海参肠道产蛋白酶菌的分离鉴定及其在海参下脚料中的应用
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摘要
以1株海参肠道产蛋白酶菌为研究对象,通过正交试验优化了该菌发酵产多肽的最佳工艺条件。对其进行形态学、生理生化特性、分子生物学16S rDNA序列同源性分析,确定该菌株隶属芽孢杆菌目,命名为Thalassobacillus sp. QDHF-1。利用QDHF-1发酵海参肠,以多肽含量为指标优化发酵条件,并对发酵产物多肽进行了抗氧化活性测定,确定对发酵海参肠产多肽的最佳工艺条件为接种量6%,料水比1∶5(g∶mL),发酵时间36 h;海参肠发酵产物多肽对·OH,DPPH·,O_2~-·3种自由基均有清除作用,其中对·OH自由基的清除活力最好,在50 g/L时清除率高达93. 94%。该研究利用海参肠道菌发酵海参下脚料来获得具有抗氧化活性的多肽,提高了下脚料的利用率及其经济价值。
A protease-producing bacterium from sea cucumber intestinal tract was isolated and identified.Through morphological,physiological,biochemical characterization and 16 S rDNA sequence homology analysis,the isolated strain was identified as Thalassobacillus sp. QDHF-1. Sea cucumber intestine waste was fermented by strain QDHF-1,and the fermentation conditions were optimized and the antioxidant activities of produced peptides were determined. The optimal fermentation condition to produce peptides was 6% inoculum,20% raw materials and 36 h fermentation. The polypeptides could scavenge ·OH,DPPH·,and O_2~-·,and the highest scavenging rate of 93. 94%was obtained on ·OH using 50 g/L polypeptides. The results indicate the potential applications of strain QDHF-1 in sea cucumber offal fermentation to produce bioactive peptides.
A protease-producing bacterium from sea cucumber intestinal tract was isolated and identified.Through morphological,physiological,biochemical characterization and 16 S rDNA sequence homology analysis,the isolated strain was identified as Thalassobacillus sp. QDHF-1. Sea cucumber intestine waste was fermented by strain QDHF-1,and the fermentation conditions were optimized and the antioxidant activities of produced peptides were determined. The optimal fermentation condition to produce peptides was 6% inoculum,20% raw materials and 36 h fermentation. The polypeptides could scavenge ·OH,DPPH·,and O_2~-·,and the highest scavenging rate of 93. 94%was obtained on ·OH using 50 g/L polypeptides. The results indicate the potential applications of strain QDHF-1 in sea cucumber offal fermentation to produce bioactive peptides.
引文
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[28]张云楚,纪宇.双辛可宁酸法测定胎盘多肽注射液中低浓度多肽的含量[J].中国生化药物杂志,2010,31(6):402-404.
[29]文镜,贺素华,杨育颖,等.保健食品清除自由基作用的体外测定方法和原理[J].食品科学,2004,25(1):190-195.
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[31]杨明琰,张晓琦,沈俭,等.超氧化物歧化酶两种邻苯三酚自氧化测定活力方法的比较[J].微生物学杂志,2006,26(3):40-42.
[2]喻世哲,刘晶晶,吴佳伦,等.外源蛋白酶对腐乳后发酵理化和感官特性的影响[J].食品科学,2018,39(8):64-68.
[3]HATANAKA T,URAJI M,FUJITA A,et al. Anti-oxidation activities of rice-derived peptides and their inhibitory effects on dipeptidylpeptidase-IV[J]. International Journal of Peptide Research&Therapeutics,2015,21(4):479-485.
[4]于亚莉,宋雪梅,王莹,等.玉米胚芽粕清蛋白酶解产物的制备及其抗氧化活性和护肝作用[J].中国食品学报,2018,18(6):93-103.
[5]OSBORN D A,SINN J K,JONES L J. Infant Formulas Containing Hydrolysed Protein for Prevention of Allergic Disease and Food Allergy[M]. The Cochrane Library. John Wiley&Sons,Ltd,2017:CD003664.
[6]刘唤明,张芷欣,洪鹏志,等.耐高温代表酶产生菌的筛选及酶学特性的初步研究[J].食品工业科技,2017,38(3):133-136; 142.
[7]HERNNDEZ-LEDESMA B,DEL M C M,RECIO I. Antihypertensive peptides:Production,bioavailability and incorporation into foods[J]. Advances in Colloid&Interface Science,2011,165(1):23-35.
[8]YARNPAKDEE S,BENJAKUL S,KRISTINSSON H G,et al. Antioxidant and sensory properties of protein hydrolysate derived from Nile tilapia(Oreochromis niloticus)by oneand two-step hydrolysis[J]. Journal of Food Science&Technology,2015,52(6):3 336-3 349.
[9]GUO Z,LIN D,GUO J,et al. In vitro antioxidant activity and in vivo anti-fatigue effect of sea horse(Hippocampus)peptides[J]. Molecules,2017,22(3):482.
[10]张海祥,魏兆军,周乐春,等.响应曲面法优化动物蛋白酶水解脱脂蚕蛹蛋白的工艺[J].食品科学,2009,30(24):188-192.
[11]陈艳辉,李超柱,黎丹戎,等.动物蛋白酶酶解制备广西产牡蛎肉抗肿瘤活性肽的实验研究[J].食品工业科技,2010,31(8):167-169.
[12]VOJCIC L,PITZLER C,KRFER G,et al. Advances in protease engineering for laundry detergents.[J]. New Biotechnology,2015,32(6):629-634.
[13]BANERJEE G,RAY A K. Impact of microbial proteases on biotechnological industries[J]. Biotechnology&Genetic Engineering Reviews,2017,33(2):119-143.
[14]BHATNAGAR I,KIM S K. Immense essence of excellence:marine microbial bioactive compounds.[J]. Marine Drugs,2010,8(10):2 673-2 701.
[15]SATPUTE S K,BANAT I M,DHAKEPHALKAR P K,et al. Biosurfactants,bioemulsifiers and exopolysaccharides from marine microorganisms[J]. Biotechnology Advances,2010,28(4):436-450.
[16]ZHANG C,KIM S K. Research and application of marine microbial enzymes:Status and prospects[J]. Marine Drugs,2010,8(6):1 920-1 934.
[17]CONTESINI F J,MELO R R,SATO H H. An overview of Bacillus proteases:From production to application[J]. Critical Reviews in Biotechnology,2018,38(3):321-334.
[18]DOS SANTOS AGUILAR J G,SATOH H. Microbial proteases:Production and application in obtaining protein hydrolysates[J]. Food Research International,2018,103:253-262.
[19]王凤舞,段书娟,仝令君,等.产低温碱性蛋白酶菌株QD-1的鉴定及产酶条件和酶学性质[J].中国食品学报,2017,17(2):85-91.
[20]李倩倩,刘双双,陈海华,等.单环刺螠内脏蛋白酶的分离纯化与酶学性质研究[J].中国食品学报,2016,16(6):88-95.
[21]BANERJEE G,MUKHERJEE S,BHATTACHARYA S,et al. Purification and characterization of extracellular protease and amylase produced by the bacterial strain,Corynebacterium alkanolyticum,ATH3 isolated from fish gut[J].Arabian Journal for Science&Engineering,2015,41(1):9-16.
[22]吴海龙.海参体壁中胶原蛋白酶的研究[D].厦门:集美大学,2013.
[23]郭晓坤,李傲婷,韩佳润,等.海参肠组织蛋白酶D的提取及酶学特性研究[J].食品工业科技,2017,38(18):135-139; 144.
[24]FU X Y,XUE C H,MIAO B C,et al. Characterization of proteases from the digestive tract of sea cucumber(Stichopus japonicus):High alkaline protease activity[J]. Aquaculture,2005,246(1-4):321-329.
[25]BEZERRA R S,LINS E J F,ALENCAR R B,et al. Alkaline proteinase from intestine of Nile tilapia(Oreochromis niloticus)[J]. Process Biochemistry,2005,40(5):1 829-1 834.
[26]WILSON K. Preparation of genomic DNA from bacteria[J]. Methods in Enzymology,2013,529(4):143-151.
[27]朱玫瑛.海参肠道产蛋白酶菌的分离鉴定及其在海参下脚料中的应用[D].山东:青岛农业大学,2014.
[28]张云楚,纪宇.双辛可宁酸法测定胎盘多肽注射液中低浓度多肽的含量[J].中国生化药物杂志,2010,31(6):402-404.
[29]文镜,贺素华,杨育颖,等.保健食品清除自由基作用的体外测定方法和原理[J].食品科学,2004,25(1):190-195.
[30]ALI B,MOHAMED H,RAFIK B,et al. Antioxidant and free radical-scavenging activities of smooth hound(Mustelus mustelus)muscle protein hydrolysates obtained by gastrointestinal proteases[J]. Food Chemistry,2009,114(4):1 198-1 205.
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