CN112971074A - 运用乳酸菌提高螺丝菜健康功能的方法 - Google Patents
运用乳酸菌提高螺丝菜健康功能的方法 Download PDFInfo
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- CN112971074A CN112971074A CN202110265425.5A CN202110265425A CN112971074A CN 112971074 A CN112971074 A CN 112971074A CN 202110265425 A CN202110265425 A CN 202110265425A CN 112971074 A CN112971074 A CN 112971074A
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Abstract
本发明公开了一种运用乳酸菌提高螺丝菜健康功能的方法,包括以下步骤:1)、配置发酵卤水:在超纯水中加入食盐、白砂糖、植物乳杆菌ZJ316菌剂,混匀,得发酵卤水;植物乳杆菌ZJ316菌剂由保藏编号CCTCC NO:M 208077的植物乳杆菌ZJ316(Lactobacillus plantarumZJ316)制备而成;2)、利用发酵卤水对螺丝菜进行发酵处理。本发明制备所得的螺丝菜具有改善肠道菌群、补充叶酸的功能。
Description
技术领域
本发明属于食品生物技术领域,具体涉及一种运用乳酸菌提高螺丝菜健康功能的方法。
背景技术
螺丝菜,又称草石蚕、螺丝菜或甘露子,为唇形科水苏属一年生或多年生短日照草本植物,广泛分布于我国山东、江苏、四川、重庆、贵州等地。螺丝菜味淡薄,熟食绵软,颜色洁净,外形美观,口感脆爽鲜嫩且无纤维,是腌制泡菜的上好食材。每1kg螺丝菜中含蛋白质55g、脂肪3g、碳水化合物203g,富含有矿物质、维生素、胆碱、寡聚糖等,几乎不含淀粉。螺丝菜不但营养成分丰富,还具有极高的药用价值,具有促进肠道蠕动、助消化、抗氧化、抗过敏、降血压、降低胆固醇等功效。
传统的螺丝菜泡菜发酵工艺主要是利用附着在螺丝菜菜体上的微生物,配合糖、醋等辅料进行自然发酵。这种腌制工艺虽然便捷,但发酵条件难控制,产品安全质量难保障,产品营养成分不稳定。在腌制过程中会产生过多亚硝酸盐,威胁人体健康;易滋生大量杂菌,导致产品霉变、腐败。
乳酸菌是发酵类食品常用的微生物之一,利用乳酸菌腌制泡菜类食品是最为常见的应用。研究表明,乳酸菌在发酵过程中能有效地降解亚硝酸盐,抑制泡菜中杂菌生长;同时,乳酸菌发酵还能产生维生素、多糖等营养成分,提高泡菜产品附加值;并且乳酸菌发酵剂具有高含量的活细菌(1010-1012CFU/g),使用量少,保质期长,产品质量稳定,无需预处理,提高泡菜发酵工业的劳动生产率与产品质量,降低发酵工业的生产成本与生产周期。
叶酸,是一种水溶性的B族维生素,参与人体内许多重要的代谢反应,如DNA的复制与修复,氨基酸的合成等。叶酸的缺乏会引起神经管缺陷、心血管疾病等。目前,人体缺乏叶酸已成为世界范围内普遍存在的问题,考虑到化学合成叶酸的潜在危害和食物中的天然叶酸含量低的问题,安全高产叶酸乳酸菌的应用将作为新的研究对象。随着对乳酸菌的深入研究,越来越多的乳酸菌被发现具有叶酸合成能力,但未见富含叶酸的乳酸菌应用于发酵泡菜的相关技术报道。
因此,运用优质乳酸菌发酵制成同时富含益生元及益生菌的绿色健康的泡菜产品,对健康食品的发展、传统工艺的创新具有重大的社会价值和研究意义。
2008100625229的发明《Lactobacillus plantarum ZJ316、产生的抗菌肽及其制备与应用》提供了一株从婴儿粪便中分离到的植物乳杆菌——Lactobacillus plantarumZJ316,保藏编号CCTCC NO:M 208077,所得的抗菌肽具有广谱的抑菌作用。
发明内容
本发明要解决的问题是提供一种运用乳酸菌提高螺丝菜健康功能的方法。
为了解决上述问题,本发明提供一种运用乳酸菌提高螺丝菜健康功能的方法,包括以下步骤:
1)、配置发酵卤水:
在1L超纯水中加入96±1g食盐、46±0.5g白砂糖、0.2g植物乳杆菌ZJ316菌剂,混匀,得发酵卤水;
所述植物乳杆菌ZJ316菌剂由保藏编号CCTCC NO:M 208077的植物乳杆菌ZJ316(Lactobacillus plantarum ZJ316)制备而成;
2)、利用发酵卤水对螺丝菜进行发酵处理。
作为本发明的运用乳酸菌提高螺丝菜健康功能的方法的改进,步骤2)为:
将螺丝菜(新鲜的螺丝菜)清洗后,沥干水分;然后放入泡菜坛先压实,再向泡菜坛注入发酵卤水,坛盖水(10%盐水)封,于34±2℃发酵7±1天。
说明:清洗后,选取完整、饱满、有光泽、无虫孔的螺丝菜菜体;沥干水分后的螺丝菜,可4℃储存后再进行发酵,也可直接进行发酵。发酵后取出菜体,调味灭菌,制成成品泡菜。注入发酵卤水时,一般直至液面距坛口6-8cm,盖上盖子,在盖子与坛口交接处加入10%的盐水使其水封。
作为本发明的运用乳酸菌提高螺丝菜健康功能的方法的进一步改进,植物乳杆菌ZJ316菌剂的制备方法为:
①、将活化后的植物乳杆菌ZJ316进行发酵培养,所得发酵液经过离心(4℃,10000×g,20min),去上清,取菌体沉淀,菌体沉淀清洗后离心,得菌泥;
②、配制保护剂:
10±0.5g脱脂乳粉、1.5±0.1g海藻糖、1±0.1g甘油、3.5±0.1g山梨醇,用水定容至100ml,均匀混合,得保护剂;
③、按照菌泥/保护剂=1g/3mL的用量比,将菌泥与保护剂混合均匀后进行冷冻,得植物乳杆菌ZJ316菌剂。
作为本发明的运用乳酸菌提高螺丝菜健康功能的方法的进一步改进,所述步骤③中:菌泥与保护剂混合均匀后,先于-80℃预冻6h,然后用真空冷冻干燥机冻干36h。
作为本发明的运用乳酸菌提高螺丝菜健康功能的方法的进一步改进,所述步骤①为:
将活化后的植物乳杆菌ZJ316按3%(体积%)的接种量接至MRS液体培养基中,于37℃发酵培养24h,所得发酵液经过离心(4℃,10000×g,20min),去上清,取菌体沉淀,菌体沉淀经过质量浓度为0.85%的生理盐水清洗(洗涤沉淀三次)后离心,得菌泥。
作为本发明的运用乳酸菌提高螺丝菜健康功能的方法的进一步改进:
制备所得的螺丝菜具有改善肠道菌群(即,有益于人体肠道菌群的调节)、补充叶酸的功能;
所述改善肠道菌群为促进(显著促进)肠道内的双歧杆菌(Bifidobacterium)和乳酸菌(Lactobacillus)的生长,从而促进肠道菌群代谢产生短链脂肪酸(乙酸、丙酸、丁酸、戊酸)。
本发明制备所得的螺丝菜中叶酸产量达69.60μg/kg。
植物乳杆菌ZJ316菌剂在发酵螺丝菜泡菜的过程中能产生大量醇类、酸类和酮类物质,赋予泡菜产品独特的发酵风味。
本发明利用乳酸菌发酵螺丝菜,产品安全无毒、风味佳、益于肠道菌群调节、补充叶酸。通过本发明获得的乳酸菌螺丝菜泡菜,具有良好的风味和稳定性,含有较高含量的植物乳杆菌及其代谢成分,在货架期内具有良好的稳定性。该乳酸菌螺丝菜泡菜可作为日常食品,在肠道调节(肠道菌群和菌群代谢产物)方面具有显著的功效,对多数人体肠道内的双歧杆菌(Bifidobacterium)和乳酸菌(Lactobacillus)的生长有一定的促进作用,且双歧杆菌为为最大差异性物种,抑制部分病原菌生长,促进人体的肠道菌群代谢产生乙酸、丙酸、戊酸有一定的促进作用。
通过产叶酸的乳酸菌发酵新鲜螺丝菜,既保留了泡菜的风味与营养功能,又显著提高了泡菜中叶酸的含量。并且本发明所产产品中的叶酸为天然叶酸,避免了化学合成叶酸补充物的副作用,且叶酸的含量较其它产品显著增高,可以替代传统工业化学合成的叶酸。本发明方法的应用,使得乳酸菌发酵所产的产物叶酸具有还原性,更易被人体吸收,同时为人体补充了益生菌,有益于维持肠道健康。通过产叶酸的乳酸菌菌剂发酵制作螺丝菜泡菜的方法,发酵条件明确,产物丰富、稳定、安全,实现了泡菜、乳酸菌、叶酸的营养功能三合一。
综上所述,本发明利用植物乳杆菌ZJ316发酵制备螺丝菜泡菜,解决了目前螺丝菜泡菜中存在的发酵条件难控制,产品质量与营养难保障的技术缺陷,获得了有助于改善肠道健康、富含叶酸的乳酸菌螺丝菜泡菜,实现了益生菌与益生元的结合应用。
附图说明
图1是体外肠道模型发酵后“门”、“属”菌群相对丰度;
图1中:a是“门”水平菌群组成及丰度;b是“属”水平菌群组成及丰度;
F代表粪便原样组的10个重复的平均水平;
N代表自然发酵组(发酵体系加入自然发酵的螺丝菜泡菜)的10个重复的平均水平;
M代表市售菌剂组(发酵体系加入市售菌剂发酵的螺丝菜泡菜)的10个重复的平均水平;
316代表植物乳杆菌ZJ316菌剂发酵组(发酵体系加入植物乳杆菌ZJ316菌剂发酵的螺丝菜泡菜)的10个重复的平均水平。
图2是体外肠道模型发酵后双歧杆菌属、乳杆菌属、布劳特氏菌属、肠杆菌科微生物的增殖情况;
图2中:a为双歧杆菌属增长情况;b为乳杆菌属增长情况;c为布劳特氏菌属增长情况;d为肠杆菌科增长情况;
F代表粪便原样组的10个重复;
N代表自然发酵组(发酵体系加入自然发酵的螺丝菜泡菜)的10个重复;
M代表市售菌剂组(发酵体系加入市售菌剂发酵的螺丝菜泡菜)的10个重复;
316代表植物乳杆菌ZJ316菌剂发酵组(发酵体系加入植物乳杆菌ZJ316菌剂发酵的螺丝菜泡菜)的10个重复。
图3是PCA肠道菌群分布情况;
F代表粪便原样组的10个重复;
N代表自然发酵组(发酵体系加入自然发酵的螺丝菜泡菜)的10个重复;
M代表市售菌剂组(发酵体系加入市售菌剂发酵的螺丝菜泡菜)的10个重复;
316代表植物乳杆菌ZJ316菌剂发酵组(发酵体系加入植物乳杆菌ZJ316菌剂发酵的螺丝菜泡菜)的10个重复。
图4是螺丝菜泡菜对体外肠道模拟系统菌群代谢生成短链脂肪酸的影响;
图4中,a为乙酸含量,b为丙酸含量,c为丁酸含量,d为戊酸含量;
F代表粪便原样组的10个重复;
N代表自然发酵组(发酵体系加入自然发酵的螺丝菜泡菜)的10个重复;
M代表市售菌剂组(发酵体系加入市售菌剂发酵的螺丝菜泡菜)的10个重复;
316代表植物乳杆菌ZJ316菌剂发酵组(发酵体系加入植物乳杆菌ZJ316菌剂发酵的螺丝菜泡菜)的10个重复。
具体实施方式
以下结合具体实施例进一步描述本发明,本发明的优点和特点将会随着描述而更为清楚。但这些实例仅是范例性的,并不对本发明的范围构成任何限制:
实施例1:直投式发酵菌剂及螺丝菜泡菜制备
(1)、取一接种环的植物乳杆菌ZJ316至10mL的MRS液体培养基中于37℃活化36小时;再按照上述方法连续传代2次,接着按照3%(体积%)的接种量接至500mL的大瓶MRS液体培养基中,37℃培养24h后将发酵液离心(4℃,10000×g,20min),去上清,取菌体沉淀,用0.85%生理盐水洗菌三次(4℃,8000×g,10min),得菌泥。
(2)、保护剂的配方为:10%脱脂乳粉、1.5%海藻糖、1%甘油、3.5%山梨醇,余量为水;即,10g脱脂乳粉、1.5g海藻糖、1g甘油、3.5g山梨醇,用水定容至100ml,得保护剂。
按菌泥/保护剂为1:3(w/v,即1g/3ml)的比例将保护剂与菌泥混合均匀,倒入玻璃培养皿,封上一层保鲜膜,并用1mL无菌注射器针头在保鲜膜上扎3到4圈小孔以便冻干时水气排出。在-80℃冰箱中预冻6h后,用真空冷冻干燥机冻干24h,干燥条件:主冻干隔板温度-5℃,冻干14h,真空度0.1mbar;解析干燥阶段:隔板温度5℃,干燥10h,真空度为0.01mbar;得植物乳杆菌ZJ316菌粉。
(3)、将新鲜的螺丝菜在清水里浸泡清洗2h,选取完整、饱满、有光泽、无虫孔的螺丝菜菜体,用硬毛刷刷去菜体上残留泥垢,再用清水漂洗三遍后晾干(即,不再滴水为止),可储存于4℃冰箱待用,也可直接进行后续步骤。
以市售菌剂作为阳性对照,不添加菌剂为空白对照。卤水配制方法如表1所示。准确称取500g螺丝菜,置入泡菜坛(泡菜坛的容积约为3L)并压实;在对应组的泡菜坛内注入不同的卤水,直至液面距坛口6-8cm;盖上盖子,在盖子与坛口交接处加入10%(质量%)的盐水使其液封;34℃发酵7天后取出菜体,作为泡菜。可后续进行调味,制成相应的成品泡菜。
表1、乳酸菌螺丝菜泡菜卤水配制方法
实验1:螺丝菜泡菜成品中叶酸含量测定
取发酵后(34℃发酵7天)的螺丝菜泡菜50g置入榨汁机,加入50mL超纯水,榨汁,将得到的菜汁经双层纱布过滤后离心(4℃,10000×g,10min),取上清用0.22μm滤膜过滤后作为样品,利用德国拜发IFP维生素B9叶酸微生物法试剂盒进行叶酸含量测定。按试剂盒中说明书要求对样品进行处理,将处理后的样品与培养基加入试剂盒自带的微孔板中,用粘合箔封紧后在37℃黑暗条件下培养48h。培养完成后从培养箱中取出微孔板,再次压紧粘合膜并在桌面上震荡,使微生物在培养基中混合均匀。随后轻轻撕下粘合膜,破坏微孔中的所有泡沫,并用酶标仪在540nm条件下读取微孔的OD540值。
经测定,空白对照组成品螺丝菜泡菜中叶酸含量为30.60μg/kg,市售菌剂组为42.60μg/kg,植物乳杆菌ZJ316组成为69.60μg/kg。可以看出,相比于自然发酵(空白对照组),人工接种乳酸菌发酵(ZJ316组和市售菌剂组)的螺丝菜泡菜产品中的叶酸含量较高;且植物乳杆菌ZJ316菌剂发酵相对于市售菌剂发酵产叶酸效果更佳。
实验2:螺丝菜泡菜成品中风味物质测定
取发酵后(34℃发酵7天)螺丝菜泡菜50g置入榨汁机,加入50mL超纯水,榨汁,将得到的菜汁经双层纱布过滤后离心(4℃,10000×g,10min),取上清用0.22μm滤膜过滤后装入15mL顶空进样瓶中,将顶空进样瓶放到50℃水浴锅内加热30min,备用。
顶空固相微萃取条件:将老化后的萃取头插入进样瓶顶空部分,50℃平衡30min、吸附30min后,将萃取头取出并插入GC进样口,同时启动仪器采集数据,解吸20min。
GC-MS条件:7890A/5975GC/MS联用仪,色谱柱:HP-5MS型(30m×0.250mm×0.25μm);载气:氦气;进样温度:230℃;进样量:1μL;升温程序:初温45℃保持2min,5℃/min上升至180℃,保持1min,25℃/min升到230℃,保持5.5min。由计算机质谱系统NSIT检索未知化合物,匹配度大于70%的结果将予以报告,面积归一法计算各成分的含量。
经GC-MS分析,如表2、3、4、5、6所示,三组样品总共检测种74种挥发性风味物质,包括酯类22种、酮类15种、醇类23种、酸类6种、酚类和呋喃类8种。可以看出,醇类、酯类、酮类物质在ZJ316组、市售菌剂组、自然发酵组样品中含量均较高,则这三类物质为螺丝菜泡菜主要风味成分。ZJ316组共检出50种物质,其中醇类14种、酯类19种、酮类9种、酸类4种、酚类和呋喃类4种;市售菌剂组共检出44种物质,其中醇类16种、酯类10种、酮类7种、酸类6种、酚类和呋喃类5种;自然发酵组共检出32种物质,其中醇类12种、酯类6种、酮类7种、酸类2种、酚类和呋喃类5种。可以看出,人工接种乳酸菌(ZJ316组和市售菌剂组)发酵的螺丝菜泡菜风味物质情况明显优于自然发酵的螺丝菜泡菜,且ZJ316组在风味物质种类和含量上优于市售菌剂组,说明植物乳杆菌ZJ316可赋予螺丝菜泡菜产品更佳的风味。其中,ZJ316组中乙酸龙脑酯、乙酸香叶酯和2-十三酮的含量显著高于其他组。研究发现,乙酸龙脑酯具有祛痰、化解粘液、缓解呼吸不适等功效,并且可增强肠道黏膜屏障,增加肠道有益微生物,减少病原菌。乙酸香叶酯是一种药物,带有玫瑰和熏衣草香气,常用于配制玫瑰、橙花、桂花等香精。2-十三酮具有果香、甜香、椰子、奶油的气味,常用于调配椰子、坚果、菠萝蜜、热带水果、牛奶、乳酪、蘑菇、鸡肉、熏肉等食用香精。
表2、醇类物质含量
表3、酯类物质含量
表4、酮类物质含量
表5、酸类物质含量
表6、其他物质含量
实验3:设置实验体系:
一、YCFA培养基、螺丝菜泡菜样品、粪便悬液配制:
氯化钙溶液:准确称取氯化钙粉末1.25g,超纯水定容至1L。
硫酸镁溶液:准确称取硫酸镁粉末0.09g,超纯水定容至1L。
维生素I溶液:准确称取维生素粉末1.25g,超纯水定容至1L。
刃天青溶液:准确称取刃天青粉末1.25g,超纯水定容至1L。
血红素溶液:准确称取血红素粉末0.50g,溶于1mL 1mol/L NaOH中,超纯水定容至100mL,4℃避光保存待用。
YCFA培养基:准确称取蛋白胨10g,酵母提取物2.5g,L-半胱氨酸1g,血红素溶液2mL,NaCl 9g,氯化钙溶液125μL,KH2PO4 0.45g,K2HPO4 0.45g,硫酸镁溶液500μL,溶于1L超纯水中,待完全溶解后,加入1mL刃天青溶液,煮沸至培养基由红色变为黄色,马上用氮吹培养基保持表面无氧,用蠕动泵分装至西林瓶中(5mL/瓶),压盖密封,高压灭菌后(121℃、15min)使用。
螺丝菜泡菜样品:取植物乳杆菌ZJ316菌剂发酵组、市售菌剂组、自然发酵组(空白对照组)成品螺丝菜泡菜50g,与50mL超纯水混合进行榨汁,双层纱布过滤,得泡菜滤液在4℃中保存待用。
粪便悬浊液:粪便样品由10位志愿者(5男5女)提供,志愿者均招募于浙江杭州,年龄20岁~40岁,作息规律,不吸烟不喝酒,日常三餐的饮食以家常饭菜或者面食为主,采样前至少两个月内没有接受过医疗机构治疗或者服用抗生素、益生元,且无胃肠疾病。取志愿者晨起新鲜粪便0.80g/人,分别加入0.1mM灭菌PBS(pH=6.8)8mL,漩涡震荡充分混匀,滤去其中的大颗粒,得粪便悬浊液。
二:人体粪便悬浮液分组发酵
取上述所得的500μL粪便悬浊液,加入100μL巴豆酸,置于一个新的2mL灭菌离心管,混合均匀,储存于-40℃冰箱,作为人体粪便发酵前分析对照样品。
分为F、N、M、316四个组,每组中10个重复样本,即10位志愿者提供的样本,每个样本3组平行。
F代表粪便原样组:500μL的粪便悬浊液;
N代表自然发酵组:500μL的粪便悬浊液、5mL的YCFA培养基、40μL的自然发酵的螺丝菜泡菜汁;
M代表市售菌剂组:500μL的粪便悬浊液、5mL的YCFA培养基、40μL的市售菌剂发酵的螺丝菜泡菜汁;
316代表植物乳杆菌ZJ316菌剂发酵组:500μL的粪便悬浊液、5mL的YCFA培养基、40μL的植物乳杆菌ZJ316菌剂发酵的螺丝菜泡菜汁。
将上述发酵样本放置到37℃的恒温培养箱中静置培养24h。培养结束后,震荡均匀,用开瓶器打开瓶盖,吸取500μL发酵悬浊液加入100μL巴豆酸,混合均匀,储存于-40℃冰箱,作为人体粪便发酵后分析样品。
实验4、粪便悬浮液发酵样品DNA提取及16S rDNA测序分析
采用凯杰的QI Aamp Power Fecal DNA Kit试剂盒对粪便原样和粪便发酵液样品(即,实验3步骤二所得的粪便发酵前样品和粪便发酵后样品)进行DNA提取。
16S rDNA V3-V4区高通量测序采用Illumina MiSeq平台,扩增上游引物为338F(ACTCCTACGGGAGGCAGCA),下游引物为(GGACTACHVGGGTWTCTAAT),目的片段长度为480bp,测序策略采用Miseq-PE250。对各样本(组)在不同分类水平的具体组成进行分析(并检验组间是否具有统计学差异)。通过多种多变量统计学分析工具,进一步衡量不同样本(组)间的菌群结构差异及与差异相关的物种。
体外发酵系统的微生物组成与丰度在“门”水平的差异,如图1a所示。厚壁菌门(Firmicutes),拟杆菌(Bacteroidetes),变形杆菌(Proteobacteria),梭杆菌门(Fusobacteria),放线菌(Acteinobacteria),软壁菌门(Tenericutes),疣微菌门(Verrucomicrobia)微生物含量较多。其中植物乳杆菌ZJ316发酵组厚壁菌门微生物的相对丰度显著多于自然发酵组、市售菌剂发酵组。拟杆菌门微生物在粪便原样组占主导地位。
微生物在“属”水平的差异,如图1b所示。在检测组中,拟杆菌属(Bacteroides)、普氏菌属(Prevotella)、双歧杆菌属(Bifidobacterium)、类杆菌属(Faecalibacterium)、梭菌属(Fusobacterium)、萨特氏菌属(Sutterella)、韦荣氏菌属(Veillonella)、类杆菌属(Dialister)、布劳特氏菌属(Blautia)等微生物含量较多。粪便原样组中,拟杆菌属与普氏菌属微生物占50%以上。植物乳杆菌ZJ316发酵组中双歧杆菌属和乳杆菌属(图1b未显示)微生物比例高于自然发酵组、市售菌剂发酵组,如图2a、2b所示。双歧杆菌属和乳杆菌属是人体肠道内的有益微生物,在抑制人体有害细菌的生长,抵抗病原菌的感染,合成人体需要的维生素,促进人体对矿物质的吸收,产生乙酸、丙酸、丁酸和等短链脂肪酸刺激肠道蠕动,刺激人体免疫系统,提高抗病能力等方面有着重要作用。ZJ316组的布劳特氏菌属(Blautia)比例较低(图2c),据研究发现肥胖儿童微生物群中Blautia物种的消耗加剧了肠道炎症和代谢表型。此外,ZJ316组的肠杆菌属(属于肠杆菌科)细菌比例低于自然发酵和市售菌剂组,如图2d所示。肠杆菌是革兰氏阴性细菌,是引起细菌性胃肠炎的病源因子及其不同表现形式(如肠炎或痢疾)的主要原因。尽管肠杆菌科细菌通常占健康肠道菌群的不到1%,但其中一些微生物会在炎症性肠道内迅速增多。肠杆菌科微生物的显著增加是肠道微生物态失衡的标志。
图3所示主成分分析(PCA),从各组检测样本点在图中分布的集散情况来看,粪便原样组与处理组之间存在明显的分离,各处理组样本点分布既有重叠也有离散的部分,且离散部分的面积大于重叠部分的面积,这表明各处理组对肠道菌群的调控作用既有相似性也有差异性,且差异性占主导地位。其中ZJ316组的样本点分布与市售菌剂组分开,自然发酵组样本点分布与市售菌剂组接近,这也说明泡菜发酵过程中无论微生物的存在与否,还是微生物组成的总体丰度,单个发酵菌剂均以不同的方式在不同程度上扰乱了肠道微生物组,而ZJ316对微生物的组成、结构及丰度的影响要大得多。
综上所述,植物乳杆菌ZJ316菌剂发酵的螺丝菜泡菜能够有益地调节肠道菌群,促进益生菌的生长并抑制部分病原菌。
实验5:气相色谱检测粪便悬浮液发酵样品中短链脂肪酸
短链脂肪酸含量是人体肠道内微生物通过代谢生成的产物,是衡量肠道菌群生长及代谢情况的一个重要指标,包括乙酸、丙酸、丁酸、戊酸等。从-40℃冰箱中取出粪便原样和粪便发酵样品(即,实验3步骤二所得的粪便发酵前样品和粪便发酵后样品),常温下解冻,待完全融化后离心(4℃,10000rpm,3min),取上清,用0.22μm滤膜过滤。取100μL样品提取液加入气相样品瓶内插管中,用盖子盖紧排除气泡后即可上样分析,其余过滤液-30℃保存备用。
色谱柱:Agilent FFAP30 m×0.25mm×0.25μm;柱温:75℃,20℃/min升至180℃保持1min,50℃/min升至220℃保持1min;进样口温度:250℃;进样量:1.0μL;分流比:5:1;载气:高纯氮;流速:2.5mL/min保持6.5min,2.8mL/min升至2.8mL/min保持2min;检测器:FID;温度:250℃;尾吹:20mL/min;氢气:30mL/min;空气:300mL/min。
图4a所示,为发酵24h后各组乙酸产量情况,其中ZJ316组的乙酸产量在均值上最高,且高于平均水平的11.33%;图4b为发酵24h后各组的丙酸产量情况,其中ZJ316组丙酸产量在均值是最高,且高于平均水平21.67%;图4c为发酵24h后各组的丁酸产量情况,其中ZJ316组的丁酸产量在均值上高于空白对照19.71%;图4d分别为发酵24h后的戊酸产量情况,其中ZJ316组的戊酸产量在均值上最高,且高于平均水平的40.11%。总的来说,从均值来看ZJ316组的乙酸、丙酸、戊酸产量高于其他各组,丁酸产量高于空白对照组,但差异性不明显,这表明植物乳杆菌ZJ316菌剂发酵的螺丝菜泡菜对人体肠道菌群的短链脂肪酸产量有一定的促进作用。
对比例1、将实施例1中的植物乳杆菌ZJ316(Lactobacillus plantarum ZJ316),改成现有的其余Lactobacillus plantarum,具体如下表7所述,其余等同于实施例1。然后按照上述实验1~实验5进行检测,所得实验结果与实施例1的对比如表7所述。
表7
对比例2-1、取消实施例1中保护剂的使用,即,将菌泥直接进行冻干,其余等同于实施例1。
对比例2-2、将实施例1中保护剂配方由“10%脱脂乳粉、1.5%海藻糖”改成“5%脱脂乳粉、5.5%海藻糖”;其余等同于实施例1。
对比例2-3、将实施例1中保护剂配方由“10%脱脂乳粉、1.5%海藻糖”改成“8%脱脂乳粉、2.5%海藻糖”;其余等同于实施例1。
对比例2-4、将实施例1卤水配方中的“植物乳杆菌ZJ316菌剂”的用量由0.2g改成0.4g,其余等同于实施例1。
对比例2-5、将实施例1卤水配方中的“植物乳杆菌ZJ316菌剂”的用量由0.2g改成0.1g,其余等同于实施例1。
然后按照上述实验1~实验5进行检测,所得实验结果与实施例1的对比如表8所述。
表8
最后,还需要注意的是,以上列举的仅是本发明的若干个具体实施例。显然,本发明不限于以上实施例,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
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<110> 浙江工商大学
<120> 运用乳酸菌提高螺丝菜健康功能的方法
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<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
actcctacgg gaggcagca 19
<210> 2
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ggactachvg ggtwtctaat 20
Claims (8)
1.运用乳酸菌提高螺丝菜健康功能的方法,其特征在于包括以下步骤:
1)、配置发酵卤水:
在1L超纯水中加入96±1g食盐、46±0.5g白砂糖、0.2g植物乳杆菌ZJ316菌剂,混匀,得发酵卤水;
所述植物乳杆菌ZJ316菌剂由保藏编号CCTCC NO:M 208077的植物乳杆菌ZJ316(Lactobacillus plantarum ZJ316)制备而成;
2)、利用发酵卤水对螺丝菜进行发酵处理。
2.根据权利要求1所述的运用乳酸菌提高螺丝菜健康功能的方法,其特征在于所述步骤2)为:
将螺丝菜清洗后,沥干水分;然后放入泡菜坛先压实,再向泡菜坛注入发酵卤水,坛盖水封,于34±2℃发酵7±1天。
3.根据权利要求1或2所述的运用乳酸菌提高螺丝菜健康功能的方法,其特征在于:植物乳杆菌ZJ316菌剂的制备方法为:
①、将活化后的植物乳杆菌ZJ316进行发酵培养,所得发酵液经过离心,去上清,取菌体沉淀,菌体沉淀清洗后离心,得菌泥;
②、配制保护剂:
10±0.5g脱脂乳粉、1.5±0.1g海藻糖、1±0.1g甘油、3.5±0.1g山梨醇,用水定容至100mL,均匀混合,得保护剂;
③、按照菌泥/保护剂=1g/3ml的用量比,将菌泥与保护剂混合均匀后进行冷冻,得植物乳杆菌ZJ316菌剂。
4.根据权利要求3所述的运用乳酸菌提高螺丝菜健康功能的方法,其特征在于:所述步骤③中:菌泥与保护剂混合均匀后,先于-80℃预冻6h,然后用真空冷冻干燥机冻干24h。
5.根据权利要求4所述的运用乳酸菌提高螺丝菜健康功能的方法,其特征在于:所述步骤①为:
将活化后的植物乳杆菌ZJ316按3%的接种量接至MRS液体培养基中,于37℃发酵培养24h,所得发酵液经过离心,去上清,取菌体沉淀,菌体沉淀经过质量浓度为0.85%的生理盐水清洗后离心,得菌泥。
6.根据权利要求1~5所述的运用乳酸菌提高螺丝菜健康功能的方法,其特征在于:制备所得的螺丝菜具有改善肠道菌群、补充叶酸的功能。
7.根据权利要求6所述的运用乳酸菌提高螺丝菜健康功能的方法,其特征在于:
所述改善肠道菌群为促进肠道内的双歧杆菌(Bifidobacterium)和乳酸菌(Lactobacillus)的生长,从而促进肠道菌群代谢产生短链脂肪酸。
8.根据权利要求7所述的运用乳酸菌提高螺丝菜健康功能的方法,其特征在于:短链脂肪酸为乙酸、丙酸、丁酸、戊酸。
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