CN1279712A - 一种具有高水平三肽生产能力的瑞士乳杆菌、发酵奶制品及其制备方法 - Google Patents
一种具有高水平三肽生产能力的瑞士乳杆菌、发酵奶制品及其制备方法 Download PDFInfo
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Abstract
本发明涉及能产生大量乳三肽的乳酸菌;以及发酵奶制品,该发酵奶制品含有多种具有降压和应激舒缓活性的成份并且该发酵奶容易吃和吞咽。更具体地说,本发明涉及瑞士乳杆菌,即瑞士乳杆菌CM4株(以保藏号FERM BP- 6060保藏在通商产业省工业技术院生命工学和人体技术研究所)其特征在于:该菌株有特定的细菌学特征,产三肽Val-Pro-Pro和/或Ile-Pro-Pro,当以含不少于9%重量(固体含量)的非脂肪乳动物乳为培养基培养时,Val-Pro-Pro的产量不少于60μg/ml,并且细胞外蛋白酶活性不少于400U/OD590;以及涉及用上述的乳酸菌发酵动物奶得到的发酵产品。
Description
技术领域
本发明涉及一种新型乳酸菌瑞士乳杆菌,该菌种用动物奶培养基培养可高效率地产生一种独特的三肽,并且具有高的胞外蛋白酶活性;含有该乳酸菌的发酵奶制品;该奶制品的制备方法。
技术背景
瑞士乳杆菌长期用作奶制品的典型乳酸菌引酵物生产发酵奶。瑞士乳杆菌具有高的蛋白水解活性,尤其是其高活性的胞外蛋白酶在动物奶发酵中起重要作用,即胞外蛋白酶消化动物奶蛋白质产生各种肽片段。所产生的肽片段经肽酶的作用进一步生成低分子量的肽。众所周知由于蛋白酶的作用,在培养液中产生的一部分肽进入乳酸菌细胞内,作为一种氮源。亦有报道在培养液中产生的某些肽对血管紧张素转化酶(ACE)有抑制活性,血管紧张素转化酶可导致高血压(奶业科学杂志78:777-783(1995))。
已有报道各种有活性的肽可抑制血管紧张素转化酶、抑制血压增高,如从奶蛋白质、黄豆蛋白质和鱼肉蛋白质降解产物分离得到的一些肽。如Val-Pro-Pro和Ile-Pro-Pro(以下分别缩写为VPP和IPP,这类肽在下文中统称为乳三肽)是存在于瑞士乳杆菌发酵奶中具有血管紧张素转化酶抑制活性的肽。采用原发性高血压大鼠(SHR)实验证实这些乳三肽具有很强的降压作用(奶业科学杂志78:1253-1257(1995))。
然而,由于普通的瑞士乳杆菌在发酵过程中产生大量的乳酸,酸性较大,因此以该菌种发酵的动物奶产生的含乳三肽发酵奶很难得到。稀释此发酵奶将导致乳三肽含量极度减少。
因此,需要生产有与发酵奶中乳酸含量相比更高的乳三肽含量的发酵奶。在不同食品和饮料中添加少量该发酵奶,可以方便地生产具有乳三肽功效的产品,并以消费者乐于接受的形式提供给消费者。但是目前已知的乳酸菌菌种没有一种能产生高效的乳三肽。
发明内容
本发明目的之一是提供一种新型乳酸菌菌种,该菌种能高效产生相对于产生的乳酸更大量的乳三肽。
本发明的另一目的是提供了一种发酵乳制品该发酵乳制品的制备方法,在该发酵乳制品中含有具有降血压活性以及预期有应激活物作用的的乳三肽和大量产生该乳三肽的乳酸菌并容易供作饮食。
根据本发明,所提供的乳酸菌瑞士乳杆菌具有下述细菌学特点,当所述的该菌种用含9%(重量百分比)非脂肪乳固形物动物奶培养基培养时,产生的三肽Val-Pro-Pro和Ile-Pro-Pro含量每毫升培养基不低于以Val-Pro-Pro计60μg,该菌种表现出的胞外蛋白质活性不低于400U/OD590 。
形态学特点
1)细胞形状:杆状
2)运动性:无
3)孢子形成:无
4)革兰氏染色:阳性
生理学特点
1)过氧化氢酶产生:阴性
2)吲哚产生:阴性
3)硝酸盐还原:阴性
4)需氧生长:兼性压氧
5)不产气乳清乳酸发酵葡萄糖生成DL-乳酸
6)碳水化合物降解
葡萄糖:+
乳糖:+
甘露糖:+
果糖:+
半乳糖:+
蔗糖:-
麦芽糖:-
木糖:-
鼠李糖:-
纤维二糖:-
海藻糖:-
蜜二糖:-
蜜三糖:-
水苏四糖:-
甘露醇:-
山梨醇:-
七叶苷:-
水杨苷:-
根据本发明,提供了乳酸菌瑞士乳杆菌,其中该乳酸菌是瑞士乳杆菌CM4菌种(1997年8月15日保藏于工业技术院生命工学和人体技术研究所,菌种保藏号FERM BP-6060)。
根据本发明,进一步提供的乳酸菌瑞士乳杆菌的染色DNA用限制性酶PstⅠ和EcoRⅠ消化后可产生15-17kb的DNA片段。
根据本发明还提供一种发酵的奶制品,该制品包含前文提及的乳酸菌和选自Val-Pro-Pro、Ile-Pro-Pro及其混合物的三肽。
根据本发明,还提供一种用该乳酸菌生产发酵奶制品的方法,该方法包括用含有选自具有Val-Pro-Pro和Ile-Pro-Pro序列的肽、蛋白质及其混合物的食品原料的培养基发酵该乳酸菌。
附图简述
图1是实施例2中不同的瑞士乳杆菌种中染色体DNA片段琼脂糖凝胶电泳带型的照片。
发明的优选实施方案
本发明的乳酸菌属瑞士乳杆菌,其特征在于用含9%(重量百分比)非脂肪乳固形物的动物奶培养基培养的乳酸菌产生Val-Pro-Pro和Ile-Pro-Pro三肽,其产量每mL培养基不低于60μg,优选地以Val-Pro-Pro的量计不低于70μg,胞外蛋白酶活性不低于400U/OD590,优选地不低于430U/OD590。所定义的乳三肽生产能力是区分本发明的乳酸菌瑞士乳杆菌与传统乳酸菌的指标。例如,用含9%(重量百分比)非脂肪乳固形物动物奶培养基培养本发明的乳酸菌,产生的乳三肽量以VPP计不低于每mL培养基60μg,该指标用传统的乳酸菌不能达到。通常,用于细菌培养的培养基中非脂肪乳固形物含量越低,产生的乳三肽数量越少。非脂肪乳固形物含量越高,产生的乳三肽数量也越大。
作为指标的乳三肽的生产能力通过下列步骤测定:接种乳酸菌于含9%(重量百分比)非脂肪乳固形物的动物奶如牛奶、山羊奶、马奶及其脱脂乳,37℃ 24小时培养制得发酵奶,15,000 rpm离心1mL发酵奶10min,分离上清测定VPP和IPP含量并换算成VPP含量。由于IPP每单位重量的ACE抑制活性是VPP的1.7倍,所以可根据下式计算换算过的以VPP计的乳三肽含量:换算后的乳三肽含量(每mLVPPμg)
=IPP量(μg/mL)×1.7+VPP量(μg/mL)
乳三肽最大生产能力并没有具体限定,但当取出培养基中包含Val-Pro-Pro和Ile-Pro-Pro序列的蛋白质时可以达到。
以Yamamoto等(Yamamoto N.et al.生物化学杂志(1993)114,740)改进的TWining等(TWining S,分析生物化学143 3410(1984))人的方法测定胞外蛋白酶活性,表示为抑制1%荧光强度的酶量定义为1U/OD590。胞外蛋白酶活性的上限亦没有具体限定,但通常为800U/OD590。
本发明的乳酸菌在发酵过程中可产生较乳酸含量为大的乳三肽。因此,采用本发明的乳酸菌发酵产生的发酵奶含有大量的乳三肽,而用传统的乳酸菌发酵产生的发酵奶含有的乳三肽量与乳酸含量相近。如此发酵产生的乳酸是DL-乳酸。用本发明的乳酸菌发酵产生的乳三肽量以VPP计优选地不低于每mL 30μg,DL-乳酸量每mL 0.01g。乳三肽量的上限没有具体限定,但对细菌来说有可能产生以VPP计每mL发酵奶约50μg,含0.01g的DL-乳酸。DL-乳酸的量大致上与乳三肽的量成比例关系。故当1mL发酵奶产生0.02g DL-乳酸时,乳三肽的产量以VPP计优选地一般不会低于60μg。与此相反,采用传统乳酸菌发酵,以VPP计,在1mL发酵奶中乳三肽的产量最高也不超过30μg,DL-乳酸为0.01g。
作为本发明乳酸菌的实例,瑞士乳杆菌CM4菌种保藏在工业技术院生命工学和人体技术研究所,编号FERM BP-6060(1997年8月15日)。瑞士乳杆菌CM4菌种的细菌学特点如下:1.形态学特点1)细胞形状:杆状2)运动性:无3)孢子形成:无4)革兰氏染色:阳性2.生理学特点1)过氧化氢酶产生:阴性2)吲哚产生:阴性3)硝酸盐还原:阴性4)需氧生长:兼性压氧5)不产气乳清乳酸发酵葡萄糖生成DL-乳酸6)碳水化合物降解
葡萄糖:+
乳糖:+
甘露糖:+
果糖:+
半乳糖:+
蔗糖:+
麦芽糖:-
木糖:-
鼠李糖:-
纤维二糖:-
海藻糖:-
蜜二糖:-
蜜三糖:-
水苏四糖:-
甘露醇:-
山梨醇:-
七叶苷:-
水杨苷:-
上述提及的CM4菌种的细菌学特点与已知的瑞士乳杆菌NCDO-99菌种用Mitsuoka等人(临床检查18,1163(1974))的方法检查时基本一致。但是CM4的下列特点可清楚地与NCDO-99菌种区分开,这些特点Mitsuoka等人的方法中没有描述。
7)胞外蛋白酶活性:不低于400U/OD590。
8)乳三肽生产能力:用含9%(重量百分比)脱脂乳37℃培养24小时产生二种三肽(VPP和IPP),以VPP计含量每mL发酵液中60Pμg或更多。
在8)中乳三肽生产能力用脱脂乳作为非脂肪乳固形物测量。
本发明的乳酸菌菌种可根据下述筛选步骤和胞外蛋白酶活性测定得到。
(1)初步筛选
(ACE抑制活性高的发酵乳的筛选)
待筛选的菌种以含9%(重量百分比)脱脂乳培养基37℃培养24小时,培养结束后测定乳酸菌数量、乳酸酸度、ACE抑制活性。选出产生1×108细胞/mL或更多的乳酸菌、乳酸酸度1.65%(重量百分比)或以上、ACE抑制活性40单位/mL或以上的菌种。ACE抑制活性参照Cushman和Cheung's方法测定(Cushman,DW and Cheung HS,药理学,20,1637(1971))。
(2)第二次筛选
(选择高乳三肽产生能力的菌种)
初筛过的菌种培养液15,000 rpm离心10分钟,所得上清经HPLC进行乳三肽定量。选出产生以VPP计每mL不低于50μg的菌株。
(3)胞外蛋白酶活性的测定
由第二次筛选选出的每种菌株用9%(重量百分比)脱脂乳培养,pH保持在6。在对数生长中期采样,以1%(重量百分比)柠檬酸钠混和,5,000 rpm离心10分钟收集细胞。收集的细胞以50mM β-磷酸甘油洗涤,以50mM Tris-HCl(pH 7.8)缓冲液悬浮,调节浊度(OD590)至1。接着测定细胞表面的蛋白酶活性。必须保证结果与第二次筛选中菌株乳三肽生产能力的相关。
根据上述方法选出的乳酸菌瑞士乳杆菌株可以加以鉴定并跟其他乳酸菌菌株通过前述乳三肽生产能力和胞外蛋白酶活性的测定相区分。
优选的本发明的乳酸菌除上述乳三肽生产能力及胞外蛋白酶活性外,其染色体DNA以限制性酶PstⅠ和EcoRⅠ消化产生15-17kb的DNA片段。因此,通过检查菌株是否具有可产生上述DNA片段的染色体DNA可将本发明的乳酸菌与同属其他菌株清楚地区分开。
根据Leenhouts等(Leenhouts K,(1990)应用环境微生物学,56:2726)人的方法可证实是否存在15-17kb DNA片段,方法如下:提取乳酸菌中的染色体DNA、以EcoRⅠ和PstⅠ消化染色体DNA、0.8%琼脂糖凝胶电泳、分析产生的电泳带型。在电泳中,通过以限制性酶HindⅢ消化λ噬菌体DNA作分子大小标记同时电泳而证实DNA片段的存在。
本发明中的发酵奶制品作为必须成分必须含有上述的乳酸菌和选自VPP、IPP及其混合物的三肽。即本发明的发酵奶制品是含有乳三肽和乳酸菌的发酵奶,经含有包含VPP和/或IPP序列的肽和/或蛋白质的食品原料的培养基用本发明的乳酸菌发酵产生。故乳酸菌和三肽的量可根据待制备的发酵奶制品的种类适当选择。本发酵奶制品可包含发酵产品本身,经稀释过的发酵产品和纯化的发酵产品。
本发明的发酵奶制品包含发酵过程中产生的DL-乳酸。本发明的发酵奶制品优选地含有乳三肽以VPP计数量为30-50μg,而DL-乳酸来说为0.01g。DL-乳酸的含量与乳三肽的含量大致相关。因此,如果发酵奶制品含有浓发酵奶及0.02g DL-乳酸,乳三肽的含量以VPP计优选地为60-100μg。如果发酵奶制品含有稀发酵奶及0.005gDL-乳酸,乳三肽的含量以VPP计优选地为15-25μg。尽管本发明的发酵奶制品可能含有L-乳酸,L-乳酸是一种调节酸度的食品添加剂,可与在发酵过程中产生的DL-乳酸相区分。
本发明发酵奶制品中的乳酸菌在发酵后可以灭活也可不灭菌而存活。
本发明发酵奶制品可以是酸奶酪、乳清乳酸菌饮料、乳酪、含发酵酸奶的加工食品、含发酵酸奶的保健食品。因此,本发明的发酵奶制品可包括除必须成分的发酵奶之外通常用于添加以产生不同产品的各种不同材料。本发明的发酵奶制品可以以颗粒、胶囊、片剂等固体或以糊状、胶状、液体等流体外形存在。
生产本发明发酵奶制品的方法,包括采用所述的乳酸菌在下列培养基中发酵:该培养基含有选自以VPP和IPP作为构成单位的肽、蛋白质及其混合物的食品原料。
培养基中的食品原料可以含有以VPP和IPP为构成单位的肽和/或蛋白质,例如,这类食品材料可以是动物奶、奶酪蛋白、谷物、谷物蛋白质、小麦、小麦蛋白质、黄豆、黄豆奶、脱脂黄豆、黄豆蛋白质及其混合物。具体而言含有动物奶的食品材料,如牛奶、羊奶、马奶或其脱脂乳的食品材料是优选的。动物奶中非脂肪乳固形成分没有特别要求,但一般为5%-20%(重量百分比)。
在培养基中接种乳酸菌的数量没有什么特别的限制。一般为培养基中1克某-特定的食品原料接种乳酸菌大约105-107个细胞。
发酵温度25-50℃,优选地在30-45℃,发酵时间6-30小时,优选地10-24小时,pH范围最好在3.0-4.0,优选地在3.0-3.5。
发酵最好进行到以VPP计每毫升发酵奶中乳三肽含量不低于60μg。具体地说,当用含非脂肪乳固形物9%(重量百分比)的牛奶做培养基时,25-40℃发酵12-48小时,得到的发酵奶含乳三肽的量以VPP计每毫升不低于70μg。培养基中非脂肪乳固形物的含量与产生的乳三肽含量大致相关。例如,如果食品材料含5%(重量百分比)非脂肪乳固形物,则以上述发酵条件发酵产生的乳三肽量以VPP计每mL约为33.3μg。
以上述发酵条件获得的发酵奶可以与其产物相混合,必要时在混合前稀释或纯化发酵奶。发酵奶可以在5℃冷冻贮藏,之后与其他成分混合生产如冷冻食品之类的产品。发酵奶也可以加热灭菌,必要时喷雾干燥制成粉末状产品以便于常温下分装。
由于本发明的发酵奶制品含有用乳酸菌发酵而得到的发酵奶,因此可以用来简便生产相对于乳酸含量有较高的乳三肽含量的产品,并以乐于接受的外形存在。发酵奶制品应用于人体时预期具有乳三肽的降血压功效和抗应激功效。
由于本发明的乳酸菌通过与特定的食品材料培养可产生大量的乳三肽,因此在各种发酵奶制品、功能食品、保健食品、用于某种特定健康用途的食品、老年人专用食品及其原料中用途很广,这些食品都具有乳三肽的降血压功效和抗应激功效。
实施例
以下通过实施例详细介绍本发明,但本发明不限于这些实施例。
在实施例采用的瑞士乳杆菌菌株中,CM4菌株保藏在工业技术院生命工学和人体技术研究所,编号FERM BP-6060。ATCC 15009、NCDO-099、JCM 1006、ATCC 10797、JCM 1062、JCM 1103、JCM 1120和JCM 1004是公开的有供应的菌株。实施例中上述菌株之外的菌株来自申请人的菌种保藏。
实施例1
(能产生高ACE抑制活性的发酵奶的菌株的选择)
筛选从不同奶制品中分离得到的36株瑞士乳杆菌。每种菌株发酵后奶中的ACE抑制活性参照下述方法测定。每种瑞士乳杆菌以9%(重量百分比)非脂肪乳固形物培养基在37℃培养24小时。将培养过的培养基加到同一类型的新鲜培养基中,新培养基含已培养的培养基3%(重量百分比)。在37℃进一步发酵24小时,发酵结束后,测定乳酸酸度(重量百分比)、乳清中肽含量(μg/mL)、细胞数目和ACE抑制活性(U/mL)。结果见表1。36种菌株中有7株发酵能力极弱。15株产生的发酵奶中乳酸酸度不低于1.6%(重量百分比)。在这15株中有8株发酵奶中乳清的ACE抑制活性不低于400U/mL。
(发酵奶ACE抑制活性的测定)
根据Cushman和Cheung方法(Cushman DW,Cheung HS,药理学,20 1637(1971))测定ACE抑制活性。即在15,000 rpm离心每一种发酵液5分钟获得上清(乳清)。将乳清作适当稀释待测。取80μL稀释的乳清于试管中,与含5mM马尿酸组氨酸亮氨酸(Hip-His-Leu)(Sigma化学公司出品)作底物的0.1M硼酸缓冲液(含0.3M NaCl,pH 7.3)相混合,再与20μL酶液(0.1U/mL,Sigma化学公司出品)混合。将混合液在37℃反应30分钟,加入250μL 1N盐酸终止反应。再加入1.7mL乙酸乙酯,振荡20秒,3000 rpm离心10分钟,分离上层1.4mL乙酸乙酯层。将该层在120℃加热40分钟以挥去溶剂,加1mL蒸馏水,振荡约20秒。提取出的马尿酸在228nm测其吸收值。酶活性以下列公式计算,50%ACE活性抑制定义为1个单位。
酶量(单位)=((A-B)/(A-C))×100×1/50
A:228nm空白对照品吸收值
B:228nm待测样品吸收值
C:228nm既无酶又无样品吸收值
(发酵奶中肽含量的定量分析)
肽的定量分析根据OPA法(Charch FC,et al.奶业科学杂志66 1219(1883))进行。采用胰酶消化的酪蛋白作为制作标准曲线的标准物质。
表1
菌株 | 酸度(重量%) | 肽含量(mg/ml) | 细胞数(×108/ml) | ACE抑制活性(U/ml) |
分离株1 | - | - | - | - |
分离株2 | - | - | - | - |
分离株3 | - | - | - | - |
分离株4 | - | - | - | - |
分离株5 | - | - | - | - |
分离株6 | - | - | - | - |
分离株7 | - | - | - | - |
分离株8分离株9分离株10分离株11分离株12分离株13分离株14分离株15分离株16分离株17分离株18分离株19分离株20分离株21分离株22分离株23分离株24分离株25分离株26分离株27分离株28分离株29JCM 1006JCM 1062JCM 1103ATCC 10797ATCC 15009NCDO-099CM4 | 0.4982.0221.7090.6150.4110.9171.0260.5171.5322.1011.7831.9552.0951.9631.7981.6041.9321.8851.8621.0630.4570.5161.8721.1091.2441.3591.4541.7691.635 | 1.591.992.101.761.351.571.711.594.692.011.941.691.742.032.852.321.771.511.463.011.982.552.352.601.362.111.812.763.12 | 0.299.538.531.280.383.635.780.565.976.095.385.317.166.056.196.817.974.915.692.780.501.136.978.503.698.565.166.594.44 | 26.434.524.529.117.619.99.426.9102.598.921.9100.661.4125.354.236.647.718.326.276.952.492.648.578.431.013.816.625.5130.0 |
(具有高乳三肽产生能力的菌株的选择)
测定前述选出的8株能产生具有高ACE抑制活性的发酵奶的菌株发酵奶中的VPP和IPP。
1mL发酵奶15,000 rpm离心10分钟。收集上清即乳清。以Sep-Pak Cartridge(Waters公司出品)吸附0.3mL乳清、蒸馏水洗涤、用5mL甲醇洗脱。在离心低压下干燥洗脱液。将干燥产物溶解于0.3mL0.05%三氟乙酸水溶液,在下述条件下进行HPLC(高效液相色谱)分析。结果见表2。采用的仪器:
日立L4000 UV检测器(检测波长215nm)
L6200智能性泵
L5030柱箱(柱温35℃)洗脱条件:流速0.5mL/min洗脱液:含0.3M NaCl和0.05 %三氟乙酸水溶液色谱柱:Asahipak GS 320(φ.9×600mm)
因每单位重量IPP的ACE抑制活性是VPP的1.7倍,以VPP计乳三肽的含量可根据下式计算出IPP和VPP的量。结果见表2。
转换后乳三肽量(以VPP计每毫升μg)
=IPP量(μg/ml)×1.7+VPP量(μg/ml)
表2
菌株 | 肽含量(μg/ml乳清) | 酸度(重量%) | ||
VPP | IPP | 以VPP计乳三肽含量 | ||
分离株17分离株19分离株20分离株21分离株22分离株24JCM 1006CM4 | 15.211.213.016.615.812.612.938.5 | 11.17.38.111.412.18.79.323.5 | 34.023.726.836.036.327.428.678.5 | 1.51.41.61.61.51.61.31.9 |
CM4发酵奶中乳三肽含量最大,以IPP计达78.5μg/mL乳清。另7种菌株乳三肽含量平均为34.2μg/mL乳清。
(胞外蛋白酶的测定)
对表1 16株具有良好发酵能力的菌株进行胞外蛋白酶活性测定。参照Yamamoto等(Yamamoto N,et al.生物化学杂志(1993)114,740)在TWining方法(TWining.S.分析生物化学143,3410(1984))基础上改进的方法进行。即以9%(重量百分比)脱脂乳培养基培养各种菌株,pH保持在6.0。在对数生长期中间采样与柠檬酸钠混和,终浓度为1%(重量百分比),以澄清奶培养基。将混合物在5,000rpm离心10min收集细胞。用50mM β-磷酸甘油洗涤,再用50mM Tris-HCl缓冲液(pH 7.8)混悬,调整浊度(OD590,即测定590nm吸收值)为1。取30μL混悬液与20μL 0.4%荧光素酪蛋白(Sigma化学公司出品)混匀,42℃温育l小时。混合液进一步与120μL 5%三氯乙酸混和,放量20分钟,15,000 rpm离心10分钟。取60μL上清加入3mL 500mM Tris-HCl缓冲液(pH 8.3),通过检查在525nm产生的荧光测定其荧光强度,激发光波长490nm。胞外蛋白酶活性单位以上述条件下1%荧光强度的酶量定义为1个单位加以计算。结果见表3。
表3
菌株 | U/OD590 |
分离株17分离株18分离株19分离株20分离株21分离株22分离株23分离株24分离株25分离株26JCM 1006JCM 1062JCM 1103ATCC 15009ATCC 10797NCDO-099CM4 | 136.7102.8103.289.980.1243.3116.6116.6192.6108.4185.796.5176.3168.1106.5229.7452.6 |
瑞士乳杆菌CM4具有最高的活性,达450U/OD590。其余16株平均活性为141U/OD590,约为CM4菌株的三分之一。
实施例2
从实施例l中36株瑞士乳杆菌中选出11株,参照Leenhouts等方法(Leenhouts K(1990)应用环境微生物学56:2726)提取染色体DNA,以几种限制性酶消化,在0.8%琼脂糖凝胶电泳上分析电泳带型。
结果,以EcoRⅠ和PsⅡ消化的CM4菌株染色体DNA片段中可观察到特征性的DNA片段(见图1箭头1)。在其余菌株的染色体片段中没有观察到,但在大多数菌株中可观察到比CM4特征性片段较短的片段(见图1箭头2)。以λ噬菌体DNA HindⅢ消化后产物作为分子大小标记(23.1kb,9.4kb,6.6kb,4.4kb,2.3kb,2.0kb,泳动性依次加快)比较电泳结果,特征性片段的分子量约为16kb。由此,以EcoRⅠ和PstⅠ酶切,可证实CM4菌株染色体DNA可产生分子量约为16kb的DNA片段,同时也证实CM4之外的其它菌株染色体DNA可产生分子量约13kb的常见的DNA片段。
实施例3
以实施例1选出的瑞士乳杆菌CM4生产发酵奶。CM4菌株在100g 9%(重量百分比)脱脂乳中37℃培养12h,之后与3kg的新鲜培养基相混合在37℃继续培养12小时。发酵之后,全部发酵奶(CM4细胞数目6.3×108/mL)作100kg 9%(重量百分比)脱脂乳的促酵物在32℃培养20小时。发酵结束后,发酵奶中含有74.8μg/mL乳三肽,乳酸含量为1.9%(重量百分比)。
取43kg已制得的发酵奶与4kg颗粒蔗糖、3kg水、0.15kg高甲氧基果胶匀浆制得50kg酸奶酪饮品。酸奶酪饮料有较好的适口感,pH 3.6,活CM4细胞数为4.6×108/mL。
实施例4
从实施例3中得到的发酵奶26.5kg与45.0kg颗粒蔗糖、4.7kg浓麦芽糖浆和13.8kg水混合,边搅拌边加入10kg 3%高甲氧基果胶溶液。所得混合物以实验室匀浆器(ATV GAULIN公司出品,型号15M-8BA)在150 kg/cm2处理压力下、流速2500mL/min匀浆。所得匀浆液与香草香精混和,加热至85℃灭菌。灭菌过的发酵奶趁热灌装到200mL玻璃瓶中。测定灭过菌的发酵奶中乳三肽含量,可发现乳三肽含量与灭菌前发酵奶中乳三肽含量相当。同时也测得乳酸含量为0.5%(重量百分比)。
Claims (11)
1.具有下列细菌学特点的乳酸菌瑞士乳杆菌种,该细菌在用含9%(重量百分比)脱脂乳固形物的动物奶培养基培养时,产生Val-Pro-Pro和Ile-Pro-Pro三肽,以Val-Pro-Pro计在培养基中不低于60μg/ml,该细菌具有不低于400U/OD590的胞外蛋白酶活性:
形态学特点
1)细胞形状:杆状
2)运动性:无
3)孢子形成:无
4)革兰氏染色:阳性
生理学特性
1)过氧化氢酶产生:阴性
2)吲哚产生:阴性
3)硝酸盐还原:阴性
4)需氧生长:兼性厌氧
5)乳清乳酸发酵萄萄糖生成DL-乳酸而不产气
6)碳水化合物降解
葡萄糖:+
乳糖:+
甘露糖:+
果糖:+
半乳糖:+
蔗糖:-
麦芽糖:-
木糖:-
鼠李糖:-
纤维二糖:-
海藻糖:-
蜜二糖:-
蜜三糖:-
水苏四糖:-
甘露醇:-
山梨醇:-
七叶苷:-
水杨苷:-
2.权利要求1的乳酸菌瑞士乳杆菌,所述的乳酸菌是瑞士乳杆菌CM4菌株(保藏在工业技术院生命工学和人体技术研究所,保藏号:FERM BP-6060)。
3.权利要求1的乳酸菌瑞士乳杆菌,其中所述的乳酸菌染色体DNA以限制性酶PstⅠ和EcoRⅠ消化时,可得到15-17kb的DNA片段。
4.一种发酵奶制品,包括权利要求1的乳酸菌和一种选自Val-Pro-Pro、Ile-Pro-Pro及其混合物的三肽。
5.权利要求4的发酵奶制品,进一步包括DL-乳酸,其中该奶制品中每0.01g DL-乳酸含有以Val-Pro-Pro计30-50μg的三肽。
6.权利要求4的发酵奶制品,其中权利要求1的乳酸菌为活菌。
7.权利要求4的发酵奶制品,其中所述的发酵奶制品选自酸奶酪、乳清乳酸菌饮料、干奶酪、含发酵酸奶的加工食品、含发酵酸奶的保健食品。
8.权利要求4的发酵奶制品的制备方法,包括用下述培养基发酵权利要求1的乳酸菌,其中该培养基含有选自以Val-Pro-Pro和Ile-Pro-Pro为构成单位的肽,蛋白质及其混合物的食品原料。
9.权利要求8的方法,其中所述的食品原料选自动物奶、奶酪蛋白、谷物、谷物蛋白质、小麦、小麦蛋白质、大豆、大豆乳、脱脂大豆、大豆蛋白质及其混合物。
10.权利要求8的方法,其中发酵方法为在25-50℃进行6-60小时。
11.权利要求8的方法,其中发酵在这种条件下进行,结果在发酵奶中产生的三肽Val-Pro-Pro和Ile-Pro-Pro量以Val-Pro-Pro计每mL不低于60μg。
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JP9277949A JP3028411B2 (ja) | 1997-09-26 | 1997-09-26 | トリペプチド高生産性ラクトバチルス・ヘルベチカス乳酸菌 |
JP277949/1997 | 1997-09-26 |
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US (2) | US6534304B1 (zh) |
EP (1) | EP1016709B1 (zh) |
JP (1) | JP3028411B2 (zh) |
KR (1) | KR100404154B1 (zh) |
CN (1) | CN1149279C (zh) |
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AT (1) | ATE294230T1 (zh) |
AU (1) | AU734422B2 (zh) |
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MY (1) | MY118157A (zh) |
PL (1) | PL188675B1 (zh) |
PT (1) | PT1016709E (zh) |
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- 1998-02-05 BR BR9813218-0A patent/BR9813218A/pt not_active Application Discontinuation
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Cited By (9)
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CN100402089C (zh) * | 2003-03-18 | 2008-07-16 | 三得利株式会社 | 血管紧张素转化酶抑制肽 |
US8097246B2 (en) | 2005-06-24 | 2012-01-17 | Calpis Co., Ltd. | Promoter of differentiation and keratinization of epidermic cell and functional beverage/food for promotion of keratinization of epidermis |
CN102098923A (zh) * | 2008-06-12 | 2011-06-15 | 雀巢产品技术援助有限公司 | 用于产生降血压肽的瑞士乳杆菌菌株 |
CN102098923B (zh) * | 2008-06-12 | 2014-01-15 | 雀巢产品技术援助有限公司 | 用于产生降血压肽的瑞士乳杆菌菌株 |
TWI551684B (zh) * | 2010-11-09 | 2016-10-01 | Asahi Group Holdings Ltd | Swiss lactic acid bacteria with high proteolytic activity |
CN102960444A (zh) * | 2012-11-19 | 2013-03-13 | 陕西科技大学 | 一种基于瑞士乳杆菌发酵的含ace抑制肽的羊奶的制备方法 |
CN104450574A (zh) * | 2014-12-10 | 2015-03-25 | 生合生物科技(南京)有限公司 | 一种瑞士乳杆菌lh43及其制作乳酸菌饮料的方法 |
CN109517763A (zh) * | 2018-12-27 | 2019-03-26 | 内蒙古农业大学 | 瑞士乳杆菌h11及其应用 |
CN109517763B (zh) * | 2018-12-27 | 2022-03-22 | 内蒙古农业大学 | 瑞士乳杆菌h11及其应用 |
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