抗氧化活性乳酸菌的筛选及其在水产品加工贮藏中的应用
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摘要
从泡菜、糟鱼等一些富含乳酸菌的样品中分离培养得到36株乳酸菌疑似菌株,经革兰氏染色、镜检、过氧化氢酶试验以及乳酸纸层析定性试验,初步确定其中20株为乳酸菌。加上实验室保存的5株菌株,共获得25株出发菌株。通过抗脂质过氧化率及超氧阴离子清除率两项指标,以Vc作阳性对照,从中复筛出两株高抗氧化性优良菌株H15、H17;并通过16S rDNA序列分析和鼠李糖发酵试验,鉴定得到H15为戊糖乳杆菌,H17为植物乳杆菌。
检测了两株优良菌株SOD活力、GSH-Px活力、还原能力及Fe2+螯合能力,结果得到:H15的菌悬液的SOD活性为2.12U/mL,无细胞抽提物的SOD活性为2.68U/mL,H17两者的活性分别为2.46U/mL,2.73U/mL;菌株H15、H17的GSH-Px活力分别为7.37(U/mL)、5.92(U/mL);两株菌株的菌悬液及无细胞抽提物皆具有一定的还原能力,H15的菌悬液及无细胞抽提物在700nm下的OD值分别为0.12及0.148,H17分别为0.073,0.113;两株菌都没有Fe2+螯合能力。
对两株优良菌株发酵剂的制备条件作了研究。研究了最佳培养温度、最适收获期及最佳保护剂的选择三个方面,结果得到最佳制备条件为:30℃下培养12h收获细胞,离心后以5%蔗糖+5%谷氨酸钠作保护剂冷冻保藏。将两株优良菌株制备成发酵剂应用于发酵鳗鱼糜制品的研制,结果表明试验菌株能很好的提高产品的感官品质,带来特有的发酵清香味,同时在一定程度上能抑制产品杂菌的生长,随着接种量的增大,发酵后的细菌数量随之减少,当接种量为0.15%时,细菌数为1.2×104cfu/g,达到卫生标准。乳酸菌发酵鳗鱼糜罐头的最佳加工工艺为:H15、H171:1混合发酵,接种0.07%(菌泥重),在30℃下密封发酵3天。
通过最优条件下发酵鳗鱼糜的过氧化值测定分析,得出两株优良菌株制备的混合发酵剂能较好的抑制鳗鱼糜发酵及贮藏过程中脂肪的氧化。发酵结束时自然组鱼糜的的过氧化值为0.12g/100g,而发酵组的则只有0.05g/100g。贮藏过程中,自然组鱼糜过氧化值升高很快,而发酵组鱼糜在初期基本保持稳定,贮藏8天后才开始出现如自然组般的持续上升,两者贮藏半个月后的过氧化值也是发酵组低于自然组。乳酸菌抗氧化活性应用于水产品的加工贮藏具有很大的发展前景。
36 strains were obtained by isolating from some samples,such as pickle and picking eel,etc.20 of them was confirmed as lactic acid bacteria by morphological examination, cataloes test and producing qualitative test of lactic acid formation. At alst, 25 strains were determined by addition of other 5 strains stored in laboratory. Two excellent strains named H15 and H17 were re-screened using inhibition ability of lipid peroxidation and scavenging capacity of superoxide anion radicals as an indicator, Vitamin C as a positive control. Finally, the strains of H15 and H17 were classified L.plantarum and L.pentosus after sequence analysis of 16S rDNA and rhamnose fermentation test.
Antioxidative mechanisms including superoxide dismutase activity, glutathione peroxidase activity, reducing activity and metal ion chelating ability for Fe2+ of H15 and H17 were studied. It demonstrated that the superoxide dismutase activity of intact cell and cell- free extracts of H15 were 2.12U/mL and 2.68U/mL,while it’s 7.37U/mL and 5.92U/mL respectively of H17.The glutathione peroxidase activity of H15 and H17 were7.37U/mL and 5.92U/mL,respectively.Both of two strains had reducing activity,and the OD value of intact cell and cell- free extracts of H15 were 0.12 and 0.148 under 700nm,while H17 were 0.073 and 0.113,respectively.The metal ion chelating ability for Fe2+ was not found in two strains.
The condition of preparation for lactobacillus starter was studied for processing application. Effects of temperature, culture time and protecting agent of lactic acid bacteria were also investigated. The optimum preparation conditions were obtained as follows: culture temperature was 30℃, harvesting time was 12h, the protectiog agent was 5% cane sugar with 5% monosodium glutamate.
The two strains of lactic acid bacteria H15 and H17 were used as culture starter in the fermented eel surimi can processing. It showed that those strains have distinct effect to improve the flavor and resist the growth of harmful bacterium.The harmful bacterium after fermentation was reduced as the inoculums size increases.The harmful bacterium was 1.2×104cfu/g when the inoculums size was 0.15%, and it met the health standards.The optimum fermentation conditions of fermented eel surimi can were obtained as follows:mixed culture fermentation as the ratio of H15 and H17 was 1:1,seed size 0.07%( weight of intact cell),incubated at 30℃for about 3d.
Finally, the peroxide value (POV) of eel surimi can was studied under the optimum fermentation condition.The results showed that the fermentation agent using H15 and H17 can resis the oxidation of lipid of fermented eel surimi during fermentation and storing time.The POV of eel surimi that not fermented was 0.12g/100g,while the fermented eel surimi was just 0.05g/100g after fermentation.The POV of eel surimi that not permented increased rapidly at an early stage,while permented eel surimi remained stable,and its POV after two weeks storage was also higher.It was promising to apply antioxidative activity of lactic acid bacteria to aquatic product processing and storing.
检测了两株优良菌株SOD活力、GSH-Px活力、还原能力及Fe2+螯合能力,结果得到:H15的菌悬液的SOD活性为2.12U/mL,无细胞抽提物的SOD活性为2.68U/mL,H17两者的活性分别为2.46U/mL,2.73U/mL;菌株H15、H17的GSH-Px活力分别为7.37(U/mL)、5.92(U/mL);两株菌株的菌悬液及无细胞抽提物皆具有一定的还原能力,H15的菌悬液及无细胞抽提物在700nm下的OD值分别为0.12及0.148,H17分别为0.073,0.113;两株菌都没有Fe2+螯合能力。
对两株优良菌株发酵剂的制备条件作了研究。研究了最佳培养温度、最适收获期及最佳保护剂的选择三个方面,结果得到最佳制备条件为:30℃下培养12h收获细胞,离心后以5%蔗糖+5%谷氨酸钠作保护剂冷冻保藏。将两株优良菌株制备成发酵剂应用于发酵鳗鱼糜制品的研制,结果表明试验菌株能很好的提高产品的感官品质,带来特有的发酵清香味,同时在一定程度上能抑制产品杂菌的生长,随着接种量的增大,发酵后的细菌数量随之减少,当接种量为0.15%时,细菌数为1.2×104cfu/g,达到卫生标准。乳酸菌发酵鳗鱼糜罐头的最佳加工工艺为:H15、H171:1混合发酵,接种0.07%(菌泥重),在30℃下密封发酵3天。
通过最优条件下发酵鳗鱼糜的过氧化值测定分析,得出两株优良菌株制备的混合发酵剂能较好的抑制鳗鱼糜发酵及贮藏过程中脂肪的氧化。发酵结束时自然组鱼糜的的过氧化值为0.12g/100g,而发酵组的则只有0.05g/100g。贮藏过程中,自然组鱼糜过氧化值升高很快,而发酵组鱼糜在初期基本保持稳定,贮藏8天后才开始出现如自然组般的持续上升,两者贮藏半个月后的过氧化值也是发酵组低于自然组。乳酸菌抗氧化活性应用于水产品的加工贮藏具有很大的发展前景。
36 strains were obtained by isolating from some samples,such as pickle and picking eel,etc.20 of them was confirmed as lactic acid bacteria by morphological examination, cataloes test and producing qualitative test of lactic acid formation. At alst, 25 strains were determined by addition of other 5 strains stored in laboratory. Two excellent strains named H15 and H17 were re-screened using inhibition ability of lipid peroxidation and scavenging capacity of superoxide anion radicals as an indicator, Vitamin C as a positive control. Finally, the strains of H15 and H17 were classified L.plantarum and L.pentosus after sequence analysis of 16S rDNA and rhamnose fermentation test.
Antioxidative mechanisms including superoxide dismutase activity, glutathione peroxidase activity, reducing activity and metal ion chelating ability for Fe2+ of H15 and H17 were studied. It demonstrated that the superoxide dismutase activity of intact cell and cell- free extracts of H15 were 2.12U/mL and 2.68U/mL,while it’s 7.37U/mL and 5.92U/mL respectively of H17.The glutathione peroxidase activity of H15 and H17 were7.37U/mL and 5.92U/mL,respectively.Both of two strains had reducing activity,and the OD value of intact cell and cell- free extracts of H15 were 0.12 and 0.148 under 700nm,while H17 were 0.073 and 0.113,respectively.The metal ion chelating ability for Fe2+ was not found in two strains.
The condition of preparation for lactobacillus starter was studied for processing application. Effects of temperature, culture time and protecting agent of lactic acid bacteria were also investigated. The optimum preparation conditions were obtained as follows: culture temperature was 30℃, harvesting time was 12h, the protectiog agent was 5% cane sugar with 5% monosodium glutamate.
The two strains of lactic acid bacteria H15 and H17 were used as culture starter in the fermented eel surimi can processing. It showed that those strains have distinct effect to improve the flavor and resist the growth of harmful bacterium.The harmful bacterium after fermentation was reduced as the inoculums size increases.The harmful bacterium was 1.2×104cfu/g when the inoculums size was 0.15%, and it met the health standards.The optimum fermentation conditions of fermented eel surimi can were obtained as follows:mixed culture fermentation as the ratio of H15 and H17 was 1:1,seed size 0.07%( weight of intact cell),incubated at 30℃for about 3d.
Finally, the peroxide value (POV) of eel surimi can was studied under the optimum fermentation condition.The results showed that the fermentation agent using H15 and H17 can resis the oxidation of lipid of fermented eel surimi during fermentation and storing time.The POV of eel surimi that not fermented was 0.12g/100g,while the fermented eel surimi was just 0.05g/100g after fermentation.The POV of eel surimi that not permented increased rapidly at an early stage,while permented eel surimi remained stable,and its POV after two weeks storage was also higher.It was promising to apply antioxidative activity of lactic acid bacteria to aquatic product processing and storing.
引文
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