益生菌发酵制备鳕鱼皮鱼皮多肽的初步研究
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摘要
胶原蛋白多肽是以胶原蛋白为原料,利用酶解法或微生物发酵法生产的多肽产品。与胶原蛋白相比胶原蛋白多肽具有良好的理化性质和生理活性。鳕鱼鱼皮含有丰富的胶原蛋白,且是目前国内主要水产品加工鱼皮下脚料,本文以鳕鱼鱼皮为原料,通过筛选具有分泌蛋白酶、脂肪酶活性和产酸活性的益生菌株进行混合发酵,制备具有较好溶解性和无鱼皮腥味的鱼皮多肽产品。
本文通过从纳豆、酒曲、醋曲和海鱼胃肠道中共分离纯化得到104株具有蛋白酶、脂肪酶等酶活力和产酸能力的菌株。通过形态学观察、特殊的细胞结构、运动性等特征和生理生化特性,依据《伯杰氏细菌鉴定手册》、魏景超的《真菌鉴定手册》和《酵母菌的特征与鉴定手册》对微生物进行分类鉴定。同时利用分子生物学技术,对细菌进行了16SrDNA序列测定,以确定其分类地位。
根据微生物鉴定结果,从中选出7株具有较高蛋白酶酶活力、脂肪酶活力和产酸能力的菌株,其中2株细菌(BNⅣ-7,JFX 80Y9),分别为蜡状芽胞杆菌和枯草芽胞杆菌;1株霉菌(H-2),鉴定为黑曲霉;2株酵母(JQⅡJM白, 4-2大),鉴定为啤酒酵母和橙黄红酵母;2株产酸菌(fGYY-3,HYW-2),鉴定为枯草芽胞杆菌。利用复合菌种发酵鳕鱼鱼皮,以水解度为指标,采用单因子试验确定了最适发酵温度、最适接种量、发酵培养基的组成、产酶细菌:霉菌:酵母菌:产酸细菌的比例和发酵时间等发酵参数,通过均匀设计法优化了复合菌种发酵制备鱼皮多肽的发酵工艺。优化实验结果表明,最佳发酵条件为:发酵温度30℃,发酵培养基中鳕鱼皮:水的比例为1:0.5,接种量为8%,产酶细菌:霉菌:酵母菌:产酸细菌的比例为3:1:1:2,发酵时间为20h。此条件下水解度可达38.5%,发酵液气味较香馥,口感较柔和,没有鱼腥味。
从发酵液中采用乙醇分离的方法提取鱼皮多肽,并且通过冷冻干燥得到鱼皮多肽粗品,采用凝胶柱进行高效液相层析,得到鱼皮多肽的分子量分布图谱,结果表明,发酵所得到的多肽集中在4000Da以下,小分子量多肽含量较高,发酵效果较好。
College peptides are products of polypeptide. It can be extracted by enzymatic hydrolysis and microorganism fermentation from collagen which is the raw material. Compared with the college, college peptides hold more physical and chemical properties and physiological activities.Skins of cods with abundant college are good material to produce college peptides.
For producing cod college peptides and removing fishy smell, 104 strains of microorganisms with protease and lipase activity and acid production ability were isolated from Natto, distiller's yeast, koji of vinegar and the guts of marine fishes. To identify the selected microorganism strains, classical identification was conducted through morphological, physiological and biochemical methods as the guide of“Yeasts: Characteristics and Identification”3rd edition,“Fungus Identification Handbook”and“Illustrated Genera of Imperfect Fungi, four edition”,“Berger's Mannal of Determinative Baceriology”eight edition. At the same time, 16SrDNA sequence was determined to confirm the classification position of bacteria by using molecular biology technology.
According to identification resulst, 7 strains of probiotic microbes with protease and lipase activity and acid production ability were selected for cod skins fermentation. The 7 strains of probiotic microbes were identified to Bacillus subtilis, Bacillus cereus, Aspergillus niger, Rhodotorula aurantiaca and Saccharomyces cerevisiae. The optium fermentation medium and culture conditions were determined through Erlenmeyer flask liquid fermentation by the test of uni-factor and uniform design, respectively. The main procedure parameter were: fermentation temperature 30℃, the ratio of cod skin to water in medium 1:0.5, inoculation quantity 8%, bacteria: mould: yeast: acid production bacteria 3:1:1:2. The hydrolysis degree of the cod skins could reach 38.5%. The fermentation liquor was smelled better, and the flavor was smooh,soft and full on the palate.
The crude polypeptides were precipitated by ethanol from fermentation liquor. Gel chromatography of molecular weight distribution was obtained by HPLC. The results indicated that the crude polypeptides had molecular weight, less than 4000Da..
本文通过从纳豆、酒曲、醋曲和海鱼胃肠道中共分离纯化得到104株具有蛋白酶、脂肪酶等酶活力和产酸能力的菌株。通过形态学观察、特殊的细胞结构、运动性等特征和生理生化特性,依据《伯杰氏细菌鉴定手册》、魏景超的《真菌鉴定手册》和《酵母菌的特征与鉴定手册》对微生物进行分类鉴定。同时利用分子生物学技术,对细菌进行了16SrDNA序列测定,以确定其分类地位。
根据微生物鉴定结果,从中选出7株具有较高蛋白酶酶活力、脂肪酶活力和产酸能力的菌株,其中2株细菌(BNⅣ-7,JFX 80Y9),分别为蜡状芽胞杆菌和枯草芽胞杆菌;1株霉菌(H-2),鉴定为黑曲霉;2株酵母(JQⅡJM白, 4-2大),鉴定为啤酒酵母和橙黄红酵母;2株产酸菌(fGYY-3,HYW-2),鉴定为枯草芽胞杆菌。利用复合菌种发酵鳕鱼鱼皮,以水解度为指标,采用单因子试验确定了最适发酵温度、最适接种量、发酵培养基的组成、产酶细菌:霉菌:酵母菌:产酸细菌的比例和发酵时间等发酵参数,通过均匀设计法优化了复合菌种发酵制备鱼皮多肽的发酵工艺。优化实验结果表明,最佳发酵条件为:发酵温度30℃,发酵培养基中鳕鱼皮:水的比例为1:0.5,接种量为8%,产酶细菌:霉菌:酵母菌:产酸细菌的比例为3:1:1:2,发酵时间为20h。此条件下水解度可达38.5%,发酵液气味较香馥,口感较柔和,没有鱼腥味。
从发酵液中采用乙醇分离的方法提取鱼皮多肽,并且通过冷冻干燥得到鱼皮多肽粗品,采用凝胶柱进行高效液相层析,得到鱼皮多肽的分子量分布图谱,结果表明,发酵所得到的多肽集中在4000Da以下,小分子量多肽含量较高,发酵效果较好。
College peptides are products of polypeptide. It can be extracted by enzymatic hydrolysis and microorganism fermentation from collagen which is the raw material. Compared with the college, college peptides hold more physical and chemical properties and physiological activities.Skins of cods with abundant college are good material to produce college peptides.
For producing cod college peptides and removing fishy smell, 104 strains of microorganisms with protease and lipase activity and acid production ability were isolated from Natto, distiller's yeast, koji of vinegar and the guts of marine fishes. To identify the selected microorganism strains, classical identification was conducted through morphological, physiological and biochemical methods as the guide of“Yeasts: Characteristics and Identification”3rd edition,“Fungus Identification Handbook”and“Illustrated Genera of Imperfect Fungi, four edition”,“Berger's Mannal of Determinative Baceriology”eight edition. At the same time, 16SrDNA sequence was determined to confirm the classification position of bacteria by using molecular biology technology.
According to identification resulst, 7 strains of probiotic microbes with protease and lipase activity and acid production ability were selected for cod skins fermentation. The 7 strains of probiotic microbes were identified to Bacillus subtilis, Bacillus cereus, Aspergillus niger, Rhodotorula aurantiaca and Saccharomyces cerevisiae. The optium fermentation medium and culture conditions were determined through Erlenmeyer flask liquid fermentation by the test of uni-factor and uniform design, respectively. The main procedure parameter were: fermentation temperature 30℃, the ratio of cod skin to water in medium 1:0.5, inoculation quantity 8%, bacteria: mould: yeast: acid production bacteria 3:1:1:2. The hydrolysis degree of the cod skins could reach 38.5%. The fermentation liquor was smelled better, and the flavor was smooh,soft and full on the palate.
The crude polypeptides were precipitated by ethanol from fermentation liquor. Gel chromatography of molecular weight distribution was obtained by HPLC. The results indicated that the crude polypeptides had molecular weight, less than 4000Da..
引文
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