Study On Preparation And Immunological Activity Of Protein Hydrolysates From Hazelnut In Changbai Mountain

Hazelnut in Changbai Mountain(Corylus heterophylla Fisch.) is nutritious and rich in protein(20% to 30%), fat(40% to 50%), carbohydrates(13% to 24%), dietary fiber(8.2% to 9.6%), vitamins, and microelements. However,the development and utilization of hazelnut is severely inadequate at present, and the utility value of hazelnut dregs(containing around 58% of protein) is very low, which leads to a waste of high-quality protein resources, so preparing functional paptides with hazelnut dregs could take full advantage of the existing resource and develop new plant protein sources. In this paper, the enzymolysis technology of Hazelnut Protein Hydrolysates(HPH) was optimized, the HPH was separated and purified and its effects on immue function in mice and the secretion of cytokines in RAW264.7 cells studied, It provided the theoretical and experimental basis for the comprehensive utilization of hazelnut protein resource in Changbai Mountain and the development of peptide products with immune activity.This assay took Changbai Mountain defatted hazelnuts powder as raw material, prepared hazelnut-isolated protein using alkaline dissolving and acid precipitating method, and then prepared the HPH using the enzymatic hydrolysis of Alcalase. With the degree of hydrolysis as the index, We selected the pH, temperature, Alcalase concentration, substrate concentration to do the single factor experiment and response surface experiment. The optimal enzyme hydrolysis conditions are as follows: substrate concentration of 2.0%, pH of 8.5, hydrolysis temperature of 53.5℃, Alcalase concentration of 10000U/g. When the hydrolysis time is 150 min, the degree of hydrolysis reaches 39.72%.Based on the optimal enzyme hydrolysis conditions, HPH with different DH(24.16%, 29.69%, 33.22%, 36.33%, 38.08% and 39.57%, respectively)were prepared according to hydrolysis time and orally gavaged 1.00g/kg bw to the test group mice. And with the immune organ, spleen lymphocyte proliferation function, phagocytic activity of macrophages, cytokine levels in serum(IFN-γ and IL-4), the activity of alkaline phosphatase in serum as evaluated indices, the immunomodulatory effect of HPH in mice was studied on 10 th, 20 th and 30 th day, respectively. The results suggested that the thymus index of DH 33.22% group was significantly higher than the control group(P<0.05); the DH 36.33, 38.08 and 39.57% groups increased the immune organ index, IFN-γ and AKP levels in serum, increased spleen lymphocyte proliferation activity and macrophage phagocytic activity(P<0.05 or P<0.01).HPH with a hydrolysis degree of 38.08% was divided into three fractions by ultrafiltration: the high molecular weight peptide(>10kDa), medium molecular weight peptide(3kDa to 10kDa), and low molecular weight peptide(<3kDa). All mice were fed daily with HPH of different molecular weights at doses of 200, 400, and 800 mg/kg body weight. On the 10 th, 20 th, and 30 th feeding day, representative immune indexes were measured. Results showed that HPH can regulate the immune system of mice, which is affected by the molecular weight of HHP and the feeding time. Overall, short-term feeding(10d to 20d) with HPH of different molecular weights can improve most immune indexes(organ index, spleen lymphocyte proliferation, macrophage activity, secretory immunoglobulin A content, and number of CD4+ and CD8+), while during long-term feeding(30d), low molecular weight HHP can better sustain immune regulation.The low molecular weight HPH(<3kDa) were separated and purified by Sephadex G-25, the A2 and A3 components of Sephadex G-25 were separated and purified by Sephadex G-15 to obtain seven components :B1、B2、B3、B4、B5、B6 and B7, the effects of the seven components on the secretion of cytokines in RAW264.7 cells were determined. The results showed that all components of Sephadex G-15 could increase the secretion level of TNF-α and IL-6 in RAW264.7 cells(P<0.05 or P<0.01). This reflected that the components of Sephadex G-15 could, to a certain degree, control the inflammatory responses and enhance the immunity in cells, the further research about the structure and composition of the hazelnut hydrolyzed peptides and the relevant immune regulation mechanism is needed.