CN115181676A - 一株通过紫外诱变获得的黄伞菌株及其应用 - Google Patents
一株通过紫外诱变获得的黄伞菌株及其应用 Download PDFInfo
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Abstract
本发明公开了一株通过紫外诱变获得的黄伞菌株及其应用，涉及食品微生物技术应用领域，该黄伞菌株已经于2022年3月21日保藏于中国典型培养物保藏中心，保藏地址为中国武汉大学，保藏编号为：CCTCCM2022295，ZJ006分类学命名Pholiotaadiposa。本发明是以购得的黄伞菌株(CGMCC5.625)作为出发菌株，在紫外诱变的条件下使之发生突变，得到能够高效发酵不额外添加其他生长因子的酶解燕麦浆的突变菌株。将此新菌株用于液态发酵酶解燕麦浆为主要成分的培养基，在不额外添加碳源、氮源的情况下，生产出富含β葡聚糖的低GI值燕麦食品，在抑制高血糖、抗氧化、抑制心血管疾病等应用方面有较好前景。
Description
技术领域
本发明涉及涉及食品微生物技术应用领域，尤其涉及一株通过紫外诱变获得的黄伞菌株，以及将其用于液态发酵酶解燕麦浆，在不额外添加生长因子的条件下，发酵获得富含β葡聚糖的低GI值的燕麦食品。
背景技术
黄伞具有非常高的研究价值。黄伞(Pholiota adiposa)，又名柳蘑、黄柳菇、多脂鳞伞，在分类学上属担子菌门，层菌纲，伞菌目，球盖菇科，鳞伞菌属，是一种价值较高的食、药用真菌，是目前最具发展前景的10种珍惜菌类。黄伞多糖是从子实体、菌体丝以及发酵醪液等中分离出的一种高分子聚合物，这种聚合物具有多种生物活性，可以为生命物质提供骨架支撑及能量，还在细胞的生命活动中起调节作用。有研究标明，黄伞发酵液经烘干后经热水提取，乙醇沉淀得到的粗多糖均为含有葡萄糖醛酸的β-D-吡喃葡聚糖，具备抗氧化、抑制心血管疾病等医疗保健作用。
燕麦，又称莜麦，被认为是2l世纪适合现代人经常食用的高营养价值谷物。燕麦富含膳食纤维，其总纤维素的含量为17％～21％，其中超过1/3是可溶性膳食纤维，主要以β-葡聚糖为主。研究表明，燕麦的摄人还与多种疾病的发病风险降低有关。虽然燕麦富含膳食纤维，但其60％的淀粉含量也是不争的事实，这与燕麦控糖的目标背道而驰，因而在原有的低GI燕麦食品中，通常使用酶解法将淀粉除去，造成燕麦的有效成分利用率不高且加工难度大、工序复杂的问题。
β-葡聚糖的活性结构是由葡萄糖单位组成的多聚糖，它能够活化巨噬细胞、嗜中性白血球等，从而提高白细胞素、细胞分裂素和特殊抗体的含量，全面刺激机体的免疫系统。大量实验表明，β-葡聚糖可促进体内IgM抗体的产生，以提高体液的免疫能力。此外，β-葡聚糖尚有清除游离基、抗辐射、溶解胆固醇，预防高脂血症及抵抗滤过性病毒、真菌、细菌等引起的感染等作用，故广泛用于医药、食品、化妆品等行业。近年研究发现，β-1,3-葡聚糖可以作为生命活动中起核心作用的遗传物质，能够控制细胞分裂和分化，调节细胞生长，在治疗肿瘤、肝炎、心血管、糖尿病和降血脂、抗衰老等方面有独特的生物活性。目前世界各国，尤其是在日本、美国、俄罗斯等国，β-1,3-葡聚已经被广泛应用于食品保健、美容护肤等行业。
β-葡聚糖广泛存在于酵母、蘑菇、燕麦和大麦等食物里，其中β-1,3/1,6存在于酵母和蘑菇葡聚糖，而β-1,3/1,4存在于燕麦和大麦葡聚糖。在目前的报道中，以单独讨论微生物中的β-葡聚糖或者燕麦β-葡聚糖为主，未见有以燕麦为培养基发酵生产微生物β-葡聚糖的报道，所以本专利具有独创性和前瞻性。
期望得到一株从未使用且具有良好的性能的黄伞菌株，并期望其能在单纯的酶解燕麦浆中生物转化方面有良好表现。这类珍稀真菌发酵纯酶解燕麦浆首先需要解决菌种的适应性问题。
发明内容
本发明的目的在于，克服现有技术中存在的缺陷，提供一株通过紫外诱变获得的黄伞菌株，通过紫外诱变的方法，提升原始菌株在不额外添加生长因子的酶解燕麦浆中的发酵能力，获得一种未见报道的黄伞新菌株。
为实现上述目的，本发明的技术方案是设计一株通过紫外诱变获得的黄伞菌株，菌株是以CCTCC M 2022295为保藏号保藏于中国典型培养物保藏中心的菌株。黄伞新菌株于2022年3月21日保藏于中国典型培养物保藏中心，保藏地址为中国武汉大学，保藏编号为：CCTCC M 2022295ZJ006分类学命名Pholiota adiposa。所述黄伞新菌株用于液态发酵酶解燕麦浆为主要成分的培养基，在不额外添加碳源、氮源的情况下，生产出富含β葡聚糖的低GI值燕麦食品，在抑制高血糖、抗氧化、抑制心血管疾病等应用方面有较好前景。所述黄伞菌株是以购得的黄伞菌株(CGMCC 5.625)作为出发菌株，在紫外诱变的条件下使之发生突变得到的。
本发明还提供的技术方案是，黄伞菌株在生产富含β葡聚糖的低GI值燕麦食品方面的应用。
进一步的技术方案是，将黄伞种子液以10％的接种量转接至酶解燕麦浆培养基中发酵，发酵罐温度为26℃，搅拌转速为120rpm，呼吸器通气量为2vvm，发酵7d。将黄伞菌株的种子液体接种至纯酶解燕麦浆培养基中，于摇瓶培养，摇瓶转速180rpm，培养温度26℃，发酵7d。
进一步的技术方案为，酶解燕麦浆培养基将燕麦与水在60℃的温度下经α淀粉酶、糖化酶酶解1h，过滤所得，其中，原料中的燕麦含量为100g/L，其余为水；固体培养基在此基础上添加重量百分比为2％的琼脂粉。纯酶解燕麦浆是以燕麦为原料，磨浆后稀释至总固形物10％后再进行液化、糖化酶解，酶解至Brix值达到8.0～9.0之间，不添加其他生长因子。
进一步的技术方案为，发酵结束后采用胶体磨对发酵液进行精细研磨以及均质，均质压力40Mpa；最后再通过UHT超高温瞬时杀菌，杀菌温度137℃，杀菌时间30s；得到富含β葡聚糖的低GI值燕麦奶产品。
本发明还提供的技术方案是，富含β葡聚糖的低GI值燕麦食品在抑制高血糖、抗氧化、抑制心血管疾病等方面的应用。
本发明的优点和有益效果在于：利用紫外诱变方法获得新菌株，在酶解燕麦浆中将碳水化合物转化为具有保健功能的多糖产物，从而进一步优化燕麦制品的保健效果。
避免使用有毒有害的化学诱变剂和在食品领域还未被大众认可的基因工程方法，将利用相对简单易行的紫外诱变技术，使出发菌株处于诱变的极端环境，最大限度扩大突变位点的范围，提高得到正突变菌株的可能性。本发明采用紫外诱变这一常用诱变方法，此方法虽然不具有创新性，但此方法得到的菌株性能却发生了创新性的变化，在发酵纯酶解燕麦浆培养基时具有独特性、创新性和实用性。本发明所得发酵纯酶解燕麦培养基的黄伞新菌株为首次发明获得。
黄伞菌株为食用菌，且改造方法为紫外诱变的方法，可应用于食品生产领域，尤其在生产富含β葡聚糖的低GI值的燕麦食品方面有较好前景。
所述黄伞菌株在纯的酶解燕麦浆中发酵7d后，菌丝体质量达到11.9g/L，相较出发菌株提高67.6％；发酵酶解燕麦浆β葡聚糖含量含量达到44.2g/L，相较出发菌株提高36.8％；发酵酶解燕麦浆GI值为30，相较出发菌株产品GI值降低12。
附图说明
图1是本发明一株通过紫外诱变获得的黄伞菌株与出发菌株对照组的初筛平板照片。
图2为本发明、出发菌株及纯酶解燕麦浆的菌丝体含量图
图3为本发明、出发菌株及纯酶解燕麦浆的β葡聚糖含量图
图4为本发明、出发菌株及纯酶解燕麦浆的GI值测定图。
具体实施方式
下面结合附图和实施例，对本发明的具体实施方式作进一步描述。以下实施例仅用于更加清楚地说明本发明的技术方案，而不能以此来限制本发明的保护范围。
本发明是一株通过紫外诱变获得的黄伞菌株，主要材料与试剂：黄伞出发菌株(CGMCC 5.625)够自中国普通微生物菌种保藏管理中心；琼脂粉购于杭州康倍斯生物科技有限公司；β葡聚糖混连检测试剂盒β-GlucanAssayKit(Mixed Linkage)购于爱尔兰Megazyme公司；β-GlucanAssay Kit(Yeast&Mushroom)购于爱尔兰Megazyme公司。
其他试剂均为国产分析纯。
所需培养基：PDA培养基(g/L)：马铃薯200；葡萄糖20；琼脂20；水。
酶解燕麦浆培养基(g/L)：燕麦100；水；60℃经α淀粉酶、糖化酶酶解1h，过滤所得。固体培养基在此基础上添加2％琼脂粉。
出发菌株孢子悬液的制备：取够得的黄伞菌株斜面菌种一支，加入5ml无菌水，轻轻将琼脂表面的孢子刮下，将孢子悬液置于50ml三角瓶内，瓶中预先放置数粒无菌玻璃球，充分震荡后用灭菌的脱脂棉进行过滤，用无菌水反复滤渣2-3次，使最终滤液体积达到10.0ml，值得孢子悬液。并用细胞计数板计数。
致死率实验：分别取5ml孢子悬液在5个平皿中，分别在紫外灯下照射10～50s，照射过程中使用磁力搅拌。紫外照射后用适当的稀释比例涂布平板进行菌落计数(避光培养)，绘制致死率曲线。选择致死率在70-80％的照射时间作为突变的照射时间。
紫外诱变：制作菌悬液，在致死率70-80％的条件下进行紫外诱变，然后稀释至适当的浓度涂布至燕麦浆培养基平板避光培养7d。
初筛：平板直径测定法：挑取培养基上生长状态良好的单菌落至新平板上培养，同时也将出发菌株接种至新平板作为对照，7日后观察其生长的菌落大小，将菌落大小明显大于出发菌株的平板记录，作为复筛对象。(见图1)本发明对黄伞菌种的发酵能力进行考察，并通过菌种诱变的方法改善其发酵性能。
复筛：摇瓶培养：将初筛中筛选的优良菌种接种至液体培养基中，摇瓶培养。并与原始菌株对照。测其菌丝体含量、发酵液β葡聚糖含量、GI值，分别如图2、图3及图4所示。
菌丝体含量测定方法：采用多层纱布过滤法将菌丝体与发酵液分离，干燥菌丝体并称重。
产品β葡聚糖含量测定方法：使用β葡聚糖检测试剂盒进行测量。
GI值的测定方法：GI＝(摄人含50g碳水化合物食物的餐后血糖曲线下面积)/(摄入50g葡萄糖(或白面包)的餐后血糖曲线下面积)×100血糖曲线下的面积＝1/2(空腹血糖+餐后3h血糖)+餐后1h血糖+餐后2h血糖GI代表了食物的生理学效应。因此和传统的物理化学方法不同，食物GI值的测定采用的是人体试验方法。具体测量方法:首先要找10～15个健康志愿者(或糖尿病患者)，在早晨空腹情况下，吃进含有50g碳水化合物的食物，测空腹和餐后5分、15分、30分、45分、60分、90分、120分时的血糖。以同一组人吃进50g葡萄糖后的血糖变化为100，计算和比较出各种食物的GI值。一种食物的GI值至少测定10个受试者才能获得足够的检验效能。研究中可以同时包括男、女受试者。
实施例一：
菌株发酵纯酶解燕麦浆生产低GI值燕麦奶
将本发明的黄伞菌株在PDA平板上活化培养到一定阶段，接种孢子到种子培养液体培养基PDB中，继续培养72h。然后以10％的接种量转接到酶解燕麦浆中，于发酵罐中发酵。发酵罐温度为26℃，搅拌转速为120rpm，呼吸器通气量为2vvm，发酵7d。发酵结束后采用胶体磨对发酵液进行精细研磨以及均质，均质压力40mpa。最后再通过UHT超高温瞬时杀菌，杀菌温度137℃，杀菌时间30s。得到富含β葡聚糖的低GI值燕麦奶产品。经检测该产品符合QBT4221-2011规定的各项理化指标。产品β葡聚糖含量4.1％，GI值33，作为食品有非常好的保健以及控糖效果。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (6)
1.一株通过紫外诱变获得的黄伞菌株，所述菌株是以CCTCC M 2022295为保藏号保藏于中国典型培养物保藏中心的菌株。
2.权利要求1所述黄伞菌株在生产富含β葡聚糖的低GI值燕麦食品方面的应用。
3.根据权利要求2所述黄伞菌株在生产富含β葡聚糖的低GI值燕麦食品方面的应用，其特征在于，将黄伞种子液以10％的接种量转接至酶解燕麦浆培养基中发酵，发酵罐温度为26℃，搅拌转速为120rpm，呼吸器通气量为2vvm，发酵7d。
4.根据权利要求3所述述黄伞菌株在生产富含β葡聚糖的低GI值燕麦食品方面的应用，其特征在于，所述酶解燕麦浆培养基将燕麦与水在60℃的温度下经α淀粉酶、糖化酶酶解1h，过滤所得，其中，原料中的燕麦含量为100g/L，其余为水；固体培养基在此基础上添加重量百分比为2％的琼脂粉。
5.根据权利要求4所述述黄伞菌株在生产富含β葡聚糖的低GI值燕麦食品方面的应用，其特征在于，发酵结束后采用胶体磨对发酵液进行精细研磨以及均质，均质压力40Mpa；最后再通过UHT超高温瞬时杀菌，杀菌温度137℃，杀菌时间30s；得到富含β葡聚糖的低GI值燕麦奶产品。
6.权利要求2所述的富含β葡聚糖的低GI值燕麦食品在抑制高血糖、抗氧化、抑制心血管疾病等方面的应用。
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