CN110885767B - 一株具有良好抗氧化活性的乳酸乳球菌霍氏亚种及其应用 - Google Patents
一株具有良好抗氧化活性的乳酸乳球菌霍氏亚种及其应用 Download PDFInfo
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- CN110885767B CN110885767B CN201910908241.9A CN201910908241A CN110885767B CN 110885767 B CN110885767 B CN 110885767B CN 201910908241 A CN201910908241 A CN 201910908241A CN 110885767 B CN110885767 B CN 110885767B
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及一株具有良好抗氧化活性的乳酸乳球菌霍氏亚种,为乳酸乳球菌霍氏亚种(Lactococcus lactis subsp.hordniae)JYZ4。本发明所述乳酸菌保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地点为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,保藏号为CGMCC No.17892,保藏日期为2019年6月3日。本发明所述乳酸菌分离自酱油渣中,通过体外益生特性研究表明其耐酸、耐人工胃液及肠液,具有良好疏水性以及抗氧化活性。其可应用于发酵酱油渣制成适口性良好的饲料,也可联合嗜热链球菌、保加利亚乳杆菌发酵酸奶等,具有广阔的应用前景以及商业价值。
Description
技术领域
本发明涉及一株具有良好抗氧化活性的乳酸乳球菌霍氏亚种及其应用,属于微生物领域。
背景技术
乳酸菌作为世界公认的益生菌,因其各种益生特性,被广泛应用于饲料发酵、食品加工及疾病防治等与人类密切相关的社会活动中。动物体内时刻都在发生氧化作用,过度的氧化会导致动物体内生物分子的损伤,导致动物免疫力下降。乳酸菌所具有的抗氧化活性有助于宿主体内维持氧化还原平衡状态,提高宿主免疫力。现有抗氧化特性的乳酸菌主要分离自传统发酵食品、宿主胃肠道系统和自然环境等。为此,从酱油渣中筛选具有一定的耐盐、耐酸、耐人工胃液及肠液以及抗氧化活性的乳酸菌,能够在宿主肠道定植并能够发挥抗氧化活性。将其应用于酱油渣饲料发酵有利于提高酱油渣饲料适口性以及增强动物免疫力,调节动物肠道平衡。
酱油渣,又称酱渣,是酱油酿造过程中产生的副产物,主要成分是酿造酱油的原料(大豆、豆饼、小麦、食盐等)在发酵结束后经淋油或抽油后产生的固体物质。国家统计局的数据显示,2018年我国酱油产量达1041万吨,按照每生产1kg酱油产出0.67kg酱油渣的比例计算,我国一年需处理的酱油渣接近700万吨。如此巨量的酱油渣若不加以充分利用,势必会造成资源的浪费和环境的污染。目前,酱油渣可作鲜味剂、肥料、饲料以及提取有效成分等,其中开发成饲料是其主要用途之一。然而酱油渣含有较高的盐分,直接作为饲料适口性较差;而且酱油渣不易保存,其应用受到制约。
微生物生长繁殖迅速,对营养物质适应性强,可利用其发酵作用来生产和调制饲料。已有相关研究表明,利用外源乳酸菌作用于酱油渣,可改善其理化性状,提高营养价值,促进其饲料化。酱油渣作为传统发酵食品的副产物,本身含有丰富的微生物资源,如曲霉、酵母菌、乳酸菌等。然而,酱油渣中乳酸菌资源的相关研究却很少。
发明内容
本发明结合抗氧化活性进行筛选研究,筛选的菌种来源是酱油渣。本发明的目的在于提供一株具有良好抗氧化活性的乳酸乳球菌霍氏亚种JYZ4,能够应用于酱油渣、酸奶、酱油、泡菜、酒糟等的发酵。
为实现上述目的,本发明采用的技术方案是:
一株具有良好抗氧化活性的乳酸乳球菌霍氏亚种JYZ4,分类名称为Lactococcuslactis subsp.hordniae。保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地点为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,保藏号为CGMCCNo.17892,保藏日期为2019年6月18号。
而且,所述乳酸乳球菌霍氏亚种JYZ4具有以下性质:
(1)具有耐酸性,在pH4~10条件下生长良好,在pH3条件下存货良好。
(2)具有耐胃液性,3小时存活率为98%。
(3)具有耐肠液性,4小时存活率为82%,8小时存活率为45%。
(4)具有抗氧化性,菌体细胞悬浮液对DPPH、羟基自由基的清除率为34%、54%。
乳酸乳球菌霍氏亚种JYZ4培养条件为MRS培养基,具体成分为:酪蛋白酶消化物1g、牛肉膏粉1g、酵母膏粉0.4g、柠檬酸三铵0.2g、乙酸钠0.5g、硫酸镁0.02g(MgSO4·7H2O)、硫酸锰0.005g(MnSO4·4H2O)、磷酸氢二钾0.2g、葡萄糖2g、吐温-80 0.108g、蒸馏水100mL,最终pH5.7±0.2(注:以上液体MRS培养基可添加1.5~2g琼脂配制成固体MRS培养基,最终pH 6.2±0.2的)。乳酸乳球菌霍氏亚种JYZ4接种上述MRS培养基后置于37℃生化培养箱培养,待有菌落长成后可进行后续操作。
乳酸乳球菌霍氏亚种JYZ4的具体筛选工艺步骤如下:
(1)收集一份酱油渣样品,称取25g酱油渣于三角瓶中,加入225mL无菌生理盐水,稍加振荡后梯度稀释成10-1~10-5共5个稀释液。
(2)分别吸取上述稀释液0.1mL涂布于MRS培养基中,用封口膜密封后置于37℃培养箱中倒置培养24h~48h。
(3)挑取疑似乳酸菌菌落于MRS培养基中三区划线纯化,操作2~3次至培养基上菌落形态一致。将纯化菌株接种于MRS液体培养基中于37℃培养箱中培养24h后与50%甘油1:1混合后置于-80℃冰箱保藏。
(4)提取上述纯化菌株进行革兰氏染色镜检、酶触试验操作进行初筛。
(5)将初筛菌株进行分子生物学鉴定(16S rRNA测序),判断所筛选菌株的种属。
(6)将鉴定为乳酸菌菌株进行耐盐性、耐酸性试验。筛选出具有较好耐盐性、耐酸性菌株。
(7)将(6)中所筛选菌株进行耐胆盐试验、模拟胃液耐受性试验、模拟肠液耐受性试验、表面疏水性测定,筛选出具有较好益生潜能菌株。
(8)将(7)中获得的具有较好益生潜能菌株进行抗氧化能力测定,分别进行清除DPPH能力测定以及清除羟基自由基能力测定,筛选出具有较好抗氧化活性菌株。
(9)最终筛选出一株综合性能较佳的乳球菌霍氏亚种JYZ4。
本发明还提供一种制备乳酸乳球菌霍氏亚种JYZ4生物制剂的方法,将乳酸乳球菌霍氏亚种JYZ4活化培养24~48h后于6000r/min离心10min后去除上清液收集菌体。将收集得到的菌体用复配保护剂重悬,梯度降温后真空冷冻干燥得到冻干菌粉。
本发明中,所述乳酸乳球菌霍氏亚种JYZ4可应用于发酵产品中,具有以下用途(1)应用于发酵制品副产物,如酱油渣饲料发酵,提高饲料适口性以及益生特性(2)应用于发酵食品,如酸奶发酵,提高酸奶品质及风味;如泡菜发酵,使得所制得泡菜酸度适宜,风味较佳。
值得注意的是,本发明适用于但不限于以上应用用途。
与现有技术相比,本发明的有益效果是:可直接用于发酵酱油渣饲料,扩充酱油渣饲料化菌种库,提高酱油渣利用率及转化率,提高酱油渣饲料适口性。同时本发明能够提高发酵食品的品质,提高发酵食品的抗氧化性。
附图说明
图1为乳酸乳球菌霍氏亚种JYZ4的菌落图。
图2为乳酸乳球菌霍氏亚种JYZ4的革兰氏染色图。
图3为乳酸乳球菌霍氏亚种JYZ4的生长曲线。
图4为乳酸乳球菌霍氏亚种JYZ4的耐盐性与耐酸性。
图5为乳酸乳球菌霍氏亚种JYZ4的益生性指标,包括胃液耐受性、肠液耐受性以及疏水率。
图6为乳酸乳球菌霍氏亚种JYZ4的抗氧化性指标,包括DPPH清除率、羟基自由基清除率。
具体实施方式
下面结合附图和实施例,对本发明进行具体描述。
实施例1:乳酸乳球菌霍氏亚种JYZ4的分离鉴定方法
(1)样品稀释培养
无菌条件下称取25g酱油渣样品于三角瓶中,加入225mL无菌生理盐水,稍加振荡后制得10-1稀释液。依次配制10-2~10-5等浓度的稀释液。分别吸取0.1mL不同梯度的酱油渣稀释液涂布于MRS培养基上。用封口膜密封,标上相关信息,于37℃条件恒温培养培养24~48h至菌落长成。
(2)分离纯化保藏
挑取疑似乳酸菌菌落于MRS培养基中三区划线纯化,操作2~3次至培养基上菌落形态一致。将纯化菌株接种于MRS液体培养基中于37℃培养箱中培养24h后与50%甘油1:1混合后置于-80℃冰箱保藏。
(3)革兰氏染色及过氧化氢酶触试验
挑取已纯化菌株按试剂盒说明书进行染色镜检,观察菌株形态。革兰氏阳性菌株呈紫色;革兰氏阴性菌株呈红色。吸取过氧化氢溶液,滴在固体培养基菌落集中的位置,用无菌牙签将过氧化氢溶液和菌苔搅浑均匀,观察30s,期间无气泡产生的菌落为过氧化氢酶阴性菌,有气泡产生的菌落则为阳性。
(4)16S rRNA分子生物学鉴定
1)菌株DNA提取
A.在酒精灯火焰附近,取乳酸菌菌体于研钵,使用液氮进行研磨。
B.将研磨完毕的菌体移至离心管(1.5mL)中,标记菌株编号,加入0.6mL TE缓冲液(pH≈8.0),使用枪头吸打成均匀菌悬液。
C.加250μL 10%十二烷基硫酸钠溶液(Sodium dodecyl sulfate,SDS),轻轻倒转使其混匀。
D.加3μL蛋白酶K(20ng/μL),使其混匀。37℃条件下水浴60min。
E.加150μL盐溶液(5mol/L),混匀。
F.加150μL 2%十六烷基三甲基溴化铵溶液(Hexadecyl trimethylammoniumBromide,CTAB),混匀,65℃条件下水浴加热20min。
G.12000RPM常温条件下离心20min。
H.小心移取上清液至新的离心管(1.5mL),加等体积异丙醇,混匀,室温放置0.5h,12000RPM,4℃条件下离心10min。
I.去上清液,用吸水纸吸干液体,加750μL 70%乙醇溶液,轻弹离心管壁,使其沉淀悬浮并倒转几次,12000RPM,4℃条件下离心2min。
J.离心管加30μL纯化水(纯化水加Rnase,使终浓度为10ng/μL),轻弹离心管壁,4℃条件下过夜溶解。
2)PCR扩增
A.PCR扩增引物采用细菌通用正向引物27F和通用反向引物1492R,DNA提取后进行PCR扩增,扩增体系:H2O 17.8μL;Buffer 3μL;d NTP 2μL;Primer1 3μL;Primer2 3μL;DNA模板1μL;酶0.2μL;总体积30μL。
B.反应条件:95℃预变性5min;95℃变性30sec;55℃退火30sec;72℃延伸1min;35个循环;72℃ 10min产物末端延伸;12℃ forever终止反应。
3)凝胶电泳与测序及比对
将PCR产物经1%琼脂糖凝胶电泳检测,目标片段在1500bp左右,将扩增成功片段送由测序公司测序。将序列经BLAST比对鉴定,结合系统发育树判断所筛选菌株的种属。
实施例2:乳酸乳球菌霍氏亚种JYZ4的生长特性研究
(1)生长曲线的测定
将本发明菌株活化三代,于MRS液体培养基中37℃培养40h,每2小时测定菌株OD600nm值。以OD600nm值为纵坐标,培养时间为横坐标绘制菌株生长曲线,实验结果见图3。接种0h~4h为菌株的生长延缓期,接种4h~12h为乳酸菌的对数生长期值,接种12h时进入稳定期,接种32h左右进入衰亡期。
(2)耐盐性研究
配制含盐的MRS培养基,盐质量分数分别为0~9%共9个梯度,灭菌待用。将本发明菌株活化三代,于MRS液体培养基中37℃培养24h,测定OD600nm值。以OD600nm值为纵坐标,盐浓度为横坐标绘制曲线,结果见图4。乳酸乳球菌霍氏亚种JYZ4具有较好耐盐性,在NaCl含量为0%~5%条件下生长良好,当NaCl含量为6%及以上时,生长受到抑制。
(3)耐酸性研究
配制不同pH的MRS液体培养基,pH值分别为2.0、3.0、4.0、5.0、6.0、7.0、8.0、9.0,灭菌待用。将本发明菌株活化三代,于MRS液体培养基中37℃培养24h,测定OD600nm值。以OD600nm值为纵坐标。以OD600nm值为纵坐标,pH值为横坐标绘制曲线,结果见图4。乳酸乳球菌霍氏亚种JYZ4具有耐酸性,在pH4~10条件下生长良好,在pH3条件下存活良好。
实施例3:乳酸乳球菌霍氏亚种JYZ4的益生特性研究
(1)模拟胃液耐受性试验
将本发明菌株活化三代,吸取3mL培养液,常温下4000r/min离心10min,去上清液,得菌体,将菌体悬浮于3mL灭菌生理盐水制成菌悬浮液备用。
无菌离心管中加入人工胃液与菌悬浮液(9:1),混合均匀。37℃培养,在0h(稀释度为10-4、10-5、10-6)和3h(稀释度为10-3、10-4、10-5)取样0.1mL,使用平板计数法计算菌株存活率。实验设置三个重复。
乳酸乳球菌霍氏亚种JYZ4具有耐胃液性,3小时存活率为98%。
(2)模拟肠液耐受性试验
取人工胃液消化3h的菌株培养液1mL,接种于9mL人工肠液中,37℃培养0h(稀释度10-3、10-4、10-5)、4h(稀释度10-2、10-3、10-4)和8h(稀释度10-2、10-3、10-4),使用平板计数法计算菌株存活率。实验设置三个重复。
乳酸乳球菌霍氏亚种JYZ4具有耐肠液性,4小时存活率为82%,8小时存活率为45%。
(3)表面疏水性试验
将本发明菌株活化三代,接种量为2%,37℃培养12h。菌液4000r/min离心5min,PBS溶液洗涤2~3次后,重悬。测定菌株OD600nm值,即为初始吸光度A0。吸取上述菌悬浮液3mL,加入1mL二甲苯,室温培养15min,震荡使其快速混合,室温静止放置1h,弃有机层与乳化层,吸取下层水相,以PBS缓冲液为空白对照,测定OD600nm即为最终吸光度A,记录并计算。
乳酸乳球菌霍氏亚种JYZ4具有表面疏水性,疏水率为25%。
(4)抗氧化能力测定
将本发明菌株活化三代,接种量为2%,37℃培养24h。4℃下4000r/min离心20min,去上清收菌体。用PBS缓冲液洗涤菌体3次,重悬。
1)清除DPPH能力测定
2mL菌体悬浮液,加2mL DPPH溶液(0.2mmol/L),室温下避光反应0.5h,测定OD517nm值,空白为无水乙醇。
测出菌体细胞悬浮液对DPPH清除率为34%。
2)清除羟基自由基能力测定
试管中加入2mL邻菲啰啉(2.5mmol/L),依次加入PBS缓冲溶液(pH=7.4,0.02mol/L)2mL和蒸馏水2mL,混匀。加硫酸亚铁溶液2mL(2.5mmol/L),2mL H2O2(20mmol/L)溶液,混匀,37℃水浴90min。4℃下5000r/min离心6min,测定OD536nm值,空白为PBS缓冲溶液。
测出菌体细胞悬浮液对羟基自由基的清除率为54%。
应用例1:制备含本发明菌株的酱油渣饲料
基料配方如下:酱油渣85%,麸皮5%,豆粕10%。投入本发明的乳酸乳球菌霍氏亚种JYZ4,待其浓度达106CFU/g后于密闭空间进行发酵5~10天,即可得到适口性好,具有一定益生功能的酱油渣饲料。
应用例2:制备含本发明菌株的酸奶
将牛乳高温瞬时灭菌后迅速冷却到室温,以重量比为1:1:1接入本发明的乳酸乳球菌霍氏亚种JYZ4、商业化保加利亚乳杆菌和商业化嗜热链球菌,总接种量为0.05%,混匀,37℃保温发酵8~12h,凝乳后置于4℃冷藏。
Claims (5)
1.一株具有良好抗氧化活性的乳酸乳球菌霍氏亚种菌株,其特征在于:该菌株为乳酸乳球菌霍氏亚种(Lactococcus lactis subsp. hordniae)JYZ4,保藏号为CGMCCNo.17892,保藏日期为2019年6月3日,保藏地点为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所;
所述乳酸乳球菌霍氏亚种JYZ4具有以下性质:
(1)具有耐酸性,在pH4~10条件下生长良好,在pH3条件下存活 良好;
(2)具有耐胃液性,3小时存活率为98%;
(3)具有耐肠液性,4小时存活率为82%,8小时存活率为45%;
(4)具有抗氧化性,菌体细胞悬浮液对DPPH、羟基自由基的清除率为34%、54%。
2.根据权利要求1所述一株具有良好抗氧化活性的乳酸乳球菌霍氏亚种菌株在发酵产品中的应用。
3.根据权利要求2所述的应用,其特征在于,所述乳酸乳球菌霍氏亚种菌株应用于发酵食品、发酵制品副产物或发酵青贮饲料中。
4.根据权利要求2所述的应用,其特征在于,所述乳酸乳球菌霍氏亚种菌株应用于酸奶、酱油、泡菜、酱油渣或酒糟。
5.根据权利要求1所述一株具有良好抗氧化活性的乳酸乳球菌霍氏亚种菌株制备生物制剂的方法,其特征在于,将乳酸乳球菌霍氏亚种JYZ4活化培养24~48 h后于6000 r/min离心10 min后去除上清液收集菌体,将收集得到的菌体用复配保护剂重悬,梯度降温后真空冷冻干燥得到冻干菌粉。
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