乳酸菌对微量元素的富集及其生物活性评估

发布时间：2018-03-08 22:07

  本文选题：乳酸菌　切入点：铁　出处：《西北农林科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：乳酸菌,作为一类世界一般公认安全的食用细菌(General Regard As Safety,GRAS),在食品工业中已被广泛应用,其所具备的突出益生特性及保健功能使之成为微生物研究中的热点。利用微生物富集微量元素一直是功能食品研究的热门方向,这其中,元素富集型酵母的研究最为广泛。而近年来的研究表明,乳酸菌也能够通过自身的理化作用,对绝大多数微量元素起到富集及有机态转化。乳酸菌的这种元素富集能力对于菌体自身以及人体营养元素需求均具有重要意义,其在益生的同时,还可以提高及改善人体健康,具有巨大的应用前景。本研究以实验室保藏的清酒乳杆菌清酒亚种WY1,WY2;植物乳杆菌WY3,WY4;短乳杆菌WY5;酒酒球菌WY6;保加利亚乳杆菌WY7及嗜热链球菌WY8为试验菌株,分别探究其对于铁、锌、硒元素的富集能力,并测试其元素富集后,菌株的体外抗氧化、还原力、胆盐耐受力及抗菌活性变化。考虑到发酵乳制品,尤其是酸奶的市场普遍性需求,结合前期试验结果,着重研究酸奶中常见的保加利亚乳杆菌WY7和嗜热链球菌WY8,对其进行元素富集条件优化,探究其最大富集率,并进一步测试其体外抗氧化活性。体外抗菌试验中,富硒后的菌株表现出优良的抗菌活性;因此,对于保加利亚乳杆菌和嗜热链球菌富硒后的抗菌活性做进一步深入研究。通过以上试验得到了以下结论:(1)来自不同种属的8株乳酸菌,对于无机态铁、锌、硒均有较好的富集作用,其元素富集率较高。样品干法消化后采用原子吸收法,在适宜的铁质量浓度下,测定菌株的富铁率由高到低依次为WY6(85.36±1.85%)、WY1(73.74±1.64%)、WY8(73.01±1.25%)、WY5(62.42±0.79%)、WY7(42.69±0.89%)、WY2(39.18±0.94%)、WY3(35.56±1.69%)和WY4(33.33±1.01%);在适宜锌质量浓度下,菌株富锌率依次为WY6(69.25±1.19%)、WY7(59.03±0.90%)、WY1(54.54±1.38%)、WY5(43.42±0.88%)、WY8(36.35±1.02%)、WY4(32.73±1.02%)、WY2(25.78±0.94%)和WY3(24.78±0.53%);同样,采用3,3’-二氨基联苯胺(3,3’-DAB)比色法,测出在适宜硒质量浓度下,菌株富硒率依次为WY8(79.19±3.01%)、WY7(78.72±1.07%)、WY6(73.42±1.61%)、WY5(67.65±3.24%)、WY1(64.27±3.31%)、WY3(61.49±4.49%)、WY2(56.29±3.01%)和WY4(34.98±1.46%)。体外试验中,元素富集后形成的富铁、富锌及富硒菌株,在1,1-二苯基-2-三硝基苯肼(DPPH)、2'-联氨-双-3-乙基苯并噻唑啉-6-磺酸(ABTS)自由基清除率以及还原力方面均有一定的提高;富集后的菌株胆盐耐受力也有所增强;此外,富铁、富锌及富硒菌株对于大肠杆菌以及鼠伤寒沙门氏菌的抗菌作用相比于对照组提高较为显著。其中,富铁及富硒菌株的体外抗氧化及抗菌活性变化最为显著。(2)以WY7和WY8为富铁测试菌株,在单因素试验基础上,确定起始pH、接种量、培养温度取值范围,通过响应面优化确定WY7最佳富铁条件为:培养液起始pH 6.7,接种量5.86%,培养温度35.55℃,富铁率为52.14%;WY8为:培养液起始pH 7.23,接种量6.06%,培养温度40℃,富铁率为97.05%。(3)以WY7和WY8为富硒测试菌株,以单因素试验结果为基础,通过响应面优化,分别确定其最佳富硒培养条件:WY7的最佳富硒条件为起始pH 5.96,接种量6.73%,培养温度33.24℃,富硒率为94.34%;WY8的最佳富硒条件为起始pH 6.37,接种量6%,培养温度40℃,富硒率为97.05%。通过傅里叶变换近红外光谱仪,对富硒乳酸菌进行定性分析,确定了富硒菌具有相同或相似的吸收峰及官能团;通过电镜扫描结合X射线光谱法,确定了富硒乳酸菌细胞表面单质硒的存在。(4)以WY7和WY8为试验菌株,通过平板菌落计数法确定适合菌株生长的富硒浓度为80μg/mL,并在此浓度下制备富硒乳酸菌,采用3,3’-二氨基联苯胺(DAB)比色法测定其富硒量分别为12.05±0.43μg/mL和11.56±0.25μg/mL。制备乳酸菌发酵滤液,菌体悬液及菌液,通过牛津杯双层平板法,测试其对于大肠杆菌、鼠伤寒沙门氏菌、单增李斯特菌及金黄色葡萄球菌的抑菌效果,结果表明乳酸菌发酵滤液和菌液表现出较强的抗菌活性,其中,富硒菌各部分的抗菌活性更为显著。通过OD值的测定考察不同乳酸菌发酵滤液对于四种致病菌生长曲线的影响,证明了富硒菌的发酵滤液能够明显延迟致病菌的对数期及稳定期,降低其生长速率。此外,同一时间,相同处理下,与未富硒的乳酸菌相比,富硒菌的发酵滤液对四种致病菌细胞结构均造成了更大的损伤。证明了富硒菌具有更强的抗菌活性。
[Abstract]:Lactic acid bacteria, as a kind of world generally recognized as safe edible bacterium (General Regard As Safety, GRAS), has been widely used in the food industry, with its outstanding probiotic properties and health care function becomes the focus of research. The use of microbial microbial enrichment of trace elements has been a hot research direction, functional food the study, enrichment type yeast is the most widely used. But recent studies show that lactic acid bacteria can also through physical and chemical effects of their own, to the enrichment and organic transformation of most trace elements. The element enrichment ability of lactic acid bacteria are of important significance for the cell itself and human nutrition in the presence of it, at the same time, also can improve and improve human health, it has great application prospect. In this study, the laboratory preserved l.sake wine subspecies WY1 WY2; plant lactobacillus Bacteria WY3 WY4; Lactobacillus brevis WY5; Oenococcus oeni WY6; Lactobacillus bulgaricus and Streptococcus thermophilus WY7 WY8 strain, respectively, for exploring the iron, zinc, selenium enrichment capability, and to test its enrichment, strains in vitro antioxidant and antibacterial activity, reducing power, changes of bile tolerance considering the fermented dairy products, especially the general requirements of yogurt Market, combined with the previous experimental results, focusing on the common yoghurt of Lactobacillus bulgaricus and Streptococcus thermophilus WY7 WY8, the enrichment conditions, explore the maximum accumulation rate, and further test its antioxidant activity in vitro. The in vitro antibacterial test. After the se strains showed excellent antibacterial activity; therefore, for Lactobacillus bulgaricus and Streptococcus thermophilus enriched after antibacterial activity to do further research. Through the above experiment was the following conclusions: (1) to 鑷�涓嶅悓绉嶅睘鐨�8鏍�涔抽吀鑿�,瀵逛簬鏃犳満鎬侀搧,閿，

本文编号：1585736