乳清抗氧化肽的制备及其生物活性研究
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摘要
乳清是工业生产干酪及干酪素的副产品,具有丰富的营养价值,利用微生物发酵法制备生物活性肽,对于乳清资源的开发具有十分重要的意义。本论文利用乳酸菌发酵法制备乳清抗氧化活性肽,从发酵菌株的筛选到发酵条件的优化、乳清多肽的分离纯化及抗氧化活性检验进行了系统的研究。主要研究内容如下：
1.本论文采用液态发酵法,以水解度和氧自由基清除率为指标,从实验室保存的6株乳酸菌中筛选出2株并建立了二元混菌体系。
2.采用单因素实验和正交实验优化了乳清抗氧化活性肽发酵工艺,得到最佳工艺条件为：接种量5%,菌种比1：1,发酵温度37℃,初始pH值6.4,时间16h,此时测得乳清水解产物O2·-清除率为39.45%,水解度为9.75%,肽含量为2.62mg/mL。
3.乳清抗氧化活性肽发酵液经6000Da膜超滤后冷冻干燥,溶解,经SephadexG-25凝胶层析柱分离纯化得到两个组分,相对分子量分别为2031Da、328Da。超滤分离的工艺条件为：操作压力0.04MPa、超滤温度25℃、超滤时间12min。与超滤液相比,透过液的氧自由基清除率提高了约29.5%,两个组分的氧自由基清除率分别提高了25.5%和39.0%。
4.体外抗氧化实验结果显示：乳清多肽具有一定的总还原能力,呈现一定的量效关系；乳清多肽的总抗氧化能力随浓度增加有较大提高,浓度由2mg/mL增加到10mg/mL时总抗氧化能力提高了近2.3倍；对羟自由基、氧自由基和DPPH自由基均有清除作用,并且清除率都与浓度有关,其半数抑制浓度IC50分别为6.31mg/mL,·6.59mg/mL,6.07mg/mL。
5.动物实验结果显示：乳清多肽中、高剂量组与衰老模型组比较,可显著提高衰老小鼠机体抗氧化酶CAT、SOD和GSH-Px的活性,明显降低衰老小鼠脂质过氧化代谢产物MDA水平,其中对血清的抗氧化作用最强;乳清多肽低剂量组则没有显著差异。实验结果说明乳清多肽具有提高衰老小鼠机体抗氧化的能力。
Whey is the production of produce cheese and casein and it is very nutrient. It is significant that the production of activity peptides fermented by microorganism in order to develop the whey resource. We used the Latic Acid Baceria Fermentation to prepare for the antioxidant peptide, a systemic research was carried on from the screening of the strains to.the optimizing of the liquid-state fermention and the purification of whey protein peptides and the'effect of antioxidant tests. The main contents are as follows:
1. In this paper, dual lactic acid bacteria system was screened out from 6 Latic Acid Bacterium in our laboratory through liquid-state fermentation according to the degree of hydrolyze and the scavenging rate of O2-.
2. The optimal condition of the production of whey antioxidant peptides with liquid fermentation was studied on the basic of the single factor test and the orthogonal test, the optimum conditions of producing antioxidative peptides were:inoculative rate 5%, strain ratiol:1, fermentation temperature 37℃, primary pH value 6.4, fermentation time 16h. Under the optimal conditions, the scavenging rate of O2- of whey hydrolysates was 39.45%, the DH was 9.75%, the content of peptides was 2.62 mg/mL.
3. The whey antioxidative peptides(WPP) were filtered by 6000Da ultrafiltration membrane and freeze-dried. Then, they were purified by SephadexG-25 Gel permeation chromatography and two compositions were obtained respectively with molecular weight of 2031Da and 328Da. Ultrafiltration process conditions for the separation was operating pressure 0.04MPa, ultrafiltration temperature 25℃, ultrafiltration time 12min. The scavenging rate of O2- of the WPP filtered by ultrafiltration membrane was increased about 29.5% compared with that in hydrolysis solution before purification, the compositions obtained by SephadexG-25 Gel was increased about 25.5%and 39.0% respectively.
4. Experimental results showed that WPP had dose-dependant deoxidization effect; the total antioxidant capacity of WPP was significantly improved while the concentration of the peptides was increased, it was improved 2.3 times when the concentration increased from 2mg/mL to 10mg/mL; WPP had the free radical-scavenging effect and the scavenging rate of OH-, O2-and DPPH·showed dose-effect relationship, their IC50 were 6.31 mg/mL,6.59mg/mL and 6.07mg/mL respectively.
5. Animal experiments showed that the CAT, SOD and GSH-Px activities in aged model group were significantly decreased, but MDA was significantly increased as comparing with the aged mice treated with WPP 200 and 400mg/kg bw-d, WPP had the most significant effect on the serum; there was no significant differences between the aged model group and the aged mice treated with WPP 100mg/kg bw·d. The results showed that WPP has the ability of improving the antioxidant capacity in aged mice.
1.本论文采用液态发酵法,以水解度和氧自由基清除率为指标,从实验室保存的6株乳酸菌中筛选出2株并建立了二元混菌体系。
2.采用单因素实验和正交实验优化了乳清抗氧化活性肽发酵工艺,得到最佳工艺条件为：接种量5%,菌种比1：1,发酵温度37℃,初始pH值6.4,时间16h,此时测得乳清水解产物O2·-清除率为39.45%,水解度为9.75%,肽含量为2.62mg/mL。
3.乳清抗氧化活性肽发酵液经6000Da膜超滤后冷冻干燥,溶解,经SephadexG-25凝胶层析柱分离纯化得到两个组分,相对分子量分别为2031Da、328Da。超滤分离的工艺条件为：操作压力0.04MPa、超滤温度25℃、超滤时间12min。与超滤液相比,透过液的氧自由基清除率提高了约29.5%,两个组分的氧自由基清除率分别提高了25.5%和39.0%。
4.体外抗氧化实验结果显示：乳清多肽具有一定的总还原能力,呈现一定的量效关系；乳清多肽的总抗氧化能力随浓度增加有较大提高,浓度由2mg/mL增加到10mg/mL时总抗氧化能力提高了近2.3倍；对羟自由基、氧自由基和DPPH自由基均有清除作用,并且清除率都与浓度有关,其半数抑制浓度IC50分别为6.31mg/mL,·6.59mg/mL,6.07mg/mL。
5.动物实验结果显示：乳清多肽中、高剂量组与衰老模型组比较,可显著提高衰老小鼠机体抗氧化酶CAT、SOD和GSH-Px的活性,明显降低衰老小鼠脂质过氧化代谢产物MDA水平,其中对血清的抗氧化作用最强;乳清多肽低剂量组则没有显著差异。实验结果说明乳清多肽具有提高衰老小鼠机体抗氧化的能力。
Whey is the production of produce cheese and casein and it is very nutrient. It is significant that the production of activity peptides fermented by microorganism in order to develop the whey resource. We used the Latic Acid Baceria Fermentation to prepare for the antioxidant peptide, a systemic research was carried on from the screening of the strains to.the optimizing of the liquid-state fermention and the purification of whey protein peptides and the'effect of antioxidant tests. The main contents are as follows:
1. In this paper, dual lactic acid bacteria system was screened out from 6 Latic Acid Bacterium in our laboratory through liquid-state fermentation according to the degree of hydrolyze and the scavenging rate of O2-.
2. The optimal condition of the production of whey antioxidant peptides with liquid fermentation was studied on the basic of the single factor test and the orthogonal test, the optimum conditions of producing antioxidative peptides were:inoculative rate 5%, strain ratiol:1, fermentation temperature 37℃, primary pH value 6.4, fermentation time 16h. Under the optimal conditions, the scavenging rate of O2- of whey hydrolysates was 39.45%, the DH was 9.75%, the content of peptides was 2.62 mg/mL.
3. The whey antioxidative peptides(WPP) were filtered by 6000Da ultrafiltration membrane and freeze-dried. Then, they were purified by SephadexG-25 Gel permeation chromatography and two compositions were obtained respectively with molecular weight of 2031Da and 328Da. Ultrafiltration process conditions for the separation was operating pressure 0.04MPa, ultrafiltration temperature 25℃, ultrafiltration time 12min. The scavenging rate of O2- of the WPP filtered by ultrafiltration membrane was increased about 29.5% compared with that in hydrolysis solution before purification, the compositions obtained by SephadexG-25 Gel was increased about 25.5%and 39.0% respectively.
4. Experimental results showed that WPP had dose-dependant deoxidization effect; the total antioxidant capacity of WPP was significantly improved while the concentration of the peptides was increased, it was improved 2.3 times when the concentration increased from 2mg/mL to 10mg/mL; WPP had the free radical-scavenging effect and the scavenging rate of OH-, O2-and DPPH·showed dose-effect relationship, their IC50 were 6.31 mg/mL,6.59mg/mL and 6.07mg/mL respectively.
5. Animal experiments showed that the CAT, SOD and GSH-Px activities in aged model group were significantly decreased, but MDA was significantly increased as comparing with the aged mice treated with WPP 200 and 400mg/kg bw-d, WPP had the most significant effect on the serum; there was no significant differences between the aged model group and the aged mice treated with WPP 100mg/kg bw·d. The results showed that WPP has the ability of improving the antioxidant capacity in aged mice.
引文
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[2]韩光烈.谈谈扩大乳清粉利用范围及其现实意义.中国乳品工业,1995,23(1)：44-47
[3]郭宇星.微生物发酵法水解乳清粉制备生物活性肽的研究.天津商学院硕士论文,2003
[4]美国乳品出口协会.美国乳清蛋白的应用研究和最新进展.中国食品添加剂,1998,3：26-31
[5]韩雪,孙冰.乳清蛋白的功能特性及应用.中国乳品工业,2003,31(3)：28-30
[6]刘静,吴晓彤.乳清蛋白及其在乳品生产中的应用.内蒙古农业科技,2007,2：92-93
[7]燕红,张兰威,朱永军.牛乳清蛋白的性质及其在食品工业中的应用.中国食物与营养,2002,3：18-20
[8]刘志广,张建军,乔淑清.乳清制品在功能食品中的应用.食品科学,2004,25(11)：376-378
[9]张守文.乳清在烘培食品中的应用.中国乳品工业,1990,1：8-14
[10]万贤生.微生物发酵生产乳清饮料的研究.中国乳品工业,1988,26(1)：7-11
[11]美国乳品出口协会,V.Lagrange.乳清蛋白及其新组分的性质、功能和新用途.中国乳品工业,1999,27(2)：24-27
[12]钟红英.乳清发酵生产方便食品调味料的研制.四川粮油科技,2000,2：37-38
[13]于嗣祥,杨林华,徐爱玲,等.乳清的营养价值及开发利用现状.农产品加工·学刊,2009,8：38
[14]王海滨.生物活性肽的应用研究概况.肉类工业,1999,8：28-32
[15]A. Jang, M. Lee. Purification and identification of angiotensin converting enzyme inhibitory peptides from beef hydrolysates. Meat Science,2005,69(4):653-661
[16]Hang Guo, Yoshiaki Kouzuma, Masami Yonekura. Isolation and Properties of Antioxidative Peptides from Water-Soluble Royal Jelly Protein Hydrolysate. Food Science and Technology Research,2005,11(2):222-230
[17]Jie Sun, Hui He, Bi Jun Xie. Novel Antioxidant Peptides from Fermented Mushroom Ganoderma lucidum.Journal of Agricultural and Food and Chemistry,2004,52:6646-6652
[18]Jae Young Je, Pyo Jam Park, Se Kwon Kim. Antioxidant activity of a peptide isolated from Alaska pollack (Theragra chalcogramma) frame protein hydrolysate. Food Research International,2005,38:45-50
[19]Jose Angel Gomez-Ruiz, Ivan Lopez-Exposito, Anne Pihlanto, et al. Antioxidant activity of ovine casein hydrolysates:identification of active peptides by HPLC-MS/MS. Europe Food Research Technology,2008,227(4):1061-1067
[20]Rhicha Sinha, C.Radha, Jamuna Prakash, et al. Whey protein hydrolysate:Functional properties, nutritional quality and utilization in beverage formulation. Food Chemistry, 2007,101:1484-1491
[21]Tugba Bayram, Murat Pekmez, Nazli Arda,et al.Antioxidant activity of whey protein fractions isolated by gel exclusion chromatography and protease treatment. Talanta, 2008,75:705-709
[22]E. A. Pena-Ramos, Y. L. Xiong. Antioxidative Activity of Whey Protein Hydrolysates in a Liposomal System. Journal of Dairy Science,2001,84:2577-2583
[23]S. P. Chawla, Ramesh Chander, Arun Shaarma. Antioxidant properties of Maillard reaction products obtained by gamma-irradiation of whey proteins. Chemistry,2009,116: 122-128
[24]张和平,郑宏旺,德力格尔桑.酶法乳清多肽饮料的研制.食品科学,1994,2：27-31
[25]谢继志,杨文,葛庆丰,等.乳清酒发酵条件及发酵动态的研究.中国乳品工业,1999,27(1)：4-6
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