Research On The Application Of Asperigillus Oryzae Recombinant Neutral Protease(rNpI) In Protein Hydrolysis

Protease can promote the hydrolysis of protein, which is broadly used in food production, tanning industry, medicine health industry and produce bioactive peptides. Different bioactive peptides can be produced by enzymatic hydrolysis of protein, such as antioxidant peptides and angiotensin-converting enzyme(ACE)inhibitory pepdides,which have an important appilication in food additive, health products, etc. Our research group had received an Asperigillus oryzae recombinant neutral protease(r Np I) bacterial strain with high yield before, which had high efficiency in hydrolysis of plant protein.The objective of the paper is to study the applications of the recombinant neutral protease(r Np I) in plant protein, such as Soy Protein Isolate(SPI), peanut protein and in casein hydrolysis. The degree of hydrolysis, bitterness, the distribution of molecular weight, DPPH radical scavenging activity, Reducing power assay, Oxygen Radical Absorbance Capacity(ORAC) assay and ACE inhibitory activity of SPI hydrolysates(SPIH) were evaluated at present. Take SPI hydrolysates for example, the results showed that r Np I had relative higher efficiency in SPI hydrolysis, the degree of hydrolysis were 7.8%、11.5% and 16.0% respectively, under the E/S ratio of 1000 U/g, 4000 U/g and 8000 U/g. For the bitterness evaluation, results showed the bitterness of SPI hydrolysates hydrolyzed by r Np I were significantly low, than that hydrolyzed by Alcalase. When the E/S ratio was 8000 U/g, HPLC measurement of SPIH results showed that the peptide fractions less than 1 k Da accounts for 44.02% of total protein hydrolyasates. The result indicates that the relative low molecular weight accounts for a large proportion of total prorein hydrolysates. After the Crucian carp acquacuture by adding r Np I in the feed of Crucian carp, the result showed that r Np I could reduce the adding amount of 5.5% rapessed meal and 5% wheat, and the carp feed conversation rate(FCR) decresed by 7.2%.The antioxidant activities of SPIH were determined by different assays, and the results showed that these SPI hydrolysates hydrolyzed by r Np I have high antioxidant activities. When the E/S ratio was 4000 U/g, SPIH had comparable higher antioxidant activities. The reducing power of 1.0 mg/ml SPIH was 0.34, its DPPH free radical scavenging activity was 80.1% and the ORAC value was 455 μmol TE/g protein. Ultrafiltration membrane was used to separate SPIH components, and their antioxidant activities were analyzed. Results showed that SPIH-I(molecular weight above 10 k Da) has the highest DPPH radical scavenging activity(82.7%), while SPIH-III(molecular weight below 3 k Da) has the highest ORAC value(722 μmol TE/g protein). When the E/S ratio of r Np I with SPI were 1000, 4000 and 8000 U/g, the protein concentration required to inhibit the original ACE activity by 50%(IC50)were 452.69 μg/ml, 136.93 μg/ml and 84.48 μg/ml respectively.From the above research, r Np I has high efficiency in hydrolysis of plant protein and animal protein, and the hydrolysates posess low bitterness. The result of Crucian carp acquaculture indicates that r Np I is suitable for adding in the aquatic animal feed. The hydrolysate not only has relative high antioxidant activity but also high ACE inhibitory activity. All these results indicate the r Np I has good application potentials in protein hydrolysis, feed addictive, food preservation and health care products.