WO2018028100A1 - 一株乳酸乳球菌及其应用 - Google Patents

一株乳酸乳球菌及其应用 Download PDF

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WO2018028100A1
WO2018028100A1 PCT/CN2016/108476 CN2016108476W WO2018028100A1 WO 2018028100 A1 WO2018028100 A1 WO 2018028100A1 CN 2016108476 W CN2016108476 W CN 2016108476W WO 2018028100 A1 WO2018028100 A1 WO 2018028100A1
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lactococcus lactis
strain
stress
medium
acid
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张娟
朱政明
杨佩珊
陈坚
堵国成
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Jiangnan University
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
    • A23L19/20—Products from fruits or vegetables; Preparation or treatment thereof by pickling, e.g. sauerkraut or pickles
    • 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; 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
    • 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
    • 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; PREPARATION THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/1203—Addition of, or treatment with, enzymes or microorganisms other than lactobacteriaceae
    • 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; 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

Definitions

  • the invention relates to a strain of Lactococcus lactis and its application, belonging to the field of food biotechnology.
  • Lactococcus lactis As an industrial microbial cell factory, Lactococcus lactis has been widely used in food, fermentation and other fields. In the fermentation process in which the industrial products required in the above-mentioned fields are carried out, the acid-producing properties become an integral part of the microorganisms. On the one hand, metabolic acid production can help promote cell energy conversion, maintain intracellular and extracellular osmotic pressure balance, and enhance the environmental competitiveness of ontological cells. On the other hand, with the accumulation of intracellular acid production, the pH in the cells of the microbial cells continues to decrease, and the activity of the enzymes that maintain the normal physiological functions of the cells is inhibited, thereby affecting the metabolic activity and production efficiency of the cells.
  • the technical problem to be solved by the present invention is to provide a strain of Jactococc ⁇ lactis WH101 which was deposited with the China Center for Type Culture Collection on April 29, 2016 under the accession number CCTCC NO: M 2016233, Address: China Wuhan, Wuhan University.
  • the Lactococcus lactis CCTCC NO: M 2016233 is based on Lactococcus lactis NZ9000 as a starting strain, and is induced by diethyl sulphate as a mutagenizing agent. Under the condition of pH 4.5, the OD6QQ value reaches 0.527, which is relatively attractive. The stress was increased by 4.5 times; the stress was 4.04% in the pH 4.0 lactic acid for 2 h; the stress was 22.4 times in the lactic acid pH 4.0, and the survival rate was 22.4 times that of the pre-mutage strain under the same treatment conditions; Coccus CTCCC NO: M 2016233 has better growth properties and acid tolerance.
  • Lactococcus lactis CTCCC NO: M 2016233 has good acid stress, ethanol stress, osmotic stress and tolerance to oxygen stress, and has great application value in food, fermentation and other fields.
  • Jactococc ⁇ /acfc WH101 has been deposited with the China Center for Type Culture Collection on April 29, 2016. The deposit address is Wuhan University, Wuhan, China, with the accession number CCTCC NO: M 2016233.
  • GM17 medium glucose 5.0 g ⁇ -1 , tryptone 5.0 g ⁇ -1 , soy peptone 5.0 gl 1 , beef extract 5.0 gL- 1 , yeast extract 2.5 gL- 1 , vitamin C 0.5 g ⁇ -1 , sulfuric acid Magnesium 0.25 g ⁇ -1 , disodium glycophosphate 19.0 g ⁇ -1 .
  • the GM17 medium at pH 4.5 was 25% lactic acid-adjusted GM17 medium with a pH of 4.5.
  • Other pH
  • GM17 medium was obtained in the same manner.
  • Lactococcus lactis actococc lactis ⁇ 9000 the cells were cultured in GM17 medium to logarithmic growth phase, adjusted to a concentration of 1 * 10 7 / CFU, and centrifuged (5000 rpm; lOmin) to remove the supernatant. 0.85% saline was resuspended and repeated twice.
  • the GM17 medium containing 0.5% v/v diethyl sulfate (DES) was resuspended in an equal volume, 100 rpm, 30.
  • the suspension was washed with 0.85% physiological saline, repeated 5 times, resuspended in an equal volume of GM17 medium, and then statically cultured at 30 ° C for 1.5 h.
  • GM17 (pH 5.0) medium to a 2.2 ml deep-well plate, transfer the culture medium after the above culture to a 96-deep well plate at 2% inoculation, and let stand for 48 h at 30 °C.
  • the growth of the mutagenized strain in the acidic medium was investigated, and the mutant mixed strain was screened according to the size of the biomass, and then the mixed strain was appropriately diluted and coated with a pH 5.0 plate to select a single colony, and the single colony was at pH 5.0.
  • the mutant strain was screened according to the biomass size, the starting strain OD 6 QQ reached 0.076, the mutant strain WH101 ODeoo reached 0.612), and the name was Lactococcus lactis WHl 01, which was deposited on April 29, 2016. China Type Culture Collection, the deposit number is CCTCC NO: M 2016233.
  • the starting strain Lactococcus lactis Z9mi which was preserved in a -80 ° C glycerol tube, and the selected Lactococcus lactis Lactococcus /acfe WHIOl were added to the GM17 medium at a 2% inoculum and allowed to stand at 30 ° C. Training 12ho
  • the activated Lactococcus lactis NZ9000 and Lactococcus lactis WH101 were transferred to GM17 medium of pH 4.5 at 2% inoculation, and cultured at 30 ° C for 48 h. After the fermentation, the fermentation broth was measured. . Under the condition of pH 4.5, the OD of the mutant strain reached 0.527, which was 4.5 times higher than that before breeding. The results are shown in Table 1.
  • the starting strain Lactococcus lactis Z9mi which was deposited in a -80 ° C glycerol tube, and the selected Lactococcus lactis Lactococcus /acfe WHIOl were added to the GM17 medium at a seeding rate of 2%, at 30.
  • C static training The activated L. lactis NZ9000 and Lactococcus lactis WH101 were added to the GM17 medium at a dose of 2%, and cultured at 30 ° C until the logarithmic growth phase.
  • the cells in the logarithmic growth phase were collected by centrifugation at 5000 rpm for 10 min.
  • the cells were washed and centrifuged twice with 0.85% physiological saline, and then resuspended in GM17 (pH 4.0) medium for sampling at different times.
  • the cells were washed twice with the same physiological saline, and resuspended in an equal volume of physiological saline.
  • the broth of the ⁇ was diluted and appropriately coated, and the plate was statically cultured at 30 ° C for 24 hours to calculate the survival rate.
  • Table 2 The mutant strain WH101 was stressed in lactic acid pH 4.0 for 1 h, 2 h, 3 h, respectively, and the survival rates were 1.2 times, 4.1 times, 22.4 times, respectively, under the same treatment conditions.
  • the starting strain Lactococcus lactis Z9mi which was deposited in a -80 °C glycerol tube, and the selected Lactococcus lactis Lactococcus /acfe WHIOl were added to the GM17 medium at a dose of 2%, at 30.
  • C static training 12ho The starting strain Lactococcus lactis Z9mi, which was deposited in a -80 °C glycerol tube, and the selected Lactococcus lactis Lactococcus /acfe WHIOl were added to the GM17 medium at a dose of 2%, at 30.
  • the activated L. lactis NZ9000 and Lactococcus lactis WH101 were added to the GM17 medium at a dose of 2%, and cultured at 30 ° C until the logarithmic growth phase.
  • the cells in the logarithmic growth phase were collected by centrifugation at 5000 rpm for 10 min. The cells were washed twice with 0.85% physiological saline and then resuspended in 15% (v/v) ethanol, 15% (v).

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Abstract

一株乳酸乳球菌及其应用，属于食品生物技术领域。分离得到的菌株命名为乳酸乳球菌WH101(Lactococcus lactis WH101)，在pH 4.5的条件下，OD600值达到0.527，相比较诱变前提高了4.5倍；在pH 4.0的乳酸中胁迫3h，存活率是同等处理条件下诱变前菌株的22.4倍。分别用15％乙醇胁迫4h，15％NaCl胁迫6h和1mmol/L H2O2胁迫3h后，其存活率分别是同等处理条件下原始菌株的5.2，2.0和1.9倍。其具有较好的酸胁迫、乙醇胁迫、渗透压胁迫和氧胁迫耐受性，在食品、发酵等领域具有巨大的应用价值。

Description

一株乳酸乳球菌及其应用 技术领域
本发明涉及一株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 (Jactococc^ lactis) WH101，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 2016233， 地址： 中国， 武汉， 武汉大学 。
所述乳酸乳球菌 CCTCC NO: M 2016233是以乳酸乳球菌 Lactococcus lactis NZ9000 为出发菌株， 以硫酸二乙酯为诱变剂诱变得到， 在 pH 4.5的条件下， OD6QQ值达到 0.527， 相 比较诱变前提高了 4.5倍； 在 pH 4.0的乳酸中胁迫 2h， 存活率为 4.04%; 在 pH 4.0的乳酸中 胁迫 3h， 存活率是同等处理条件下诱变前菌株的 22.4倍； 可见， 乳酸乳球菌 CCTCC NO: M 2016233具有更好的生长性能和酸耐受性。 分别用 15%乙醇胁迫 4 h， 15% NaCl胁迫 6 h和 lmmol/L H202胁迫 3 h后， 其存活率分别是同等处理条件下原始菌株的 5.2， 2.0禾卩 1.9倍。 可见， 乳酸乳球菌 CCTCC NO: M 2016233具有较好的酸胁迫、 乙醇胁迫、渗透压胁迫和氧胁 迫耐受性， 在食品、 发酵等领域具有巨大的应用价值。
生物材料保藏
乳酸乳球菌 (Jactococc^ /acfc) WH101，已于 2016年 4月 29日保藏于中国典型培养物保 藏中心， 保藏地址为中国武汉 武汉大学， 保藏编号为 CCTCC NO: M 2016233。
附图说明
图 1 不同环境胁迫条件下菌株的存活率实验
具体实施方式 GM17培养基：葡萄糖 5.0 g丄 -1, 胰蛋白胨 5.0 g丄 -1，大豆蛋白胨 5.0 gl 1 ,牛肉浸膏 5.0 g.L-1 ,酵母提取物 2.5 g.L-1 ,维生素 C 0.5 g丄 -1， 硫酸镁 0.25 g丄 -1，甘油磷酸二钠 19.0 g丄 -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 2016233)的选育方法
以乳酸乳球菌 actococc lactis Ζ9000 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; lOmin)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， lOOrpm, 30。C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°C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°C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 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 OD6QQ达到 0.076， 突变菌株 WH101 ODeoo达到 0.612 )， 命名为乳酸乳球菌 WH101 (Lactococcus lactis WHl 01) ,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 2016233。
实施例 2 乳酸乳球菌 (CCTCC NO: M 2016233)酸胁迫条件下的生长性能
将保藏于 -80°C甘油管中的出发菌株乳酸乳球菌 Lactococcus lactis Z9mi)及选育到的 乳酸乳球菌 Lactococcus /acfe WHIOl ) 以 2%的接种量接入 GM17培养基， 在 30°C静置培 养 12ho
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 WH101转接到 pH 4.5 的 GM17培养基中， 于 30°C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 OD,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
Figure imgf000003_0001
实施例 3 酸耐受性实验
将保藏于 -80°C甘油管中的出发菌株乳酸乳球菌 Lactococcus lactis Z9mi)及选育到的 乳酸乳球菌 Lactococcus /acfe WHIOl ) 以 2%的接种量接入 GM17培养基， 在 30。C静置培 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 WH101到 GM17培养 基中， 30°C静置培养至对数生长期。
取对数生长期的细胞， 经 5000rpm离心 lOmin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 ΙΟΟμΙ的菌液经适当稀释后涂布 平板，于 30°C静置培养 24h，分别计算存活率大小（如表 2所示）。突变菌株 WH101在 pH 4.0 的乳酸中分别胁迫 lh, 2h, 3h， 存活率分别是同等处理条件下下选育前菌株的 1.2倍， 4.1倍， 22.4倍。
表 2 酸耐受性实验
Figure imgf000004_0001
实施例 4 其它环境耐受性实验
将保藏于 -80°C甘油管中的出发菌株乳酸乳球菌 Lactococcus lactis Z9mi)及选育到的 乳酸乳球菌 Lactococcus /acfe WHIOl ) 以 2%的接种量接入 GM17培养基， 在 30。C静置培 养 12ho
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 WH101到 GM17培养 基中， 30°C静置培养至对数生长期。取对数生长期的细胞，经 5000rpm离心 lOmin收集菌体， 菌体经 0.85%的生理盐水洗涤离心 2次后， 分别等体积重悬于含 15% (v/v) 乙醇， 15% (v/v) NaCl和 lmmol/L H202的 GM17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 ΙΟΟμΙ的菌液经适当稀释后涂布平板，于 30。C 静置培养 24h，分别计算存活率大小（如图 1所示）。突变菌株 WH101在含 15%乙醇的 GM17 培养基中胁迫 4 h后，存活率是同等处理条件下原始菌株的 5.2倍。其在含 15% NaCl的 GM17 培养基中胁迫 6 h后， 存活率是同等处理条件下原始菌株的 2.0倍。 而在含 lmm0l/L H202的 GM17培养基中胁迫 3 h后， 存活率是同等处理条件下原始菌株的 1.9倍。
Figure imgf000005_0001

Claims

权 利 要 求 书
1、 一株乳酸乳球菌 iLactococc lactis WH101 , 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 2016233，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 iLactococc lactis) WH101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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CN107227279B (zh) * 2017-07-18 2019-09-03 江南大学 一株乳酸片球菌及其应用
CN107964524B (zh) * 2018-01-18 2020-11-06 海南大学 一种具有乳酸活性的乳酸乳球菌hks2及其分离筛选方法和应用
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