CN115551991A - 乳酸菌增殖促进剂 - Google Patents
乳酸菌增殖促进剂 Download PDFInfo
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- CN115551991A CN115551991A CN202180034794.1A CN202180034794A CN115551991A CN 115551991 A CN115551991 A CN 115551991A CN 202180034794 A CN202180034794 A CN 202180034794A CN 115551991 A CN115551991 A CN 115551991A
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- peptide
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- lactic acid
- lactobacillus
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Abstract
提供具有乳酸菌增殖促进作用的乳酸菌增殖促进剂。乳酸菌增殖促进剂包含选自由第1肽和第2肽组成的组中的至少1种肽,其中所述第1肽的总残基数为9以下,在C末端具有疏水性氨基酸残基,所述第2肽的总残基数为9以下,疏水性氨基酸残基及脯氨酸残基的总数相对于总残基数的比为2/3以上。
Description
技术领域
本发明涉及乳酸菌增殖促进剂。
背景技术
已知乳酸菌表现出复杂的营养要求性。在乳酸菌的培养中,由于培养基原料的品质的偏差等,有时所得到的乳酸菌的产量等产生偏差。关于此,提出了促进乳酸菌等的增殖的因子。例如,国际公开第2017/034011号中,提出了包含特定环二肽的肠内有益菌的增殖剂。此外,国际公开第2011/027719号中,提出了以κ-酪蛋白糖巨肽作为有效成分包含的乳杆菌属乳酸菌的增殖促进剂。
发明内容
发明要解决的课题
本发明的目的在于提供具有乳酸菌的增殖促进作用的乳酸菌增殖促进剂。
用于解决课题的方案
用于解决上述课题的具体方案如下,本发明包含以下方式。第1方式为一种乳酸菌增殖促进剂,其包含选自由第1肽和第2肽组成的组中的至少1种肽,上述第1肽的总残基数为9以下,在C末端具有疏水性氨基酸残基,上述第2肽的总残基数为9以下,疏水性氨基酸残基及脯氨酸残基的总数相对于总残基数的比为2/3以上。
在一个实施方式中,上述疏水性氨基酸残基可以为选自由丙氨酸(A)、甘氨酸(G)、缬氨酸(V)、异亮氨酸(I)、亮氨酸(L)、苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)、甲硫氨酸(M)及半胱氨酸(C)组成的组中的氨基酸的残基,也可以为选自由丙氨酸(A)、甘氨酸(G)、苯丙氨酸(F)及酪氨酸(Y)组成的组中的氨基酸的残基。
在一个实施方式中,上述第1肽的总残基数为6以下,上述疏水性氨基酸残基可以为选自由苯丙氨酸(F)及酪氨酸(Y)组成的组中的氨基酸的残基。在一个实施方式中,上述第1肽的疏水性氨基酸残基及脯氨酸残基的总数相对于总残基数的比可以为2/3以上。
在一个实施方式中,上述第2肽的疏水性氨基酸残基及脯氨酸残基的总数相对于总残基数的比可以为3/4以上。在一个实施方式中,上述第2肽的总残基数可以为4以下。在一个实施方式中,上述第2肽可以由疏水性氨基酸残基及脯氨酸残基组成。在一个实施方式中,上述第2肽可以在C末端具有疏水性氨基酸残基。
发明效果
根据本发明,能够提供具有乳酸菌的增殖促进作用的乳酸菌增殖促进剂。
附图简单说明
图1为示出对于加氏乳杆菌(Lactobacillus gasseri)的各种肽的增殖促进效果的图。
图2为示出对于加氏乳杆菌的二肽和氨基酸混合物的增殖促进效果的图。
图3为示出对于嗜酸乳杆菌(Lactobacillus acidophilus)的各种肽的增殖促进效果的图。
图4为示出对于食淀粉乳杆菌(Lactobacillus amylovorus)的各种肽的增殖促进效果的图。
图5为示出对于弯曲乳杆菌(Lactobacillus curvatus)的各种肽的增殖促进效果的图。
图6为示出对于植物乳杆菌(Lactobacillus plantarum)的各种肽的增殖促进效果的图。
图7为示出对于短乳杆菌(Lactobacillus brevis)的各种肽的增殖促进效果的图。
图8为示出对于加氏乳杆菌的二肽的增殖促进效果的图。
发明的具体实施方式
本说明书中,组成物中的各成分的含量在组成物中存在多个与各成分相应的物质时,意味着组成物中存在的该多个物质的合计量,除非另外说明。以下,详细说明本发明的实施方式。但是,以下示出的实施方式仅是用于具体说明本发明的技术思想而例示的乳酸菌增殖促进剂,本发明并不限定于以下示出的乳酸菌增殖促进剂。
乳酸菌增殖促进剂
乳酸菌增殖促进剂可以包含第1肽的至少1种,上述第1肽的总残基数为9以下,在C末端具有疏水性氨基酸残基。此外,乳酸菌增殖促进剂可以包含第2肽的至少1种,上述第2肽的总残基数为9以下,疏水性氨基酸残基及脯氨酸残基的总数相对于肽的总残基数的比为2/3以上。具有特定结构的肽具有乳酸菌增殖作用。通过包含选自由具有乳酸菌增殖作用的第1肽及第2肽组成的组中的至少1种肽,乳酸菌增殖促进剂能够示出优秀的乳酸菌增殖促进效果。
在此,本说明书中的氨基酸包括蛋白质合成中所用的氨基酸,这些优选为L-氨基酸。蛋白质合成中所用的氨基酸为在基因上作为密码子编码的20种氨基酸。蛋白质合成中所用的氨基酸包括疏水性氨基酸、亲水性氨基酸及脯氨酸(P)。疏水性氨基酸包括由丙氨酸(A)、甘氨酸(G)、缬氨酸(V)、异亮氨酸(I)及亮氨酸(L)组成的脂肪族疏水性氨基酸,由苯丙氨酸(F)、酪氨酸(Y)及色氨酸(W)组成的芳香族疏水性氨基酸,由甲硫氨酸(M)及半胱氨酸(C)组成的含硫疏水性氨基酸。此外,亲水性氨基酸包括由赖氨酸(K)、精氨酸(R)及组氨酸(H)组成的碱性亲水性氨基酸,由天冬氨酸(D)、谷氨酸(E)组成的酸性亲水性氨基酸,由丝氨酸(S)、苏氨酸(T)、天冬酰胺(N)及谷氨酰胺(Q)组成的中性亲水性氨基酸。需要说明的是,脯氨酸(P)为亚氨基酸,在本说明书中,有时称作特殊氨基酸。
构成乳酸菌增殖促进剂中所包含的肽的氨基酸,可以包括蛋白质合成中所用的上述20种氨基酸,可构成包含除了蛋白质合成中所用的氨基酸以外的硒代半胱氨酸、吡咯赖氨酸、N-甲酰甲硫氨酸、焦谷氨酸、胱氨酸、羟脯氨酸、羟赖氨酸、甲状腺素、O-磷酸丝氨酸等翻译后修饰所生成的氨基酸的蛋白质的氨基酸,β-丙氨酸、肌氨酸、鸟氨酸、瓜氨酸、肌酸、γ-氨基丁酸(GABA)、冠瘿碱、三甲基甘氨酸等通常不含在蛋白质中的氨基酸。
乳酸菌增殖促进剂中所包含的第1肽及第2肽的总残基数分别可以为9以下,优选为8以下、7以下、6以下、5以下、或4以下。此外,第1肽及第2肽的总残基数分别可以为2以上,优选为3以上、或4以上。第1肽及第2肽的总残基数可以是相同的,也可以是不同的。
第1肽在C末端具有疏水性氨基酸残基。第1肽的C末端所具有的疏水性氨基酸残基可以为选自由丙氨酸(A)、甘氨酸(G)、缬氨酸(V)、异亮氨酸(I)、亮氨酸(L)、苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)、甲硫氨酸(M)及半胱氨酸(C)组成的组中的至少1种。此外,第1肽的C末端所具有的疏水性氨基酸残基优选为选自由丙氨酸(A)、甘氨酸(G)、苯丙氨酸(F)及酪氨酸(Y)组成的组中的至少1种,更优选为由苯丙氨酸(F)及酪氨酸(Y)组成的组中的至少1种。
第1肽的总残基数为6以下,在C末端可以具有选自由苯丙氨酸(F)及酪氨酸(Y)组成的组中的至少1种疏水性氨基酸残基。此外,第1肽的疏水性氨基酸残基及脯氨酸残基的总数相对于总残基数的比(疏水性氨基酸残基数+脯氨酸残基数)/总残基数,以下,也称作“特定残基比”)可以为2/3以上,优选特定残基比为3/4以上。
以下例示第1肽的具体例,但本发明并不限定于此。例如,第1肽可以包括在以下具体例的氨基酸序列中任意的1残基或2残基被插入、缺失、取代或添加了的肽且具有乳酸菌增殖促进作用的肽。此外,乳酸菌增殖促进剂可以单独包含1种第1肽,也可以组合包含2种以上。
[表1]
肽序列 | C末端 | 总残基数 | 特定残基比 | 序列号 |
VPAF | F | 4 | 4/4 | 1 |
LPWG | G | 4 | 4/4 | 2 |
APKPDYPIA | A | 9 | 7/9 | 3 |
PVF | F | 3 | 3/3 | - |
PSVF | F | 4 | 3/4 | 4 |
AGPQTF | F | 6 | 4/6 | 5 |
PVY | Y | 3 | 3/3 | - |
LPLG | G | 4 | 4/4 | 7 |
SEYPPLG | G | 7 | 5/7 | 8 |
IPGI | I | 4 | 4/4 | 9 |
LF | F | 2 | 2/2 | - |
IF | F | 2 | 2/2 | - |
LPVL | L | 4 | 4/4 | 11 |
VPI | I | 3 | 3/3 | - |
FL | L | 2 | 2/2 | - |
VPL | L | 3 | 3/3 | - |
VPVG | G | 4 | 4/4 | 12 |
第2肽的总残基数为9以下,疏水性氨基酸残基及脯氨酸残基的总数相对于总残基数的比(特定残基比)为2/3以上,优选特定残基比为3/4以上、或为1。在此,特定残基比为1是意味着第2肽仅由疏水性氨基酸残基及脯氨酸残基构成。第2肽的总残基数可以为6以下,优选为4以下。此外,第2肽的总残基数可以为2以上,优选为3以上。进而,第2肽可以在C末端具有疏水性氨基酸残基。
第2肽中的疏水性氨基酸残基可以为选自由丙氨酸(A)、甘氨酸(G)、缬氨酸(V)、异亮氨酸(I)、亮氨酸(L)、苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)、甲硫氨酸(M)及半胱氨酸(C)组成的组中的至少1种。此外,第2肽中的疏水性氨基酸残基优选为选自由丙氨酸(A)、甘氨酸(G)、苯丙氨酸(F)及酪氨酸(Y)组成的组中的至少1种,更优选为由苯丙氨酸(F)及酪氨酸(Y)组成的组中的至少1种。
以下举出第2肽的具体例,但本发明并不限定于此。例如,第2肽可以包括在以下具体例的氨基酸序列中任意的1残基或2残基被插入、缺失、取代或添加了的肽且具有乳酸菌增殖促进作用的肽。此外,乳酸菌增殖促进剂可以单独包含1种第2肽,也可以组合包含2种以上。
[表2]
肽序列 | C末端 | 总残基数 | 特定残基比 | 序列号 |
VPAF | F | 4 | 4/4 | 1 |
LPWG | G | 4 | 4/4 | 2 |
APKPDYPIA | A | 9 | 7/9 | 3 |
PVF | F | 3 | 3/3 | - |
PSVF | F | 4 | 3/4 | 4 |
AGPQTF | F | 6 | 4/6 | 5 |
PVY | Y | 3 | 3/3 | - |
IPIT | T | 4 | 3/4 | 6 |
LPLG | G | 4 | 4/4 | 7 |
SEYPPLG | G | 7 | 5/7 | 8 |
IPGI | I | 4 | 4/4 | 9 |
LF | F | 2 | 2/2 | - |
IPIQ | Q | 4 | 3/4 | 10 |
IF | F | 2 | 2/2 | - |
LPVL | L | 4 | 4/4 | 11 |
VPI | I | 3 | 3/3 | - |
FL | L | 2 | 2/2 | - |
VPL | L | 3 | 3/3 | - |
LVE | E | 3 | 2/3 | - |
LPT | T | 3 | 2/3 | - |
VPVG | G | 4 | 4/4 | 12 |
乳酸菌增殖促进剂除了选自由第1肽及第2肽组成的组中的至少1种以外,还可以包含具有乳酸菌增殖促进作用的其它肽的至少1种。以下举出其它肽的具体例,但本发明并不限定于此。需要说明的是,乳酸菌增殖促进剂可以单独包含1种其它肽,也可以组合包含2种以上。
[表3]
肽序列 | C末端 | 总残基数 | 特定残基比 | 序列号 |
WDESRFQ | Q | 7 | 2/7 | 13 |
HDDKHIIVD | D | 9 | 3/9 | 14 |
PNVLPETE | E | 8 | 4/8 | 15 |
乳酸菌增殖促进剂中所包含的肽可以是通过例如对于制造批次不同的乳酸菌用培养基的成分的差分分析来选择的。成分的差分分析能够用例如液相层析-质量分析法(LC-MS)来实施。
作为本说明书中的乳酸菌,能够举出属于乳杆菌、乳球菌的种,例如加氏乳杆菌(Lactobacillus gasseri;例如FERM BP-11331)、嗜酸乳杆菌(Lactobacillusacidophilus;FERM BP-4981)、食淀粉乳杆菌(Lactobacillus amylovorus;FERM BP-11255)、弯曲乳杆菌(Lactobacillus curvatus;NITE P-02033)、植物乳杆菌(Lactobacillus plantarum)、短乳杆菌(Lactobacillus brevis)、干酪乳杆菌(Lactobacillus casei)、副干酪乳杆菌(Lactobacillus paracasei)、鼠李糖乳杆菌(Lactobacillus rhamnosus)、瑞士乳杆菌(Lactobacillus helveticus)、发酵乳杆菌(Lactobacillus fermentum)、短乳杆菌(Lactobacillus brevis)、植物乳杆菌(Lactobacillus plantarum)、唾液乳杆菌(Lactobacillus salivarius)、罗伊氏乳杆菌(Lactobacillus reuteri)、约氏乳杆菌(Lactobacillus johnsonii)、詹氏乳杆菌(Lactobacillus jensenii)、卷曲乳杆菌(Lactobacillus crispatus)、德氏乳杆菌(Lactobacillus delbrueckii)、玉米乳杆菌(Lactobacillus zeae)、母鸡乳杆菌(Lactobacillus gallinarum)、乳酸乳球菌(Lactococcus lactis)等,优选为选自由这些组成的组中的至少1种。更优选乳酸菌可以为选自由加氏乳杆菌、嗜酸乳杆菌、食淀粉乳杆菌、弯曲乳杆菌、植物乳杆菌及短乳杆菌组成的组中的至少1种。
将对照(control)的增殖率设为100%时,乳酸菌增殖促进剂对乳酸菌的增殖促进效果可以例如大于100%,优选为105%以上、110%以上、或120%以上。乳酸菌的增殖促进效果能够用向培养基中以0.25mg/mL添加乳酸菌增殖促进剂、在37℃厌氧培养18小时后的菌体数的增加率来进行评价。
乳酸菌增殖促进剂中所包含的肽能够通过例如使合适的蛋白酶与食用等蛋白质进行作用而得到。此外,能够从食用肉提取物、酵母提取物等中分离而得到。进而,也能够通过化学合成而得到。肽的化学合成中能够适当采用通常所使用的方法。
乳酸菌增殖促进剂能够用于乳酸菌的制造、使用乳酸菌的发酵物的制造等。乳酸菌的制造中,在例如以规定量添加了乳酸菌增殖促进剂的培养基中培养乳酸菌。向培养基的乳酸菌增殖促进剂的添加量可以为例如0.001mg/mL以上且25mg/mL以下,优选为0.01mg/mL以上且10mg/mL以下,更优选为0.1mg/mL以上且2.5mg/mL以下。培养基中,能够根据需要适宜选择肉提取物、酵母提取物、豆乳、脱脂乳粉、蔗糖、无机盐类而以规定量含有。此外,作为培养条件,例如能够将温度设为约25℃以上且约40℃以下,将pH设为约5以上且约6.5以下,设成氮气氛围等的厌氧条件下。乳酸菌的初始浓度可以为例如1×104个以上/mL培养基,优选为1×105个以上/mL培养基。培养时间可以为例如4小时以上,优选为6小时以上且48小时以下。作为培养方式,能够从静置培养、搅拌培养、振动培养等通常使用的培养方式中适宜选择。乳酸菌能够使用离心分离等分离装置从培养后的培养基回收。
作为使用乳酸菌的发酵物,能够举出例如发酵乳、青贮饲料等。发酵乳能够通过向乳中添加乳酸菌和乳酸菌增殖促进剂,使乳发生乳酸发酵而得到。青贮饲料为将收割了的饲料作物(牧草、玉米等)装入仓库、使其乳酸发酵后贮藏了的动物饲料。制造青贮饲料时,通过向饲料作物中添加乳酸菌增殖促进剂,能够有效地增殖乳酸菌,促进乳酸发酵。由此,能够有效地抑制腐败菌的增殖,能够制造高品质的青贮饲料。
因此,本发明还包含乳酸菌增殖促进剂在制备乳酸菌或使用乳酸菌的发酵物中的应用。进而,本发明作为另一方式还包含选自由第1肽及第2肽组成的组中的至少1种肽在制备乳酸菌增殖促进剂中的应用,选自由第1肽及第2肽组成的组中的至少1种肽在乳酸菌培养中的应用,用于乳酸菌培养的选自由第1肽及第2肽组成的组中的至少1种肽。
实施例
以下,通过实施例更具体说明本发明,但本发明并不限定于这些实施例。
(1)评价用培养基的准备
研究了为了使加氏乳杆菌(Lactobacillus gasseri:FERM BP-11331)、嗜酸乳杆菌(Lactobacillus acidophilus:FERM BP-4981)、食淀粉乳杆菌(Lactobacillusamylovorus:FERM BP-11255)、弯曲乳杆菌(Lactobacillus curvatus:NITE P-02033)、植物乳杆菌(Lactobacillus plantarum)及短乳杆菌(Lactobacillus brevis)的6菌株分别培养后的总菌数成为1.0×108个/mL以上且1.0×109个/mL以下,从MRS培养基减少了氮源的培养基组成,将该组成作为“修改的MRS培养基”用于评价中。在此,MRS培养基的组成参考了BD公司(Becton,Dickinson and Company)的培养基目录所记载的组成。以下示出MRS培养基及修改的MRS培养基的组成。
[表4]
实施例1
对于加氏乳杆菌(FERM BP-11331)的增殖促进效果
通过化学合成准备了示出以下序列的候选肽28种。向修改的MRS培养基中添加候选肽,使其浓度成为0.25mg/mL,准备了评价用培养基。在准备好的评价用培养基中,以1.0×107个/mL接种乳酸菌加氏乳杆菌,在温度37℃以培养时间18小时进行了培养。回收培养后的培养液,作为成菌体增殖的指标的特性值,使用粒子计数分析装置(CDA-1000、Sysmex)测定了粒子浓度(个/mL)。求出将没有添加肽的培养基的对照值设为100%时的增加率(%)。将结果示于下表中。
[表5]
比较例1
使用二肽LF(Leu-Phe)和亮氨酸(L)与苯丙氨酸(F)的等摩尔混合物,与实施例1同样地进行培养,评价了二肽和氨基酸混合物对于乳酸菌的增殖促进效果。将结果示于下表中。
[表6]
实施例2
对于嗜酸乳杆菌(FERM BP-4981)的增殖促进效果
关于示出以下序列的候选肽8种,除了代替加氏乳杆菌使用了嗜酸乳杆菌以外,与实施例1同样地进行,评价了对于乳酸菌的增殖促进效果。将结果示于下表中。
[表7]
实施例3
对于食淀粉乳杆菌(FERM BP-11255)的增殖促进效果
关于示出以下序列的候选肽8种,除了代替加氏乳杆菌使用了食淀粉乳杆菌以外,与实施例1同样地进行,评价了对于乳酸菌的增殖促进效果。将结果示于下表中。
[表8]
实施例4
对于弯曲乳杆菌(NITE P-02033)的增殖促进效果
关于示出以下序列的候选肽8种,除了代替加氏乳杆菌使用了弯曲乳杆菌以外,与实施例1同样地进行,评价了对于乳酸菌的增殖促进效果。将结果示于下表中。
[表9]
实施例5
对于植物乳杆菌的增殖促进效果
关于示出以下序列的候选肽8种,除了代替加氏乳杆菌使用了植物乳杆菌以外,与实施例1同样地进行,评价了对于乳酸菌的增殖促进效果。将结果示于下表中。
[表10]
实施例6
对于短乳杆菌的增殖促进效果
关于示出以下序列的候选肽8种,除了代替加氏乳杆菌使用了短乳杆菌以外,与实施例1同样地进行,评价了对于乳酸菌的增殖促进效果。将结果示于下表中。
[表11]
实施例7
作为肽,使用二肽(LF),将添加浓度变更为0.1mg/mL、0.25mg/mL或1.0mg/mL,除此之外,同样地进行,评价了对于乳酸菌的增殖促进效果。将结果示于下表中。
[表12]
日本专利申请2020-096288号(申请日:2020年6月2日)所公开的全部内容通过参照并入本说明书中。本说明书中所记载的全部文献、专利申请及技术规格均通过参照并入本说明书中,其程度与各个文献、专利申请和技术规格被具体和单独标明为通过参照而并入相同。
序列表
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Claims (9)
1.一种乳酸菌增殖促进剂,其包含选自由第1肽和第2肽组成的组中的至少1种肽,
所述第1肽的总残基数为9以下,在C末端具有疏水性氨基酸残基,
所述第2肽的总残基数为9以下,疏水性氨基酸残基及脯氨酸残基的总数相对于总残基数的比为2/3以上。
2.根据权利要求1所述的乳酸菌增殖促进剂,其中,
所述疏水性氨基酸残基为选自由丙氨酸(A)、甘氨酸(G)、缬氨酸(V)、异亮氨酸(I)、亮氨酸(L)、苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)、甲硫氨酸(M)及半胱氨酸(C)组成的组中的氨基酸的残基。
3.根据权利要求1所述的乳酸菌增殖促进剂,其中,
所述疏水性氨基酸残基为选自由丙氨酸(A)、甘氨酸(G)、苯丙氨酸(F)及酪氨酸(Y)组成的组中的氨基酸的残基。
4.根据权利要求1~3中任一项所述的乳酸菌增殖促进剂,其中,
所述第1肽的总残基数为6以下,所述疏水性氨基酸残基为选自由苯丙氨酸(F)及酪氨酸(Y)组成的组中的氨基酸的残基。
5.根据权利要求1~4中任一项所述的乳酸菌增殖促进剂,其中,
所述第1肽的疏水性氨基酸残基及脯氨酸残基的总数相对于总残基数的比为2/3以上。
6.根据权利要求1~5中任一项所述的乳酸菌增殖促进剂,其中,
所述第2肽的疏水性氨基酸残基及脯氨酸残基的总数相对于总残基数的比为3/4以上。
7.根据权利要求1~6中任一项所述的乳酸菌增殖促进剂,其中,
所述第2肽的总残基数为4以下。
8.根据权利要求1~7中任一项所述的乳酸菌增殖促进剂,其中,
所述第2肽由疏水性氨基酸残基及脯氨酸残基组成。
9.根据权利要求1~8中任一项所述的乳酸菌增殖促进剂,其中,
所述第2肽在C末端具有疏水性氨基酸残基。
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