Characterization of a recombinant Aspergillus niger GZUF36 lipase immobilized by ionic liquid modification strategy.

Enzyme immobilized on magnetic nanomaterials is a promising biocatalyst with efficient recovery under applied magnets. In this study, a recombinant extracellular lipase from Aspergillus niger GZUF36 (PEXANL1) expressed in Pichia pastoris GS115 was immobilized on ionic liquid-modified magnetic nano ferric oxide (Fe3O4@SiO2@ILs) via electrostatic and hydrophobic interaction. The morphology, structure, and properties of Fe3O4@SiO2@ILs and immobilized PEXANL1 were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, x-ray diffraction, vibration sample magnetometer, and zeta potential analysis. Under optimized conditions, the immobilization efficiency and activity recovery of immobilized PEXANL1 were 52 ± 2% and 122 ± 2%, respectively. The enzymatic properties of immobilized PEXANL1 were also investigated. The results showed that immobilized PEXANL1 achieved the maximum activity at pH 5.0 and 45 °C, and the lipolytic activity of immobilized PEXANL1 was more than twice that of PEXANL1. Compared to PEXANL1, immobilized PEXANL1 exhibited enhanced tolerance to temperature, metal ions, surfactants, and organic solvents. The operation stability experiments revealed that immobilized PEXANL1 maintained 86 ± 3% of its activity after 6 reaction cycles. The enhanced catalytic performance in enzyme immobilization on Fe3O4@SiO2@ILs made nanobiocatalysts a compelling choice for bio-industrial applications. Furthermore, Fe3O4@SiO2@ILs could also benefit various industrial enzymes and their practical uses. KEY POINTS: • Immobilized PEXANL1 was confirmed by SEM, FT-IR, and XRD. • The specific activity of immobilized PEXANL1 was more than twice that of PEXANL1. • Immobilized PEXANL1 had improved properties with good operational stability.

Effect of freeze-dried protectants on the survival rate and fermentation performance of fermented milk's directed vat set starters.

A directed vat set (DVS) starter was proposed to improve the drawbacks of liquid starters in fermented production and enhance the survival rates of B. animalis subsp. lactis BZ11, S. thermophilus Q-1, and Lactiplantibacillus plantarum LB12. The protective agent formula was optimized using the response surface method (RSM), with the survival rate as the benchmark. The best combination of cryoprotectants was determined to be BZ11: 10 % skimmed milk powder, 3 % sodium glutamate, and 15 % trehalose; LB12: 10 % skim milk powder, 5 % glutamate sodium, and 10 % trehalose; Q-1: 10 % skimmed milk powder, 3 % sodium glutamate, and 10 % trehalose. The survival rate of BZ11 significantly increased to 92.87 ± 1.25 %. The DVS fermented milk did not differ significantly from the control group regarding cholesterol removal, live cell counts and pH (p > 0.05). All DVS can be stored for at least 2500 d at -20 °C-this DVS starter for fermented milk benefits from its large-scale and automated commercial production.

Magnesium deficiency in liver cirrhosis: a retrospective study.

BACKGROUND: Magnesium, known as "the forgotten electrolyte", is an essential element of life. Magnesium deficiency is implicated in many diseases, including liver cirrhosis. This study aimed to explore the prevalence of magnesium deficiency in liver cirrhosis and investigate the relationship between magnesium levels and complication of liver cirrhosis and clinical outcomes. PATIENTS AND METHODS: Cirrhotic patients with serum magnesium levels measured were retrospectively identified from 2016 to 2017. Demographics, laboratory parameters, complications were collected. The Child-Pugh class, MELD score, and ALBI score were calculated. RESULTS: The mean serum magnesium level of all 152 patients was lower than the normal, including 92 patients diagnosed with magnesium deficiency. Compared to Child-Pugh class A, magnesium levels were significantly lower in the patients with Child-Pugh class B or C (F = 10.26, p < .05). Magnesium levels were also considerably lower in the group with MELD score ≥21, compared to the other two groups with MELD score < 15 or 15-20 (F = 6.59, p < .05). Similarly, magnesium levels were significantly lower in the group with ALBI score > -1.39 (grade 3), compared to the other two groups with ALBI with score ≤ -2.6 (grade 1) or > -2.6, ≤ -1.39 (grade 2) (F = 8.44, p<.001). Furthermore, magnesium levels were lower in cirrhotic patients with infection. Magnesium-deficient patients had lower transplant-free survival rates than non-deficient patients. CONCLUSION: Magnesium deficiency is highly prevalent in cirrhotic patients. Magnesium deficiency is related to worse transplant-free survival, infection and the severity of liver cirrhosis.

Effect of cross-linked enzyme aggregate strategy on characterization of sn-1,3 extracellular lipase from Aspergillus niger GZUF36.

The sn-1,3 extracellular lipase from Aspergillus niger GZUF36 (EXANL1) has important potential applications. The cross-linked enzyme aggregate (CLEA) of purified EXANL1 (CLEA-EXANL1) achieved optimum activity recovery (148.5 ± 0.9%), immobilization yield (100 ± 0%), and recovered activity (99.7 ± 0.6%) with 80% tert-butanol as the precipitant, glutaraldehyde (GA) concentration of 30 mM, GA treatment time of 1.5 h, and centrifugal speed of 6000×g. The effect of CLEA strategy on the characterization of EXANL1 was evaluated in this work. CLEA-EXANL1 exhibited a broader optimum pH range (4-6) compared with free EXANL1 (6.5). CLEA-EXANL1 presented optimum activity at 40 °C, which was 5 °C higher than that of free EXANL1. CLEA strategy decreased the maximum reaction rate and increased the Michaelis-Menten constant of EXANL1 when olive oil emulsion was used as a substrate. Moreover, after 30 days, free EXANL1 lost more than 80.0% of its activity, whereas CLEA-EXANL1 retained more than 90.0% of its activity. CLEA strategy improved the tolerance of EXANL1 in polar organic solvents. Fourier transform infrared spectroscopy results showed that the CLEA technique increased the contents of ß-sheets and ß-turns in EXANL1 and reduced those of α-helixes and irregular crimps. CLEA strategy caused no change in the sn-1,3 selectivity of EXANL1. Therefore, EXANL1 in the form of CLEA is a valuable catalyst in the synthesis of 1,3-diacylglycerol. KEY POINTS: • Cross-linked enzyme aggregate (CLEA) strategy broadened the optimum pH range of sn-1,3 extracellular lipase from Aspergillus niger GZUF36 (EXANL1). • CLEA strategy improved the tolerance of EXANL1 in polar organic solvents. • CLEA strategy caused no change in the positional selectivity of EXANL1.

[Virtual screen of effective AChE inhibitory constituents from Glycyrrhizae Radix et Rhizoma based on pharmacophore and molecular docking].

This research is to predict anti-Alzheimer's disease active constituents on the target of acetylcholinesterase(AChE) from Glycyrrhizae Radix et Rhizoma with the help of pharmacophore and molecular docking. AChE ligand-based pharmacophore model was set up and the molecular library of the constituents from Glycyrrhizae Radix et Rhizoma were established by collecting literature. Then the constituents from Glycyrrhizae Radix et Rhizoma were screen for the potential AChE inhibitory potency in silico through matching with the best pharmacophore model. The flexible docking was used to evaluate the interactions between compounds screened from pharmacophore model and AChE protein(PDB ID:4 EY7). The interactions were expressed including but not limited to CDOCKER interaction energy, hydrogen bonds and non-bonding interactions. The molecular library of Glycyrrhizae Radix et Rhizoma contains 44 chemical constituents. As for the pharmacophore model, six kinds of potential AChE inhibitory constituents from Glycyrrhizae Radix et Rhizoma were considered to be the promising compounds according to the results of searching 3 D database of pharmacophore model. The molecular docking was possessed and the interaction patterns were given to show the detail interactions. The compounds screening from the pharmacophore model were consistent with the existing studies to some degree, indicating that the virtual screen protocols of AChE inhibitory constituents from Glycyrrhizae Radix et Rhizoma based on pharmacophore and molecular docking was reliable.

Effects of different strains and fermentation method on nattokinase activity, biogenic amines, and sensory characteristics of natto.

Nattokinase activity (NK), biogenic amine content and sensory properties of natto are of great significance to consumers, which are affected by strains and fermentation methods. In this study, changes in the pH, biogenic amine and free amino nitrogen (FAN) contents, NK and protease activities, and sensory characteristics of natto prepared using Bacillus subtilis GUTU09 combined with different strains (Lactobacillus, Bifidobacterium and Mucor) and fermentation methods were investigated. The combination of two strains showed the best fermentation performance among all samples. The NK and protease activity and FAN content in double-strain fermentation increased by 10.33 FU/g, 88.78 U/g, and 2.34 g/kg, respectively, compared with those in single-strain fermentation. Sensory evaluation demonstrated that mixed fermentation primarily affected the sensory acceptance. This method also reduced the contents of various biogenic amines in natto compared with single-strain fermentation. Tyramine, cadaverine, spermine, and spermidine were significantly reduced, whereas histamine was slightly increased. The total biogenic amines decreased from 390.76 mg/kg to a minimum of 16.16 mg/kg. Some Mucor strains also reduced the contents of various biogenic amines. In the dual-bacteria fermentation of Mucor and GUTU09, co-fermentation has advantages over stage-fermentation, with higher NK and protease activity and higher sensory scores. Correlation analysis showed that the formation and accumulation of some biogenic amines in natto prepared using different microbial combinations were related to NK activity and pH. All these results showed that the quality of natto was improved by mixed fermentation and suitable fermentation methods, which laid a foundation for its potential industrial application.

Gene cloning, expression, purification and characterization of a sn-1,3 extracellular lipase from Aspergillus niger GZUF36.

Sn-1,3 extracellular Aspergillus niger GZUF36 lipase (EXANL1) has wide application potential in the food industry. However, the A. niger strain has defects such as easy degradation and instability in the expression of sn-1,3 lipase. To obtain a stable expression of this lipase and its subsequent enzymatic properties, the gene encoding EXANL1 was cloned and expressed in Escherichia coli BL21 (DE3) cells using pET-28a as the expression vector. The temperature-induced conditions were optimized, and we successfully achieved its active expression in E. coli. These conditions significantly influenced the active expression of EXANL1 (P < 0.05), and the highest enzyme activity of the supernatant of lysis cells expressed at 20 °C was at 7.02 ± 0.05 U/mL. The expressed recombinant EXANL1 was purified using Ni-NTA, showing an estimated relative molecular mass of 35 kDa. The recombinant EXANL1 exhibited maximum activity at 35 °C and pH 4.0, with a wide acid pH range. Thin-layer chromatography analysis showed that the enzyme displayed sn-1,3 positional selectivity toward triolein. The recombinant EXANL1 could maintain its relative activities (> 80%) after 24 h of incubation at pH 3-10, suggesting its suitability for a wide range of industrial applications. After comparing these properties with those of the other A. niger lipases, we found that some key amino acids may play a decisive role in enzymology. This work laid a foundation for the stable expression of the EXANL1 gene and its potential industrial application.

Synthesis and anti-tyrosinase mechanism of the substituted vanillyl cinnamate analogues.

This study aimed to synthesize and screen tyrosinase inhibitors for delay fruit browning. A series of vanillyl cinnamate analogues were designed and synthesized by simple processes, and the inhibitory effects of all the synthesized derivatives on mushroom tyrosinase were evaluated. In the enzymatic activity test, compounds 21, 22, and 26 had significant (P < 0.05) effect on mushroom tyrosinase at a preliminary screening dose (1 mg/mL in vitro). IC50 analysis showed that the IC50 values of compounds 21, 22 and 26 were 268.5 µM, 213.2 µM and 413.5 µM, respectively. In the cytotoxicity evaluation, Cell Counting Kit-8 (CCK-8) assay showed that compounds 21, 22 and 26 had no significant effect on the proliferation of hepatocyte L02 and B16 melanoma cells at the dosage of 25-200 µM. Inhibition of tyrosinase activity and melanin content in B16 melanoma cells investigations indicated that compounds 21, 22 and 26 inhibited both cellular tyrosinase activity and melanin content dose-dependently and more strongly than the reference standard arbutin. The UV-visible spectra showed compound 22 inhibits the formation of dopamine quinone, further the molecular docking analysis of compound 22 with tyrosinase (PDB: 2Y9X) indicated that compound 22 interacted with the amino acid residues of tyrosinase. The results of anti-browning test showed that compounds 21, 22 and 26 had significant tyrosinase inhibition and anti-browning effects on fresh-cut apple slices at 4 °C in 48 h. Compound 22 could be used as novel tyrosinase inhibitor to delay fruit browning.

Influence of different parameters on reverse micelle extraction combined with acetone precipitation to purify sn-1,3 extracellular lipase from Aspergillus niger GZUF36.

There are few reports on the feasibility of combined reverse micelle extraction and acetone precipitation to obtain electrophoretic pure enzymes. We aimed to purify a sn-1,3 extracellular lipase from a novel Aspergillus niger GZUF36 through this combination in this work. This lipase preliminarily purified by controlling the volume ratio (1:2.5) of crude enzyme solution and acetone. Then, we studied effects of different parameters on reverse micelle extraction. The suitable surfactant, pH, salt and cosolvent and extraction time for forward extraction were 125 mM cetyl trimethylammonium bromide (CTAB), 9.0, 0.075 M NaCl, 10% n-hexanol and 30 min, respectively. Under these conditions, the forward extraction rate reached 90.3% ± 3.2%. The suitable salt, pH, extraction time and short chain alcohol for backward extraction were consecutively 1.5 M KCl, 6.5, 60 min and 10% ethanol. Adding 10% ethanol shows a significant advantage of improvement the extraction rate. Under these optimal conditions, the total extraction rate and purification factor of lipase reached 76.8% and 10.14, respectively. SDS-PAGE showed that molecular weight of the pure protein was 42.7 kDa and TLC exhibited sn-1,3 selectivity of this lipase. LC-MS/MS analysis revealed that the lipase had 297 amino acid residues and was likely to glycosylate. Through the study of different parameters, it demonstrated that the new and simple combination of reverse micelle extraction using CTAB as surfactant and n-hexanol as cosolvent for forward extraction and adding ethanol for backward extraction and acetone precipitation is a promising method to get almost an electrophoretically pure sn-1,3 lipase.

Effects of organic solvent, water activity, and salt hydrate pair on the sn-1,3 selectivity and activity of whole-cell lipase from Aspergillus niger GZUF36.

We previously screened a whole-cell lipase EC 3.1.1.3 from the novel strain Aspergillus niger GZUF36, which exhibited 1,3-selectivity in the synthesis of 1,3-diacylglycerol via glycerolysis. However, the mechanism of lipase selectively in catalyzing the sn-1,3 position remains ambiguous. This work was performed to investigate the 1,3-selective mechanism of lipase using glycerolysis to synthesize 1,3-diacylglycerol (1,3-DG) as a model reaction by changing solvent(s) and water activity (aw), and addition of salt hydrate pair. The measured diacylglycerol yield was also used to examine lipase activity. Results indicated that not only organic solvent and aw have strong effect on the sn-1,3 selectivity, but also ions of salt hydrate pair also affected selectivity. Lipase conformation was altered by hydrophobic interactions of the solvent, aw, or ions of salt hydrate, resulting in distinct sn-1,3 selectivity of the lipase. The salt hydrate pair changed the lipase conformation and selectivity not only by aw but also by static interactions, which was rarely reported. These parameters also affected lipase activity. The lipase displayed the highest selectivity (about 88%) and activity in solvents of t-butanol and n-hexane (1:29, v/v) at aw 0.43. The results demonstrated that the sn-1,3 selectivity and activity of the lipase from A. niger GZUF36 may be improved by control of some crucial factors. This work laid a foundation for the application of lipase in the synthesis of 1,3-DG and other structural and functional lipids.

Total Flavonoids of Drynariae Rhizoma Prevent Bone Loss Induced by Hindlimb Unloading in Rats.

Drynariae Rhizoma is a kidney-tonifying herb that has a long history in clinical practice for the treatment of bone fractures and joint diseases in China. Flavonoids are considered to be its major active ingredients and are reported to ease bone loss in ovariectomized rats. However, the beneficial effects of the total flavonoids of Drynariae Rhizoma on osteoporosis caused by microgravity or mechanical inactivity remain unknown. This study assessed the effects of total Drynariae Rhizoma flavonoids (DRTF, Qihuang, Beijing, China, national medicine permit No. Z20030007, number of production: 04080081, content of DRTF ≥80%) against bone loss induced by simulated microgravity. A hindlimb unloading tail-suspended rat model was established to determine the effect of DRTF on bone mineral density (BMD), biomechanical strength and trabecular bone microarchitecture. Twenty-eight male Sprague-Dawley rats were divided into four groups: the baseline, control, hindlimb unloading with vehicle (HLU), and hindlimb unloading treated with DRTF (HLU-DRTF, 75 mg/kg/day) groups. Oral DRTF was administered for 4 weeks. The underlying mechanisms of the DRTF actions on disuse-induced osteoporosis are discussed. The results showed that DRTF treatment significantly increased the BMD and mechanical strength of tail-suspended rats. Enhanced bone turnover markers with HLU treatment were attenuated by DRTF administration. Deterioration of trabecular bone induced by HLU was prevented through elevated bone volume/tissue volume (BV/TV), trabecular number (Tb. N), trabecular thickness (Tb. Th) and decreased trabecular separation (Tb. Sp). The present study provides the first evidence that DRTF prevents bone loss induced by HLU treatment, indicating its potential application in the treatment of disuse-induced osteoporosis.

[SPME-GC-MS combined with Kovat's retention index analysis for volatile components in Pistacia chinensis].

OBJECTIVE: To analyze and compare the volatile components in fruits and leaves of Pistacia chinesis. METHODS: The volatile components were extracted from the fruits and leaves of Pistacia chinesis by solid-phrase microextration, and were analyzed and identified by gas chromatography-mass spectrometry(GC-MS) combined with Kovat's retention index. The relative content of each component was calculated by normalization method. RESULTS: 29 and 17 volatile components were identified from the fruits and leaves respectively, representing the relative content of 95. 30% and 96. 91% of the volatile components. 13 common components were identified in both the fruits and leaves. CONCLUSION: The volatile components in the fruits vary from that in the leaves in type and content, terpenoids are major components in the fruits and leaves of Pistacia chinesis in Shaanxi. Monoterpenes(76. 32%) are the major components of the fruits, while sesquiterpenes(65. 42%) are the major components of the leaves.

Effect of Zanthoxylum bungeanum extract on the quality and cathepsin L activity of Niuganba.

Niuganba (NGB) is a traditional fermented beef product. Protease activity typically significantly affects the quality of NGB. Some natural food extracts may markedly influence NGB's protease activity and performance. This study aims to investigate the effect of Zanthoxylum bungeanum extract (ZBE) on the quality and cathepsin L activity of NGB. Following ZBE treatment, the myofibril fragmentation index (MFI), the content of TCA-soluble peptides, surface hydrophobicity, disulfide bond content, and cathepsin L activity of NGB significantly decrease. The content of free thiol groups and ß-sheet significantly increases. Scanning electron microscopy (SEM) reveals that the arrangement of muscle fibers in the cross-section of NGB is more compact after ZBE treatment. The research results indicate that ZBE effectively inhibits cathepsin L activity, alleviates the degradation of myofibrillar proteins, improves the physicochemical characteristics of NGB, and enhances its structural stability.

Changes in food quality and characterization under thermal accumulation conditions during Chinese cooking.

Chinese cooking is the primary treatment method for table food in China. The process is complex and large-scale, which is important to the macroeconomy and national nutrition and health. First, this article puts forward the concept of thermal accumulation for Chinese cooking by taking pork tenderloin fried at different oil temperatures, explaining changes in moisture content, hardness, and color with different thermal accumulation conditions, and measuring kinetic parameters. The variations of L* and b* obtained by the experimental results belong to the first-order reaction kinetic model, while the changes in water content and shear force belong to the zero-order reaction kinetic model. Simultaneously, the superheat value is used as a thermal accumulation indicator, combined with sensory evaluation to determine that the Z value of the human sensory overheating of pork tenderloin is 99°C, and O s,max (Z = 99°C, the reference temperature is 110°C) is 5.86 min.

Effect of different conditions on the germination of coix seed and its characteristics analysis.

Coix seed (CS) has high nutritional value, but the deep processing of CS is relatively limited. Sprouting can significantly improve nutritional value, laying the foundation for efficient consumption or further processing. The optimal conditions for the germination of CS are a soaking temperature of 36 °C for 10 h and a germination temperature of 29 °C for 24 h. Under these conditions, the final germination rate of CS reached 90%. Additionally, the content of γ-aminobutyric acid was 21.205 mg/100 g; soluble protein, free amino acids, γ-aminobutyric acid, and other essential substances increased in CS. Especially after germination, the γ-aminobutyric acid (GABA) content increased by 7.8 times compared with the GABA content of ungerminated CS. Therefore, the nutritional value and flavor of germinated CS are better than those of ungerminated ones, which establishs a solid foundation for its application in developing various products such as compound health drinks, coix yogurt, and others.

C57BL/6 mice need MHC class II Aq to develop collagen-induced arthritis dependent on autoreactive T cells.

INTRODUCTION: Collagen-induced arthritis (CIA) has traditionally been performed in MHC class II A(q)-expressing mice, whereas most genetically modified mice are on the C57BL/6 background (expressing the b haplotype of the major histocompatibility complex (MHC) class II region). However, C57BL/6 mice develop arthritis after immunisation with chicken-derived collagen type II (CII), but arthritis susceptibility has been variable, and the immune specificity has not been clarified. OBJECTIVE: To establish a CIA model on the C57BL/6 background with a more predictable and defined immune response to CII. RESULTS: Both chicken and rat CII were arthritogenic in C57BL/6 mice provided they were introduced with high doses of Mycobacterium tuberculosis adjuvant. However, contaminating pepsin was strongly immunogenic and was essential for arthritis development. H-2(b)-restricted T cell epitopes on chicken or rat CII could not be identified, but expression of A(q) on the C57BL/6 background induced T cell response to the CII260-270 epitope, and also prolonged the arthritis to be more chronic. CONCLUSIONS: The putative (auto)antigen and its arthritogenic determinants in C57BL/6 mice remains undisclosed, questioning the value of the model for addressing T cell-driven pathological pathways in arthritis. To circumvent this impediment, we recommend MHC class II congenic C57BL/6N.Q mice, expressing A(q), with which T cell determinants have been thoroughly characterised.

Mixed fermentation with Lactobacillus plantarum, Bifidobacteriµm animalis subsp. lactis and Candida utilis improves the fermentation quality of Hong Suan Tang.

Lactobacillus plantarum NR1-7, Bifidobacteriµm animalis subsp. lactis BZ11 and Candida utilis RY were selected as specific starter cultures to ferment Hong Suan Tang (HST), a traditional hot and sour soup of southwest China made from tomatoes and red peppers. The fermentation conditions were optimized with response surface methodology (RSM). The changes in the physicochemical index, flavor quality, organic acids, and bioactive substances were evaluated. The results showed that the mixed starter fermented HST was quickly acidified, promoting product maturity and significantly reducing the nitrite content, which shortened the fermentation time and improved product safety. Moreover, lactic acid concentration was significantly increased, about 3 times higher than the control group. Sixteen compounds were confirmed to be the critical aroma-active compounds present in the HST. Furthermore, mixed inoculation improved the content of bioactive substances by lowering their degradation. This research sets the basis for the standardized production of HST products.

Adsorption-precipitation-cross-linking immobilization of GDSL-type esterase from Aspergillus niger GZUF36 by polydopamine-modified magnetic clarity tetroxide nanocouplings and its enzymatic characterization.

Recombinant INANE1 (rINANE1), a recombinant intracellular GDSL-type esterase from Aspergillus niger GZUF36, has high acetate substrate specificity. Here, rINANE1 was successfully immobilized on polydopamine (PDA)-modified magnetic ferric oxide nanoparticles (Fe3O4NPs) by adsorption-precipitation-cross-linking to obtain cross-linked enzyme aggregate (CLEA)-rINANE1-Fe3O4@PDA. Fe3O4, Fe3O4@PDA, and CLEA-rINANE1-Fe3O4@PDA were characterized by scanning electron microscopy, X-ray diffraction, vibrating-sample magnetometry, Fourier transform infrared (FTIR) spectroscopy, and zeta potential analysis. Upon immobilization, CLEA-rINANE1-Fe3O4@PDA, with a protein loading of 72.72 ± 1.01 mg/g, reached optimal activity recovery of 104.40 % ± 1.14 %. FTIR analysis showed that immobilization increased the relative content of ß-folding in rINANE1 by 12.25 % and reduced irregular curl by 4.16 %, rendering the structure more orderly. Specifically, under an alkaline condition (pH 10), CLEA-rINANE1-Fe3O4@PDA performed over 100 % of initial activity. The optimum temperature increased by 5 °C, and over 55 % of the initial activity was observed after 12 h at 55 °C. CLEA-rINANE1-Fe3O4@PDA showed over 40 % of its relative activity, whereas free rINANE1 showed <10 % in acetonitrile. In addition, the relative activity of CLEA-rINANE1-Fe3O4@PDA was retained at about 80 % after eight cycles and maintained at 109 % after 45 days. The PDA-modified magnetic ferrite nanoparticles exhibited excellent stability and recyclability, providing a new avenue for developing industrial biocatalysts.

Purification of fibrinolytic enzyme from Bacillus amyloliquefaciens GUTU06 and properties of the enzyme.

A producing-fibrinolytic enzyme strain was isolated with high yield. The strain was identified as Bacillus amyloliquefaciens. B. amyloliquefaciens GUTU06 fibrinolytic enzyme was purified by acetone precipitation and reverse micelle. Acetone precipitation condition and reverse micelle condition were examined. Results showed that the total reverse micelle extraction efficiency was 64.49 % ± 1.6 %. The purification fold of the entire process reached 13.38. The optimum pH of purified enzyme is 5, and the optimum temperature is 45 °C. Fe3+ and K+ can enhance the fibrinolytic activity of the enzyme. Compared to commercial fibrinolytic enzymes such as urokinase and lumbrukinase, GUTU06 fibrinolytic enzymes have a lower pH optimal range and higher temperature stability. The molecular weight of the enzyme was approximately 28 kDa. Reverse micelle extraction with cetyl trimethylammonium bromide as a surfactant combined with acetone precipitation is suitable for separating and purifying fibrinolytic enzymes and a promising technique for obtaining active proteins.

The effects of Trametes versicolor fermented Rosa roxburghii tratt and coix seed quild on the nutrition, sensory characteristics and physical and chemical parameters of yogurt.

Trametes versicolor can produce aromatic flavor in growth and development, widely used in food fermentation. This study used a One-Factor-at-a-Time (OFAT) test and response surface analysis to study the optimum fermentation parameters of Rosa roxburghii tratt and coix seed yogurt by Trametes versicolor. The best fermentation process is as follows: skim milk powder 17 %, sucrose content 4 %, Rosa roxburghii tratt and coix seed liquid 36 %, fermentation temperature 39 °C, inoculum 2 %, strain ratio 2:1:1(LB12: BLH1: Q-1), fermentation time 9.5 h. Under this fermentation process, the sensory score was 82.11, the contents of vitamin C, GABA, and total live bacteria in this yogurt were 13.89, 2.58, and 1.02 times higher than in common yogurt. Correlation analyses showed a significant contribution of the leavening agent to the GABA content of yogurt. This study provides a foundation for producing Rosa roxburghii tratt and coix seed yogurt with high sensory and nutritional value.