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Organic-inorganic hybrid nanoflowers (hNFs) with commercial protease "Neutrase" is proposed and characterized as efficient and green biocatalysts for promiscuous catalysis in aldol-type and multicomponent reactions. Neutrase hNFs [Neutrase-(Cu/Ca/Co/Mn)3(PO4)2] are straightforwardly prepared through mixing metal ion (Cu2+, Ca2+, Co2+ or Mn2+) aqueous solutions with Neutrase in phosphate buffer (pH 7.4, 10 mM) resulting in precipitation (3 days). The hNFs were characterized by various techniques including scanning electron microscopy (SEM), energy dispersive X-ray (EDX), element mapping, X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). In SEM images, the metal-Neutrase complexes revealed flower-like or granular structures after hybridization. The effect of metal ions and enzyme concentrations on the morphology and enzyme activity of the Neutrase-hNFs was examined. The synthesized Neutrase-Mn hNFs showed superior activity and stability compared to free Neutrase. Traditional organic CC coupling reactions such as aldol condensation, decarboxylative aldol, Knoevenagel, Hantzsch-type reactions and synthesis of 4H-pyran derivatives were used to test the generality and scope of Neutrase promiscuity, while optimizing conditions for the Neutrase-Mn hNF biocatalyst. Briefly, Neutrase-Mn3(PO4)2 hNFs showed excellent enzyme activity, stability and reusability, qualifying as effective reusable catalysts for coupling reactions under mild conditions.

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BACKGROUND AND AIM: Gelatin is a dissolved protein that results from partial extraction of collagen, commonly from pig and bovine skin. There was no study on gelatin production from Kacang goat bones through enzymatic extraction. This study aimed to evaluate the chemical, physical, and functional properties of gelatin from bones of Kacang goat using alcalase and neutrase enzymes. MATERIALS AND METHODS: Male Kacang goat bones aged 6-12 months and two commercial enzymes (alcalase and neutrase) were used for this study. Descriptive analysis and completely randomized design (one-way analysis of variance) were used to analyze the chemical, physical, and functional properties of gelatin. Kacang goat bone was extracted with four concentrations of alcalase and neutrase enzymes, namely, 0 U/g (AG-0 and NG-0), 0.02 U/g (AG-1 and NG-1), 0.04 U/g (AG-2 and NG-2), and 0.06 U/g (AG-3 and NG-3) with five replications. RESULTS: The highest yield of gelatin extraction with alcalase obtained on AG-3 was 9.78%, and that with neutrase on NG-3 was 6.35%. The moisture content of alcalase gelatin was 9.39-9.94%, and that of neutrase gelatin was 9.15-9.24%. The ash and fat content of gelatin with alcalase was lower than that without enzyme treatment with higher protein content. The lowest fat content was noted in AG-1 (0.50%), with protein that was not different for all enzyme concentrations (69.65-70.21%). Gelatin with neutrase had lower ash content than that without neutrase (1.61-1.90%), with the highest protein content in NG-3 (70.89%). The pH of gelatin with alcalase and neutrase was 6.19-6.92 lower than that without enzymes. Melting points, gel strength, and water holding capacity (WHC) of gelatin with the highest alcalase levels on AG-1 and AG-2 ranged from 28.33 to 28.47°C, 67.41 to 68.14 g bloom, and 324.00 to 334.67%, respectively, with viscosity that did not differ, while the highest foam expansion (FE) and foam stability (FS) were noted in AG-1, which were 71.67% and 52.67%, respectively. The highest oil holding capacity (OHC) was found in AG-2 (283%). FS and OHC of gelatins with the highest neutrase levels in NG-2 were 30.00% and 265.33%, respectively, while gel strength, viscosity, FE, and WHC of gelatins with the highest neutrase levels did not differ with those without enzymes at all enzyme concentrations. B chain was degraded in all gelatins, and high-intensity a-chains in gelatin with alcalase and peptide fraction were formed in gelatin with neutrase. Extraction with enzymes showed loss of the triple helix as demonstrated by Fourier transform infrared spectroscopy. CONCLUSION: Based on the obtained results, the Kacang goat bone was the potential raw source for gelatin production. Enzymatic extraction can increase the quality of gelatin, especially the alcalase (0.02-0.04 U/g bone) method. This can be used to achieve the preferable quality of gelatin with a higher yield.

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The protein fraction of Brewers' spent grain (BSG) was used as substrate to obtain hydrolysates with antioxidant activity. Three enzymatic approaches were applied: brewer's spent yeast (BSY) proteases, Neutrase® and Alcalase®, at the same proteolytic activity (1U/mL), using an enzyme/substrate ratio of 10:100 (v/v), at 50°C, 4h. Total Phenolic Content (TPC) and Ferric Ion Reducing Antioxidant Power (FRAP) of hydrolysates and fractions <10kDa and <3kDa were assayed. Additionally, the protective ability of <10kDa fractions against oxidative stress on Caco-2 and HepG2 cells was investigated. Alcalase® hydrolysate presented significantly (p<0.05) higher TPC and FRAP (0.083mgGAE/mgdw; 0.101mgTE/mgdw, respectively) than Neutrase® and BSY hydrolysates. The three BSG protein hydrolysates (fraction <10kDa) exerted protective effect against free-radical induced cytotoxicity in Caco-2 and HepG2 cell lines, but the strongest effect was observed for BSY hydrolysates, therefore, it presents greater potential as functional ingredient.

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The neutrase (EC 3.4.24.4) and papain (EC 3.4.22.2) were together immobilized ascross-linked enzyme aggregates (N-P-CLEAs) and their properties were characterized. The influence of the precipitant, cross-linking ratio of glutaraldehyde and cross-linking time were investigated. Ethanol was selected as the more efficient precipitant compared with ammonium sulfate. The proper cross-linking ratio of enzyme and glutaraldehyde was 1:5 (v/v) and the optimized cross-linking time was 4h. N-P-CLEAs showed obvious improvement in thermal stability and pH stability than the free enzyme (P<0.05) and could hold relatively high activity retention in nonpolar and hydrophilic solvents and without activity loss at 4°C for more than six months. The cross-linking reaction had been appeared in N-P-CLEAs and more orderly microscopic surface morphology of N-P-CLEAs was observed. The molecular weight and thermal denaturation temperature of N-P-CLEAs were increased while the isoelectric point was decreased compared with those of the free enzymes. Application of N-P-CLEAs in bean proteins and zein showed a higher degree of hydrolysis, such as the hydrolysis degree of mung bean protein hydrolyzed by N-P-CLEAs was 12%, increased by approximately 4.5% compared to that of free enzyme. The results demonstrated that the N-P-CLEAs was suitable for application in food protein hydrolysis.

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Casein hydrolysate was prepared by hydrolyzing casein with Neutrase and then modified by a Neutrase-catalyzed plastein reaction. The prepared hydrolysate had a degree of hydrolysis of 13.0% and exhibited ACE inhibition in vitro with an IC50 value of 40.4 µg⋅mL(-1). With the decreased amount of free amino groups of the modified hydrolysate as the response, some conditions of the plastein reaction including substrate concentration, enzyme to substrate ratio, reaction temperature and time were studied by single factor experiments and response surface methodology, and optimized finally as 62% (w/w), 3.0 kU⋅g(-1) peptides, 30 °C and 6.3 h, respectively. The maximum decreased amount of free amino groups of the modified hydrolysate prepared under these optimized conditions was 210.0 µmol⋅g(-1) peptides, while corresponding IC50 value was lowered to 14.7 µg⋅mL(-1). The present result indicates that Neutrase-catalyzed plastein reaction was capable of enhancing ACE-inhibitory activity in vitro of casein hydrolysate, and also highlights the importance of a forthcoming study to investigate the peptide compositions of the modified hydrolysate and the role of protease used in the plastein reaction.

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This study utilized commercially available proteolytic enzymes to prepare egg-white protein hydrolysates (EPHs) with different degrees of hydrolysis. The antioxidant effect and functionalities of the resultant products were then investigated. Treatment with Neutrase yielded the most α-amino groups (6.52 mg/mL). Alcalase, Flavourzyme, Protamex, and Ficin showed similar degrees of α-amino group liberation (3.19-3.62 mg/mL). Neutrase treatment also resulted in the highest degree of hydrolysis (23.4%). Alcalase and Ficin treatment resulted in similar degrees of hydrolysis. All hydrolysates, except for the Flavourzyme hydrolysate, had greater radical scavenging activity than the control. The Neutrase hydrolysate showed the highest 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity (IC50=3.6mg/mL). Therefore, Neutrase was identified as the optimal enzyme for hydrolyzing egg-white protein to yield antioxidant peptides. During Neutrase hydrolysis, the reaction rate was rapid over the first 4 h, and then subsequently declined. The IC50 value was lowest after the first hour (2.99 mg/mL). The emulsifying activity index (EAI) of EPH treated with Neutrase decreased, as the pH decreased. The EPH foaming capacity was maximal at pH 3.6, and decreased at an alkaline pH. Digestion resulted in significantly higher 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ABTS radical scavenging activity. The active peptides released from egg-white protein showed antioxidative activities on ABTS and DHHP radical. Thus, this approach may be useful for the preparation of potent antioxidant products.