阅读说明：本技术 产高活性纤溶酶的植物乳杆菌及用其发酵的果蔬汁饮料 (Lactobacillus plantarum for producing high-activity plasmin and fruit and vegetable juice beverage fermented by same ) 是由 胡欣洁 朱琦琦 何维 曹阳 吴任之 余东梅 赵俊梅 刘书亮 秦文 韩国全 *** 于 2019-11-19 设计创作，主要内容包括：本发明提供了一株产高活性纤溶酶的植物乳杆菌,所述产高活性纤溶酶的植物乳杆菌的分类命名为植物乳杆菌Lactobacillus plantarum,保藏于中国科学院微生物研究所的中国普通微生物菌种保藏管理中心,保藏号为CGMCC No.17372,保藏时间为2019年3月20日。本发明还提供了利用上述植物乳杆菌发酵制备得到的果蔬汁饮料。本发明所得的产高活性纤溶酶的植物乳杆菌发酵上清液纤溶酶的酶活性为497.72U/mL；菌株发酵上清液具有较强抑菌能力；并且菌株本身对多种抗生素均无多重耐药,对于胃肠液具有较高耐受性,还具有潜在的肠道附着能力。本发明饮料达到国家相关活菌数标准,具有良好商业化价值。(The invention provides a Lactobacillus plantarum for producing high-activity plasmin, which is classified and named as Lactobacillus plantarum and is preserved in China general microbiological culture collection management center of institute of microbiology, China academy of sciences, with the preservation number of CGMCC No.17372 and the preservation time of 2019, 3 months and 20 days. The invention also provides a fruit and vegetable juice beverage prepared by fermenting the lactobacillus plantarum. The enzymatic activity of the lactobacillus plantarum fermented supernatant plasmin capable of producing the high-activity plasmin is 497.72U/mL; the strain fermentation supernatant has strong bacteriostatic ability; and the strain has no multiple drug resistance to various antibiotics, has higher tolerance to gastrointestinal fluids, and also has potential intestinal attachment capability. The beverage of the invention reaches the national relevant viable count standard and has good commercial value.)

1. The Lactobacillus plantarum for producing the high-activity plasmin is classified and named as Lactobacillus plantarum, is preserved in China general microbiological culture collection management center of institute of microbiology, China academy of sciences, has a preservation number of CGMCC No.17372, and has a preservation time of 3 months and 20 days in 2019.

2. A fruit and vegetable juice beverage, which is prepared by using fruits and vegetables as raw materials and fermenting the lactobacillus plantarum producing high-activity plasmin according to claim 1.

3. The fruit and vegetable juice beverage according to claim 2, wherein the fruit and vegetable is kiwi.

4. A method of preparing the juice beverage of claim 3, the steps of the method comprising:

juicing kiwi fruits, adding water for blending, and fermenting by using the lactobacillus plantarum producing the high-activity plasmin to obtain the high-activity plasmin.

5. Method according to claim 4, characterized in that it comprises the following steps:

(1) selecting and treating kiwi fruits: selecting 8-9-minute-maturity red-yang kiwi fruits, and removing pests and rotten fruits for later use; juicing the selected red kiwi fruits by using a spiral juicer, separating peels and fruits, and reserving fruit pulp;

(2) blending: uniformly mixing the red kiwi fruit pulp and water according to the volume ratio of 1:1, adding 80mg/100g of sodium carboxymethylcellulose and 12g/100g of white granulated sugar, and adjusting the pH value to 5.5 by using sodium citrate;

(3) and (3) sterilization: heating the prepared red-yang kiwi fruit pulp at 83 ℃ for 10min to kill harmful microorganisms and pathogenic bacteria;

(4) fermentation: cooling the sterilized red-yang kiwi fruit pulp to room temperature, adding 1% of activated lactobacillus plantarum producing high-activity plasmin for fermentation, and standing and fermenting at 37 ℃ for 28h to obtain the high-activity plasmin.

Technical Field

The invention relates to the technical field of biotechnology and food, in particular to lactobacillus plantarum for producing high-activity plasmin and fruit and vegetable juice beverage fermented by the same

Background

Lactobacillus plantarum (lactobacillus plantarum) is one of the lactobacilli that are commonly found in fermented vegetables and fruit juices. The lactobacillus plantarum is used as probiotics of human gastrointestinal tracts and has multiple functions of maintaining the balance of flora in intestinal tracts, improving the immunity of organisms, promoting the absorption of nutrient substances and the like. In the food fermentation process, lactobacillus plantarum can convert sugars in food raw materials into lactic acid, and simultaneously produce antibacterial peptides, exopolysaccharides and other metabolites. The application of the lactobacillus plantarum in the food is mainly in the aspects of dairy products, meat products, fruit and vegetable products, bean products, grains, potato products and the like, and has a wide market. According to the relevant regulations of ' list of strains available for food ' (ministry of health and supervision issue [ 2010 ] 65 '), lactobacillus plantarum can be directly used for food production. However, the plasmin production by lactobacillus plantarum and related research reports are few at present.

Thrombotic diseases are frequently generated in old people before, but the threat of thrombotic diseases to people's health is no longer limited to the old people only because the unhealthy living habits and dietary patterns of contemporary people. According to data in reports of nutrition and chronic disease status of Chinese residents (2015), the death rate of adults aged 2012 and older than eighteen years old in the country is 271.8/10 ten thousand due to cardiovascular and cerebrovascular diseases belonging to thrombotic diseases, and the death rate is a main cause of death. Thrombolysis by means of thrombolytic agents is one of the main therapeutic methods for thrombotic diseases, and prevention of thrombotic chronic diseases by means of thrombolytic agents is also attracting much attention. The thrombolytic agent mainly used for treatment at present has the defects of large side effect or high price and the like, and is not suitable for preventing thrombotic diseases. The microorganisms are various in types, the metabolites are rich and diverse, and the microorganisms are easy to culture and fast to reproduce, so the microorganisms become important resources for exploring novel thrombolytic agents. Microbial plasmin such as streptokinase, glucokinase, nattokinase and the like all show good thrombolysis characteristics, and particularly, nattokinase secreted by bacillus subtilis is already prepared into health-care food for commercialization. The screened lactobacillus which can be directly used for food fermentation production and can produce high-activity plasmin is used for researching and developing foods with plasmin activity, and can be used as effective measures for preventing thrombotic diseases and reducing the morbidity of the diseases.

Disclosure of Invention

In view of the disadvantages and needs of the prior art, it is an object of the present invention to obtain a lactobacillus plantarum strain that produces high-activity plasmin, has good bacteriostatic properties, and is not multi-resistant to multiple antibiotics.

In order to achieve the aim, the invention provides a Lactobacillus plantarum for producing high-activity plasmin, which is classified and named as Lactobacillus plantarum and is preserved in the China general microbiological culture collection management center of the institute of microbiology of China academy of sciences (the postal code 100101 of the institute of microbiology of China academy of sciences No. 3 of West Lu No.1 of Beijing sunward Xilun, Beijing city), the preservation number is CGMCC No.17372, and the preservation time is 2019, 3 and 20 days.

Unless otherwise specified, in the specification of the present invention, the lactobacillus plantarum producing high-activity plasmin in the present invention is simply referred to as lactobacillus plantarum HQ-3.

The invention also aims to provide a fruit and vegetable juice beverage, which is prepared by using fruits and vegetables as raw materials and fermenting the lactobacillus plantarum producing high-activity plasmin.

As an optional embodiment of the present invention, the fruit and vegetable is kiwi.

The invention also provides a method for preparing the fruit and vegetable juice beverage, which comprises the following steps:

juicing kiwi fruits, adding water for blending, and fermenting by using the lactobacillus plantarum producing the high-activity plasmin to obtain the high-activity plasmin.

As an alternative embodiment of the invention, the method comprises the steps of:

(1) selecting and treating kiwi fruits: selecting 8-9-minute-maturity red-yang kiwi fruits, and removing pests and rotten fruits for later use; juicing the selected red kiwi fruits by using a spiral juicer, separating peels and fruits, and reserving fruit pulp;

(2) blending: uniformly mixing the red kiwi fruit pulp and water according to the volume ratio of 1:1, adding 80mg/100g of sodium carboxymethylcellulose and 12g/100g of white granulated sugar, and adjusting the pH value to 5.5 by using sodium citrate;

(3) and (3) sterilization: heating the prepared red-yang kiwi fruit pulp at 83 ℃ for 10min to kill harmful microorganisms and pathogenic bacteria;

(4) fermentation: cooling the sterilized red-yang kiwi fruit pulp to room temperature, adding 1% of activated lactobacillus plantarum producing high-activity plasmin for fermentation, and standing and fermenting at 37 ℃ for 28h to obtain the high-activity plasmin.

The invention has the beneficial effects that:

the enzymatic activity of plasmin fermentation supernatant of the lactobacillus plantarum producing high-activity plasmin is 497.72U/mL; the fermented supernatant has stronger bacteriostatic ability; meanwhile, the lactobacillus plantarum producing the high-activity plasmin has no multiple drug resistance to various antibiotics; has high tolerance to gastrointestinal fluid and potential intestinal adhesion capability.

The beverage obtained by the invention reaches the national standard of viable count of the lactobacillus fermented viable bacteria beverage, and meanwhile, the beverage contains high-activity plasmin, so that the beverage has good commercial value.

Drawings

FIG. 1 is a graph showing preliminary screening results of screening of plasmin-producing lactic acid bacteria;

FIG. 2 is a diagram showing the results of rescreening of the screening of plasmin-producing lactic acid bacteria;

FIG. 3 is a urokinase standard curve for determining plasmin activity of Lactobacillus plantarum HQ-3 according to the present invention;

FIGS. 4 and 5 are graphs showing the morphological identification results of Lactobacillus plantarum HQ-3 according to the present invention;

FIG. 6 is a 16S rDNA amplification electrophoretogram of Lactobacillus plantarum HQ-31 of the present invention;

FIG. 7 is a phylogenetic tree of Lactobacillus plantarum HQ-3 according to the invention;

FIG. 8 is a graph showing the growth of Lactobacillus plantarum HQ-3 according to the present invention;

FIG. 9 is a PCR electrophoretogram of specific adhesin gene of Lactobacillus plantarum HQ-3 according to the present invention;

FIGS. 10 and 11 are graphs showing the results of experiments for determining the way of hydrolyzing fibrin in fermented supernatant of Lactobacillus plantarum HQ-3 according to the present invention.

Detailed Description

The present invention is described in detail below by way of examples, and it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.