of neutral protease and trypsin hydrolysates of EBN was lower than that of flavor enzymes, alkaline protease and pepsin hydrolysates of EGF. This study was the first to determine the type and content of EGF in EBN, and provided a theoretical basis for the selection and processing of EBN and using EBN as a source of EGF.

Simultaneous inhibition of two neutrophil serine proteases by the S. aureus innate immune evasion protein EapH2

Extracellular adherence protein domain (EAP) proteins are high-affinity, selective inhibitors of neutrophil serine proteases (NSP), including cathepsin-G (CG) and neutrophil elastase (NE). Most Staphylococcus aureus isolates encode for two EAPs, EapH1 and EapH2, that contain a single functional domain and share 43% identity with one another. Although structure/function investigations from our group have shown that EapH1 uses a globally similar binding mode to inhibit CG and NE, NSP inhibition by EapH2 is incompletely understood due to a lack of NSP/EapH2 cocrystal structures. To address this limitation, we further studied NSP inhibition by EapH2 in comparison with EapH1. Like its effects on NE, we found that EapH2 is a reversible, time-dependent, and low nanomolar affinity inhibitor of CG. We characterized an EapH2 mutant which suggested that the CG binding mode of EapH2 is comparable to EapH1. To test this directly, we used NMR chemical shift perturbation to study EapH1 and EapH2 binding to CG and NE in solution. Although we found that overlapping regions of EapH1 and EapH2 were involved in CG binding, we found that altogether distinct regions of EapH1 and EapH2 experienced changes upon binding to NE. An important implication of this observation is that EapH2 might be capable of binding and inhibiting CG and NE simultaneously. We confirmed this unexpected feature by solving crystal structures of the CG/EapH2/NE complex and demonstrating their functional relevance through enzyme inhibition assays. Together, our work defines a new mechanism of simultaneous inhibition of two serine proteases by a single EAP protein.

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.