CN108330082A - 一株副干酪乳杆菌及其应用 - Google Patents
一株副干酪乳杆菌及其应用 Download PDFInfo
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- CN108330082A CN108330082A CN201711381421.3A CN201711381421A CN108330082A CN 108330082 A CN108330082 A CN 108330082A CN 201711381421 A CN201711381421 A CN 201711381421A CN 108330082 A CN108330082 A CN 108330082A
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- lactobacillus paracasei
- bacterial strain
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- moringa
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
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- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
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- A—HUMAN NECESSITIES
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/38—Other non-alcoholic beverages
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- A—HUMAN NECESSITIES
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- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
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- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一株副干酪乳杆菌及其应用。本发明提供了一株在大理白族自治州地区泡梨中分离到的副干酪乳杆LSE‑2014ZA001‑M012‑A‑03(a),它的保藏编号为CGMCC No.14751。本发明的具有下述有益效果:菌株具有很强的耐酸性、耐胆盐性能和较强的降解胆固醇的能力。菌株在4℃贮藏条件下能够维持很好的活力,用其制备的褐色益生菌饮料4℃贮藏24天,离心沉淀率最大不超过1.5%,且体系无分层、絮凝的现象。本发明菌株具有较高的木聚糖酶活性,适合做发酵果蔬汁或植物性发酵酸奶等。
Description
技术领域
本发明属于微生物技术领域,尤其涉及一株副干酪乳杆菌及其应用。
背景技术
益生菌是指当人体摄入足够数量的、某种特定活的微生物时,能够促进机体的健康,对胃肠液有耐受性、且在胆汁的作用下可以生长。副干酪/干酪乳杆菌是目前研究最为深入、应用最为广泛的益生菌之一,与嗜酸乳杆菌、双歧杆菌一同被认为是目前最为经典的三种益生菌。与其他两种益生菌相比,副干酪/干酪乳杆菌具有较强的加工性能,并且在调节人体免疫系统,及调节人体肠道环境、降解胆固醇等方面具有突出的功能。
胆固醇是人体维持正常机能必须的物质,但摄入过多会积聚在血管壁上,导致血管硬化和变窄,如果胆固醇在血管壁上积得太多,阻塞了血管,人体器官便会因为无法从血液中吸取足够的氧气和养料而坏死。由于人类生活方式及饮食结构的改变,胆固醇过高引起的健康问题,已经成为威胁人类健康的普遍问题,所以筛选出降解胆盐效果显著的益生菌并将其应用到食品加工领域,对于人类健康具有重要的意义。
木聚糖主要存在于植物细胞壁,在自然界中是除纤维素以外第二丰富的可再生资源,植物细胞壁中含有大量的木聚糖和纤维素,在果蔬汁加工的过程中细胞壁中的果胶、纤维素、木聚糖等形成的网状结构可以阻止细胞内容物的渗出。传统加工工艺难以分解上述物质并造成营养的大量流失。
本发明通过耐酸、耐胆盐试验筛选出的副干酪乳杆菌LSE-2014ZA001-M012-A-03(a)具有较强的耐酸、耐胆盐性能并具有较强的降胆固醇能力,而且还具有较高的木聚糖酶活性,能将菌株应用于益生菌饮料和发酵果蔬汁及果蔬酸奶等生产中。
发明内容
针对现有技术存在的上述不足,本发明的目的是提供一株副干酪乳杆菌LSE-2014ZA001-M012-A-03(a),其要解决的技术问题是该菌株具有很强的耐酸性能、耐胆盐性能,同时能降解胆固醇和木聚糖,本发明菌株在贮藏期间能保持较高的活菌数和更高的益生功能,适合做益生菌饮料的发酵剂及植物性发酵酸奶。
本发明的第二个目的是提供一株副干酪乳杆菌LSE-2014ZA001-M012-A-03(a) 在制备活性乳酸菌饮料中应用。
本发明的第三个目的是基于一株副干酪乳杆菌LSE-2014ZA001-M012-A-03(a)的木聚糖酶活性,提供一种其在制备辣木酸奶中的应用。
为实现上述发明目的,本发明采用下述技术方案:一株副干酪乳杆菌(Lactobacillus paracasei)LSE-2014ZA001-M012-A-03(a),它的保藏编号为CGMCCNo.14751,保藏日期为2017年09 月26 日。
与现有技术相比,本发明的有益效果有:
1、本发明副干酪乳杆菌LSE-2014ZA001-M012-A-03(a)具有很强的耐酸性,进入人体后能够对胃酸具有较强的抵抗能力,其在pH1.5中作用3h后活菌数仍能达到104CFU/mL,比同种属的商业菌种高一个数量级。
2、本发明副干酪乳杆菌LSE-2014ZA001-M012-A-03(a)具有很强耐胆盐性能,0.03g/100mL胆盐中作用3h菌株的存活率为100%,活菌数达到109 CFU/mL;0.15 g/100mL胆盐中作用3h活菌数达到107CFU/mL数量级;0.3g/100mL胆盐中作用3h菌株的活菌数达到104 CFU/m数量级。
3、本发明副干酪乳杆菌LSE-2014ZA001-M012-A-03(a)具有胆盐水解酶活性。
4、本发明副干酪乳杆菌LSE-2014ZA001-M012-A-03(a)具有较强的降解胆固的能力,发酵24h能够降解39%的胆固醇、发酵48h能够降解44.2%的胆固醇、发酵72h可以降解49.6%的胆固醇。采用统一检测方法比很多文献报道的菌株的降胆固醇能力强。
5、本发明副干酪乳杆菌LSE-2014ZA001-M012-A-03(a)有较高的木聚糖酶活性,适合做发酵果蔬汁或植物性发酵酸奶等。
本发明保藏说明:
分类命名:副干酪乳杆菌;
拉丁名:Lactobacillus paracasei;
参椐的生物材料:LSE-2014ZA001-M012-A-03(a);
保藏机构:中国微生物菌种保藏管理委员会普通微生物中心;
保藏机构简称:CGMCC;
地址:北京市朝阳区北辰西路 1 号院 3 号。
保藏日期:2017年 9 月 26 日
保藏中心登记入册编号:CGMCC No.14751。
附图说明
图1为本发明所述副干酪乳杆菌LSE-2014ZA001-M012-A-03(a)的菌落照片。
图2为本发明所述副干酪乳杆菌 LSE-2014ZA001-M012-A-03(a)的镜检照片。
图3为本发明所述副干酪乳杆菌 LSE-2014ZA001-M012-A-03(a)胆盐水解酶的定性测定结果。
图4为本发明在胆固醇降解能力测定中绘制的胆固醇标准曲线。
图5为本发明在发酵液木聚糖酶活力测定中绘制的木聚糖标准曲线。
图6为本发明所述副干酪乳杆菌 LSE-2014ZA001-M012-A-03(a)在蛋白含量为3.5%的脱脂乳中的产酸曲线。
具体实施方式
下面结合实施例,进一步阐述本发明,但不限于实施例。下述实施例中所使用的实验方法如无特殊说明,均为常规方法。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1:菌种的分离纯化鉴定
1.菌种的分离纯化
称取采集到的水牛奶样品10g,用生理盐水进行梯度稀释最高稀释度为8,采用采用加了碳酸钙的MRS固体培养基进行分离,取5、6、7、8四个梯度的稀释样品进倒板(37℃,48h)。初步根据菌落形态、大小、溶钙圈大小、颜色、培养基中生长位置(表面、内部、及底部)挑取不同表观特征且溶钙圈较大的菌落平板划线培养,进行纯化。再用同样的方法反复划线纯化数次, 使最终平板上为同一形态菌落为止。挑取单菌落接菌于 MRS 液体培养基中 37℃培养18h,以备保藏和鉴定使用。
2.菌种的鉴定
2.1菌种的形态学特
见图1的菌落照片,MRS固体培养基上的菌落为乳白色中等大小。见图2镜检照片,显微镜下为短杆成链。
2.1菌种的分子生物学鉴定
将初步获得革兰氏阳性、过氧化氢酶阴性的菌株暂定为乳酸菌,然后根据UNI-Q柱式细菌DNA抽提试剂盒的实验步骤提取细菌基因组,用0.8%的琼脂糖凝胶电泳观察提取到的基因组。以提取到的菌株的基因组为模板,以采用细菌通用引物:27F (5'- AGAGTTTGATCCTGGCTCAG -3') 和1541R (5'-TAAGGAGGTGATCCAGCC-3')为上、下游引物,采用PCR扩增试剂盒,以50µL的反应体系进行PCR反应。反应参数为:95℃预变性5min,35个循环的参数设置为94℃30s,55℃30s,72℃90s,72℃延伸10min。用0.8%琼脂糖凝胶电泳检测扩增DNA片段的大小,测序由上海生物工程技术服务有限公司完成。将经过测序所得到的序列用Blast软件在Genebank中搜索相似序列,经ClustalX序列比对后,采用MEGA4.0中的邻接法(Neighbour-joining)进行系统发育树构建,分析样品中乳酸菌的遗传距离。
据设计的引物进行16SrDNA扩增,电泳显示在1500bp左右有阳性条带,与目的片段大小一致,将PCR产物送至测序公司进行测序。16SrDNA 全长1422bp,全长序列如SEQ IDNO.1所示。测序结果通过Blasten比对,菌株为乳杆菌属副干酪乳杆菌种。
结合菌株的生理生化特征和分子生物学16S rDNA 鉴定结果,本发明菌株LSE-2014ZA001-M012-A-03(a)鉴定为副干酪乳杆菌 Lactobacillus paracasei,并将其命名为副干酪乳杆菌 Lactobacillus paracasei LSE-2014ZA001-M012-A-03(a),保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为:CGMCC No.14751。
3 LSE-2014ZA001-M012-A-03(a)的耐酸性能测定
将LSE-2014ZA001-M012-A-03(a)菌株按照2%的接种量,接种于5 mL MRS 液体培养基中, 37℃恒温培养18h,3000 r/min 离心10 min,弃去上清液液后将菌泥悬浮于5 mL无菌生理盐水中,取1 mL悬液3000 r/min 离心10 min,弃去上清液液后将菌泥悬浮于1 mL过滤除菌的pH为2.5、2.0、1.5的MRS液体培养基中,置37℃恒温培养0h、3h后取样测定活菌数,用MRS 琼脂培养基倾注平板,于 37℃培养48 h 后进行活菌计数,每一处理 3 个重复,计算存活率。
存活率=培养3h活菌数/培养0h活菌数*100%
通过三次重复试验, LSE-2014ZA001-M012-A-03(a)在不同梯度的pH中作用3h的存活率以及活菌数见表1 。而商业菌种在不同梯度的pH中作用条件下活菌数菌低于LSE-2014ZA001-M012-A-03(a),尤其是在pH1.5中作用3h,商业菌种的活菌数仅达到103CFU/mL,比LSE-2014ZA001-M012-A-03(a)低一个数量级。表明副干酪乳杆菌LSE-2014ZA001-M012-A-03(a)具有很强的耐酸性,进入人体后能够对胃酸具有较强的抵抗能力。
表 1 :LSE-2014ZA001-M012-A-03(a)在不同梯度pH中作用3h
的存活率及活菌数
4.LSE-2014ZA001-M012-A-03(a)的耐胆盐性能测定
将LSE-2014ZA001-M012-A-03(a)按照2%的接种量,接种于10mL MRS 液体培养基中,37℃恒温培养18h,3000 r/min 离心10 min,弃去上清液后将菌泥悬浮于10 mL无菌生理盐水中,取1 mL悬液3000 r/min 离心10 min,弃去上清液后将菌泥悬浮于1 mL 120℃ ,15min高压灭菌的牛磺胆酸钠浓度为0.3g/100mL、0.15 g/100mL、0.03 g/100mL、0 g/100mL的MRS液体培养基中,置37℃恒温培养0h、3h后取样测定活菌数,用MRS 琼脂培养基倾注平板,于 37℃培养48 h 后进行活菌计数,每一处理 3 个重复,计算存活率。
存活率=培养0h活菌数/培养3h活菌数*100%
通过三次重复试验,LSE-2014ZA001-M012-A-03(a)在不同梯度胆盐中作用3h的存活率及活菌数见表2。0.03 g/100mL胆盐中作用3h菌株的存活率为100%,活菌数达到109 CFU/mL;0.15 g/100mL胆盐中作用3h活菌数达到107CFU/mL;0.3g/100mL胆盐中作用3h菌株的活菌数达到104 CFU/mL。而商业菌种在0.03 g/100mL胆盐中作用3h菌株的存活率为84.6%,活菌数达到109 CFU/mL;0.15g/100mL胆盐中作用3h活菌数仅达到103 CFU/mL;0.3 g/100mL胆盐中作用3h菌株的活菌数达到102CFU/mL。该菌株的具有显著的耐胆盐性能。
表 2 :LSE-2014ZA001-M012-A-03(a)在不同梯度胆盐中
作用3h的存活率及活菌数
5.菌株胆盐水解酶定性分析
胆盐水解酶表达的定性分析
在新鲜配制的MRS液体培养基中添加0.3g/100mL脱氧牛胆酸钠,0.20g/100mL巯基乙酸钠,0.37g/LCacl2,
1.5g/100mL的琼脂,121℃灭菌15min,倾倒入灭菌平皿中,待凝固后把无菌滤纸片均匀放入平板中,在每个滤纸片上滴加10L的菌液,于37℃培养48h。若纸片周围有白色的沉淀物则认为有胆盐水解酶活性。平皿培养结果见图3,菌液周围的培养基中产生白色沉淀,表明菌株具有胆盐水解酶活性。
6.胆固醇降解能力测定
6.1 胆固醇标准曲线的绘制配制浓度为0 mg/mL、0.04 mg/mL、0.08 mg/mL、0.12 mg/mL、0.16 mg/mL 、0.2 0 mg/mL的胆固醇标准溶液,采用邻苯二甲醛法测定不同浓度胆固醇溶液在550nm处的OD值。按照表3顺序表依次加入如下制剂。
表3 制作标准曲线加入试剂
6.2 胆固醇标准曲线见图4胆固醇标准曲线,所得的方程为y=7.23x R2=0.9991
Y:为吸光度X:为胆固醇标准品的质量。
6.3 菌种的胆固醇降解能力测定
胆固醇胶束溶液的配制:胆固醇0.2g、牛胆盐0.3g、蔗糖酯0.1g、吐温80 1.2 ml,搅拌均匀超声15min,再加5mL冰乙酸水浴加热溶解,用0.45µm无菌滤膜快速过滤至1000mLMRS灭菌培养基中,边加边搅拌,使其形成均匀稳定的胶体溶液。
按照2%接种量接种于胆固醇胶束溶液中,37℃培养分别培养24h、48h、72h、96h。取1mL发酵液于25mL比色管中,测定方法同6.1所述,在550nm处测定吸光度。根据胆固醇标准曲线计算发酵液中胆固醇浓度C1,计算胆固醇降解率。
6.4胆固醇降解率=0.2-C1/0.2*100%。
该菌株发酵96h内的胆固醇降解率见表4。
表4:LSE-2014ZA001-M012-A-03(a)胆固醇降解率
6.5 胆盐水解酶可以将胆盐水解为胆酸和氨基酸,胆酸可以与胆固醇形成形成沉淀从人体内排出。副干酪乳杆菌LSE-2014ZA001-M012-A-03(a)可以表达胆盐水解酶基因,具有胆盐水解酶活性,通过体外试验研究具有降胆固醇的作用。
7.菌种的木聚糖酶基因的扩增与木聚糖酶活性分析
7.1菌种的木聚糖酶基因的扩增
搜索Genebank数据库副干酪乳杆菌木聚糖解酶基因序列设计引物:上游引物AGCATCGCATCCTCAGTCTT,下游引物:CACTGGTGGC TCATTTTCCT。由上海生物工程技术服务有限公司合成,采用PCR扩增试剂盒,以50L的反应体系进行PCR反应。反应参数为:95℃预变性10min,36个循环的参数设置为95℃90s,59℃30s,72℃90s,72℃延长10min。用0.8%琼脂糖凝胶电泳检测扩增DNA片段的大小,测序由上海生物工程技术服务有限公司完成。将经过测序所得到的序列用Blast软件在BLASTX进行比对。
根据设计的引物进行进行木聚糖酶基因的扩增,获得的扩增产物进行电泳,在目的基因处有阳性条带。将扩增产物送至测序公司测序,全长序列如SEQ ID NO.2所示。测序结果通过Blasten比对为副干酪乳杆菌木聚糖酶基因,说明菌株具有表达木聚糖酶的基因。
7.2菌株发酵液中木聚糖酶活力测定
A 木聚糖标准曲线的绘制:取6支25mL比色管,按表5数据分别准确移取木聚糖母液、1mol/L柠檬酸钠缓冲溶液和DNS溶液摇匀沸水浴显色5min,冰水浴迅速冷却后加缓冲溶液摇匀。用分光光度计在540nm处测定标准溶液的OD值,以OD540为纵坐标、木聚糖浓度为横坐标绘制木聚糖标准曲线。木聚糖标准曲线见图5。
表5:木聚糖标准曲线配制
B 木聚糖酶活力测定:按照2%接种量接入MRS液体培养基,37℃培养24 h后离心,取上清液液过滤。取0.1mL的该滤液加入0.4mL,0.8%的木聚糖底物, 1mol/L柠檬酸钠缓冲溶液0.6mL和2mL DNS溶液摇匀沸水浴显色5min加10mL去离子水,混匀后置540nm出测定OD值。酶活单位定义为在40℃、PH4.5时每分钟催化产生1moL还原糖(木聚糖)的酶量。该菌株的酶活为634 Uint/mL。
实施例2: LSE-2014ZA001-M012-A-03(a)作为发酵剂在制备益生菌饮料中的应用
(1)产酸曲线绘制:将活化3代的LSE-2014ZA001-M012-A-03(a)按照2%的接种量,接入蛋白含量3.5%脱脂复原乳中,每24h测定一次酸度,通过产酸曲线评价菌株的发酵能力。LSE-2014ZA001-M012-A-03(a)的产酸曲线见图6。结果表明LSE-2014ZA001-M012-A-03(a)在脱脂复原乳中发酵24小时酸度可以达到176oT,96h可以达到295 oT,本发明菌株按照褐饮的生产工艺LSE-2014ZA001-M012-A-03(a)产酸能力,可以用于褐色活性益生菌饮料的制备。
(2)母种的制备:将活化3代的LSE-2014ZA001-M012-A-03(a),按照2%的接种量,接入蛋白含量为3.7%+0.2的脱脂复原乳中,37℃恒温发酵24 h。
(3)发酵基料制备:
配制蛋白含量为4.5%+0.2%、果葡糖浆含量为1.3% 的脱脂乳700mL,20~30℃,20MPa均质,94℃褐变3 h。
酸液配制:将乳酸和柠檬酸分别配成20% 及12% 的酸液,95 ℃杀菌5 min用于后续调酸。
糖液制备:白糖11%、果葡糖浆1.4%、大豆多糖4%以终产量2000mL计。
(4)接种发酵:母种按照1.5%的接种量接入发酵基料中,37℃发酵培养72 h。酸度达到190 oT破乳,停止发酵。加入制备好的糖液将发酵液补足至2000mL,蛋白含量为1.5%。用酸液调节酸度至63 oT,20 MP均质后无菌灌装,灌装产品于4 ℃贮存,制成褐色活性益生菌饮料。(4)感官评价:褐色活性益生菌饮料4℃冷藏24 h后进行感官评定。
感官评价指标:口感具有该类产品的正常色泽、无肉眼可见的外物、无异味、无异臭、无分层。
口感品尝结果:产品无分层,口感爽滑,质地醇厚,酸香味纯正,无异味,可以作为益生菌饮料的生产发酵剂。
(5)离心沉淀率测定:
A测定方法:褐色活性益生菌饮料4℃冷藏24 h后,称取100 g活性益生菌饮料,3200 r/min离心15 min,弃去上清液液,烘干至恒重,称取沉淀的质量,计算沉淀率(sr)。Sr(%)=m/M* 100,其中M为样品质量,m为沉淀质量。
B测定结果:褐色活性益生菌饮料离心沉淀率测定结果为1.3%,明显低于采用常规菌株的产品。沉淀离心率越低,体系中的不稳定蛋白含量越少,产品稳定性越好。
(6)4℃冷藏24天活菌数及离心沉淀率测定:
A测定方法:在24天4℃冷藏期内,每6天取样一次,用生理盐水进行梯度稀释,用MRS固体培养基进行倒板,37℃培养48h,计算褐色活性益生菌饮料的活菌数。同时采用(5)中所述方法进行离心沉淀率测定。
B测定结果:4℃条件贮藏1周活菌数并无下降趋势,在前期还有轻微的上升趋势,第24天活菌数仍达到1*108CFU/mL,菌株在4 ℃贮藏条件下短期内维持缓慢的生长,到贮藏的后期活菌数减少也不明显,说明本发明菌株在4 ℃贮藏条件下能够维持很好的活力。4℃贮藏24天离心沉淀率最大不超过1.5%,且体系无分层、絮凝的现象,说明LSE-2014ZA001-M012-A-03(a)作为发酵剂制备褐色益生菌饮料的体系稳定,不需要添加果胶、明胶等稳定剂。
实施例3: LSE-2014ZA001-M012-A-03(a)作为发酵剂在制备辣木酸奶中的应用
1、辣木发酵液的制备
辣木干粉添加量:2.5‰(1吨辣木酸奶添加2.5kg辣木干粉)
发酵剂:副干酪乳杆菌LSE-2014ZA001-M012-A-03(a)活菌数109cfu/mL
(1)辣木粉:水:MRS培养液=2.5kg:37.5kg:12.5kg的比例进行配制。
(2)用60~70℃热水将辣木粉溶解,通过胶体磨进行细化处理10~15分钟,直至无结块,将辣木水溶液、营养液移入菌种罐混合;
(3)巴氏杀菌:将菌种罐中物料升88℃,开始计时,温度达到要求后保温60分钟;
(4)巴氏消毒结束,降温至36-38℃,接种发酵剂,接种量1.5%,搅拌均匀后进行发酵.
(5)发酵时间10~15小时,发酵终点酸度95~130°T,PH值:3.70~3.80(32~36℃)
(6)发酵结束后立即冷却降温,冷却达到4-6℃,待用。
2、辣木发酵液PH值调整
(1)生产投入调配前要先将辣木发酵液的PH值调至中性,PH值为6.50~6.70;
(2)先用辣木发酵液重量的2%柠檬酸钠进行调整,将1 kg柠檬酸钠用3~4kg 60~70℃的热水完全溶解,然后边开启搅拌边加入辣木发酵液中;
(3)再用辣木发酵液重量的(2%±0.3%)的磷酸氢二钠溶液调整PH值,1—1.15公斤磷酸氢二钠用4~5kg 60~70℃的热水溶解,待磷酸氢二钠溶解完全,边开启搅拌边将其加入辣木发酵液中;
3、将最终辣木发酵液称重以待用;
4、辣木酸奶调配、杀菌、发酵、后熟冷却、冷藏。
(1)发酵基料制备:
辣木发酵液添加量:所制备的辣木酸奶辣木发酵液浓度为5%,即1吨辣木酸奶产品中添加50kg辣木发酵液;
鲜牛奶以及乳清蛋白粉的添加量:以辣木酸奶的总蛋白质含量为3.4%计算,蛋白质含量不足部分添加乳清蛋白粉;
白糖:8.5%
乳化稳定剂:采用换热板片加热后的热奶来处理,化胶温度要求在80~85℃,时间10分钟。在乳化稳定剂投入后6-7分钟后才可将白糖投入其中溶解为止。
混合以上物料,调节物料的PH值至6.45~6.60;酸度:16~20°T,18-20MPa均质,95℃灭菌300s。
(2)接种发酵剂菌种:嗜热链球菌(活菌数109cfu/mL)与保加利亚乳杆菌(活菌数109cfu/mL)按照3:1制成辣木发酵剂,按照接种量为1.5%接入发酵物料中,搅拌均匀,灌装;
(3)发酵: 42 ℃发酵4~6小时,凝固状态良好,发酵终点酸度65~72°T;
(4)发酵终止,后熟冷却、冷藏。
5. 实施效果
(1)辣木的叶子中含丰富蛋白质、维生素C、胡萝卜素、铁、钙等有 “植物牛奶”之称。但是将辣木叶与牛奶、奶粉等直接混合无法解决辣木口感粗糙和有涩辣味的问题,通过发酵后很好的解决了辣木带来的口感粗糙和苦涩问题,而且提供了一种风味独特、组织状态好、营养健康的辣木酸奶。本发明将从泡梨中分离出来的副干酪乳杆菌LSE-2014ZA001-M012-A-03(a)应用于辣木的发酵, 由于LSE-2014ZA001-M012-A-03(a)发酵产生木聚糖酶且具有较高的活性,能够使辣木更加彻底的发酵。
(2)基于副干酪乳杆菌副干酪乳杆菌LSE-2014ZA001-M012-A-03(a)发酵辣木基料研制的辣木酸奶织状态与色泽良好,既有酸奶固有香味,又有辣木特有风味。成功解决了辣木乳制品苦涩、粗糙等不良的口感问题,赋予酸奶产品更加丰富的氨基酸、维生素、多肽等营养物质。
序列表
[001]
SEQUENCE LISTING
<110> 云南皇氏来思尔乳业有限公司
<120> 一株副干酪乳杆菌及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1422
<212> DNA
<213> 副干酪乳杆菌(Lactobacillus paracasei)
<400> 1
ctcgctccct aaaagggtta cgccaccggc ttcgggtgtt acaaactctc atggtgtgac 60
gggcggtgtg tacaaggccc gggaacgtat tcaccgcggc gtgctgatcc gcgattacta 120
gcgattccga cttcgtgtag gcgagttgca gcctacagtc cgaactgaga atggctttaa 180
gagattagct tgacctcgcg gtctcgcaac tcgttgtacc atccattgta gcacgtgtgt 240
agcccaggtc ataaggggca tgatgatttg acgtcatccc caccttcctc cggtttgtca 300
ccggcagtct tactagagtg cccaactaaa tgctggcaac tagtcataag ggttgcgctc 360
gttgcgggac ttaacccaac atctcacgac acgagctgac gacaaccatg caccacctgt 420
cattttgccc ccgaagggga aacctgatct ctcaggtgat caaaagatgt caagacctgg 480
taaggttctt cgcgttgctt cgaattaaac cacatgctcc accgcttgtg cgggcccccg 540
tcaattcctt tgagtttcaa ccttgcggtc gtactcccca ggcggaatgc ttaatgcgtt 600
agctgcggca ctgaagggcg gaaaccctcc aacacctagc attcatcgtt tacggcatgg 660
actaccaggg tatctaatcc tgttcgctac ccatgctttc gagcctcagc gtcagttaca 720
gaccagacag ccgccttcgc cactggtgtt cttccatata tctacgcatt tcaccgctac 780
acatggagtt ccactgtcct cttctgcact caagtttccc agtttccgat gcgcttcctc 840
ggttaagccg agggctttca catcagactt aaaaaaccgc ctgcgctcgc tttacgccca 900
ataaatccgg ataacgcttg ccacctacgt attaccgcgg ctgctggcac gtagttagcc 960
gtggctttct ggttggatac cgtcacgccg acaacagtta ctctgccgac cattcttctc 1020
caacaacaga gttttacgac ccgaaagcct tcttcactca cgcggcgttg ctccatcaga 1080
cttgcgtcca ttgtggaaga ttccctactg ctgcctcccg taggagtttg ggccgtgtct 1140
cagtcccaat gtggccgatc aacctctcag ttcggctacg tatcatcgcc ttggtgagcc 1200
attacctcac caactagcta atacgccgcg ggtccatcca aaagcgatag cttacgccat 1260
ctttcagcca agaaccatgc ggttcttgga tctatgcggt attagcatct gtttccaaat 1320
acttaagggc aggttaccca cgtgttactc acccgtccgc cactcgttcc atgttgaatc 1380
tcggtgcaag caccgatcat caacgagaac tcgttcgact gc 1422
序列表
[002]
SEQUENCE LISTING
<210> 2
<211> 830
<212> DNA
<213> 副干酪乳杆菌(Lactobacillus paracasei)
<400> 2
atggcagcaa gcaagacgca cgcgaagtcg gtgagtgagc atgaggcggc tgtggcttct 60
tcgcggcggc acgcggcgag gaaggcatca aagcggcatg ttgcggctgt gtcatctcgg 120
cgacgggcag cagcggaggc ttctagggaa gaggcgcagt cgaaagcggc acaggtgggc 180
caaaatcata ttgctgaggc caatcaatat gcttatccag ttgcccaagt caaacaagag 240
atggatgcac cgtatacaag tccaattaaa gagaaggtgg tatttttaac tttcgatgat 300
ggtccgaata cggttaattc gcctaaggtt ttggatattc tcagtcaggc tggcgttcat 360
gggacttttt tcgtcgttgg caagcaggtt agtccagaga cagcgccggt tcttaaggcg 420
gaatatgatg ctgggcatgc aattggatta catagcatga cgcacgatta tagtttgctt 480
tatcctagtc gggtgggagc cacggcagta atcgagaatg aagcgaagag tgcccaagcc 540
cattttagct ggaaggactt ggctgccgca gatgccgcgc ttagtcggct tggtctcgat 600
tggattgatt gggatgcggc agtcggcgat gcgcttggtc cggcgcagga gccgaagact 660
gaggatgcga tgctgcaata ccatctacgt tccctgaatg tttatccggc cagtaacgta 720
cgggttgtct taatgcatga tgcccttggt aaggatctga cggttaaaac gttgccgcgg 780
atcattcagt attatcgtga caatggttat aaatttggcg ttttggaata 830
序列表
<110> 云南皇氏来思尔乳业有限公司
<120> 一株副干酪乳杆菌及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1422
<212> DNA
<213> 副干酪乳杆菌(Lactobacillus paracasei)
<400> 1
ctcgctccct aaaagggtta cgccaccggc ttcgggtgtt acaaactctc atggtgtgac 60
gggcggtgtg tacaaggccc gggaacgtat tcaccgcggc gtgctgatcc gcgattacta 120
gcgattccga cttcgtgtag gcgagttgca gcctacagtc cgaactgaga atggctttaa 180
gagattagct tgacctcgcg gtctcgcaac tcgttgtacc atccattgta gcacgtgtgt 240
agcccaggtc ataaggggca tgatgatttg acgtcatccc caccttcctc cggtttgtca 300
ccggcagtct tactagagtg cccaactaaa tgctggcaac tagtcataag ggttgcgctc 360
gttgcgggac ttaacccaac atctcacgac acgagctgac gacaaccatg caccacctgt 420
cattttgccc ccgaagggga aacctgatct ctcaggtgat caaaagatgt caagacctgg 480
taaggttctt cgcgttgctt cgaattaaac cacatgctcc accgcttgtg cgggcccccg 540
tcaattcctt tgagtttcaa ccttgcggtc gtactcccca ggcggaatgc ttaatgcgtt 600
agctgcggca ctgaagggcg gaaaccctcc aacacctagc attcatcgtt tacggcatgg 660
actaccaggg tatctaatcc tgttcgctac ccatgctttc gagcctcagc gtcagttaca 720
gaccagacag ccgccttcgc cactggtgtt cttccatata tctacgcatt tcaccgctac 780
acatggagtt ccactgtcct cttctgcact caagtttccc agtttccgat gcgcttcctc 840
ggttaagccg agggctttca catcagactt aaaaaaccgc ctgcgctcgc tttacgccca 900
ataaatccgg ataacgcttg ccacctacgt attaccgcgg ctgctggcac gtagttagcc 960
gtggctttct ggttggatac cgtcacgccg acaacagtta ctctgccgac cattcttctc 1020
caacaacaga gttttacgac ccgaaagcct tcttcactca cgcggcgttg ctccatcaga 1080
cttgcgtcca ttgtggaaga ttccctactg ctgcctcccg taggagtttg ggccgtgtct 1140
cagtcccaat gtggccgatc aacctctcag ttcggctacg tatcatcgcc ttggtgagcc 1200
attacctcac caactagcta atacgccgcg ggtccatcca aaagcgatag cttacgccat 1260
ctttcagcca agaaccatgc ggttcttgga tctatgcggt attagcatct gtttccaaat 1320
acttaagggc aggttaccca cgtgttactc acccgtccgc cactcgttcc atgttgaatc 1380
tcggtgcaag caccgatcat caacgagaac tcgttcgact gc 1422
<210> 2
<211> 830
<212> DNA
<213> 副干酪乳杆菌(Lactobacillus paracasei)
<400> 2
atggcagcaa gcaagacgca cgcgaagtcg gtgagtgagc atgaggcggc tgtggcttct 60
tcgcggcggc acgcggcgag gaaggcatca aagcggcatg ttgcggctgt gtcatctcgg 120
cgacgggcag cagcggaggc ttctagggaa gaggcgcagt cgaaagcggc acaggtgggc 180
caaaatcata ttgctgaggc caatcaatat gcttatccag ttgcccaagt caaacaagag 240
atggatgcac cgtatacaag tccaattaaa gagaaggtgg tatttttaac tttcgatgat 300
ggtccgaata cggttaattc gcctaaggtt ttggatattc tcagtcaggc tggcgttcat 360
gggacttttt tcgtcgttgg caagcaggtt agtccagaga cagcgccggt tcttaaggcg 420
gaatatgatg ctgggcatgc aattggatta catagcatga cgcacgatta tagtttgctt 480
tatcctagtc gggtgggagc cacggcagta atcgagaatg aagcgaagag tgcccaagcc 540
cattttagct ggaaggactt ggctgccgca gatgccgcgc ttagtcggct tggtctcgat 600
tggattgatt gggatgcggc agtcggcgat gcgcttggtc cggcgcagga gccgaagact 660
gaggatgcga tgctgcaata ccatctacgt tccctgaatg tttatccggc cagtaacgta 720
cgggttgtct taatgcatga tgcccttggt aaggatctga cggttaaaac gttgccgcgg 780
atcattcagt attatcgtga caatggttat aaatttggcg ttttggaata 830
Claims (3)
1.一株副干酪乳杆菌(Lactobacillus paracaseii)LSE-2014ZA001-M012-A-03(a),其特征在于:它的保藏编号为CGMCC No.14751。
2.根据权利要求1所述的一株副干酪乳杆菌LSE-2014ZA001-M012-A-03(a)在制备益生菌饮料中的应用。
3.根据权利要求1所述的一株副干酪乳杆菌LSE-2014ZA001-M012-A-03(a)在制备辣木酸奶中的应用。
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CN116059258A (zh) * | 2022-11-22 | 2023-05-05 | 重庆市天友乳业股份有限公司 | 副干酪乳杆菌在降低血清胆固醇水平制剂中的应用 |
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