A substrate protection approach to applying the calcium ion for improving the proteolysis resistance of the collagen.

Enzymatic dehairing, as a crucial part of cleaner leather processing, has reached processive advancement with potentially replacing the traditional hair removal due to increasing pressure from environmental demand. However, this cleaner technology based on proteases has a problem that the hide grain (collagen-rich structure) is susceptible to be hydrolyzed, decreasing the quality of finished leather. From the perspective of improving the stability of collagen fibers and their resistance to proteolysis, a method for protecting the hide grain during the enzymatic dehairing process was developed. The results showed that calcium ions had a swelling effect on collagen fibers under near-neutral conditions (pH 6.0-10.0), decreasing the thermal stability of collagen and the proteolysis resistance of collagen significantly. The alkaline environment (pH 10.0-12.0) will promote the dissociation of carboxyl groups in hide collagen, promoting the combination of calcium ions and carboxyl groups. This strategy can change the surface charge of collagen fibers and strengthen the connection between collagen fibers, thus improving protease resistance and the thermal stability of collagen. However, collagen fibers could swell violently once the alkalinity of the solution environment was extreme. Despite the above situation, calcium ion was still conducive to maintain the structural stability of collagen fibers. At pH 10.0-12.0, pretreating animal hide with a solution containing calcium ions can improve the protease resistance of hide grain, making the hide grain well-protected. This method provided an effective way to establish a safer enzymatic unhairing technology based on substrate protection. KEY POINTS: • A collagen protection method for hair removal of animal hide was developed. • This method applied calcium ions to collagen at alkaline conditions (pH 10.0-12.0). • Pretreatment results of calcium ions at different pH values on animal hide were compared.

Development and validation of a label-free method for measuring the collagen hydrolytic activity of protease.

Collagen is the most abundant fibrous structural protein, and therefore, the quantitative evaluation of the effect of protease on collagen has a profound influence on enzyme application. In this research, unlabeled native bovine hide powder was utilized to detect collagen hydrolytic activity of the protease. The optimum conditions of the determination method were as follows: 30 mg/mL substrate concentration, 30 min reaction time, and 2-9 U/mL enzyme concentration. Then, several typical industrial protease preparations were chosen to measure collagenolytic activities at different temperatures and pH values, whose change trends were quite distinct from those of proteolytic activity assay method based on casein or dye-labeled hide powder substrate. Especially, in the pH 5-7, casein hydrolytic activities of these proteases showed sharper peaks with relative activity from 6% to 100%, whereas, their collagen hydrolytic activities based on native hide powder exhibited 30-100% with broader peaks. And collagen hydrolytic activities resulted from using dye-labeled substrate reached a lower optimum pH value than that of other methods. Besides, the results of these measurements displayed a moderate degree of reproducibility. This method is more reasonable than the protease assay method using casein or labeled hide powder as the substrate in many fields.

Resource Utilization of Bovine Neck Ligament: Enzymatic Preparation of Elastin Peptide and Its Antioxidant Activity.

Elastin is considered an excellent resource for obtaining antioxidant peptides due to unique amino acid composition. However, it is hardly soluble in water or in dilute acid or alkali; most of the elastases have low yields for preparing elastin peptides, making it difficult to meet industrial applications. To address above problems, enzymes capable of hydrolyzing elastin into soluble peptides were preferred from typical commercial protease preparations. The optimal enzymatic hydrolysis process conditions for elastin peptides were obtained by response surface optimization design. The molecular weight, amino acid composition, and antioxidant activity of the enzymatic hydrolysis products were determined. The results show that the alkaline protease NUE has a strong hydrolysis effect. The optimized enzymatic hydrolysis conditions are as follows: substrate concentration is 5%, enzyme concentration is 650 U/mL, pH is 10.0, temperature is 60 °C, time is 6 h. The degree of hydrolysis of elastic protein peptides obtained through this method is 14.42%. The distribution of molecular weight is 200-6500 Da, more than 85% of the component molecular amount is greater than 800 Da; the amino acid content related to antioxidant activity has reached 68 mg/100 mg, so it has extremely high free radical clearance. Compared with acid and alkali methods, the anti-oxidation capacity of enzyme-based peptide is better, the reaction conditions are milder, the yield is higher, and by-products and pollutants are fewer. It provides an effective way to industrialized production of elastin peptides with high antioxidant activity and a basis for its widespread application in the food and pharmaceutical industries.