CN106282053A - 一株耐酸乳酸乳球菌及其应用 - Google Patents
一株耐酸乳酸乳球菌及其应用 Download PDFInfo
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- 241000194035 Lactococcus lactis Species 0.000 title claims abstract description 34
- 235000014897 Streptococcus lactis Nutrition 0.000 title claims abstract description 34
- 238000004321 preservation Methods 0.000 claims description 2
- 235000021107 fermented food Nutrition 0.000 claims 3
- 235000021110 pickles Nutrition 0.000 claims 1
- 235000013618 yogurt Nutrition 0.000 claims 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 abstract description 12
- 239000002253 acid Substances 0.000 abstract description 8
- 230000001580 bacterial effect Effects 0.000 abstract description 8
- 235000014655 lactic acid Nutrition 0.000 abstract description 6
- 239000004310 lactic acid Substances 0.000 abstract description 6
- 230000004083 survival effect Effects 0.000 abstract description 6
- 238000000855 fermentation Methods 0.000 abstract description 4
- 230000004151 fermentation Effects 0.000 abstract description 4
- 235000013305 food Nutrition 0.000 abstract description 4
- 230000035772 mutation Effects 0.000 abstract description 4
- 239000001963 growth medium Substances 0.000 description 12
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 238000009395 breeding Methods 0.000 description 5
- 239000002054 inoculum Substances 0.000 description 5
- 230000006518 acidic stress Effects 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 239000012267 brine Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000003505 mutagenic effect Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- CATWCRUATKKAAQ-UHFFFAOYSA-N 2,3-dihydroxypropyl dihydrogen phosphate;sodium Chemical compound [Na].[Na].OCC(O)COP(O)(O)=O CATWCRUATKKAAQ-UHFFFAOYSA-N 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229940099352 cholate Drugs 0.000 description 1
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical compound CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 description 1
- 238000011143 downstream manufacturing Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000006353 environmental stress Effects 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 239000003471 mutagenic agent Substances 0.000 description 1
- 231100000707 mutagenic chemical Toxicity 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 230000000529 probiotic effect Effects 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000029219 regulation of pH Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- 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
- A23C9/1236—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt using Leuconostoc, Pediococcus or Streptococcus sp. other than Streptococcus Thermophilus; Artificial sour buttermilk in general
-
- 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
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/21—Streptococcus, lactococcus
- A23V2400/231—Lactis
-
- 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
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
Abstract
本发明公开了一株耐酸乳酸乳球菌及其应用,属于食品生物技术领域。本发明分离得到的菌株命名为乳酸乳球菌WH102,在pH 4.5的条件下,OD600值达到0.371,相比较诱变前提高了2.6倍;在pH 4.0的乳酸中胁迫2h,存活率为6.09%;在pH 4.0的乳酸中胁迫2h,存活率是同等处理条件下诱变前菌株的6.1倍。具有较好的酸耐受性,在食品、发酵等领域具有巨大的应用价值。
Description
技术领域
本发明涉及一株耐酸乳酸乳球菌及其应用,属于食品生物技术领域。
背景技术
作为工业化的微生物细胞工厂,乳酸乳球菌已广泛应用于食品,发酵等领域。在开展上述领域中所需工业产品的发酵法生产过程中,产酸特性成为微生物必不可少的组成部分。一方面代谢产酸可帮助促进细胞能量转化、维持细胞内外渗透压平衡、增强本体细胞的环境竞争性。另一方面,随着胞内产酸的积累,导致微生物菌体细胞内的pH持续下降,胞内维持细胞正常生理功能的相关酶类活性受到抑制,进而影响细胞的代谢活性及其生产效率,并为生产成本、下游加工及后续工业排放物环境治理埋下诸多隐患。同时,作为益生菌中的一种,在人体胃肠道中也会遭受酸胁迫、胆盐胁迫等环境胁迫。因此,提高微生物菌株的酸胁迫耐受性,成为学术界和产业界亟待解决的重要问题。
发明内容
本发明所要解决的技术问题是提供一株乳酸乳球菌WH102(Lactococcus lactisWH103),其于2016年4月29日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M2016234,地址:中国,武汉,武汉大学。
所述乳酸乳球菌WH102是以乳酸乳球菌(Lactococcus lactis NZ9000)为出发菌株,以硫酸二乙酯(DES)为诱变剂诱变得到的,在pH 4.5的条件下,OD600值达到0.371,相比较诱变前提高了2.6倍;在pH 4.0的乳酸中胁迫2h,存活率为6.09%;在pH 4.0的乳酸中胁迫2h,存活率是同等处理条件下诱变前菌株的6.1倍。
本发明选育得到的乳酸乳球菌WH102(CCTCC NO:M 2016234)在较低pH条件下能够更好地生长,而出发菌株不能在较低pH条件下生长,突变菌株WH102具有更好的生长性能和酸耐受性。本发明提供的选育方法,操作简单易行,且效果较明显。
生物材料保藏
一株乳酸乳球菌WH102(Lactococcus lactis WH103),其于2016年4月29日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M 2016234,地址:中国,武汉,武汉大学。
具体实施方式
GM17培养基为:葡萄糖5.0g.L-1,胰蛋白胨5.0g.L-1,大豆蛋白胨5.0g.L-1,牛肉浸膏5.0g.L-1,酵母提取物2.5g.L-1,维生素C 0.5g.L-1,硫酸镁0.25g.L-1,甘油磷酸二钠19.0g.L-1。
pH 4.5的GM17培养基为25%的乳酸调节的pH值为4.5的GM17培养基。其他pH的GM17培养基以同样方法得到。
实施例1乳酸乳球菌(CCTCC NO:M 2016234)的选育方法
以乳酸乳球菌(Lactococcus lactis NZ9000)为出发菌株,将其在GM17培养基中培养至对数生长期,调整菌液浓度1*107个/CFU,取样离心(5000rpm;10min)后去上清,用0.85%的生理盐水洗涤重悬,重复两次。加入含0.5%v/v硫酸二乙酯(DES)的GM17培养基等体积重悬,100rpm,30℃处理30min后立即用0.85%的生理盐水洗涤重悬,重复5次,加入等体积的GM17培养基重悬后在30℃下静置培养1.5h。
在2.2ml的96深孔板中加入1ml的GM17(pH 5.0)培养基,取上述后培养的培养液以2%的接种量转接到96深孔板中,30℃下静置培养48h,考察诱变菌株在酸性培养基中的生长情况,根据生物量的大小筛选出突变混合菌株,然后将混合菌株经适当稀释涂布pH 5.0的平板,挑选单菌落,将单菌落于pH为5.0条件下发酵,根据生物量大小筛选出突变菌株,出发菌株OD600达到0.076,突变菌株乳酸乳球菌WH102的OD600达到0.441,于2016年4月29日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M 2016234。
实施例2乳酸乳球菌(CCTCC NO:M 2016234)酸胁迫条件下的生长性能
将保藏于-80℃甘油管中的出发菌株乳酸乳球菌(Lactococcus lactis NZ9000)及选育到的乳酸乳球菌WH102以2%的接种量接入GM17培养基,在30℃静置培养12h。
以2%的接种量分别取活化好的乳酸乳球菌NZ9000和乳酸乳球菌WH102转接到GM17(pH 4.5)培养基中,于30℃下静置培养48h,发酵结束后测定发酵液的菌浓。在pH 4.5的条件下,突变菌株OD600值达到0.527,相比较选育前提高了4.5倍。结果如表1所示。
表1酸胁迫条件下菌体生长性能
菌株 | Lactococcus lactis NZ9000 | Lactococcus lactis WH102 |
OD600 | 0.081 | 0.371 |
实施例3酸耐受性实验
将保藏于-80℃甘油管中的出发菌株乳酸乳球菌(Lactococcus lactis NZ9000)及选育到的乳酸乳球菌WH102以2%的接种量接入GM17培养基,在30℃静置培养12h。
以2%的接种量分别取活化好的乳酸乳球菌NZ9000和乳酸乳球菌WH102到GM17培养基中,30℃静置培养至对数生长期。
取对数生长期的细胞,经5000rpm离心10min收集菌体,菌体经0.85%的生理盐水洗涤离心2次后,等体积重悬于GM17(pH 4.0)培养基中胁迫不同的时间取样,重新用相同的生理盐水离心洗涤细胞2次,并重悬于等体积的生理盐水中,取100μl的菌液经适当稀释后涂布平板,于30℃静置培养24h,分别计算存活率大小(如表2所示)。突变菌株在pH 4.0的乳酸中分别胁迫1h,2h,3h,存活率分别是同等处理条件下下选育前菌株的0.9倍,6.1倍,2.2倍。
表2酸耐受性实验
Claims (4)
1.一株乳酸乳球菌WH102(Lactococcus lactis WH103),其特征在于,于2016年4月29日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M 2016234,保藏地址为中国武汉武汉大学。
2.权利要求1所述的乳酸乳球菌WH102在制备发酵食品中的应用。
3.根据权利要求2所述的应用,其特征在于,所述发酵食品是泡菜。
4.根据权利要求2所述的应用,其特征在于,所述发酵食品是酸奶。
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