阅读说明：本技术 植物乳杆菌胞外多糖及其在制备ace抑制剂组合物中的用途 (Lactobacillus plantarum exopolysaccharide and application thereof in preparation of ACE inhibitor composition ) 是由 韩美琪 于 2019-09-17 设计创作，主要内容包括：本发明提供植物乳杆菌胞外多糖及其在制备ACE抑制剂组合物中的用途,属于微生物技术领域,植物乳杆菌胞外多糖,具有如下1)和/或2)的特征：1)胞外多糖重均分子量为1.15×10<Sup>6</Sup>Da；2)胞外多糖由摩尔比为5.6:7.3:1的甘露糖、半乳糖和葡萄糖组成。本发明还提供一种植物乳杆菌胞外多糖的磷酸化衍生物。本发明植物乳杆菌胞外多糖及其磷酸化衍生物均显示出显著的ACE抑制活性且无细胞毒性。本发明还提供一种用于预防或治疗心血管疾病的组合物,包含上述胞外多糖和/或其磷酸化衍生物,该心血管疾病是选自于以下疾病所组成的组的一种或多种疾病：高血压、心脏病、中风、血栓、动脉硬化、心绞痛、心力衰竭以及心肌梗塞。(The invention provides lactobacillus plantarum exopolysaccharide and application thereof in preparation of an ACE inhibitor composition, belonging to microorganismsThe technical field, the extracellular polysaccharide of lactobacillus plantarum has the following characteristics 1) and/or 2): 1) the extracellular polysaccharide has weight average molecular weight of 1.15 × 10 6 Da; 2) the exopolysaccharide consists of mannose, galactose and glucose in a molar ratio of 5.6:7.3: 1. The invention also provides a phosphorylation derivative of the lactobacillus plantarum exopolysaccharide. The lactobacillus plantarum exopolysaccharide and the phosphorylated derivatives thereof show remarkable ACE inhibitory activity and have no cytotoxicity. The present invention also provides a composition for preventing or treating cardiovascular diseases, comprising the above exopolysaccharide and/or phosphorylated derivative thereof, wherein the cardiovascular diseases are one or more diseases selected from the group consisting of: hypertension, heart disease, stroke, thrombosis, arteriosclerosis, angina pectoris, heart failure and myocardial infarction.)

1. The lactobacillus plantarum exopolysaccharide is characterized in that: has the following characteristics 1) and/or 2):

1) the weight average molecular weight of the extracellular polysaccharide is 1.35 multiplied by 10⁶Da；

2) The exopolysaccharide is composed of mannose, galactose and glucose with a molar ratio of 5.6:7.3: 1.

2. A lactobacillus plantarum exopolysaccharide according to claim 1, characterized in that: the exopolysaccharide inhibits Angiotensin Converting Enzyme (ACE).

3. Use of a lactobacillus plantarum exopolysaccharide according to claim 1 or 2 for the preparation of an ACE inhibitor composition.

4. Phosphorylated derivatives of extracellular polysaccharides of lactobacillus plantarum as claimed in claim 1 or 2.

5. Phosphorylated derivatives of extracellular polysaccharides of lactobacillus plantarum according to claim 4, characterized in that: the substitution degree of the lactobacillus plantarum exopolysaccharide is 0.24.

6. A composition for preventing or treating cardiovascular disease, characterized by: comprising the exopolysaccharide according to claim 1 or 2 and/or the phosphorylated derivative according to claim 4 or 5.

7. The composition for preventing or treating cardiovascular disease according to claim 6, wherein: the cardiovascular disease is one or more selected from the group consisting of: hypertension, heart disease, stroke, thrombosis, arteriosclerosis, angina pectoris, heart failure and myocardial infarction.

8. The composition for preventing or treating cardiovascular disease according to claim 6, wherein: the composition is an oral composition.

9. The composition for preventing or treating cardiovascular disease according to claim 6 or 8, wherein: the composition is used in a manner that 0.1-500mg of extracellular polysaccharide and/or phosphorylated derivative of Lactobacillus plantarum is taken per day by an adult.

10. The composition for preventing or treating cardiovascular disease according to claim 6 or 8, wherein: the composition is administered by ingestion for 9-12 weeks or more.

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus plantarum exopolysaccharide and application thereof in preparation of an ACE inhibitor composition.

Background

Polysaccharides are biopolymers that occur widely in nature. They are present in all organisms, i.e. animals, plants and microorganisms. All polysaccharides are homopolysaccharides or heteropolysaccharides consisting of the same or different monosaccharides. They may also be substituted with non-sugar units, forming linear or branched structures. In recent years, the research on the activity of polysaccharides has been greatly advanced, and a large number of polysaccharides such as lentinan and ganoderan are proved to have anti-tumor activity, and ginseng polysaccharide is proved to have the function of enhancing immunity. The microbial polysaccharide mountain has also emerged a great research hot trend due to stable yield and small influence by climate and geography. The biological activity of polysaccharides is related to their structure, and four major factors can influence the biological activity of modified polysaccharides: many researches on water solubility, molecular weight (Mw), substitution degree and substituent groups find that the biological activity of chemically modified polysaccharides can be obviously enhanced or new activity is generated, and some chemically modified polysaccharides such as sulfated, phosphorylated, methylated or carboxymethylated polysaccharides have strong biological activity and can be developed into novel medicines. Therefore, the molecular modification and structural modification of the polysaccharide have important significance.

The immunomodulatory activity of microbial polysaccharides is mainly manifested in their ability to activate macrophages, which attack dead cells and intracellular pathogens; can activate neutrophils, thereby effectively inhibiting suppurative cells; can activate natural killer cells circulating in blood, thereby lysing cancer cells and virus-infected cells. The high-quality Guo acid bacteria resources exist in China, and many traditional fermented foods contain lactic acid bacteria, so that research and development of the resources can make a contribution to improving the lives of the nations. Some scholars believe that the probiotic function of exopolysaccharide-producing lactic acid bacteria is mainly dependent on their exopolysaccharide. Exopolysaccharides of lactic acid bacteria (EPS) generally exist in two forms: one of the Cell-bound exopolysaccharides (C-EPS) tightly bound to the surface of the bacterial cells; another release into the surrounding liquid environment is the release of polysaccharides (r-EPS). Most of the reported lactic acid bacteria produce only r-EPS, however, some lactic acid bacteria can produce both C-EPS and r-EPS. At present, about 30 kinds of extracellular polysaccharide-producing lactic acid bacteria are mainly reported. Among these, we are familiar with lactobacillus casei (l.casei), lactobacillus bulgaricus (l.delbrueckiibull), lactobacillus plantarum (l.plantarum), lactobacillus acidophilus (l.acidiophilus), lactobacillus rhamnosus (l.rhamnous), lactobacillus helveticus (l.helveticus) and lactobacillus johnsonii (l.johnsonil). The extracellular polysaccharide of lactobacillus has the functions of enhancing the immunoregulation action, such as proliferation of lymphocytes, activation of macrophages, increase of intracellular lysosome content, induction of cytokine (interferon and interleukin 1 alpha) production and the like. The extraction of the extracellular polysaccharide of the lactobacillus is relatively simple, the thalli and the liquid are separated only by separation, then the liquid is concentrated and is directly added with process alcohol to precipitate the r-EPS, and part of the podogenic bacteria can also be used for extracting the C-EPS; meanwhile, compared with other microbial polysaccharides, the extracellular polysaccharide of the lactic acid bacteria has better safety and can be widely applied to various foods and medicines. Therefore, it is necessary to research exopolysaccharides of lactic acid bacteria.

Disclosure of Invention

It is a first object of the present invention to provide a lactobacillus plantarum exopolysaccharide showing a significant ACE inhibitory activity and no cytotoxicity.

The technical scheme adopted by the invention for realizing the purpose is as follows:

the extracellular polysaccharide of lactobacillus plantarum has the following characteristics 1) and/or 2):

1) the extracellular polysaccharide has weight average molecular weight of 1.35 × 10⁶Da；

2) The exopolysaccharide consists of mannose, galactose and glucose in a molar ratio of 5.6:7.3: 1.

The above exopolysaccharide inhibits Angiotensin Converting Enzyme (ACE). The exopolysaccharide of the present invention exhibits Angiotensin Converting Enzyme (ACE) inhibitory activity, has effects of regulating blood pressure and preventing hypertension, and thus makes it possible to effectively use the exopolysaccharide of the present invention as an active ingredient of a composition for preventing or treating cardiovascular diseases. The exopolysaccharide is not cytotoxic.

The second purpose of the invention is to provide the application of the lactobacillus plantarum exopolysaccharide in preparing the ACE inhibitor composition. The exopolysaccharide of the present invention shows remarkable ACE inhibitory activity without cytotoxicity, and is useful as an ACE activity inhibitor composition.

The third purpose of the invention is to provide a phosphorylated derivative of the extracellular polysaccharide of lactobacillus plantarum. The phosphorylation derivative of the lactobacillus plantarum exopolysaccharide also shows Angiotensin Converting Enzyme (ACE) inhibition activity, has the effects of regulating blood pressure and preventing hypertension, and has no cytotoxicity.

The substitution degree of the lactobacillus plantarum exopolysaccharide is 0.24.

It is a fourth object of the present invention to provide a composition for preventing or treating cardiovascular diseases, comprising the above exopolysaccharide and/or phosphorylated derivative thereof. The composition is a food composition or a pharmaceutical composition, and comprises 0.1-80 wt% of exopolysaccharide and/or phosphorylated derivative relative to the total weight of the composition.

The cardiovascular disease is one or more selected from the group consisting of: hypertension, heart disease, stroke, thrombosis, arteriosclerosis, angina pectoris, heart failure and myocardial infarction.

The composition described above is an oral composition.

The composition is administered in a manner that 0.1-500mg of extracellular polysaccharide and/or phosphorylated derivative of Lactobacillus plantarum is taken per day by an adult. Preferably 50-100mg/kg per day, and the administration frequency is preferably 1-3 times per day.

The above composition is administered by ingestion for 9-12 weeks or more.

Compared with the prior art, the invention has the beneficial effects that: the exopolysaccharide and its phosphorylated derivatives of the present invention exhibit Angiotensin Converting Enzyme (ACE) inhibitory activity, and have the effects of regulating blood pressure and preventing hypertension, so that the exopolysaccharide and its phosphorylated derivatives of the present invention can be effectively used as active ingredients of compositions for preventing or treating cardiovascular diseases. The extracellular polysaccharide and the phosphorylated derivative thereof have no cytotoxicity and have wide application prospect.

The invention adopts the technical scheme to provide the lactobacillus plantarum exopolysaccharide and the application thereof in preparing the ACE inhibitor composition, makes up the defects of the prior art, and has reasonable design and convenient operation.

Drawings

FIG. 1 is a graph showing the growth of Lactobacillus plantarum ATCC8014 in test example 1 of the present invention;

FIG. 2 is a standard curve of molecular weight of extracellular polysaccharide of Lactobacillus plantarum measured by HPLC in test example 2 of the present invention;

FIG. 3 is a high performance liquid chromatogram of extracellular polysaccharide of Lactobacillus plantarum in test example 2 of the present invention;

FIG. 4 is a gas chromatogram of extracellular polysaccharide of Lactobacillus plantarum and standard monosaccharide in test example 2 of the present invention;

FIG. 5 shows the ACE inhibitory effect of extracellular polysaccharide of Lactobacillus plantarum and its phosphorylated derivatives in test example 4 of the present invention;

FIG. 6 shows the cytotoxicity of extracellular polysaccharide of Lactobacillus plantarum and its phosphorylated derivative in Experimental example 4 of the present invention;

FIG. 7 is a graph showing the effect of extracellular polysaccharide of Lactobacillus plantarum and its phosphorylated derivatives on systolic blood pressure in spontaneously hypertensive rats in test example 4 of the present invention;

FIG. 8 is a graph showing the effect of extracellular polysaccharide of Lactobacillus plantarum and its phosphorylated derivative on diastolic blood pressure of spontaneously hypertensive rats in test example 4 of the present invention.

Detailed Description

The present invention will be described in detail below.

The embodiment provides a lactobacillus plantarum exopolysaccharide having the following characteristics 1) and/or 2):

1) the extracellular polysaccharide has weight average molecular weight of 1.35 × 10⁶Da；

2) The exopolysaccharide consists of mannose, galactose and glucose in a molar ratio of 5.6:7.3: 1.

As a preferred specific example, the exopolysaccharide inhibits Angiotensin Converting Enzyme (ACE). The exopolysaccharide of the present invention exhibits Angiotensin Converting Enzyme (ACE) inhibitory activity, has effects of regulating blood pressure and preventing hypertension, and thus makes it possible to effectively use the exopolysaccharide of the present invention as an active ingredient of a composition for preventing or treating cardiovascular diseases. The exopolysaccharide is not cytotoxic.

The exopolysaccharide of the present embodiment is prepared by the following method:

s1: fermenting lactobacillus plantarum, and collecting fermentation liquor;

s2: mixing trichloroacetic acid water solution and the fermented liquid, reacting and collecting the supernatant in the reacted liquid;

s3: mixing the supernatant with anhydrous ethanol, standing, and collecting precipitate;

s4: dissolving the precipitate in water, dialyzing, and purifying to obtain exopolysaccharide.

As a preferred embodiment, the preparation method of the exopolysaccharide comprises the following steps:

s1: continuously activating Lactobacillus plantarum ATCC8014 for two generations, transferring to MRS culture medium containing tetraethyl thiuram disulfide of 0.02-0.1 μ M and lactose of 0.1-0.5mM, standing and fermenting at 30-40 deg.C for 12-24h, and collecting fermentation liquor; the step uses MRS culture medium containing tetraethyl thiuram disulfide and lactose to change the chemical structure of the exopolysaccharide, obtain the exopolysaccharide, and is beneficial to prolonging the logarithmic phase and the stationary phase of the lactobacillus plantarum, synthesizing the exopolysaccharide in a large amount, improving the exopolysaccharide producing capacity of the lactobacillus plantarum, and finally improving the yield of the exopolysaccharide;

s2: uniformly mixing 70-80% trichloroacetic acid aqueous solution with the fermentation liquor, stirring and reacting for 1-2h at normal temperature, and collecting supernatant in the reaction liquor; removing cells and proteins in the fermentation liquor;

s3: adding 2-3 times volume of anhydrous ethanol into the supernatant, mixing, standing at 4 deg.C for 10-15 hr, centrifuging at 4 deg.C and 8000-;

s4: dissolving the precipitate in water, transferring into dialysis bag, changing deionized water every 5-10 hr, dialyzing for 12-24 hr, collecting product, and freeze drying to obtain crude extracellular polysaccharide;

s5: purifying the crude extracellular polysaccharide by ion exchange column chromatography and gel column chromatography, and freeze drying to obtain the lactobacillus plantarum extracellular polysaccharide.

The embodiment also provides application of the lactobacillus plantarum exopolysaccharide in preparing the ACE inhibitor composition. After the polysaccharide molecule is modified by acidification, the philosomal group on the branched chain is substituted by acid group, thereby enhancing the water solubility of the polysaccharide and changing the chain conformation.

The present embodiment also provides a phosphorylated derivative of lactobacillus plantarum exopolysaccharide.

As a preferred embodiment, phosphorylated derivatives of extracellular polysaccharides of Lactobacillus plantarum are prepared by the following method:

dissolving the lactobacillus plantarum extracellular polysaccharide in dimethyl sulfoxide according to the solid-to-liquid ratio of 1:10-30g/mL, heating at 50-70 ℃ for 20-40min to fully dissolve the lactobacillus plantarum extracellular polysaccharide, then adding urea and phosphoric acid, reacting at 50-70 ℃ for 2-5h, adding deionized water to terminate the reaction, neutralizing the reaction solution with 1M NaOH, dialyzing, and freeze-drying to obtain the phosphorylated derivative of the lactobacillus plantarum extracellular polysaccharide. The weight ratio of the exopolysaccharide to the urea is 1:15-20, the solid-liquid ratio of the exopolysaccharide to the phosphoric acid is 1:5-10g/mL, and the solid-liquid ratio of the exopolysaccharide to the deionized water is 1:8-12 g/mL.

As a preferred embodiment, the degree of substitution of the extracellular polysaccharide of Lactobacillus plantarum is 0.24.

The present embodiment also provides a composition for preventing or treating cardiovascular diseases, comprising the above exopolysaccharide and/or its phosphorylated derivative. The composition is a food composition or a pharmaceutical composition, and comprises 0.1-80 wt% of exopolysaccharide and/or phosphorylated derivative relative to the total weight of the composition. The food composition of the present invention may be added with the exopolysaccharide of the present invention and/or its phosphorylated derivative to beverages, meats, sausages, breads, biscuits, rice cakes, chocolates, candies, desserts, cookies, pizza, ramen, chewing gums, dairy products (including ice cream), special nutritional foods (such as formula milk or baby diet), processed meat products, fish products, tofu, starch gel products, health supplements, savory foods (such as soy sauce, thick broad bean paste, chili paste, or assorted paste), sauces, other processed foods, pickles (such as kimchi or pickles), soups, beverages, alcoholic beverages, and vitamin complexes, and may include almost every food suitable for the production of health foods in a broad sense. The pharmaceutical composition of the present invention may further comprise various nutrients, vitamins, minerals, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid viscosity increasing agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents for addition to soda water, and the like. The carrier, excipient or diluent may be selected from the group consisting of: lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin, and saline, but is not always limited thereto. All the mentioned ingredients can be added individually or together.

As a preferred embodiment, the cardiovascular disease is one or more selected from the group consisting of: hypertension, heart disease, stroke, thrombosis, arteriosclerosis, angina pectoris, heart failure and myocardial infarction.

As a preferred embodiment, the composition is an oral composition.

As a preferred embodiment, the composition is used in such a way that 0.1-500mg of the extracellular polysaccharide and/or phosphorylated derivative of Lactobacillus plantarum is taken per day by an adult. Preferably 50-100mg/kg per day, and the administration frequency is preferably 1-3 times per day.

As a preferred embodiment, the composition is administered by ingestion for 9-12 weeks or more.

The invention is further illustrated by the following examples. It is to be understood that the examples are for illustrative purposes only and are not intended to limit the scope and spirit of the present invention.