牛骨髓蛋白的酶解工艺优化及其理化性质和抗氧化特性

Optimization of Enzymatic Hydrolysis of Bovine Bone Marrow Protein and Its Physicochemical and Antioxidant Properties

探讨牛骨髓蛋白(Bovine bone marrow protein,BBMP)酶解工艺并评价其理化性质和抗氧化活性,挖掘其潜在药用和保健功效物质基础,提升牛骨的综合利用价值。以水解度(DH)、蛋白含量、1,1-二苯基-2-三硝基苯肼自由基(DPPH·)清除率为评价指标,结合结构表征,筛选最佳酶种。以酶解时间、酶添加量、pH、酶解温度为自变量,采用响应面法优化牛骨髓蛋白的酶解工艺,并研究其酶解物的理化性质和抗氧化活性。结果表明,最适蛋白酶为胃蛋白酶(Bovine bone marrow protein pepsin hydrolysate,BBMP-PH),最佳酶解工艺参数为酶解时间2 h,酶添加量2%,pH为3,酶解温度为37℃。在此条件下,DH、蛋白含量及DPPH·清除率分别为36.07%、37.05%及39.57%;四种因素对酶解效率的影响为:加酶量>酶解时间>酶解温度>pH。理化性质实验结果表明,BBMP-PH的等电点在pH6左右,并在等电点处溶解性、乳化性及乳化稳定性、持水性最弱,远离等电点时较高;当pH为2时,BBMP-PH溶解性最好,达94.72%,当pH为8时乳化性及乳化稳定性达1.11 m^(2)/g和101.37%。BBMP-PH具有较强的DPPH·、羟自由基(·OH)、超氧阴离子自由基(O_(2)^(-)·)、2,2’-联氮-双-3-乙基苯并噻唑啉-6-磺酸自由基(ABTS^(+)·)清除能力,半抑制浓度IC_(50)分别为0.65、1.16、0.35、0.57 mg/mL,蛋白浓度为1 mg/mL时,总还原能力为0.88。本文为牛骨髓资源的高值化利用和多肽类功能食品的开发提供一定的参考。

The enzymatic hydrolysis of bovine bone marrow protein(BBMP)was optimized and its physicochemical properties and antioxidant activities were studied,aimed to explore the material basis of its potential medicinal and health benefits and enhance the comprehensive utilization value of beef bone.The degree of hydrolysis(DH),protein content,and 1,1-diphenyl-2-trinitrophenylhydrazine free radical(DPPH·)scavenging rate were used as evaluation indexes,combined with the results of structural characterization,the suitable enzyme was screened.The response surface methodology was used to the optimization of enzymatic hydrolysis of bovine bone marrow protein as the hydrolysis time,enzyme concentration,pH,and temperature were independent variables.Then,the physicochemical properties and antioxidant activities of the obtained hydrolysates were investigated.The results showed that pepsin was selected as the optimal protease for the preparation of BBMP pepsin hydrolysate(BBMP-PH).The optimum enzymatic hydrolysis conditions were time 2 h,enzyme concentration 2%,pH3,and temperature of 37℃.Under these conditions,the DH,protein content,and DPPH·scavenging rate were 36.07%,37.05%,and 39.57%,respectively.The effects of four factors on the efficiency of enzymatic hydrolysis were ordered as:Enzyme concentration>enzymatic hydrolysis time>enzymatic hydrolysis temperature>pH.The results of physicochemical properties showed that the isoelectric point(pI)of BBMP-PH was about pH6,and the solubility,emulsifying activity,emulsion stability,and water holding capacity of BBMP-PH were up to their lowest value at pI,and the better when far from the pI.When pH was 2,BBMP-PH has the highest solubility(94.72%),whereas pH8 for the emulsifying activity and emulsion stability(1.11 m^(2)/g and 101.37%).BBMP-PH had strong scavenging ability of DPPH·,hydroxyl(·OH),superoxide anion(O_(2)^(-)·),and 2,2'-azo-bis-3-ethyl benzothiazoline 6-sulfonic acid(ABTS+·)free radicals,and the half inhibitory concentration IC_(50) were 0.65,1.16,0.35,0.57 mg/mL,respectively.When the protein concentration was 1 mg/mL,the total reducing power was 0.88.This paper provides a reference for the high-value utilization of bovine bone marrow resources and the development of peptide-originated functional foods.