Characteristics And Biologic Activities Of Enzymatic Hydrolysates From Royal Jelly And Their Mechanisms

With the rapid development of the medicine, food and cosmetic industries as well as demands of agriculture and animal husbandry for energy, more and more modified proteins and their processing products are applied. Compared to the raw materials, these products have excellent physical and chemical characteristics, nutritional properties and biological activity that can meet the needs of the industrial production. These have become the new type of industrial raw materials and energy. Hydrolysis technique is one of the effective ways to obtain these proteins products. Through the hydrolysis modification, hydrolysates from the proteins become more valuable than the raw protein to some extent. Currently, hydrolysates from some plant protein (such as soy), animal protein (such as fish and shrimp) and microbial protein (such as yeast) have been developed and utilized widely.Royal jelly (RJ) is a secretion of the hypopharyngeal and mandibular glands of worker honeybee and is used to feed the queen and larvae which are less than3days old. RJ is rich in proteins and has high value in health care such as immunity adjustment, antioxidant activity, antiaging activity, antibacterial activity, antitumor activity and cardiovascular system regulation. The production and exportation of RJ in China rank first in the international trade. But the products processing of RJ in our country still at a primary level with the restriction of characteristics of RJ (the slightly pungent odor and taste, homogenate texture and poor solubility) and the technological development. The situation limits the market prospects and the economic value of RJ, and holds back the application of RJ.Our study focused on the physical and chemical characteristics as well as the biological activities of the hydrolysates from RJ. We also investigated the correlation between hydrolysis condition and antioxidant activity of the hydrolysates from RJ. The colorimetric index and viscosity of RJ hydrolysates was reported. The optimal hydrolysis conditions and optimized enzymatic RJ hydrolysate were obtained after the single factor test and orthogonal experiment were carried out by taking the impact order of different hydrolysis factors into account. It was also the first report about the antiaging activity of the optimized enzymatic RJ hydrolysate in D-galactose induced aging mouse model in vivo. Moreover, the cell model was added into the evaluation of immunity adjustment of optimized RJ hydrolysate. The results are as follows:(1) Through the enzymatic hydrolysis, RJ hydrolysates were abundant in protein content and recovery, as well as containing a great variety and content of amino acids. The relative molecular weight and viscosity of RJ hydrolysates were significantly decreased. Besides, the enzymatic also change the color of RJ and the permeability of water solution of optimized RJ hydrolysates was enhanced.(2) The in vitro antioxidant activities of RJ hydrolysates were tested in both2,2-diphenyl-l-picrylhydrazyl free radical scavenging (DPPH) system and ferric reducing antioxidant power (FRAP) system. The enzymatic hydrolysis process significant changed in vitro antioxidant activity of RJ. The optimized RJ hydrolysate possessed higher antioxidant activity than RJ did. Results of single factor test showed that enzymatic hydrolysis conditions would affect the scavenging activity of DPPH and reducing power of ferric ion of RJ hydrolysates. Orthogonal experiment showed that the impact order of different hydrolysis factors on scavenging activity of RJ hydrolysates was:hydrolysis time> substrate concentration> enzyme concentration> pH. The impact order of different hydrolysis factors on reducing power of RJ hydrolysates was:substrate concentration> pH> hydrolysis time> enzyme concentration. Finally, taking the impact order into account, we got the optimal hydrolysis condition of RJ was as follows:conditions as substrate concentration125g kg-1; enzyme concentration1.5%[w/w]; temperature45℃; hydrolysis time4h and pH8.(3) The antiaging activity of the optimized RJ hydrolysate was evaluated using the D-galactose induced aging mouse model in vivo. In the respects of improving external aging performance, low doses of optimized RJ hydrolysate could significantly prevent the body weight loss of the aging mouse. Both low and high doses of optimized RJ hydrolysate improved the age-related locomotor decline. Moreover, the administration of optimized RJ hydrolysate improved both spatial and non-spatial long term memory of mice, help them maintain good spatial orientation judgment, excellent learning and memory about the training after a long period of time. In the respects of improving internal aging performance, given optimized RJ hydrolysate help to delay the atrophy of thymus to prevent immune function diminished with aging. Optimized RJ hydrolysate also increased antioxidant enzyme activity and inhibit the production of lipid peroxides of mice, which was equal to or even better than RJ did. (4) The immunity adjustment of RJ hydrolysate was tested in two parts. Concanavalin A (ConA) and lipopolysaccharide (LPS) were used to stimulate proliferation and transformation of T lymphocyte and B lymphocyte from mice spleen, respectively. Effects of optimized RJ hydrolysate on the stimulatory index of both T lymphocyte and B lymphocyte were determined by3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Lactate dehydrogenase (LDH) release assay was used for evaluation of natural killer (NK) cell activity by the influence of optimized RJ hydrolysate. The effect of optimized RJ hydrolysate on LPS stimulated RAW264.7cells was also tested. Results showed that, Comparing with the aging mice, given low dose of optimized RJ hydrolysate promoted the proliferation and transformation of B lymphocyte induced by LPS. RJ hydrolysate with high dose had better performance, which was significantly more efficient than RJ. Moreover, the degradation of inhibitor of kappa B alpha (IκBα) was partly restrained by the optimized RJ hydrolysate. Besides, no significant difference was found between normal mice and those treated with the optimized RJ hydrolysate on the proliferation and transformation of T lymphocyte induced by ConA and the activity of NK cell.