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BACKGROUND: The purpose of this research was to stratify the level of frailty to examine the risk factors associated with reversible cognitive frailty (RCF) and potentially reversible cognitive frailty (PRCF) in nursing homes to provide a basis for hierarchical management in different stages of frailty. METHODS: The study was a cross-sectional study conducted from September to November 2022; 504 people were selected by stratified random sampling after convenience selection from the Home for the Aged Guangzhou. The structured questionnaire survey was conducted through face-to-face interviews using the general data questionnaire, Fried Frailty Phenotype, Montreal Cognitive Assessment Scale. RESULTS: In total, 452 individuals were included for analysis. A total of 229 cases (50.7%) were PRCF, 70 (15.5%) were RCF. Multivariate logistic regression analysis showed that in pre-frailty, the Geriatric Depression Scale (GDS-15) score (odds ratio (OR) 1.802; 95% CI 1.308-2.483), Instrumental Activities of Daily Living Scale (IADL) score (0.352; 0.135-0.918) and energy (0.288; 0.110-0.755) were influencing factors of RCF. GDS-15 score (1.805; 1.320-2.468), IADL score (0.268; 0.105-0.682), energy (0.377; 0.150-0.947), lack of intellectual activity (6.118; 1.067-35.070), admission time(>3 years) (9.969; 1.893-52.495) and low education (3.465; 1.211-9.912) were influencing factors of PRCF. However, RCF with frailty was associated with the Short-Form Mini-Nutritional Assessment (MNA-SF) score (0.301; 0.123-0.739) and low education time (0 ~ 12 years) (0.021; 0.001-0.826). PRCF with frailty was associated with age (1.327; 1.081-1.629) and weekly exercise time (0.987; 0.979-0.995). CONCLUSIONS: The prevalence of RCF and PRCF was high among pre-frail and frail older adults in nursing homes. Different levels of frailty had different influencing factors for RCF and PRCF. Depression, daily living ability, energy, intellectual activity, admission time, education level, nutrition status, age and exercise time were associated with RCF and PRCF. Hierarchical management and intervention should be implemented for different stages of frailty to prevent or delay the progression of cognitive frailty.

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China faces a persistent deficiency in feed protein resources. Enhancing the utilization efficiency of indigenous feed protein resources emerges as a viable strategy to alleviate the current deficit in protein feed supply. Corn gluten meal (CGM), characterized by a high proportion of crude protein and glutamine, is predominantly employed in animal feed. Nonetheless, the water-insolubility of CGM protein hampers its protein bioavailability when utilized as feed material. The aim of this study was to augment protein bioavailability, liberate glutamine peptides from CGM, and produce glutamine-enriched CGM fermented feed. We executed a co-fermentation protocol using Bacillus subtilis A5, Lactobacillus 02002, and acid protease to generate the CGM fermented feed. Subsequent in vivo experiments with broilers were conducted to assess the efficacy of the fermented product. The findings revealed that the soluble protein, glutamine, small peptides, and lactic acid contents in the fermented feed increased by 69.1%, 700%, 47.6%, and 125.9%, respectively. Incorporating 15% and 30% CGM fermented feed into the diet markedly enhanced the growth performance and intestinal health of broilers, positively modulated the cecal microbiota structure, and augmented the population of beneficial bacteria, specifically Lactobacillus. These results furnish both experimental and theoretical foundations for deploying CGM fermented feed as an alternative protein feed resource.

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A novel fibrinolytic enzyme, ACase was isolated from fruiting bodies of a mushroom, Agrocybe aegerita. ACase was purified by using ammonium sulfate precipitation, gel filtration, ion exchange and hydrophobic chromatographies to 237.12 fold with a specific activity of 1716.77 U/mg. ACase was found to be a heterodimer with molecular mass of 31.4 and 21.2 kDa by SDS-PAGE and appeared as a single band on Native-PAGE and fibrin-zymogram. The N-terminal sequence of the two subunits of ACase was AIVTQTNAPWGL (subunit 1) and SNADGNGHGTHV (subunit 2). ACase had maximal activity at 47 °C and pH 7.6. It's activity was improved by Cu2+, Na+, Fe3+, Zn2+, Ba2+, K+ and Mn2+, but inhibited by Fe2+, Mg2+ and Ca2+. PMSF, SBTI, aprotinine and Lys inhibited the enzyme activity, which suggested that ACase was a serine protease. ACase could degrade all three chains (α, ß and γ) of fibrinogen. Moreover, the enzyme acted as both, a plasmin-like fibrinolytic enzyme and a plasminogen activator. It could hydrolyze human thrombin slightly, which indicated that the ACase could inhibit the activity of thrombin and acted as an anticoagulant to prevent thrombosis. Based on these results, ACase might act as a therapeutic agent for treating thrombosis, or as a functional food. Further investigation of the enzyme is underway.

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In this study, we have isolated and characterized a fibrinolytic enzyme from the GRAS (Generally Recognized as Safe) fungus, Neurospora sitophila. The enzyme was purified by fractional ammonium sulfate precipitation, hydrophobic interaction, ion exchange and gel filtration chromatography to 45.2 fold with a specific activity of 415.6U/mg protein. The native molecular mass of the enzyme was 49kDa, while the denatured molecular mass was 30kDa and 17.5kDa, indicating that the enzyme was a hetero-dimer. It was optimally active at 50°C and pH 7.4 and stable at human physiological temperature and pH. It was found to be a chymotrypsin-like serine protease which cleaved the synthetic chromogenic substrate, N-Succinyl-Ala-Ala-Pro-Phe-pNA for which the apparent Km and Vmax values were 0.24mM and 4.17×10-5mM/s, respectively. The enzyme hydrolyzed all the chains of fibrinogen by cleaving α chain first, followed by ß chain and then γ chain. Moreover, the enzyme possessed dual function of direct fibrinolysis as well as plasminogen activation. Due to its attractive biochemical and fibrinolytic properties and being from a GRAS fungus, the fibrinolytic enzyme has application as a safe and efficient thrombolytic drug.

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The focus of the present work was to investigate the glycosylation of zein, partial properties of the glycosylated zein (GZ) and its retarding effect on lipid oxidation of ground pork. Zein was glycosylated with chitosan (MW 1500Da) by microbial transglutaminase, the reaction was verified by FT-IR. Under the optimized conditions, 97.48mg of glucosamine was covalently conjugated to 1g of zein, determined by HPLC. The solubility and the surface hydrophobicity of GZ were significantly improved. In vitro studies of GZ showed a dose-dependent scavenging activity against free radicals of DPPH, superoxide and hydroxyl radical, and the EC50 value for DPPH radical was 1.99µg TE/mg protein. In addition, reducing power and Fe2+-chelating capacity of it were 16.60 and 12.96µg TE/mg protein, respectively. GZ resulted in low levels of thiobarbituric acid-reactive substances and peroxide value of ground pork. These results suggest that GZ is a potential natural antioxidant.

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A fibrinolytic enzyme was produced by the medicinal mushroom, Cordyceps militaris using submerged fermentation. The enzyme was purified from culture supernatant by hydrophobic interaction, ion exchange and gel filtration chromatographies. It was purified by 36 fold, with a specific activity of 1,467.4U/mg protein and the final yield was 5.8%. The molecular weight of the enzyme as determined by SDS-PAGE and gel filtration was 28kDa and 24.5kDa, respectively, and its isoelectric point (pI) was 9.0±0.2. It was found to be a glycoprotein with carbohydrate content of 1.67% (w/v). The enzyme was optimally active at 37°C and pH 7.2. The enzyme activity was strongly inhibited by soybean trypsin inhibitor (SBTI) and aprotinin which indicated it to be a serine protease, while other inhibitors like N-α-tosyl-l-phenylalanine chloromethyl ketone (TPCK), phenyl methane sulfonyl fluoride (PMSF), pepstatin and metal chelator EDTA did not inhibit its activity. Amino acid sequences of the purified enzyme were determined partially by Q-TOF2 and they were IEDFPYQVDLR; ANCGGTVISEK; YVLTAGHCAEGYTGLNIR; TNYASVTPITADMICAGFPEGK; KDSCSGDSGGPLVTGGK; VVGIVSFGTGCAR; ANKPGVYSSVASAEIR. Sequences of the seven peptides completely matched with those of a trypsin-like serine protease from Cordyceps militaris CM01 (accession no. EGX95217.1). The purified enzyme degraded α chains of fibrinogen first and then ß and γ chains and also activated plasminogen into plasmin. It can act as an anticoagulant and prevent clot formation by degrading fibrinogen. Based on these studies, the purified enzyme has great potential to be developed as a natural agent for prevention and treatment of thrombolytic diseases.

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A novel fibrinolytic enzyme from Cordyceps militaris was produced by submerged culture fermentation, purified, and biochemically characterized. The enzyme was purified to homogeneity, with an overall yield of 4.0% and a specific activity of 1682 U/mg. The molecular weight and pI of the enzyme were 32 kDa and 9.3 ± 0.2, respectively. The optimal pH and temperature of the enzyme were 7.4 and 37 °C, respectively. The enzyme activity was inhibited by Fe(2+), phenylmethane sulfonyl fluoride (PMSF), aprotinin, and pepstatin but not by N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and ethylenediamine tetracetic acid (EDTA). Three internal peptides of the enzyme, APQALTVAAVGATWAR, EKNVGSTVNLLSYDGNK, and TDATSVLLDGYNVSAVNDLVAK, were obtained. The enzyme could hydrolyze fibrin(ogen) directly and cleave the α-chains more efficiently than ß- and γ-chains, suggesting that it is a plasmin like protein. It degraded thrombin, which indicated that it can act as an anticoagulant and prevent thrombosis. Intravascular thrombosis is one of the major reasons of cardiovascular diseases. On the basis of these results, the purified enzyme can be developed as a natural agent for oral fibrinolytic therapy or prevention of thrombosis.

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A fibrinolytic enzyme was produced by an edible mushroom of Pleurotus ostreatus using submerged culture fermentation. The enzyme was purified from the culture supernatant by applying a combination of freeze-thaw treatment, ammonium sulfate precipitation, hydrophobic interaction, and gel filtration chromatographies. The enzyme was purified by a 147-fold, with a yield of 7.54%. The molecular masses of the enzyme an determined by gel filtration and SDSPAGE were 13.6 and 18.2 kDa, respectively. The isoelectric point of the enzyme was 8.52. It hydrolyzed fibrinogen by cleaving the α and ß chains of fibrinogen followed by the γ chains, and also activated plasminogen into plasmin. The enzyme was optimally active at 45°C and pH 7.4. The enzyme activity was completely inhibited by EDTA, whereas protease inhibitors of TPCK, SBTI, PMSF, aprotinin and pepstatin showed no inhibition on its activity. The partial amino acid sequences of the enzyme as determined by Q-TOF2 were ATFVGCSATR, GGTLIHESSHFTR, and YTTWFGTFVTSR. These sequences showed a high degree of homology with those of metallo-endopeptidases from P. ostreatus and Armillaria mellea. The purified enzyme can also be applied as a natural agent for oral fibrinolytic therapy or prevention of thrombosis.

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Increasing evidence shows that sortilin (encoded by SORT1 gene), a member of the vacuolar protein sorting 10 family of sorting receptors, can modulate amyloid-ß peptides (Aß) metabolism and clearance, as well as mediate the neurotoxicity of the Aß oligomer and proneurotrophins, thus playing diverse roles in the pathogenesis of Alzheimer's disease. To assess the association between single nucleotide polymorphism (SNP) of the SORT1 gene and sporadic Alzheimer's disease (sAD) in the Chinese Han population, a case-control study was carried out including 220 sAD patients and 245 controls. One tag SNP was selected from the entire SORT1 gene through construction of linkage disequilibrium blocks, and three SNPs located in the vicinity of SORT1 that affect its expression were also selected. The four target SNPs were genotyped using a multiplex PCR-ligase detection reaction method, yielding no significant association between them or haplotypes containing three of them, and the risk of sAD. The results of this study indicate that polymorphisms of the SORT1 gene are unlikely to confer the risk of sAD in the Chinese Han population.

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