TWI766184B - A kind of Lactobacillus plantarum and its use for preparing vegetable condensed yogurt and improving intestinal flora - Google Patents

Abstract

A novel Lactobacillus plantarum strain, which is deposited in the Food Industry Development Research Institute of the Republic of China, with the deposit number BCRC910891, and deposited in the General Microorganism Center of the China Microorganism Culture Collection and Management Committee, with the deposit number CGMCC17716, the plant milk Bacillus can be added to plant pulp to prepare vegetable condensed yogurt with good taste and high nutritional value. The vegetable condensed yogurt has high content of gamma aminobutyric acid and deglycosyl isoflavones. It can improve the intestinal flora of healthy people and diabetic patients. Improving the intestinal flora refers to increasing beneficial bacteria and opportunistic bacteria, especially Ekman Mucophilus in the intestine.

Description

A kind of Lactobacillus plantarum and its use for preparing vegetable condensed yogurt and improving intestinal flora

The present invention relates to a Lactobacillus plantarum that can be used for preparing vegetable condensed yogurt, and has the effect of improving intestinal bacteria.

Lactic acid bacteria (LAB) is a general term for microorganisms that can convert sugars into lactic acid as the main metabolite. Lactic acid bacteria are widely used in traditional foods all over the world, and are generally recognized as safe (General Recognized as Safe; GRAS) microbial strains.

Lactic acid bacteria can provide the manufacture and flavor of fermented food mainly through three metabolic pathways, including: glycolysis (fermentation of sugars), lipolysis (degradation of fat), and proteolysis (decomposition of proteins into small molecular peptides or amino acids) ). Lactic acid is the result of the main carbohydrate metabolism, but some microbial strains convert intermediate metabolites such as pyruvate into diacetyl, acetoin, and acetaldehyde. Or acetic acid, which has become the main source of sour taste in lactic acid fermentation of common dairy products; however, the main flavor of protein-based lactic acid fermentation food comes from the volatile gases generated during protein degradation, including different alcohols ( alcohols), aldehydes, esters, etc.

Although commercially available lactic acid bacteria foods, yogurt or drink yogurt made from animal milk as the main raw material for fermentation is the bulk. However, according to statistics, the number of people with lactose intolerance in the world is as high as 70%, of which Asians are as high as 95%. In addition, for vegetarians or vegetarians who choose not to use dairy products because of their beliefs, protein fermented foods that provide lactic acid bacteria have become an alternative. Trends in the development of yogurt as a substitute for animal milk.

When lactic acid bacteria are cultivated in protein solution, the organic acid released by lactic acid bacteria will cause the pH value to drop, and at the same time, it will cause the aggregation of protein molecules to form a coagulation state. This state occurs in yogurt products, and is susceptible to changes in pH, temperature, external force, etc., resulting in different degrees of water separation, serious water separation, resulting in product appearance loss and quality variation. In commercial curd products, in order to reduce the phenomenon of coagulation from water, it is often improved by adding additional solids (increasing whey protein, etc.) or adding stabilizers (such as modified starch, pectin, agarin, etc.).

Some lactic acid bacteria can produce exopolysaccharides (EPS) structures under specific ratios and culture conditions, which can slow down the phenomenon of water separation from protein coagulation, thereby improving texture and taste. However, with the difference of lactic acid bacteria species and culture conditions, the yield, composition and properties of these exopolysaccharides vary greatly. The extracellular polysaccharides of some strains can help the strains resist the pH value of the gastrointestinal tract and survive safely in the gastrointestinal tract.

The "gut-brain axis" is an important way for the gut and the brain to communicate with each other, in which the microbes in the gut play an important role, including irritable bowel disorder, depression, anxiety, autism, chronic Fatigue, type 2 diabetes and other diseases with a high prevalence are all related to the brain-gut axis. The bacterial phyla in the human gut are dominated by Firmicutes and Bacteroidetes, followed by Actinobacteria, Proteobacteria, Fusobacteria and Verrucobacteria, etc., can be classified into as many as 50 phyla depending on the living area, diet, and health status.

The interaction between the dynamics of gut microbes and the human body is often characterized by beneficial bacteria, harmful bacteria, and opportunistic microorganisms. Among them, opportunistic microorganisms change with the ratio of beneficial and harmful bacteria. However, when the proportion of beneficial bacteria is higher than that of harmful bacteria, the proportion of opportunistic microorganisms increases; and vice versa.

Commonly considered beneficial bacteria in gut microbes include: Bifidobacterium bifidum , Bifidobacterium pseudolongum , Bifidobacterium animalis, Bifidobacterium animalis , Bifidobacterium bifidum in the order Bifidobacteriales Bifidobacterium thermoacidophilum , Bifidobacterium breve , Bifidobacterium adolescentis , Bifidobacterium longum , etc.; Lactobacillus in Lactobacillaceae reuteri ), Lactobacillus plantarum , Lactobacillus salivarius , Lactobacillus paraplantarum , Lactobacillus acidophilus , Lactobacillus delbrueckii bulgaricus , Lactobacillus delbrueckii bulgaricus Lactobacillus brevis ), Lactobacillus casei ( Lactobacillus casei ), Lactobacillus gasseri ( Lactobacillus gasseri ), Lactobacillus helveticus ( Lactobacillus helveticus ), Lactobacillus jensenii ( Lactobacillus jensenii ) etc .; Mucinophilic bacteria ( Akkermansia muciniphila ) and so on.

The gut microbes that are often considered harmful include: Clostridium difficilie , Clostridium perfringes , pathogenic Escherichia coli , Staphylococcus aureus , green Pseudomonas aeruginosa , Klebseilla , Salmonella , Shigella , Campylobacter , Vibrio cholerae , Bacillus cereus ), Clostridium ( Fusobacteria ), Streptococcus ( Streptococcus ), etc.

Therefore, maintaining or promoting a healthy intestinal flora through diet or medicine is worth looking forward to maintaining a higher proportion of probiotics. At present, the mechanism of why the proportion of beneficial bacteria in animals can be increased through specific dietary supplements is still unknown. Therefore, what kind of probiotics, or what combination of probiotics, and what kind of substrate is used for fermentation products, There is still a need to explore how to increase the proportion of beneficial bacteria in the animal gut. In view of this, the purpose of the present invention is to provide a plant fermentation product that can increase the ratio of beneficial bacteria in animals and its application.

Among the beneficial flora in the gut, Akkermansia muciniphila is considered to be an important species. Ekmanmucinophilus is an absolutely anaerobic bacterium that uses intestinal mucosal proteins as the sole carbon and nitrogen source in animals. It was first reported in 2004 by the laboratory of Dutch scholar Willemde Vos. This bacterium is abundant in the human body and can be as high as 1-3%. Because it can decompose intestinal mucosal proteins, it can affect the physiology of intestinal endothelial cells that grow tightly under the intestinal mucosa. Since 2007, successive studies have found that Ekman Mucophilus may be a next-generation probiotic strain with physiological functions and worthy of application and development. It is now known that Ekmanella is negatively associated with many health problems. For example, individuals with less Ekmanophilus are more likely to gain weight, develop inflammatory symptoms, and type 2 diabetes. The more animals with more Ekmania muciniphila, the proportion of obesity, inflammatory symptoms and type 2 diabetes is relatively reduced. The Ekmani mucinophilus in the feces of diabetic mice will be reduced to less than 3000 times compared with normal mice; the feces of obese mice In Ekman mucinophilus, it will be reduced to less than 100 times compared with normal mice. The growth of Ekman Muxophilus in the intestines of mice on a high-fat diet can protect mice from the metabolic and immune risks caused by high-fat diets. It has a very significant effect on increasing the effect of cancer immunotherapy. Therefore, providing a proper diet to increase the number of Ekmanmucinophilus in the gastrointestinal tract is expected to be beneficial in maintaining physical health.

Previous studies have shown that adding Bifidobacterium animalis subsp.lactis to the diet directly for 14 days can increase the number of Ekmanmucinophilus in the feces, but Lactobacillus rhamnosus has no such effect; Sugar given to mice receiving a high-fat diet can effectively increase the number of Ekman Mucophila in the feces; supplementation with cranberry or Concord grape extract can effectively increase the amount of Ekman Mucophila in the feces. Quantity, however, supplementation with pomegranate extract, green tea extract, or California grape extract had no effect on changes in the number of Ekmanmucinophilus in healthy humans or mice.

At present, the mechanism of why the number of Ekmanmucinophilus in animals can be increased by specific dietary supplements is not known. Therefore, what kind of probiotics, or what kind of combination of probiotics, and under what kind of substrate are fermented The latter product still needs to be explored to increase the number of Ekman Muxophilus in animals.

In view of this, how to provide a plant-based fermentation product that can increase the number of Ekmanyl muxophilus in animals or in vitro and its application has become an important issue to be solved by the present invention.

The object of the present invention is to provide a novel lactic acid strain, which is a Lactobacillus plantarum , which is deposited in the Food Industry Development Research Institute of the Republic of China, and the deposit number is BCRC910891, and is deposited in the China Microorganism Culture Collection Management Committee General Microbiology Center, deposit number CGMCC17716.

Another object of the present invention is to provide a vegetable condensed yogurt, which is prepared by adding probiotics to plant pulp, wherein the probiotics are Lactobacillus plantarum , which is registered in the Food Industry Development Research of the Republic of China Institute, the deposit number is BCRC910891, and the deposit number is CGMCC17716, and the deposit number is CGMCC17716; the water-free water of the plant condensed yogurt is less than 25%.

In order to achieve the aforementioned purpose of the invention, the probiotics may further comprise Pediococcus pentosaceus .

In order to achieve the aforementioned purpose of the invention, the plant pulp is obtained by homogenizing water and whole grains.

For the purpose of the invention, wherein the whole grains and stone fruits are selected from the group consisting of millet, rice, wheat, soybean, sorghum, peanut, coix seed, quinoa, coix seed, almond, sesame, red bean, mung bean, and cashew nut.

In order to achieve the aforementioned purpose of the invention, the content of gamma aminobutyric acid in every 100 grams of the vegetable condensed yogurt exceeds 15 mg.

In order to achieve the aforementioned purpose of the invention, the content of gamma aminobutyric acid in each 100 grams of the vegetable condensed yogurt exceeds 25 mg.

In order to achieve the aforementioned purpose of the invention, the deglycosylation rate of isoflavones in the vegetable condensed yogurt exceeds 80%.

In order to achieve the aforementioned object of the invention, the vegetable condensed yogurt can be used as a culture component for culturing Akkermansia muciniphilia .

Another object of the present invention is to provide a use of the aforementioned vegetable condensed yogurt for improving intestinal flora.

In order to achieve the above-mentioned object of the invention, wherein the improvement of the intestinal flora is to improve the diabetic patients. of gut bacteria.

In order to achieve the aforementioned purpose of the invention, the improvement of the intestinal flora is the increase of beneficial bacteria or opportunistic bacteria in the intestinal tract.

In order to achieve the aforementioned purpose of the invention, the beneficial bacteria include but are not limited to Akkermansia muciniphilia .

To sum up, compared with the prior art. The present invention has the following advantages:

(1) BCRC 910891 has a good production capacity of viscous polysaccharides. It can be used alone or in combination with other probiotics to agglomerate soy protein (soymilk) into a vegetable condensed state yogurt that is not easy to separate from water-soymilk lactic acid bacteria fermentation product.

(2) The soymilk lactic acid bacteria fermented product made by BCRC 910891 can improve the beneficial bacteria and opportunistic bacteria in the intestine and effectively promote the ratio of Akkermansia muciniphila strain in the intestinal flora, which can improve the intestinal flora, especially the intestinal flora of diabetic patients. Fungal phase.

(3) The content of γ -aminobutyric acid and the proportion of deglycosyl isoflavones in the total isoflavones in the soymilk lactic acid bacteria fermented products made by BCRC 910891 were greatly increased.

In addition to soy fermented products, lactic acid bacteria fermented products made from oats, rice, and barley with BCRC 910891 can also promote the growth of Akkermansia muciniphila strains.

Figure 1 shows the lactic acid bacteria fermentation products of soybean milk containing L. plantarum BCRC 910891 (A), Pediococcus pentosaceus BCRC 11064 (B) and Lactobacillus pentosus BCRC 11053 (C) alone or in combination. Effects of Akkermansia muciniphila BCRC 81048 ^T growth.

Figure 2 shows the fermentation of mixed L. plantarum BCRC 910891 (A), Pediococcus pentosaceus BCRC 11064 (B) and Lactobacillus pentosus BCRC 11053 (C) using commercially available plant sources as substrates The effect of the product on the growth of Akkermansia muciniphila BCRC 81048 ^T.

Figure 3 shows the changes of intestinal flora in the diabetic model mice successfully induced by STZ, respectively, after 28 days of feeding with cooked soy milk (comparison group) and cooked soy milk lactic acid bacteria fermented by L. plantarum BCRC 910891 (experimental group).

Figure 4 shows the diabetic model mice successfully induced by STZ. Ekman mucin in the intestines of the cooked soy milk (comparative group) and the cooked soy milk lactic acid bacteria fermented by L. plantarum BCRC 910891 (experimental group) were fed for 28 days. proportion of bacteria.

The present invention is exemplified by the following examples, but the present invention is not limited by the following examples.

Microorganisms and culture medium: L. plantarum BIFT -LP-588, which is unique to this experiment, is a strain selected from Taiwan's alpine tea planting and is deposited in the Food Industry Development Research Institute, a consortium, and the deposit date is May 3, 2019 The date, the deposit number is BCRC 910891; and in accordance with the Budapest Treaty, the deposit date is May 6, 2019, and the deposit number is CGMCC17716. Other used lactic acid bacteria were purchased from the Biological Resource Conservation and Research Center (BCRC), Taiwan Institute of Food Industry, including L. plantarum BCRC 10069, Pediococcus pentosaceus BCRC 11064, and Lactobacillus pentosus BCRC 11053 . Add 50 mL of MRS culture solution to 1 mL of the above frozen bacterial solution of lactic acid bacteria, and cultivate at 37° C. for 8-24 hours to activate the bacterial solution, so that the number of bacteria reaches 1.0*10 ⁸ CFU/mL.

Embodiment 1 The manufacturing method of vegetable condensed state yogurt

A vegetable protein mixture is provided, wherein the vegetable protein mixture is composed of whole grains or stone fruits and water; a homogenization step and a heating step are performed to make the vegetable protein mixture form a vegetable pulp, wherein the heating step is higher than 90 ℃; And carry out a fermenting step, add a fermenting strain to the vegetable pulp, and carry out a fermenting time at a fermenting temperature, when the pH value of the vegetable protein mixed pulp reaches 4.5, immediately at 4 ℃ Carry out low temperature treatment, whereby the vegetable pulp is formed into the vegetable condensed state yogurt; wherein the whole grains and stone fruits comprise millet, rice, wheat, soybean, sorghum, peanut, barley, quinoa, barley, almond, sesame, red bean , mung beans, cashew nuts, the Lactobacillus plantarum is deposited in the Food Industry Development Research Institute of the Republic of China, and the deposit number is BCRC910891, and is deposited in the General Microorganism Center of the China Microbial Culture Collection and Management Committee, and the fermentation time is 6-36 hours, the fermentation temperature was 27-45°C.

The vegetal condensed state yogurt obtained by the aforementioned method has a water separation of less than 25%, has a good taste, and the gamma aminobutyric acid content per 100 grams of the vegetable condensed state yogurt exceeds 15 mg, and the isoflavones are desugarized. The base rate is more than 80%, and the nutritional value is high.

Example 2 Characteristics of L. plantarum BCRC 910891

L. plantarum BCRC 910891 , which is unique to this experiment, was screened out from Taiwan alpine tea plants. The 16rDNA sequence was identified by molecular biology as appendix. The partial sequence similarity with the 16S ribosomal RNA of Lactobacillus plantarum disclosed so far is less than 100%, as follows ; plantarum strain CIP 103151 16S ribosomal RNA、(99.52718676%)、 Lactobacillus plantarum strain JCM 1149 16S ribosomal RNA(99.52718676%)、 Lactobacillus plantarum strain NRRL B-14768 16S ribosomal RNA(99.52718676%)、 Lactobacillus plantarum strain NBRC 15891 16S ribosomal RNA( 99.52038369%), and Lactobacillus plantarum subsp. argentoratensis strain DKO 22 16S ribosomal RNA (98.81982691%) and so on.

Example 3 Differences in the ability of probiotics to produce polysaccharides

The results in Table 1 show the difference in the ability of probiotics to produce polysaccharides. Lactobacillus plantarum BCRC 910891 was cultured in MRS medium for 24 hours, and the polysaccharide content was analyzed by the phenol sulfuric acid method . plantarum ) BCRC 10069 under the same culture conditions, the polysaccharide produced at 3222ppm is high; compared with other Lactobacillus plantarum screened from different sources, the polysaccharides obtained under the same culture conditions are also relatively high.

L. plantarum BCRC 910891 was cultured in MRS plate medium for 24 hours. After 24 hours, the colonies were picked vertically with a toothpick . Under the same culture conditions, the length of the colony sticky filaments was 120 mm; for Lactobacillus plantarum screened from different sources, under the same culture conditions, the measured colony sticky filaments extended lengths ranging from 0 to 210 mm, while other lactic acid bacteria such as Lactobacillus rhamnosus (Lactobacillus rhamnosus ( Lactobacillus rhamnosus TS6), Lactobacillus brevis BCRC 12310, Pediococcus pentosaceus BCRC 11064, under the same culture conditions, the measured length of colony sticky filaments ranged from 0 to 95 mm.

The amount of polysaccharide produced by L. plantarum BCRC 910891 in MRS medium is lower than that of L. plantarum G30 or P30, but the polysaccharide ductility exhibited by L. plantarum BCRC 910891 is much higher than that of L. plantarum G30 or P30. L. plantarum G30 or P30 is better, showing that different Lactobacillus plantarum still have differences in polysaccharide properties, and there is not necessarily a positive correlation between polysaccharide content and polysaccharide extension properties.

Example 4 Lactic acid bacteria fermented product

4-1 Preparation of lactic acid bacteria fermentation products

Soymilk production: Soymilk includes cooked soybean milk and cooked whole soybean milk; Cooked soybean milk production: After soaking soybeans in water at a ratio of 1:5, use commercially available grinding equipment to homogenize the soybean pulp, then use commercially available filtering equipment, filter After removing the soybean dregs with cloth or filter, the obtained raw soybean milk is put into an autoclave for staged heat treatment: heated at 105°C for 5 minutes to remove the activity of soybean lipoxygenase, and the temperature is lowered to 90°C and heated for 15 minutes . After the cooked soy milk is cooled, put it at 4°C for later use; for the production of cooked whole soy milk: soak the soybeans in water at a ratio of 1:5, filter, weigh the filtered water and record it, put the drained soybeans in the Autoclave for staged heat treatment: heat at 121°C for 10 minutes, reduce the temperature to 105°C and heat for 25-60 minutes, take it out and place it in a commercially available wet homogenizer machine, such as a juicer, etc., and add the filtered water weight recorded before heating, and then homogenize. The homogenized whole soybean milk was heated at 90°C for 15 minutes, and after the whole soybean milk was cooled, it was placed at 4°C for use.

Soymilk lactic acid bacteria fermentation: The refrigerated cooked soybean milk or whole soybean milk is heated at 65°C for 30 minutes, and the temperature is lowered to below 37°C, which can be used as the substrate for lactic acid bacteria fermentation. Add 0.1-5% (v/v) of activated lactic acid bacteria liquid to the fermentation substrate, so that the initial bacterial count of each lactic acid bacteria strain in the fermentation substrate is at least 1.0*10 ⁵ CFU per milliliter, at 30-37 ℃ After culturing for 8-24 hours, when the pH value of the soymilk lactic acid bacteria fermented product reaches 4.6, the fermentation end point is reached, and it is moved to 4°C for low-temperature aging treatment. The fermented product is freeze-dried with a freeze dryer, and then crushed into powder with a homogenizer to obtain a powdered soymilk lactic acid bacteria fermented product, which is then reduced to a soymilk lactic acid bacteria fermented product solution with sterile water.

Lactobacillus fermentation of plant pulp: buy commercially available coix seed pulp, almond milk, rice milk, and wheat milk, add L. plantarum BCRC 910891 , L. plantarum BCRC 10069 ^T , Pediococcus pentosaceus BCRC 11064 and pentose lactic acid Bacillus Lactobacillus pentosus BCRC 11053 ^T was 1% each, and cultured at 30-37°C for 8-24 hours. When the pH value of the fermented product of Lactobacillus pentosus in plant plasma reached 4.6, the fermentation end point was reached, and it was transferred to 4°C for low-temperature aging treatment. After the fermented product is freeze-dried with a freeze dryer, it is crushed into powder with a homogenizer to obtain a powdered lactic acid bacteria fermentation product, which is then reduced to a solution of the lactic acid bacteria fermentation product with sterile water.

The lactic acid bacteria fermented product of the aforementioned low-temperature aging treatment is freeze-dried with a freeze dryer, and then crushed into powder with a homogenizer to obtain a powdered lactic acid bacteria fermented product, which is then reduced to a lactic acid bacteria fermented product solution with sterile water.

4-2 Quality inspection of soymilk lactic acid bacteria fermentation products

Characteristics of soymilk lactic acid bacteria fermentation products

The cooked soybean milk lactic acid bacteria fermented products and the cooked whole soybean soybean milk lactic acid bacteria fermented products completed with different probiotics were refrigerated at 4 °C, and the characteristics of the lactic acid bacteria bacteria count, acidity (relative lactic acid content), and water separation were measured regularly, and the characteristics of the soybean milk lactic acid bacteria fermented products were evaluated. The results are shown in Table 2 .

Water separation rate detection

A 20 g sample was centrifuged at 1000 x g for 10 min, the supernatant was decanted and weighed. During the storage period of soymilk lactic acid bacteria fermented products, because the lactic acid bacteria continue to produce acid, the structure of protein coagulation is constantly changed, and the water in the structure is released, resulting in syneresis. Excessive occurrence of this phenomenon will cause inconsistencies in the overall organization and quality, which will affect the taste of the product, so the less water separation, the better. The calculation method of the degree of water separation is as follows: degree of water separation = weight of supernatant / original weight of soybean lactic acid bacteria fermentation product (20g) x 100%

All combinations can reach the concentration of lactic acid bacteria above 10 ⁸ CFU/mL within 24 hours of fermentation. Compared with the combination without L. plantarum BCRC 910891 , in the combination with L. plantarum BCRC 910891 , the pH at the end of fermentation was slightly higher, and the pH value decreased more than 7 days after refrigerating at 4°C. Small, but also has a lower water separation rate. Soymilk was fermented with L. plantarum BCRC 910891 alone. After 24 hours of fermentation, the pH was still above 4.5, showing a semi-fluid coagulation shape, so the water separation rate was not tested.

Analysis of 4-3 gamma aminobutyric acid (γ-aminobutyric acid; GABA)

GABA is a substance that mainly inhibits nerve conduction in the central nervous system. In recent years, studies have confirmed that GABA, whether by injection or oral administration, can control the phenomenon of blood pressure increase, sleep quality and emotional depression through physiological mechanisms. the goal of. The "Japan Health and Nutritional Foods Association" has clearly identified GABA as one of the materials recommended for use in "special health food" for hypertensive patients. In this type of food, the recommended daily intake of 10-20mg of GABA has the effect of reducing blood pressure. Blood pressure function; and the "Japan Functional Food Research Association" recommends that GABA has the potential to become a functional material. Many studies have pointed out that ingesting GABA 26.4mg has the effect of improving insomnia and anxiety. In this study, different lactic acid bacteria were added to soy milk for fermentation, and the results are summarized in Table 2: L. plantarum BCRC 910891 alone can significantly increase the GABA content in soy milk from 9.36±0.06mg/100g to 25.97±0.08mg/100g; adding Lactic acid bacteria including L.plantarum BCRC 910891, L.plantarum BCRC 10069 ^T , L.plantarum G30, Pediococcus pentosaceus BCRC 11064 and Lactobacillus pentosus BCRC 11053 ^T are fermented in soybean milk, so the GABA in the fermented product can be increased to 35.72±0.15mg/ 100g; if L.plantarum BCRC 910891, Pediococcus pentosaceus BCRC 11064 and Lactobacillus pentosus BCRC 11053 ^T are added, the GABA content in the fermented product is 26.34±0.13mg/100g; if the above lactic acid bacteria combination, Lactobacillus plantarum only uses plantarum BCRC 10069 And the self-screened L. plantarum G30 combined with Pediococcus pentosaceus BCRC 11064 and Lactobacillus pentosus BCRC 11053 ^T , the GABA content in the fermented product was 20.46±0.07mg/100g; the experimental results showed that L. plantarum and Pediococcus pentosaceus And Lactobacillus pentosus in the process of fermenting soybean milk, the interaction between lactic acid bacteria can also increase the GABA content in the fermentation result; In this study, L. plantarum BCRC 10069 ^T or self-selected L. plantarum G30 can also produce GABA, but the production capacity of L. plantarum BCRC910891 is still higher than that of L. plantarum BCRC910891 . good.

4-4 Isoflavones (Isoflavones) Deglycosylation Analysis

Soy isoflavones are a kind of phytoestrogens contained in soybeans, and more scientific literatures have pointed out that soy isoflavones are effective in breast cancer, rectal cancer, or It has a preventive effect on osteoporosis. Long-term consumption of foods containing soy isoflavones can alleviate the symptoms of menopausal syndrome. In soy isoflavones, there are many compounds with similar structures, which can be roughly divided into three groups of aglycones, such as daidzein, genistein, and glycitein. The main structure is connected with different sugar groups to form daidzin, genistin, glycitin, daidzein, genistein genistein, glycitein, acetyldaidzin, acetylgenistin, acetylglycitin, malonyldaidzin ), malonylgenistin and malonylglycitin and other glycosyl isoflavones are stable in soybean. After ingestion, soybeans are hydrolyzed into deglycosyl isoflavones by glycosidases produced by intestinal microorganisms. These deglycosyl isoflavones can be further metabolized in the body to form compounds such as equol; due to the chemical structure of these metabolites It is very similar to estrogen in mammals, so it can exhibit the properties of "Selective Estrogen Receptor Modulator (S.E.R.M.)" in the body.

However, because the microorganisms distributed in the human intestinal tract do not necessarily have good glycosyl hydrolysis enzymes, it may affect the bioactive utilization of soybean isoflavones. Therefore, through the processing process, selecting appropriate microorganisms to convert the glycosyl isoflavones in soybeans can effectively provide fermented foods with deglycosylated isoflavones with physiological activity.

The L. plantarum BCRC910891 used in this study has excellent production capacity of glycosyl hydrolase. When used alone, the proportion of deglycosylated isoflavones in soy milk can reach 88.62±0.13%; L.plantarum BCRC 10069 ^T , the proportion of deglycosylated isoflavones in soy milk can reach 61.62±0.13%, but the self-selected L. plantarum G 30 only has deglycosylated isoflavones in soymilk ratio of 24.32±0.06%, Pediococcus pentosaceus Although BCRC 11064 or Lactobacillus pentosus BCRC 11053 ^T , etc., can make the ratio of deglycosylated isoflavones in soymilk reach 50.62±0.13%, but if combined with L. plantarum BCRC910891 , it can make soymilk deglycosylated isoflavones The proportion of deglycosylated isoflavones was 92.15±0.23%.

The effect of embodiment 5 fermentation product on the growth of Ekman Mucophilus

Pretreatment of fermented products: After homogenization of lactic acid bacteria fermented products such as cooked soybean milk, cooked whole soybean soybean milk or plant pulp, the supernatant was obtained by centrifugation in a water bath at 65°C for 30 minutes, and the supernatant was stored at 4°C for later use.

Akkermansia muciniphila BCRC 81048 ^T (purchased from BCRC), cultured in GAM medium, 37°C, in an absolute anaerobic incubator with N ₂ : CO ₂ (80:20 ratio) replacing air For 24 hours, centrifuge when the absorbance at 600 nm reaches 2.0, remove the culture medium, collect the cells, add freshly prepared GAM medium, and adjust the absorbance to 0.05. Take 10% (v/v) of the supernatant of the pretreated fermentation product, add it, and incubate it in an absolute anaerobic incubator with N ₂ : CO ₂ (80:20 ratio) at 37°C for 24 hours. The growth of Ekman Muxophila was detected by the change of absorbance value.

Calculation of relative growth amount = absorbance value at 24 hours - absorbance value at 0 hours / absorbance value at 0 hours

GAM Medium is formulated to contain per liter: Proteose Peptone 5.0g, Digested Serum 10.0g, Yeast Extract 2.5g, Meat Extract 2.2g, Liver Extract 1.2g, Dextrose 0.5g, Soluble Starch 5.0g, L-Tryptophan 0.2g, L-Tryptophan Cysteine Hydrochloride 0.3g, Sodium Thioglycollate 0.3g, L-Arginine 1.0g, Vitamin K1 5mg, Hemin 10mg, Potassium Dihydrogen Phosphate 2.5g, Sodium Chloride 3.0g, Peptone 5.0g, Soya Peptone 3.0g, pH adjusted to 7.1.

L. plantarum BCRC 910891, Pediococcus pentosaceus BCRC 11064 or Lactobacillus pentosus BCRC 11053 ^T in different combinations were added to cooked soybean milk for fermentation. The supernatant was added to GAM medium at a ratio of 10% to observe the effect of various lactic acid bacteria combinations on the growth of Akkermansia muciniphila BCRC 81048 ^T.

Fig. 1 The results show that the fermentation of soymilk lactic acid bacteria containing Lactobacillus plantarum BCRC 910891 (A), Pediococcus pentosaceus BCRC 11064 (B) and Lactobacillus pentosaceus BCRC 11053 (C) alone or in combination, for Ekman Muxophilus Effects of BCRC 81048 ^T growth. It can be seen from the results in Figure 1 that the components of the soymilk that have not been fermented by lactic acid bacteria have very little effect on the growth stimulation of Ekman Mucophilus BCRC 81048 ^T. However, the soymilk fermented products fermented with different combinations of Lactobacillus plantarum BCRC 910891 and Pediococcus pentosaceus BCRC 11064 or Lactobacillus pentosus BCRC 11053 ^T , the growth of Ekman Muxophilus BCRC 81048 ^T was higher than that of the original GAM. The medium is better.

Lactobacillus plantarum BCRC 910891, Pediococcus pentosus BCRC 11064 and Lactobacillus pentosus BCRC 11053 ^T were added together in commercially available substrates of different plant sources for fermentation. Proportionally added into GAM medium to observe the effect of various combinations of lactic acid bacteria on the growth of Ekman Muxophilus BCRC 81048 ^T.

Fig. 2 is the product that contains mixed Lactobacillus plantarum BCRC 910891 (A), Pediococcus pentosaceus BCRC 11064 (B) and Lactobacillus pentosus BCRC 11053 ^T (C) with commercially available different plant sources as substrates for fermentation. Effects of Mucinophilus BCRC 81048 ^T growth. From the results in Figure 2, it can be seen that the components in the barley juice, oat drink and rice milk that have not been fermented by lactic acid bacteria are beneficial to the growth stimulation of Ekman Muxophilus BCRC 81048 ^T compared to the GAM medium. However, the fermented product after combining Lactobacillus plantarum BCRC 910891 with Pediococcus pentosus BCRC 11064 or Lactobacillus pentosus BCRC 11053 ^T was better than the original GAM medium for the growth of Ekman Muxophilus BCRC 81048 ^T. The aforementioned commercially available vegetable pulp is replaced with the vegetable pulp of whole grains or stone fruits and water homogenization, and similar results can also be obtained. The whole grains and stone fruits include millet, rice, wheat, soybean, sorghum, peanut, barley, quinoa, barley, almond , sesame, red beans, mung beans, cashews.

Through this study, it was shown that the products obtained from the fermentation of lactic acid bacteria from different plant-derived substrates are conducive to the growth of Ekman Muxophila, which can be used for the cultivation of Ekman Mucophila in the future.

Example 6 The effect of soy milk probiotic fermentation product on the intestinal flora of diabetic mice

Yuan Hongde's master's thesis "High-value-added fermented soybean milk products in Streptozotocin (STZ)-induced hyperglycemia mice model" taught that L. plantarum fermented soybean milk products have significant protective effects on hyperglycemic mice. The results of the thesis Shows the changes of blood glucose concentration during 28 days after feeding with sterile water (control group), soy milk (comparison group) and fermented soymilk lactic acid bacteria product (experimental group) in mice with streptozotocin (Streptozotocin; STZ)-induced diabetic rat model. , the blood glucose of the mice in the experimental group was significantly different from the control group from the 7th day, and this phenomenon continued to be stable until the 28th day after the end of the experiment. In the STZ diabetic mice, the blood sugar increased from 220mg/dL to 400mg/dL continuously during the 28-day feeding period; while in the group fed with soy milk, the blood sugar slowly increased from 220mg/dL to 320mg/dL. Around dL, but after being fed with fermented soy milk products, the blood sugar of STZ diabetic mice was stably maintained at around 200-220 mg/dL during the feeding period. From the changes in blood sugar, it can be clearly seen that fermented soybean milk products can effectively control the rapid rise in blood sugar. Therefore, the present invention further knows whether the soymilk probiotic fermented product prepared by L. plantarum BCRC 910891 can improve the intestinal flora of diabetic patients.

Thirty 6-week-old 6-week-old Specified Pathogen-Free (SPF) mice (strain: C57/BL6; source: National Laboratory Animal Center) were used. injection) 40 mg/kg body weight of streptozotocin (Streptozotocin or Streptozocin; STZ for short; dissolved in 100 mM sodium citrate, pH 4.5). During the first seven days, streptozotocin was administered once a day for a total of 7 times to establish a diabetic model in mice. Seven days later, blood glucose was measured by the glucose oxidase method to confirm whether diabetes was successfully induced. When the blood glucose level of the unfasted mice reached 250 mg/dL during the experiment, the establishment of the disease model was considered to be successful. During the subsequent experiments, blood glucose was measured every three days, and the blood glucose concentration of the diabetic mice was evaluated by using a commercially available Glucose Assay Kit (Sigma, USA) to evaluate the glucose content in the blood. The blood glucose concentration test was performed when the mice were fasted for 4 hours.

Each group consisted of 3 male mice and 3 female mice, randomly assigned. The mice were fed 100 μL of sterile water by tube every day according to the weight of the mice. The daily intake of the lactic acid bacteria fermented product per kilogram of cooked soybean milk for the mice in the experimental group was 250 mg/Kg body weight/day or 100 mg/Kg body weight/day. The daily intake of cooked soybean milk per kilogram of mice in the comparison group was 250 mg/Kg body weight/day or 100 mg/Kg body weight/day. The control group was given sterile water. Since the injection of streptozotocin completed the establishment of the mouse model of diabetes, it was administered once a day for 30 consecutive days. C57/BL6 mice were kept in a positive pressure environment without specific concurrency, and the temperature was kept at 24°C, and the The light-dark cycle field was maintained at a 12-hour switch. Feed and water supply were not restricted during the feeding period. The rearing conditions of C57/BL6 mice were carried out in accordance with the relevant laboratory animal management guidelines announced by the National Institutes of Health. Mice in all groups were treated with an induced diabetes model. At the end of the experiment, the mice were sacrificed, and whole small intestine sections were obtained, which were frozen at -20°C.

The mouse small intestine frozen at -20°C was cut open with sterilized scissors, and then the intestinal mucosa was scraped with a sterilized spatula and placed in a PBS solution. Use vortex to fully shake out the bacteria on the mucosa, centrifuge at 12,000 rpm, remove the supernatant, and then wash the precipitate with PBS solution. The set of reagents QIAmp DNA Stool Mini kit (Qiagen) was used for DNA extraction. The resulting DNA extract was stored at -20°C for later use. For the analysis of intestinal bacteria, the 16S rRNA primer of bacterial universal ribosome is used, and the primer sequence is shown in Table 3 for the V4 region. For example, the forward primer F1 can be combined with the four reverse primers R1, R2, R3, and R4. It is added to the extracted DNA sample, and the polymerase chain reaction is carried out, and the analysis is carried out by the next generation gene sequencing (Next General Sequencing) method.

A: Adenine; C: Cytosine; G: Guanine; T: Thymine; R: Guanine/Adenine(purine); Y: Cytosine/Thymine(pyrimidine); M: Adenine/Cytosine; S: Guanine/Cytosine D: Guanine/Adenine /Thymine; H: Adenine/Cytosine/Thymine; V: Guanine/Cytosine/ Adenine; N: Adenine/Guanine/Cytosine/Thymine.

The intestinal flora is classified into beneficial bacteria, harmful bacteria and opportunistic bacteria. Figure 3 shows the results of STZ-induced diabetes model mice, which were fed with cooked soybean milk (comparative group) and L. plantarum BCRC 910891 fermented lactic acid bacteria fermentation product (experimental group) for 28 days after feeding the intestinal bacteria phase changes: feeding In the group of cooked soybean milk lactic acid bacteria fermented by BCRC 910891, the proportion of beneficial bacteria in the intestinal tract of mice reached 77.6%, and the proportion of opportunistic bacteria was 20.0%; while in the group fed cooked soybean milk, the proportion of beneficial bacteria in the intestinal tract of mice reached 77.6%. The proportion is only 3.8%, which is obviously lower than the 49.4% of harmful bacteria, and the proportion of opportunistic bacteria ( 46.8 %) is similar to the proportion of harmful bacteria. The fermented cooked soybean milk lactic acid bacteria fermented product, after 28 days of feeding, the beneficial bacteria in the intestinal bacteria will increase by more than 15 times compared with the intestinal beneficial bacteria in the group fed only cooked soybean milk. Figure 4 shows the results of STZ-induced diabetes model mice, respectively, comparing the levels of Ekmania in the intestines of cooked soymilk (comparison group) and cooked soymilk lactic acid bacteria fermented with L. plantarum BCRC 910891 (experimental group) after feeding for 28 days The proportion of Mucinobacteria in the intestinal tract of mice in the experimental group fed with BCRC 910891 fermented soy milk was 400 times higher than that in mice fed only with soy milk.

To sum up, the Lactobacillus plantarum strain BCRC 910891 screened by Taiwan alpine tea planting in the present invention can achieve the following effects:

(1) BCRC 910891 has a good production capacity of viscous polysaccharides. It can be used alone or in combination with other probiotics to agglomerate soy protein (soymilk) into a vegetable condensed state yogurt that is not easy to separate from water-soymilk lactic acid bacteria fermentation product.

(2) The soymilk lactic acid bacteria fermented product made by BCRC 910891 can effectively promote the ratio of Akkermansia muciniphila in the intestinal flora, increase the content of lactic acid bacteria in the intestinal tract and improve the bacterial phase.

(3) The content of γ -aminobutyric acid in the soymilk lactic acid bacteria fermentation product made by BCRC 910891 can reach more than 20mg/100g, and the deglycosylated isoflavones can reach 92% of the total isoflavones.

(4) In addition to bean fermented products, the fermented products of plant pulp lactic acid bacteria made from oat, rice, barley, etc. according to BCRC 910891 can also promote the growth of Akkermansia muciniphila strains.

The above detailed descriptions are specific descriptions of feasible embodiments of the present invention, but the embodiments are not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not depart from the technical spirit of the present invention shall be included in this case. within the scope of the patent.

The above-mentioned functions are indeed in full compliance with the statutory patent requirements of novelty and progress, and you can file an application in accordance with the law.

【Biological Material Deposit】 Domestic storage information [please note in the order of storage institution, date and number]

1. The Food Industry Development Research Institute, a consortium, the deposit date is May 3, 2019, and the deposit number is BCRC 910891

Foreign deposit information [please note in the order of deposit country, institution, date and number]

1. General Microbiology Center of China Microorganism Culture Collection Management Committee, the deposit date is May 6, 2019, and the deposit number is CGMCC17716

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Claims (5)

A novel lactic acid strain, the bacterium is a Lactobacillus plantarum , which is deposited in the Food Industry Development Research Institute of the Republic of China, the deposit number is BCRC910891, and is deposited in the General Microorganism Center of the China Microorganism Culture Collection Management Committee, the deposit number for CGMCC17716. A vegetable condensed yogurt is prepared by adding probiotics to plant pulp, wherein the probiotics comprise Lactobacillus plantarum , which is deposited in the Food Industry Development Research Institute of the Republic of China, and the deposit number is BCRC910891, and the deposit number is BCRC910891. In the General Microbiology Center of the China Microbial Culture Collection Management Committee, the deposit number is CGMCC17716; wherein the water-free water of the vegetable condensed state yogurt is less than 25%, and the vegetable pulp is obtained by homogenizing water, whole grains and stone fruits; wherein each 100 The GABA content in this vegetable condensed yogurt exceeds 25 mg. A vegetable condensed yogurt is prepared by adding probiotics to plant pulp, wherein the probiotics comprise Lactobacillus plantarum, which is deposited in the Food Industry Development Research Institute of the Republic of China, and the deposit number is BCRC910891, and the deposit number is BCRC910891. In the General Microbiology Center of the China Microorganism Culture Collection Management Committee, the deposit number is CGMCC17716; wherein the water-free water of the vegetable condensed state yogurt is less than 25%, wherein the vegetable pulp is obtained by homogenizing water and soybean; wherein per 100 grams of the plant The content of gamma aminobutyric acid in the condensed yogurt exceeds 25 mg; the deglycosylation rate of isoflavones in the vegetable condensed yogurt exceeds 80%. The plant-based condensed yogurt described in the second or third claim of the patent application can be used for culturing Akkermansia muciniphilia . A use of plant-based condensed yogurt as claimed in claim 2 or 3 of the scope of the application, which is used to prepare a pharmaceutical composition for improving intestinal bacteria, wherein the improving intestinal bacteria is Ekman's viscous admixture in the intestinal tract. Proteobacteria (Akkermansia muciniphilia) increased, wherein the improvement of intestinal flora is to improve the intestinal flora of diabetic patients.

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