CN110904000A - 一株具有降胆固醇作用的植物乳杆菌及其在哈尔滨风干肠中的应用 - Google Patents
一株具有降胆固醇作用的植物乳杆菌及其在哈尔滨风干肠中的应用 Download PDFInfo
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Abstract
本发明公开了一株具有降胆固醇作用的植物乳杆菌及其应用，属于微生物技术领域。本发明的菌株命名为植物乳杆菌KLDS1.0391(L.plantarum KLDS1.0391)，保藏于中国微生物菌株保藏管理委员会普通微生物中心，保藏号为：CGMCC No.3151。通过在培养基中对六种乳酸菌进行降胆固醇能力测定，发现接种植物乳杆菌KLDS1.0391的培养基中胆固醇降解率最高。将其接种到哈尔滨风干肠中，发现风干肠在发酵过程中胆固醇含量逐渐降低，且在风干肠发酵的第3‑12天，接种植物乳杆菌KLDS1.0391的风干肠的胆固醇含量显著低于未接种的风干肠。因此，该菌株的发现为生产低胆固醇产品提供了有效的解决办法。
Description
技术领域
本发明属于微生物技术领域，涉及一株具有降胆固醇作用的植物乳杆菌KLDS1.0391及其在哈尔滨风干肠中的应用。
背景技术
机体内胆固醇水平的高低与心脑血管疾病的发生有关，高血清胆固醇易引起动脉粥样硬化、冠心病和脑中风等，严重影响人体健康。膳食中的胆固醇摄入过多会导致血清中胆固醇含量升高，这也是造成人们心血管疾病发作的主要原因。肉类制品富含脂肪，一定含量的脂肪可以为肉制品提供良好的风味、嫩度和多汁性，但脂肪含量多的肉制品中胆固醇含量往往较高，成为人体的安全隐患。哈尔滨风干肠是我国最受欢迎的传统发酵肉制品之一，因其独特的口感和风味深受消费者喜爱。哈尔滨风干肠在制作过程中通常加入质量分数为10-20％的猪背脂，导致终产品中胆固醇含量较高。因此，如何降低风干肠中的胆固醇含量受到了消费者的广泛关注。
乳酸菌作为发酵肉制品中的优势细菌之一，在风干肠的发酵过程中可以通过代谢碳水化合物、降低pH等产生良好的风味，而且乳酸菌的代谢可以降低肉制品中的胆固醇含量。体外研究表明，部分乳酸菌可以通过膜吸附的途径，同化环境中的胆固醇到自身细胞中去。此外，一些乳酸菌可能代谢产生胆固醇氧化酶或还原酶，改变其分子结构。其次，一些乳酸菌还可以降低机体内胆固醇含量，乳酸菌产生的胆汁酸水解酶能使胆汁盐从人体的胆肠循环中解偶联，生成游离胆汁酸和氨基酸。部分胆汁酸通过消化系统排出体外，为稳定体内胆汁酸水平，部分胆固醇被机体利用合成胆汁酸，从而导致机体内胆固醇含量降低。所以，通过乳酸菌发酵降低肉制品中胆固醇含量是一条安全、有效的途径。
发明内容
本发明的目的是选择一株具有降胆固醇能力的乳酸菌，并应用到哈尔滨风干肠中，研究风干肠发酵过程中胆固醇降解率，以生产低胆固醇发酵肉制品。
本发明的植物乳杆菌KLDS1.0391由东北农业大学乳品重点实验室所提供，保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，地址：北京市朝阳区北辰西路1号院中科院微生物研究所，其菌种保藏编号为：CGMCC No.3151，保藏日期为2009年6月29日。
本发明的植物乳杆菌KLDS1.0391在体外降胆固醇试验中呈现良好的降胆固醇能力，其胆固醇降解率显著高于其它五株乳酸菌。因此，植物乳杆菌KLDS1.0391可以作为胆固醇降解剂应用到发酵肉制品中，生产低胆固醇肉制品。
附图说明
图1为6种乳酸菌的体外胆固醇降解率；
图2为接种植物乳杆菌KLDS1.0391的风干肠在发酵过程中胆固醇含量变化。
具体实施方式
下面结合附图对本发明的技术方案作进一步的说明，但并不局限于此，凡是对本发明技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，均应涵盖在本发明的保护范围中。
一、6株乳酸菌降胆固醇能力测定
(1)胆固醇标准曲线
分别取0.1mL、0.2mL、0.3mL、0.4mL、0.5mL、0.6mL胆固醇标准溶液(1mg/mL)于试管中，60℃氮吹，吹尽溶剂无水乙醇后，加入2mL邻苯二甲醛试剂(0.5mg/mL)，混匀后室温静置10min，加入1mL浓硫酸，混匀后室温静置10min，于波长550nm处测定吸光值，以胆固醇质量浓度(μg/mL)为横坐标，吸光度为纵坐标绘制胆固醇标准曲线。
(2)胆固醇降解率测定
将6株乳酸菌(植物乳杆菌、清酒乳杆菌、乳酸片球菌、弯曲乳杆菌、戊糖片球菌和嗜酸乳杆菌)接种于MRS培养基中活化3代后，分别以体积比为2％的接种量接种至MRS-CHOL-THIO培养基(在MRS培养基中加入100ug/mL胆固醇、0.25％巯基乙酸钠和0.35g/100mL胆盐)，37℃培养24h，将6种菌液于4℃、5000r离心10min，取1mL上清液于试管内，加入4mL无水乙醇和3mL质量分数为3％的氢氧化钾溶液，混匀后，60℃水浴15min，迅速冷却至室温，加入正己烷7mL，混匀后加入蒸馏水4mL，漩涡振荡后室温静置至分层。取3mL上层正己烷，60℃氮吹，挥发尽溶剂，加入5mL邻苯二甲醛试剂(0.5mg/mL)，混匀后室温静置10min，加入3mL浓硫酸，混匀后室温静置10min，测定波长550nm处的吸光值，以未接菌的MRS-CHOL-THIO培养基作为空白组。
胆固醇降解率的计算方法：
式中：C0为空白组在550nm波长处的胆固醇质量浓度(μg/mL)；C1为6株试验菌株处理组在550nm波长处的胆固醇质量浓度(μg/mL)
二、风干肠的制作
(1)原辅料
瘦肉(猪臀肉，猪里脊)4kg，肥肉(猪背脂)1kg，麯酒(玉泉大麯)0.15kg，亚硝酸钠0.004kg，葡萄糖0.25kg，味素0.075kg，盐0.125kg，混合调料0.025kg(花椒、大料、桂皮和丁香等)。
(2)发酵剂的制备
将活化后的植物乳杆菌接种到MRS培养基中进行发酵培养，37℃培养12h。发酵液于4℃、10,000g离心5min后，备用。添加发酵剂时，乳酸菌添加量为107cfu/g肉馅。
(3)风干肠制作工艺流程
原料肉(剔除淋巴，筋腱，血管等结缔组织)→切丁(1cm见方的肉丁)→腌制(加盐和调味料等)→加发酵剂(对照组不添加)→拌馅→灌制→发酵风干。
三、风干肠胆固醇含量的测定
(1)胆固醇标准曲线
分别取0.0mL、0.5mL、1.0mL、1.5mL、2.0mL胆固醇标准工作液(100ug/mL)置于试管中，加入冰乙酸使总体积均达4mL，加入2mL铁矾显色液，混匀后室温静置15min，于560nm处测定吸光度。以胆固醇质量浓度(μg/mL)为横坐标，吸光度为纵坐标制作标准曲线。
(2)胆固醇含量的测定
用索氏脂肪提取法提取风干肠的脂肪，并计算出每100g风干肠中的脂肪含量。取3滴提取的脂肪置于试管中，准确记录其质量。加入4mL无水乙醇，0.5mL 50％氢氧化钾溶液，混匀，65℃皂化1h，每隔20min振摇一次使皂化完全，皂化完毕后立即冷却至室温。加入3mL5％氯化钠溶液，10mL石油醚，振荡2min，静置后取上层石油醚2mL，65℃氮吹，吹尽溶剂后，加入4mL冰乙酸，2mL铁矾显色液，混匀后室温静置15min，于560nm处测定吸光值，在标准曲线上计算出相应的胆固醇含量。
风干肠中胆固醇含量的计算方法:
式中：X为风干肠中胆固醇含量(mg/100g)；A为测得的吸光度值在标准曲线上对应的胆固醇含量(μg)；C为风干肠脂肪含量(g/100g)；V1为石油醚总体积(mL)；V2为取出的石油醚体积(mL)；M为称取风干肠油脂试样量(g)；1000为换算系数。
四、结果与分析
从图1可知，6株乳酸菌都能够降低培养基中的胆固醇，降解率为16.6-45.63％。另外，植物乳杆菌的胆固醇降解率显著高于其它5株乳酸菌(P<0.05)，说明该菌株具有较强的降低胆固醇能力。
图2是接种植物乳杆菌与未接种的风干肠在发酵过程中胆固醇含量的变化，在发酵过程中，两个处理组的胆固醇含量都不断下降，但接种植物乳杆菌的处理组从发酵第3d开始，胆固醇含量显著低于对照组。试验结果表明，该植物乳杆菌KLDS1.0391具有显著的降胆固醇能力，能够应用于低胆固醇发酵肉制品的生产。
Claims (2)
1.一株具有体外降胆固醇作用的植物乳杆菌KLDS1.0391，保藏于中国微生物菌种保藏管理委员会普通微生物中心，菌种保藏编号为：CGMCC No.3151。
2.如权利要求1所述的植物乳杆菌KLDS1.0391在哈尔滨风干肠中降低胆固醇含量的应用。
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