蜂王浆酶解产物的理化特性、生物学活性及其作用机理研究
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摘要
蜂王浆是哺育蜂咽下腺和上颚腺分泌的,用以饲喂蜂王和1-3日龄幼虫的浆状物质。蜂王浆富含蛋白质,且具有免疫调节、抗氧化、抗衰老、抗菌、抗肿瘤和调节心血管系统等多种生物学功能。我国是世界第一蜂王浆生产国和输出国,但长期以来,受到蜂王浆本身辛辣口感、浆状质地和溶解性欠佳等理化特性的局限和研究水平、加工技术的限制,我国出口和销售的大部分蜂王浆产品均为初级加工或原料产品。这在很大程度上制约了蜂王浆的市场空间和经济价值,滞缓了蜂王浆推广应用的速度。因此,采用一些技术手段对蜂王浆进行改造显得尤为重要。
水解技术是蛋白类材料改性的一种有效手段。经过水解改性所得的产物往往比原料蛋白具有更优异的理化特性、营养特性和生物学活性,能够更好地满足相关行业的需要。目前,一些植物蛋白(如大豆)、动物蛋白(如鱼虾)及微生物蛋白(如酵母)的水解产物已经得到开发和利用,成为新型的工业原材料和农业新能源。
本研究从酶解的角度入手,对蜂王浆酶解产物的理化特性和生物学活性进行了详细分析,并探讨了酶解工艺与产物特性的相关性。理化特性部分首次界定了蜂王浆酶解产物的色度和粘度特性。酶解工艺部分,通过单因素分析和正交分析,探寻了各酶解条件对于产物抗氧化性的影响次序和影响力,为明确产物抗氧化性与酶解工艺条件的相关性提供理论依据,并获取了具有优异抗氧化性能的蜂王浆酶解优化产物。随后,通过构建D-半乳糖诱导的衰老小鼠模型,首次全面衡量了蜂王浆酶解产物在该模型条件下的体内抗衰老活性。最后,还结合细胞模型对蜂王浆酶解产物的免疫调节作用进行了分析。研究结果如下：
(1)蜂王浆在经过酶解后,其粗蛋白含量和蛋白回收率能维持较高水平,氨基酸种类丰富、含量高。酶解产物的相对分子量显著下降,粘度下降,表现出良好的流动性。此外,酶解还改变了蜂王浆的色泽,酶解产物水溶液的通透性相比蜂王浆显著提高。
(2)采用2,2-二苯基-1-苦基苯肼(DPPH)法和铁离子还原抗氧化能力(FRAP)法测定蜂王浆酶解优化产物的体外抗氧化活性。结果发现,酶解能够显著改变蜂王浆的体外抗氧化活性,优化的蜂王浆酶解产物具有显著高于蜂王浆本身的抗氧化活性。单因素试验测定结果表明,酶解工艺条件的改变会影响蜂王浆酶解产物清除DPPH的活性和还原铁离子的能力。正交试验结果显示,酶解工艺条件对酶解产物清除DPPH的影响次序为：酶解时间>底物浓度>酶与底物浓度比>pH；对酶解产物还原力的影响的次序为：底物浓度>pH>酶解时间>酶与底物浓度比。综合各因素影响主次及影响力大小,最终获得的最佳酶解工艺为：在pH8.0条件下,加入胰蛋白酶对蜂王浆进行酶解,选择酶与底物浓度比为1.5%,控制底物浓度为125g kg-1,以45℃恒温磁力搅拌酶解4h。
(3)通过构建D-半乳糖诱导的衰老小鼠模型,首次检测了优化的蜂王浆酶解产物对于该类衰老模型小鼠的外部衰老表征和内部衰老表征的改善作用,全面衡量了蜂王浆酶解产物的体内抗衰老活性。结果表明,在改善外部衰老方面,低剂量的蜂王浆酶解优化产物能够干预D-半乳糖诱导的小鼠体重下降的状况。高低剂量的蜂王浆酶解优化产物均能改善抗衰老小鼠活动能力下降的情况。与此同时,蜂王浆酶解优化产物还能够提高小鼠非空间性长期学习记忆能力和空间性长期学习记忆能力,使之保持良好的记忆力、对空间和方位的快速判断力以及对学习和训练结果的长期巩固能力,具有优异的回避危害的学习能力及长期记忆能力。在改善内部衰老方面,蜂王浆酶解优化产物能延缓胸腺等器官的萎缩,表现出对器官衰老的抑制作用。蜂王浆酶解优化产物还能够提高小鼠血清超氧化物歧化酶(SOD)活力、提高小鼠肝脏过氧化氢酶(CAT)的活力、提高小鼠肝脏谷胱甘肽过氧化物酶(GSH-px)的活力并抑制小鼠脑中丙二醛(MDA)的积累,高剂量效果等同或显著优于蜂王浆。
(4)采用刀豆蛋白A(ConA)刺激小鼠脾脏T细胞转化,用脂多糖(LPS)刺激小鼠脾脏B细胞的转化,以四甲基戊唑蓝(MTT)法检测蜂王浆酶解产物对于衰老小鼠脾T/B淋巴细胞转化的影响。同时,采用乳酸脱氢酶(LDH)释放法测定蜂王浆酶解产物对于自然杀伤细胞(NK)活力的影响,研究了蜂王浆酶解产物对于免疫调节的作用。并进一步从NF-κB信号通路的角度评价蜂王浆及其优化的酶解产物对于LPS刺激下小鼠单核巨噬细胞RAW264.7的免疫调控作用。结果发现,相比衰老小鼠,低剂量的蜂王浆优化酶解产物表现出对LPS刺激下小鼠脾脏B淋巴细胞转化的显著促进作用。高剂量效果更佳,与蜂王浆相比达到显著差异。优化的蜂王浆酶解产物还能部分抑制NF-κB抑制蛋白(IκBα)的降解。而对于ConA刺激下小鼠脾脏T细胞的转化和NK细胞活性影响,给予蜂王浆酶解产物组小鼠与正常小鼠无显著性差异。
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.
水解技术是蛋白类材料改性的一种有效手段。经过水解改性所得的产物往往比原料蛋白具有更优异的理化特性、营养特性和生物学活性,能够更好地满足相关行业的需要。目前,一些植物蛋白(如大豆)、动物蛋白(如鱼虾)及微生物蛋白(如酵母)的水解产物已经得到开发和利用,成为新型的工业原材料和农业新能源。
本研究从酶解的角度入手,对蜂王浆酶解产物的理化特性和生物学活性进行了详细分析,并探讨了酶解工艺与产物特性的相关性。理化特性部分首次界定了蜂王浆酶解产物的色度和粘度特性。酶解工艺部分,通过单因素分析和正交分析,探寻了各酶解条件对于产物抗氧化性的影响次序和影响力,为明确产物抗氧化性与酶解工艺条件的相关性提供理论依据,并获取了具有优异抗氧化性能的蜂王浆酶解优化产物。随后,通过构建D-半乳糖诱导的衰老小鼠模型,首次全面衡量了蜂王浆酶解产物在该模型条件下的体内抗衰老活性。最后,还结合细胞模型对蜂王浆酶解产物的免疫调节作用进行了分析。研究结果如下：
(1)蜂王浆在经过酶解后,其粗蛋白含量和蛋白回收率能维持较高水平,氨基酸种类丰富、含量高。酶解产物的相对分子量显著下降,粘度下降,表现出良好的流动性。此外,酶解还改变了蜂王浆的色泽,酶解产物水溶液的通透性相比蜂王浆显著提高。
(2)采用2,2-二苯基-1-苦基苯肼(DPPH)法和铁离子还原抗氧化能力(FRAP)法测定蜂王浆酶解优化产物的体外抗氧化活性。结果发现,酶解能够显著改变蜂王浆的体外抗氧化活性,优化的蜂王浆酶解产物具有显著高于蜂王浆本身的抗氧化活性。单因素试验测定结果表明,酶解工艺条件的改变会影响蜂王浆酶解产物清除DPPH的活性和还原铁离子的能力。正交试验结果显示,酶解工艺条件对酶解产物清除DPPH的影响次序为：酶解时间>底物浓度>酶与底物浓度比>pH；对酶解产物还原力的影响的次序为：底物浓度>pH>酶解时间>酶与底物浓度比。综合各因素影响主次及影响力大小,最终获得的最佳酶解工艺为：在pH8.0条件下,加入胰蛋白酶对蜂王浆进行酶解,选择酶与底物浓度比为1.5%,控制底物浓度为125g kg-1,以45℃恒温磁力搅拌酶解4h。
(3)通过构建D-半乳糖诱导的衰老小鼠模型,首次检测了优化的蜂王浆酶解产物对于该类衰老模型小鼠的外部衰老表征和内部衰老表征的改善作用,全面衡量了蜂王浆酶解产物的体内抗衰老活性。结果表明,在改善外部衰老方面,低剂量的蜂王浆酶解优化产物能够干预D-半乳糖诱导的小鼠体重下降的状况。高低剂量的蜂王浆酶解优化产物均能改善抗衰老小鼠活动能力下降的情况。与此同时,蜂王浆酶解优化产物还能够提高小鼠非空间性长期学习记忆能力和空间性长期学习记忆能力,使之保持良好的记忆力、对空间和方位的快速判断力以及对学习和训练结果的长期巩固能力,具有优异的回避危害的学习能力及长期记忆能力。在改善内部衰老方面,蜂王浆酶解优化产物能延缓胸腺等器官的萎缩,表现出对器官衰老的抑制作用。蜂王浆酶解优化产物还能够提高小鼠血清超氧化物歧化酶(SOD)活力、提高小鼠肝脏过氧化氢酶(CAT)的活力、提高小鼠肝脏谷胱甘肽过氧化物酶(GSH-px)的活力并抑制小鼠脑中丙二醛(MDA)的积累,高剂量效果等同或显著优于蜂王浆。
(4)采用刀豆蛋白A(ConA)刺激小鼠脾脏T细胞转化,用脂多糖(LPS)刺激小鼠脾脏B细胞的转化,以四甲基戊唑蓝(MTT)法检测蜂王浆酶解产物对于衰老小鼠脾T/B淋巴细胞转化的影响。同时,采用乳酸脱氢酶(LDH)释放法测定蜂王浆酶解产物对于自然杀伤细胞(NK)活力的影响,研究了蜂王浆酶解产物对于免疫调节的作用。并进一步从NF-κB信号通路的角度评价蜂王浆及其优化的酶解产物对于LPS刺激下小鼠单核巨噬细胞RAW264.7的免疫调控作用。结果发现,相比衰老小鼠,低剂量的蜂王浆优化酶解产物表现出对LPS刺激下小鼠脾脏B淋巴细胞转化的显著促进作用。高剂量效果更佳,与蜂王浆相比达到显著差异。优化的蜂王浆酶解产物还能部分抑制NF-κB抑制蛋白(IκBα)的降解。而对于ConA刺激下小鼠脾脏T细胞的转化和NK细胞活性影响,给予蜂王浆酶解产物组小鼠与正常小鼠无显著性差异。
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.
引文
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