阅读说明：本技术 一种高免疫活性酵母细胞壁多糖的提取方法 (Method for extracting yeast cell wall polysaccharide with high immunocompetence ) 是由 江建梅 徐婷婷 李丹丹 姜峰 刘立娟 周雪玲 杨文娇 刘银升 陈志颖 于 2020-12-29 设计创作，主要内容包括：本发明涉及天然产物有效成分的提取领域,提出了一种高免疫活性酵母细胞壁多糖的提取方法,包括以下步骤：S1、啤酒酵母预处理S2、冻融法破壁：将预处理的啤酒酵母泥置于-20℃下冷冻1～2h,再置于80℃水中骤然升温使其融化,后4000r/min离心5～10min,如此反复3次,得沉淀物；S3、酶解：将S2所得的沉淀物与蒸馏水按1:(10～15)的体积混合,用盐酸调pH值为6～8,加入蛋白酶、多糖酶、复配促进剂,进行超声波处理,将混合物5000r/min离心5～10min,收集上清液；S4、洗涤干燥：上清液用1～2倍体积的质量浓度为60％～85％乙醇沉淀,沉淀物丙酮洗涤两次,真空干燥至恒重即得酵母细胞壁多糖干品。通过上述技术方案,解决了现有技术中的纯度、提取率低,免疫活性不佳的问题。(The invention relates to the field of extraction of active ingredients of natural products, and provides a method for extracting yeast cell wall polysaccharide with high immunocompetence, which comprises the following steps: s1, beer yeast pretreatment S2, freeze-thaw method wall breaking: freezing the pretreated beer yeast paste at-20 ℃ for 1-2 h, then placing the frozen beer yeast paste in 80 ℃ water to suddenly raise the temperature to melt the beer yeast paste, centrifuging the beer yeast paste for 5-10 min at 4000r/min, and repeating the steps for 3 times to obtain a precipitate; s3, enzymolysis: mixing the precipitate obtained in the step S2 with distilled water according to the volume of 1 (10-15), adjusting the pH value to 6-8 by using hydrochloric acid, adding protease, polysaccharidase and a compound accelerator, carrying out ultrasonic treatment, centrifuging the mixture at 5000r/min for 5-10 min, and collecting supernatant; s4, washing and drying: precipitating the supernatant by using 1-2 times volume of 60-85% ethanol, washing the precipitate twice by using acetone, and drying in vacuum to constant weight to obtain the dried yeast cell wall polysaccharide. Through the technical scheme, the problems of low purity, low extraction rate and poor immunocompetence in the prior art are solved.)

1. The extraction method of the yeast cell wall polysaccharide with high immunocompetence is characterized by comprising the following steps:

s1, pretreatment of beer yeast: mixing beer yeast and water according to a mass-to-volume ratio of 1 (10-20), fully and uniformly stirring, centrifuging for 5-10 min at 4000r/min, repeatedly washing, filtering through a 100-mesh sieve, removing visible impurities, adding a 5% tartaric acid solution with the same volume as that of the water, stirring, standing for deodorization, and centrifuging for 5-10 min at 4000r/min to obtain a beer yeast pretreatment substance;

s2, breaking the wall by a freeze-thaw method: freezing the pretreated beer yeast paste at-20 ℃ for 1-2 h, then placing the frozen beer yeast paste in 80 ℃ water to suddenly raise the temperature to melt the beer yeast paste, centrifuging the beer yeast paste for 5-10 min at 4000r/min, and repeating the steps for 3 times to obtain a precipitate;

s3, enzymolysis: mixing the precipitate obtained in the step S2 with distilled water according to the volume of 1 (10-15), adjusting the pH value to 6-8 by using hydrochloric acid, adding protease, polysaccharidase and a compound accelerator, carrying out ultrasonic treatment, centrifuging the mixture at 5000r/min for 5-10 min, and collecting supernatant;

s4, washing and drying: precipitating the supernatant by using 1-2 times volume of 60-85% ethanol, washing the precipitate twice by using acetone, and drying in vacuum to constant weight to obtain the dried yeast cell wall polysaccharide.

2. The method for extracting yeast cell wall polysaccharide with high immunological activity as claimed in claim 1, wherein the ultrasonic treatment in step S3 is performed at 45 deg.C under 50W ultrasonic power for 4S and 5S pause for 30 min.

3. The method for extracting yeast cell wall polysaccharide with high immunological activity as claimed in claim 1, wherein the polysaccharase of step S3 includes 2.5-5% per mill mannanase, 0.2-1% per mill glucanase, 0.2-1% per mill cellulase, the ratio is calculated according to the dry material mass ratio of beer yeast.

4. The method for extracting yeast cell wall polysaccharide with high immunological activity as claimed in claim 1, wherein the protease comprises one or more of ficin, papain, and neutral protease.

5. The method for extracting yeast cell wall polysaccharide with high immunological activity as claimed in claim 1, wherein the protease is added in an amount of 0.1-0.5% of dry beer yeast.

6. The method for extracting the yeast cell wall polysaccharide with high immunocompetence according to claim 1, wherein the compound accelerant is manganese gluconate and cobalt gluconate, and the mass ratio of the manganese gluconate to the cobalt gluconate is (4-5): 1.

7. The method for extracting the yeast cell wall polysaccharide with high immunological activity according to claim 1, wherein the mass of the compound accelerator is 5-10% of that of the protease.

Technical Field

The invention relates to the field of extraction of active ingredients of natural products, in particular to an extraction method of yeast cell wall polysaccharide with high immunocompetence.

Background

The yeast cell wall is mainly composed of carbohydrates, mainly glucans and mannans, and proteins. The yeast cell wall polysaccharide can be used as a functional oligosaccharide which is utilized by beneficial bacteria in animals, is a green non-nutrient regulating substance, influences the glycometabolism, promotes the intestinal function, has good stability and high activity in the human digestive tract, and has a certain immunoregulation function, such as good inhibition effect on salmonella, salmonella typhimurium, clostridium botulinum and clostridium botulinum. In addition, the yeast cell wall polysaccharide has 100 percent interception effect on salmonella enteritidis, proteus morganii and citrobacter; it does not affect other components by physical adsorption or direct binding of mycotoxins.

Since substances such as proteins and lipids in yeast cells and cell wall polysaccharides exist in the form of macromolecular carbohydrate polymers, the cell wall polysaccharides need to be separated, extracted and reused. At present, the extraction methods of the active ingredients of the zymosan are more, such as a grinding method, a freeze-thawing method, a high-pressure homogenization method, an ultrasonic method, an alkali dissolution method, a conventional water extraction method, a Soxhlet extraction method and the like, and the traditional methods have the defects of low extraction rate, low purity and the like.

Disclosure of Invention

The invention provides an extraction method of yeast cell wall polysaccharide with high immunocompetence, which solves the problems of low purity, low extraction rate and poor immunocompetence in the prior art.

The technical scheme of the invention is as follows:

a method for extracting yeast cell wall polysaccharide with high immunocompetence comprises the following steps:

s1, pretreatment of beer yeast: mixing beer yeast and water according to a mass-to-volume ratio of 1 (10-20), fully and uniformly stirring, centrifuging for 5-10 min at 4000r/min, repeatedly washing, filtering through a 100-mesh sieve, removing visible impurities, adding a 5% tartaric acid solution with the same volume as that of the water, stirring, standing for deodorization, and centrifuging for 5-10 min at 4000r/min to obtain a beer yeast pretreatment substance;

s2, breaking the wall by a freeze-thaw method: freezing the pretreated beer yeast paste at-20 ℃ for 1-2 h, then placing the frozen beer yeast paste in 80 ℃ water to suddenly raise the temperature to melt the beer yeast paste, centrifuging the beer yeast paste for 5-10 min at 4000r/min, and repeating the steps for 3 times to obtain a precipitate;

s3, enzymolysis: mixing the precipitate obtained in the step S2 with distilled water according to the volume of 1 (10-15), adjusting the pH value to 6-8 by using hydrochloric acid, adding protease, polysaccharidase and a compound accelerator, carrying out ultrasonic treatment, centrifuging the mixture at 5000r/min for 5-10 min, and collecting supernatant;

s4, washing and drying: precipitating the supernatant by using 1-2 times volume of 60-85% ethanol, washing the precipitate twice by using acetone, and drying in vacuum to constant weight to obtain the dried yeast cell wall polysaccharide.

As a further technical scheme, the ultrasonic treatment conditions in the step S3 are that the temperature is 45 ℃, the ultrasonic power is 50W, the time is 4S, the pause is 5S, and the ultrasonic time is 30 min.

According to a further technical scheme, the polysaccharose enzyme obtained in the step S3 comprises 2.5-5% per mill mannanase, 0.2-1% per mill glucanase and 0.2-1% per mill cellulase, and the proportion is calculated according to the dry material mass ratio of the beer yeast.

As a further technical scheme, the protease comprises one or more of ficin, papain and neutral protease.

As a further technical scheme, the addition amount of the protease is 0.1-0.5% of the dry beer yeast.

As a further technical scheme, the compound accelerant is manganese gluconate and cobalt gluconate, and the mass ratio of the manganese gluconate to the cobalt gluconate is (4-5): 1.

As a further technical scheme, the mass of the compound accelerator is 5-10% of that of the protease.

The invention has the beneficial effects that:

1. the invention extracts polysaccharide from the cell wall of the beer yeast through the synergistic effect of ultrasonic wave and enzymolysis, the extraction rate is high, the purity of the obtained polysaccharide is high, and the immunocompetence of the polysaccharide is high.

2. The invention has the advantages that the cell wall of the raw material is easy to break through the cavitation of the ultrasonic wave, thereby being beneficial to the dissolution of the polysaccharide and improving the extraction rate of the polysaccharide, and the ultrasonic wave assisted extraction can reduce the extraction time and reduce the extraction temperature.

3. The invention adds protease, and through the hydrolysis of enzyme, in order to accelerate the disintegration of cell wall, the protein layer of cell wall is hydrolyzed, and the macromolecular protein in cell is hydrolyzed, so that the macromolecular protein becomes molecule which can penetrate to the outside of cell and is smaller than the pore on cell wall. By adding the promoters, the manganese gluconate and the cobalt gluconate can dissolve lipid substances and other components on the cell wall and permeate out from the cell, the extraction rate and the purity of the polysaccharide are promoted, and meanwhile, the metal ions of the manganese gluconate and the cobalt gluconate can promote the enzymolysis of protease in the cell wall on protein, so that the enzymolysis efficiency is accelerated, and the purity is improved.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any inventive step, are intended to be within the scope of the present invention.

Example 1

Adding 100g of beer yeast into 1L of water, fully and uniformly stirring, centrifuging at 4000r/min for 5min, repeatedly washing, filtering by a 100-mesh screen to remove visible impurities, then adding 1L of 5% tartaric acid solution, stirring, standing for deodorizing, and centrifuging at 4000r/min for 5min to obtain a beer yeast pretreatment substance; freezing the pretreated beer yeast paste at-20 deg.C for 1h, placing in 80 deg.C water, rapidly heating to melt, centrifuging at 4000r/min for 5min, and repeating the above steps for 3 times to obtain precipitate; adding 15 times of distilled water into the precipitate, adjusting pH to 6 with hydrochloric acid, adding ficin 100mg, manganese gluconate 4mg, cobalt gluconate 1mg, mannase 250mg, glucanase 20mg and cellulase 20mg, performing ultrasonic treatment at 45 deg.C for 30min with ultrasonic power of 50W for 4S and pause of 5S, centrifuging the mixture for 5min at 5000r/min, and collecting the supernatant; precipitating the supernatant with 1-2 times volume of 95% absolute ethyl alcohol, washing the precipitate with acetone twice, and vacuum drying to constant weight to obtain the dried yeast cell wall polysaccharide product with the polysaccharide purity of 91.24% and the polysaccharide extraction rate of 25.13%.

Wherein the purity of the polysaccharide is measured by adopting a phenol-sulfuric acid colorimetric method, and the polysaccharide extraction rate is calculated according to the following formula:

polysaccharide extraction rate ═ C × V/mx 100%

In the formula: c is the mass concentration of polysaccharide in the supernatant, mg/L; v is the total volume of the supernatant, L; m is the dry mass of the yeast paste, g.

Example 2

Adding 100g of beer yeast into 1L of water, fully and uniformly stirring, centrifuging at 4000r/min for 10min, repeatedly washing, filtering by a 100-mesh sieve, removing visible impurities, adding 1L of 5% tartaric acid solution, stirring, standing, deodorizing, and centrifuging at 4000r/min for 10min to obtain a beer yeast pretreatment substance; freezing the pretreated beer yeast paste at-20 deg.C for 2h, placing in 80 deg.C water, rapidly heating to melt, centrifuging at 4000r/min for 10min, and repeating the above steps for 3 times to obtain precipitate; adding 15 times of distilled water into the precipitate, adjusting pH to 7 with hydrochloric acid, adding 500mg of papain, 41.6mg of manganese gluconate, 8.4mg of cobalt gluconate, 500mg of mannase, 100mg of glucanase and 100mg of cellulase, performing ultrasonic treatment at 45 ℃ for 30min with ultrasonic power of 50W for 4S and pause of 5S, centrifuging the mixture for 10min at 5000r/min, and collecting the supernatant; precipitating the supernatant by using 1-2 times volume of 95% absolute ethyl alcohol, washing the precipitate twice by using acetone, and drying in vacuum to constant weight to obtain a dried yeast cell wall polysaccharide product, wherein the purity of the polysaccharide is 92.19%, and the extraction rate of the polysaccharide is 25.88%.

Example 3

Adding 100g of beer yeast into 1L of water, fully and uniformly stirring, centrifuging at 4000r/min for 10min, repeatedly washing, filtering by a 100-mesh sieve, removing visible impurities, adding 1L of 5% tartaric acid solution, stirring, standing, deodorizing, and centrifuging at 4000r/min for 10min to obtain a beer yeast pretreatment substance; freezing the pretreated beer yeast paste at-20 deg.C for 2h, placing in 80 deg.C water, rapidly heating to melt, centrifuging at 4000r/min for 10min, and repeating the above steps for 3 times to obtain precipitate; adding 15 times of distilled water into the precipitate, adjusting pH to 7 with hydrochloric acid, adding 250mg of neutral protease, 20mg of manganese gluconate, 5mg of cobalt gluconate, 400mg of mannase, 80mg of glucanase and 80mg of cellulase, performing ultrasonic treatment at 45 ℃ for 30min, performing ultrasonic power of 50W for 4S at an interval of 5S, centrifuging the mixture for 10min at 5000r/min, and collecting the supernatant; precipitating the supernatant by using 1-2 times volume of 95% absolute ethyl alcohol, washing the precipitate twice by using acetone, and drying in vacuum to constant weight to obtain a dried product of the yeast cell wall polysaccharide, wherein the purity of the polysaccharide is 92.67%, and the extraction rate of the polysaccharide is 26.24%.

Comparative example 1

The difference from example 3 is that the purity of the yeast cell wall polysaccharide obtained is 87.34% and the extraction rate of the polysaccharide is 19.96% in the same process without ultrasonic treatment.

Comparative example 2

The difference from the example 3 is that the purity of the obtained yeast cell wall polysaccharide is 89.61% and the polysaccharide extraction rate is 22.52% by adding 25mg of manganese gluconate without adding cobalt gluconate.

Comparative example 3

The difference from the example 3 is that the purity of the obtained yeast cell wall polysaccharide is 87.12% and the polysaccharide extraction rate is 19.42% without adding manganese gluconate and cobalt gluconate, and the other processes are the same.

Experiments on immunological Activity

The experiment was conducted by selecting only representative examples of the present invention and comparative examples.

90 mice were randomly divided into 3 groups of 3 replicates each of 3 replicates of normal control group, trial 1 group (example 3 group), and trial 2 group (comparative example 3 group). All mice were fed basal diet, and after 10 days of adaptive feeding, mice in experiment 1 group were administered with 0.2ml of 80mg/ml zymosan solution prepared in example 3 every morning 1 time, mice in experiment 2 group were administered with 0.2ml of 80mg/ml zymosan solution prepared in comparative example 2 every morning 1 time, and mice in normal control group were administered with 0.2ml of 0.9% sodium chloride solution every morning 1 time for 15 days.

After 15 days of administration, 2 mice were taken per each replicate and weighed, spleen and thymus, immune organs were dissected and harvested, and after removal of fascia and adipose tissue, weighed, spleen index and thymus index were calculated according to the formula:

immune organ index ═ immune organ weight (mg)/body weight (g)

After 15 days of administration, 2 mice per group were each repeatedly intraperitoneally injected with 1ml of 4% starch solution to induce macrophage chemotaxis toward the abdominal cavity. After 24h, each mouse was injected intraperitoneally with 1ml of a fresh 10% suspension of chicken red blood cells, 30min later, the mice were sacrificed by cervical dislocation and were quickly fixed on a mouse operating plate. Inject 2ml of 0.9% NaCl solution into the abdomen, and gently knead the abdomen for 1 min. The abdominal cavity of the mouse was then aseptically opened, 1ml of peritoneal lavage fluid was aspirated, dropped onto a clean slide, and moved to a 37 ℃ incubator for 30 min. Rinsing with 0.9% sodium chloride solution to remove non-patch cells, and air drying. After being fixed by methanol solution, the chicken red blood cells are stained by Giemsa-phosphate buffer solution for 20min, then rinsed by distilled water, dried in the air, observed by a light microscope to phagocytose and clear the chicken red blood cells by macrophages, and the phagocytosis percentage and the phagocytosis index are calculated according to the following formula:

percent phagocytosis ═ number of macrophages engulping chicken red blood cells/number of macrophages counted × 100%;

phagocytosis index is the total number of chicken erythrocytes phagocytosed per number of macrophages counted.

TABLE 13 comparison of data from groups of experiments on immune Activity

The comparative example 1 of the invention does not carry out ultrasonic treatment, and under the same other conditions as the example 3, the extraction rate and purity of the polysaccharide are lower, because the action of ultrasonic wave can reduce enzymolysis time and enzymolysis temperature, so when ultrasonic treatment is not carried out, the same temperature and time are not enough to completely carry out enzymolysis, and the process of ultrasonic treatment and the enzymolysis temperature and time play a mutual auxiliary synergistic effect to improve the purity and the extraction rate.

The purity and the extraction rate of the polysaccharide obtained in the comparative example 3 are lower than those of the polysaccharide obtained in the comparative example 2, and the purity and the extraction rate of the polysaccharide obtained in the comparative example 3 are both lower than those of the polysaccharide obtained in the comparative example 2, while the amount of the accelerant added in the comparative example 2 are the same as those in the example 3, but the purity and the extraction rate of the polysaccharide obtained in the comparative example 2 are lower than those of the polysaccharide obtained in the example 3, so that the enzymolysis effect is best when the two components are compounded, and the purity and the extraction rate are improved.

The comparative example 3 is added with no accelerant, the extraction rate of the obtained polysaccharide is low, the purity is low, meanwhile, compared with the example 3, the immune activity of the extracted polysaccharide is low, the example 3 has a remarkable improvement effect on the phagocytic capacity of phagocytic phagocytes of mice, and the immune organ index is higher than that of the comparative example 3, which shows that the manganese gluconate and the cobalt gluconate are added to promote the enzymolysis of cell wall proteins, accelerate the enzymolysis efficiency and improve the purity, and the immune activity of the obtained polysaccharide is also high due to the improvement of the purity.

The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.