WO2019214062A1 - 一种植物乳杆菌中分离纯化苯基乳酸的方法 - Google Patents

一种植物乳杆菌中分离纯化苯基乳酸的方法 Download PDF

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WO2019214062A1
WO2019214062A1 PCT/CN2018/095752 CN2018095752W WO2019214062A1 WO 2019214062 A1 WO2019214062 A1 WO 2019214062A1 CN 2018095752 W CN2018095752 W CN 2018095752W WO 2019214062 A1 WO2019214062 A1 WO 2019214062A1
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lactic acid
lactobacillus plantarum
phenyl lactic
fermentation
liquid
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顾青
郦萍
周青青
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浙江工商大学
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  • the invention belongs to the field of food biotechnology, and particularly relates to a method for separating and purifying phenyl lactic acid from a fermentation supernatant of a strain of Lactobacillus plantarum ZJ316 deposited under the accession number CCTCC NO: M 208077.
  • antibacterial agents are essential in preventing food spoilage and extending the shelf life of foods.
  • the chemical synthetic antibacterial agents especially the types and quantities of non-food-based chemical antibacterial agents, have doubled, and while promoting the development of the food industry, serious food safety hazards have been buried.
  • Long-term studies have found that certain synthetic preservatives are carcinogenic, teratogenic and prone to food poisoning.
  • the development of broad-spectrum, high-efficiency, safe, stable and natural antibacterial agents is an inevitable requirement for the development of the food industry.
  • Food-grade microbial lactic acid bacteria can be fermentatively metabolized to produce a variety of antibacterial substances, such as organic acids, bacteriocins, rosin, hydrogen peroxide and the like.
  • phenyl lactic acid of organic acid is a novel natural antibacterial agent with a molecular weight of 166 Da and a molecular formula of C 9 H 10 O 3 .
  • the second carbon atom in the molecular structure is a chiral carbon atom.
  • Phenyl lactic acid has the characteristics of being easily soluble in water, good in stability, and not damaged at 121 ° C for 20 min.
  • Nisin a bacteriocin
  • phenyl lactic acid can effectively inhibit food-borne pathogens and spoilage bacteria, such as Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, Bacillus subtilis, Salmonella, Aspergillus niger, Penicillium and the like.
  • phenyl lactic acid has high safety and a wide range of inhibition, which makes it have good development and application prospects.
  • Lactobacillus brevis Pseudomonas, Geotrichum candidum, Lactobacillus plantarum, F. faecalis, etc.
  • the methods of separation and purification are different, mainly including main solid phase extraction, Thin layer chromatography, capillary electrophoresis, chiral column high performance liquid chromatography, and the like.
  • the invention finds that the Lactobacillus plantarum ZJ316 strain has the function of producing phenyl lactic acid, and establishes high performance liquid chromatography to separate and purify the phenyl lactic acid and determine the enantiomer, and screen and characterize the phenyl lactic acid producing strain. Quantitative determination is of great significance.
  • the object of the present invention is to establish a rapid and efficient method for separating and purifying phenyl lactic acid from the fermentation supernatant of Lactobacillus plantarum ZJ316, and to determine the broad-spectrum antibacterial effect of the phenyl lactic acid.
  • Lactobacillus plantarum ZJ316 has been deposited in the China Center for Type Culture Collection. Address: Wuhan University, Wuhan, China, deposit number CCTCC NO: M 208077, date of deposit, May 23, 2008.
  • the Lactobacillus plantarum ZJ316 strain was activated and fermented in MRS medium at a culture temperature of 37 ° C and a culture time of 24 h.
  • the fermentation broth was centrifuged, and the supernatant was taken and stored at 4 °C.
  • the fermentation supernatant of Lactobacillus plantarum ZJ316 was penetrated by XAD-2 macroporous resin, and the membrane (0.22 ⁇ m) was separated and purified by high performance liquid chromatography.
  • the high performance liquid chromatography column model was YMC-Pack ODS-AQ 150 ⁇ 20 mm LD, mobile phase 0.05% trifluoroacetic acid/water and 0.05% trifluoroacetic acid/methanol (v/v).
  • the content of phenyl lactic acid in the fermentation supernatant of Lactobacillus plantarum ZJ316 was determined by high performance liquid chromatography (column type YMC-Pack ODS-AQ 150 ⁇ 4.6mm LD) for a series of concentration gradients of phenyl lactic acid. (Sigma) analyzed and established a linear relationship between peak area and retention time to obtain a regression equation.
  • the content of phenyl lactic acid in the fermentation supernatant of Lactobacillus plantarum ZJ316 was determined to be 108.87 mg/mL, and the purity of the phenyl lactic acid obtained by separation and purification was 98.14%.
  • the broad-spectrum bacteriostatic and minimum inhibitory concentration (MIC) of the purified phenyl lactic acid were determined by the Oxford Cup method and the 96-well plate micro method. The results showed that the phenyl lactic acid produced by the Lactobacillus plantarum has antibacterial activity against various pathogenic bacteria and spoilage bacteria, and has broad-spectrum antibacterial properties.
  • the invention has the advantages of determining the function of producing phenyl lactic acid by Lactobacillus plantarum ZJ316 strain, establishing a method for separating and purifying phenyl lactic acid by high performance liquid chromatography and determining the enantiomer thereof, and determining the broad-spectrum antibacterial property of phenyl lactic acid, Screening and qualitative and quantitative determination of phenyl lactic acid producing strains are of great significance.
  • Figure 1 is the first HPLC elution chromatogram
  • Figure 2 is a secondary HPLC elution chromatogram
  • Figure 3 is the purity of separation and purification of phenyl lactic acid by high performance liquid chromatography
  • Figure 4 shows the peak area-retention time standard curve of the phenyl lactic acid standard
  • Figure 5 Lactobacillus plantarum ZJ316 production of enantiomer ratio in phenyl lactic acid
  • Example 1 Activation of Lactobacillus plantarum ZJ316 strain and preparation of fermentation supernatant
  • Lactobacillus plantarum ZJ316 strain activation The strain was taken out from -80 ° C, thawed at 4 ° C, streaked in MRS solid medium, statically cultured at 37 ° C for 24 h; single colony was picked in MRS liquid medium at 37 ° C The culture was carried out for 24 hours, and serially passaged twice to obtain a seed liquid. The seed solution was inserted into the fermentation medium (MRS liquid) at a volume fraction of 2%, and cultured at 37 ° C for 24 hours to obtain a fermentation broth. The fermentation broth was centrifuged at 8000 rpm for 15 min (4 ° C) to obtain a supernatant, which was stored at 4 ° C until use.
  • MRS liquid fermentation medium
  • Example 3 Production and separation purity of phenyl lactic acid in the supernatant of Lactobacillus plantarum ZJ316
  • Example 4 Enantiomeric ratio of phenyl lactic acid in Lactobacillus plantarum ZJ316 supernatant
  • Example 5 Broad-spectrum antibacterial activity of purified phenyl lactic acid in fermentation supernatant of Lactobacillus plantarum ZJ316
  • the purified phenyl lactic acid was subjected to broad-spectrum bacteriostatic determination using the Oxford Cup method.
  • the size of the inhibition zone is shown by the Oxford Cup method to determine the effect of the antibacterial substance on the indicator bacteria.
  • the 96-well plate was cultured under appropriate conditions for the culture of the indicator bacteria, and the OD 600 value was measured by a microplate reader every 1 hour. Compared with the initial OD 600 value, cultured for 24 hours OD 600 value is not increased, this condition is defined as the concentration of phenyl lactic acid phenyl lactic acid indicates the minimum inhibitory concentration of bacteria (MIC).
  • Table 1 The antibacterial effect of phenyllactate produced by Lactobacillus plantarum ZJ316 on indicator bacteria and its minimum inhibitory concentration
  • diameter of inhibition zone (mm): “+++”, d>16.00, “++” 14.00 ⁇ d ⁇ 16.00, “+”: d ⁇ 14.00, “-”: no antibacterial effect, “/”: Not tested.

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Abstract

本发明公开了一种植物乳杆菌中分离纯化苯基乳酸的方法，属于食品生物技术领域。本发明公开的植物乳杆菌ZJ316，已保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M 208077。本发明以植物乳杆菌ZJ316为目标菌株，未经任何条件优化的情况下，高效液相色谱法测定MRS液体培养基发酵上清液中苯基乳酸含量达108.87mg/mL(0.66mmol/L)，纯化后苯基乳酸的纯度高达98.14％。利用手性色谱柱-高效液相分析得到其中L-苯基乳酸的质量百分比范围为92％-98％。本发明所得产品安全可靠，可有效抑制食源性致病菌和腐败菌，可应用于药用中间体，食品添加剂、化妆品等。

Description

一种植物乳杆菌中分离纯化苯基乳酸的方法 技术领域
本发明属于食品生物技术领域，具体涉及从保藏号为CCTCC NO：M 208077的植物乳杆菌ZJ316(Lactobacillus plantarum ZJ316)菌株发酵上清液中分离纯化苯基乳酸的方法。
背景技术
在食品工业的发展过程中，抗菌剂在防止食品的腐败、延长食品的保质期方面是必不可少的。化学合成抗菌剂特别是非食源的化学抗菌剂的种类和数量倍增，在促进食品工业发展的同时，埋下了严重的食品安全隐患。经长期研究发现某些人工合成的防腐剂具有诱癌性、致畸性并且容易引起食物中毒。研制广谱、高效、安全、稳定、天然的抗菌剂是食品工业发展的必然要求。
食品级微生物乳酸菌，作为一类传统的发酵菌株，能发酵代谢产生多种抗菌物质，如有机酸、细菌素、罗氏菌素、过氧化氢等。其中有机酸类的苯基乳酸，是一种新型的天然抗菌剂，分子量为166Da，分子式为C 9H 10O 3，分子结构中的第二个碳原子为手性碳原子。苯基乳酸具有易溶于水、稳定性好、121℃保持20min不被破坏等特点。
Figure PCTCN2018095752-appb-000001
目前，只有Nisin一种细菌素被商业化应用，由于其抑菌谱窄，只抑制部分革兰氏阳性菌，使应用范围受到了极大的限制。相关研究表明，苯基乳酸可有效抑制食源性致病菌和腐败菌，如大肠杆菌、单增李斯特氏菌、金黄色葡萄球菌、枯草芽孢杆菌、沙门氏菌、黑曲霉、青霉等。与大多数阳离子抗菌肽相比， 苯基乳酸安全性高、抑菌范围广，使其具有良好的开发和应用前景。
目前，研究工作者已从短乳杆菌、假单胞菌、白地霉、植物乳杆菌、费氏杆菌等中分离纯化得到苯基乳酸，分离纯化的方法各不相同，主要包括主要固相萃取、薄层层析、毛细管电泳、手性柱高效液相色谱法等。本发明发现了植物乳杆菌ZJ316菌株具有生产苯基乳酸的功能，并建立高效液相色谱法对该苯基乳酸进行分离纯化及对映异构体测定，对苯基乳酸生产菌株的筛选及定性定量测定具有重大意义。
发明内容
本发明目的是建立从植物乳杆菌ZJ316发酵上清液中快速、高效的分离纯化苯基乳酸的方法，并测定该苯基乳酸广谱抗菌效果。
本发明的上述目的是通过以下技术方案得以实现的：
植物乳杆菌ZJ316，已保藏于中国典型培养物保藏中心，地址：中国武汉武汉大学，保藏编号CCTCC NO:M 208077，保藏日期2008年5月23日。
植物乳杆菌ZJ316菌种于MRS培养基活化及发酵，培养温度为37℃，培养时间为24h。发酵液离心，取上清储存于4℃。
植物乳杆菌ZJ316发酵上清液经XAD-2大孔树脂穿透，过滤膜(0.22μm)，经两次高效液相色谱分离纯化，高效液相色谱柱型号为YMC-Pack ODS-AQ 150×20mm L.D.，流动相为0.05％三氟乙酸/水和0.05％三氟乙酸/甲醇(v/v)。
植物乳杆菌ZJ316发酵上清液中苯基乳酸的含量检测，是利用高效液相色谱(色谱柱型号为YMC-Pack ODS-AQ 150×4.6mm L.D.)对一系列浓度梯度的苯基乳酸标品(Sigma公司)进行分析，建立峰面积-保留时间的线性关系，得到回归方程。测定植物乳杆菌ZJ316发酵上清液中苯基乳酸的含量为108.87mg/mL，分离纯化得到的苯基乳酸纯度为98.14％。
选用大赛路
Figure PCTCN2018095752-appb-000002
IG(IG00CE-UC058)(0.46cm I.D.×25cm L×5μm)手性色谱柱，以正己烷/乙醇/三氟乙酸＝92/8/0.1(v/v/v)为流动相等度洗脱。该苯基乳酸中D/L型-苯基乳酸的保留时间分别为11.288min、12.309min。
采用牛津杯法和96孔板微量法分别对纯化得到的苯基乳酸进行广谱抑菌性 和最小抑菌浓度(MIC)测定。结果显示该植物乳杆菌产生的苯基乳酸对多种致病菌和腐败菌有抗菌作用，具有广谱抗菌性。
本发明的优点：确定植物乳杆菌ZJ316菌株具有生产苯基乳酸的功能，建立高效液相色谱法分离纯化苯基乳酸及测定其对映异构体的方法，测定苯基乳酸广谱抗菌性，对苯基乳酸生产菌株的筛选及定性定量测定具有重大意义。
附图说明
图1首次HPLC洗脱色谱图；
图2二次HPLC洗脱色谱图；
图3高效液相色谱分离纯化苯基乳酸的纯度；
图4苯基乳酸标准品测定峰面积-保留时间标准曲线；
图5植物乳杆菌ZJ316生产苯基乳酸中对映异构体比例(a)：D-苯基乳酸标准品进样的色谱图；(b)：D-苯基乳酸标准品和L-苯基乳酸标准品等比例混合进样的色谱图；(c)：分离纯化苯基乳酸进样的色谱图。
具体实施方式
下面结合具体实施例对本发明进行进一步描述，但对本发明的保护范围并不仅限于此：
实施例1：植物乳杆菌ZJ316菌种活化及发酵上清液制备
植物乳杆菌ZJ316菌种活化：将菌种从-80℃取出，于4℃解冻，划线于MRS固体培养基，37℃静置培养24h；挑取单菌落于MRS液体培养基中37℃静置培养24h，连续传代两次得到种子液。将种子液以体积分数2％的接种量接入发酵培养基(MRS液体)中扩培，37℃静止培养24h，得到发酵液。发酵液经8000rpm离心15min(4℃)，得到上清液，储存于4℃待用。
实施例2：植物乳杆菌ZJ316发酵上清液中苯基乳酸的分离纯化
离心后的上清部分经过XAD-2大孔树脂吸附，收集流穿液，经过微孔滤膜过滤(0.22μm)。滤液经过高效液相色谱连续两次分离纯化。首次分离制备HPLC条件：色谱柱型号为YMC-Pack ODS-AQ 150×20mm L.D.，流动相：0.05％三氟乙酸/水(A)和0.05％三氟乙酸/甲醇(B)，紫外检测条件：检测器型号Waters 2998、 测器波长：215nm，柱温：25℃，流速：4mL/min，进样量：5mL。梯度洗脱方法：0、5、25、30、40、46分钟的洗脱液A/B体积比组成分别为95:5、95:5、5:95、5:95、95:5、95:5。其中保留时间为24.854min的色谱峰为苯基乳酸粗品峰，高效液相色谱图见图1。
二次高效液相色谱分离纯化条件：色谱柱型号为YMC-Pack ODS-AQ 150×20mm L.D.，流动相：0.05％三氟乙酸/水(A)和0.05％三氟乙酸/甲醇(B)，紫外检测条件：检测器型号Waters 2998、测器波长：215nm，柱温：25℃，流速：5mL/min，进样量：5mL。梯度洗脱方法：0、20、35、40、46、50分钟的洗脱液A/B体积比组成分别为80:20、80:20、60:40、5:95、80:20、80:20。其中保留时间为24.918min的色谱峰为苯基乳酸纯品峰，高效液相色谱图见图2。
实施例3：植物乳杆菌ZJ316上清液中苯基乳酸产量及分离纯度
检测苯基乳酸纯度(见图3)，分析型高效液相色谱条件：色谱柱型号YMC-Pack ODS-AQ 150×4.6mm L.D.，紫外检测条件：检测器型号Waters 2498、检测器波长：215nm，柱温：25℃，流速：0.8mL/min，进样量：50μL。流动相和梯度洗脱方法与实施例2中二次高效液相色谱分离纯化相同。
采用同样的方法，对一系列浓度梯度(0，0.05mg/mL，0.10mg/mL，0.25mg/mL，0.50mg/mL，0.75mg/mL)的苯基乳酸标准品(购买于Sigma公司)测定。建立出峰保留时间和峰面积的标准曲线，并计算得到回归方程(见图4)。
实施例4：植物乳杆菌ZJ316上清液中苯基乳酸的对映异构体比例
植物乳杆菌ZJ316发酵生产苯基乳酸中手性分子分析(见图5)，液相色谱仪LC-20A(岛津，日本)，手性色谱柱型号：大赛路
Figure PCTCN2018095752-appb-000003
IG(IG00CE-UC058)(0.46cm I.D.×25cm L×5μm)，流动相：正己烷/乙醇/三氟乙酸＝92/8/0.1(v/v/v)，柱温25℃，紫外检测波长为215nm，流速1.0mL/min。进样量为5μL，等度洗脱20min。
实施例5：植物乳杆菌ZJ316发酵上清液中纯化的苯基乳酸的广谱抑菌性
采用牛津杯法对纯化得到的苯基乳酸进行广谱抑菌性测定。牛津杯法显示的抑菌圈大小来测定抑菌物质对指示菌的作用效果。采用96孔板微量法对该苯 基乳酸的最小抑菌浓度测定。在96孔板中依次加入100μL指示菌(起始OD 600＝0.05)，将苯基乳酸按50μL体积加入到相应的96孔板中，形成苯基乳酸终浓度梯度：0.50mg/mL、1.00mg/mL、1.25mg/mL、1.5mg/mL、1.75mg/mL、2.00mg/mL、2.50mg/mL、3.00mg/mL、3.50mg/mL、4.00mg/mL。将96孔板置于适宜的指示菌培养条件下培养，每隔1小时用酶标仪测定OD 600值。与起始OD 600值相比，培养24小时OD 600值不增加，则该条件下苯基乳酸的浓度定义为苯基乳酸对指示菌的最小抑菌浓度(MIC)。
结果显示该植物乳杆菌产生的苯基乳酸对多种致病菌和腐败菌有抗菌作用(见表1)，具有广谱抗菌性。
表1植物乳杆菌ZJ316产生苯基乳酸对指示菌的抗菌效果及其最小抑菌浓度
Figure PCTCN2018095752-appb-000004
注：抑菌圈直径(mm)：“+++”，d>16.00，“++”14.00<d<16.00,“+”：d<14.00,“-”：无抗菌效果，“/”：未测。

Claims (9)

  1. 一株产苯基乳酸的植物乳杆菌，已保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：M 208077。
  2. 如权利要求1所述的植物乳杆菌发酵产生的苯基乳酸，其特征在于，该苯基乳酸中L-苯基乳酸的质量百分数范围为92％-98％。
  3. 制备权利要求2所述的苯基乳酸的方法，其特征在于，该方法包括：
    活化植物乳杆菌生长出单菌落，由单菌落得到种子液，根据种子液得到发酵液，离心发酵液取得上清液；
    利用高效液相色谱法从发酵上清液中分离纯化得到苯基乳酸。
  4. 如权利要求3所述的方法，其特征在于，所述活化植物乳杆菌生长出单菌落具体为：将植物乳杆菌在MRS固体培养基上划线，37℃培养24h，生长出植物乳杆菌的单菌落，所述MRS固体培养基按如下组成配制：无水葡萄糖20g，吐温-80 1mL/L，七水硫酸镁0.2g，硫酸锰0.05g，胰蛋白胨10g，酵母提取物5g，磷酸氢二钾2g，柠檬酸三铵2g，牛肉浸膏10g，无水乙酸钠5g，琼脂15g，pH7.0，超纯水补足至1000mL，121℃灭菌15min。
  5. 如权利要求3所述的方法，其特征在于，所述由单菌落得到种子液具体为：挑取生长出的单菌落于MRS液体培养基中，连续传代2次得到种子液，所述MRS液体培养基按如下组成配制：无水葡萄糖20g、吐温-80 1mL/L、七水硫酸镁0.2g、硫酸锰0.05g、胰蛋白胨10g、酵母提取物5g、磷酸氢二钾2g、柠檬酸三铵2g、牛肉浸膏10g、无水乙酸钠5g，pH7.0，超纯水补足至1000mL，121℃灭菌15min。
  6. 如权利要求3所述的方法，其特征在于，所述根据种子液得到发酵液具体为：将种子液以体积分数2％的接种量接入到发酵培养基(MRS液体)中，37℃静止培养24h，得到发酵液。
  7. 如权利要求3所述的方法，其特征在于，所述离心发酵液的条件为8000rpm，15min，4℃。
  8. 如权利要求3所述的方法，其特征在于，所述分离纯化方法为：
    取发酵上清液，经XAD-2大孔树脂吸附，收集流穿液；
    以0.05％三氟乙酸/水(A)和0.05％三氟乙酸/甲醇(B)(v/v)为流动相，利用YMC-Pack ODS-AQ 150×4.6mm L.D.型号色谱柱，经两次高效液相色谱梯度洗脱，分离纯化得到苯基乳酸；首次高效液相色谱梯度洗脱方法为：0、5、25、30、40、46分钟的洗脱液A/B体积比组成分别为95:5、95:5、5:95、5:95、95:5、95:5，流速为4.0mL/min；二次高效液相色谱梯度洗脱方法为：0、20、35、40、46、50分钟的洗脱液A/B体积比组成分别为80:20、80:20、60:40、5:95、80:20、80:20，流速为5.0mL/min；
    经手性色谱柱-高效液相色谱等度洗脱20min得到苯基乳酸中L-苯基乳酸和D-苯基乳酸的比例；流动相为正己烷/乙醇/三氟乙酸＝92/8/0.1(v/v/v)，手性色谱柱型号为大赛路
    Figure PCTCN2018095752-appb-100001
    IG(IG00CE-UC058)(0.46cm I.D.×25cm L×5μm)，流速为1.0mL/min。
  9. 如权利要求2所述的苯基乳酸在抗菌中的应用，该苯基乳酸对金黄色葡萄球菌D48、瓦氏葡萄球菌、蝇葡萄球菌、肉葡萄球菌pCA 44、肉葡萄球菌pot 20、模仿葡萄球菌、甲型副伤寒沙门氏菌CMCC 50093、乙型副伤寒沙门氏菌CMCC 50094、肠炎沙门氏菌肠炎亚种ATCC 14028、肠炎沙门氏菌亚利桑那亚种CMCC(B)47001、猪霍乱沙门氏菌ATCC 13312、鼠伤寒沙门氏菌CMCC 50015、白假丝酵母、烟曲霉ATCC 13073均有抗菌作用。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117903998A (zh) * 2024-03-01 2024-04-19 朗恒科技集团有限公司 一株高产苯乳酸的植物乳杆菌st10-12及其应用

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109468285B (zh) * 2018-10-29 2022-05-27 润盈生物工程（上海）有限公司 一种植物乳杆菌gs高酶活性发酵方法
CN110205267A (zh) * 2019-05-31 2019-09-06 上海应用技术大学 一株产细菌素的植物乳杆菌及其应用
CN111197015B (zh) * 2019-12-24 2022-06-10 浙江工商大学 直投式乳酸菌发酵剂和制备方法
CN111281895A (zh) * 2019-12-24 2020-06-16 顾青 用于治疗结肠炎的乳酸菌及其应用
CN111264817B (zh) * 2019-12-24 2023-02-21 浙江工商大学 直投式乳酸菌发酵剂在榨菜腌制中的应用
CN114044750B (zh) * 2021-08-06 2022-09-23 浙江工商大学 植物乳酸菌发酵上清中纯化吲哚-3-乳酸的方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101308124A (zh) * 2008-06-17 2008-11-19 江南大学 一种d-型和/或l-型苯乳酸快速检测方法
CN101657192A (zh) * 2007-03-30 2010-02-24 三得利控股株式会社 具有副交感神经活动亢进作用的医药组合物或饮食物
CN101906392A (zh) * 2010-06-23 2010-12-08 常熟理工学院 一株乳酸杆菌菌株及其应用
CN104845904A (zh) * 2015-02-27 2015-08-19 河北科技大学 一种植物乳杆菌菌株及其应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101353633B (zh) * 2008-06-13 2011-12-28 浙江工商大学 Lactobacillus plantarum ZJ316、产生的抗菌肽及其制备与应用
CN101440382A (zh) * 2008-12-19 2009-05-27 江南大学 一种生物防腐剂4-羟基苯乳酸的制备方法
CN105274023A (zh) * 2015-09-30 2016-01-27 厦门和美科盛生物技术有限公司 植物乳杆菌L.plantarum HM6055及其用途
WO2017104880A1 (ko) * 2015-12-18 2017-06-22 (주)일위 항균, 항진균 및 항바이러스제를 생산하는 락토바실러스 플란타룸 irwee-01
CN106434483B (zh) * 2016-11-04 2019-08-06 浙江工业大学 一种布氏乳杆菌及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101657192A (zh) * 2007-03-30 2010-02-24 三得利控股株式会社 具有副交感神经活动亢进作用的医药组合物或饮食物
CN101308124A (zh) * 2008-06-17 2008-11-19 江南大学 一种d-型和/或l-型苯乳酸快速检测方法
CN101906392A (zh) * 2010-06-23 2010-12-08 常熟理工学院 一株乳酸杆菌菌株及其应用
CN104845904A (zh) * 2015-02-27 2015-08-19 河北科技大学 一种植物乳杆菌菌株及其应用

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117903998A (zh) * 2024-03-01 2024-04-19 朗恒科技集团有限公司 一株高产苯乳酸的植物乳杆菌st10-12及其应用

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