CN109207384B

CN109207384B - Modified yeast cell wall and preparation method and application thereof

Info

Publication number: CN109207384B
Application number: CN201710532216.6A
Authority: CN
Inventors: 胡骏鹏; 俞学锋; 李知洪; 姚鹃; 廖汉江; 谢智文; 黄鑫; 聂琴; 龚发源
Original assignee: Angel Yeast Co Ltd
Current assignee: Angel Yeast Co Ltd
Priority date: 2017-07-03
Filing date: 2017-07-03
Publication date: 2021-05-18
Anticipated expiration: 2037-07-03
Also published as: CN109207384A

Abstract

The invention relates toModified yeast cell wall and its preparation process and application. The content of beta-glucan in the yeast cell wall is 25-50%, wherein the relative molecular weight of more than 99% of beta-glucan is 3.0 multiplied by 10⁵‑1.0×10⁶(ii) a The content of mannan is not less than 20%, wherein the relative molecular weight of mannan above 99% is 8.0 × 10⁵‑2.0×10⁶. The invention also provides a preparation method of the yeast cell wall, which comprises the following steps: (1) carrying out autolysis, wall breaking and separation on yeast to obtain yeast cell wall milk; (2) the yeast cell wall milk is subjected to enzymolysis through mannanase, alkaline protease, papain, cellulase and beta-glucanase, and dried to obtain the yeast cell wall. The modified yeast cell wall produced by the method has the characteristics of high dissolution rate and good bacteriostatic effect, has no drug residue and no pollutant in the production process, and is a pure natural safe product.

Description

Modified yeast cell wall and preparation method and application thereof

Technical Field

The invention relates to the technical field of microorganism application, in particular to a modified yeast cell wall and a preparation method and application thereof.

Background

In intensive culture, the morbidity and mortality of livestock and poultry are high, and antibiotics are widely applied to feeds and culture sites as feed pharmaceutical additives and veterinary drugs. However, the problems of drug residues, drug resistance to germs, double infection of animal organisms and the like caused by long-term use of antibiotics have attracted much attention of society, and thus countermeasures against antibiotics for feeding have become strong. In 2015, 7 months and 9 months, the state successively goes out of 'antibacterial comprehensive treatment for 5 years' plan and '2292 bulletin' to forbid four sarin antibiotics. Whether the antibiotics in the feed are replaced or the medicines used on the culture site are reduced, certain influence is brought to the culture performance, and the development of products for effectively replacing the antibiotics for the feed is urgently needed in the market.

The yeast cell wall is a product obtained by concentrating and drying cell walls obtained by separating cell walls after autolysis or exogenous enzyme catalytic hydrolysis or mechanical crushing of thalli obtained by fermenting saccharomyces cerevisiae. The functional components in the yeast cell wall are mainly mannan and beta-glucan. A large number of studies at home and abroad show that the yeast cell wall has the effects of enhancing the immunity of animal organisms and adsorbing mycotoxin and pathogenic bacteria. The beta-glucan in the yeast cell wall acts on macrophages to stimulate a series of immune reactions, release downstream signal molecules, stimulate the organism to produce more macrophages, T cells, neutrophils and the like, and induce the organism to generate nonspecific and specific immune response reactions. Mannan in yeast cell wall can combine with lectin of pathogenic bacteria to prevent adhesion of pathogenic bacteria on intestinal wall and prevent propagation of pathogenic bacteria in intestinal tract. Meanwhile, based on a specific spatial structure, the yeast cell wall can stably adsorb various mycotoxins through intermolecular forces such as hydrogen bonds, van der waals force and the like. Based on the biological safety and functionality of yeast cell walls, the Ministry of agriculture has moved yeast cell walls from feed additive variety catalog to feed material catalog (2013), laying the policy foundation for their wider application in animal husbandry.

The yeast cell wall has the using effects of improving immunity and promoting the health of animal organisms, is gradually accepted by people, and is an ideal antibiotic substitute for feeding. The yeast cell wall has a special triple helix structure, is insoluble in water, is in an aggregated granular state, and is slightly soluble in dimethyl sulfoxide (DMSO). The use of yeast cell wall products is greatly restricted due to their insolubility, and the yeast cell wall products on the market today are based on yeast cell walls that are not further processed. In order to expand the application range of yeast cell walls, scholars at home and abroad carry out a great deal of research on physical, chemical and biological modification and the like.

The physical modification method of the yeast cell wall mainly comprises the steps of breaking the main chain of the yeast cell wall polysaccharide by applying an external force mode on the basis of not damaging the basic molecular structure of the yeast cell wall polysaccharide, so that the main chain is changed into soluble micromolecules to increase the solubility of the yeast cell wall polysaccharide, and currently, ultrasonic depolymerization, irradiation treatment and the like are mainly used; the biological modification technology is mainly enzymatic modification, and the spatial structure of the polysaccharide is correspondingly changed under the action of corresponding polysaccharase, so that long molecular chains are hydrolyzed into short molecular chains, and the polysaccharide is partially converted into oligosaccharide or oligosaccharide; the chemical modification is to introduce or remove some groups on the molecular chain of the polysaccharide of the yeast cell wall to change the molecular structure of the polysaccharide, so as to obtain the derivative with various special properties. Through modification, the solubility and the immunocompetence of the yeast cell wall can be improved to a certain degree, and the application range of the yeast cell wall is expanded.

Disclosure of Invention

The invention solves the problems of the prior art that: the existing yeast has low cell wall solubility and no bacteriostatic effect, and limits the application of the yeast in the field of feed addition. According to the invention, the solubility and the bacteriostatic effect of the yeast cell wall are improved by modifying the yeast cell wall, so that the animal immunity can be improved when the yeast cell wall is used in the field of feed addition, and the solubility of the yeast cell wall is improved at the same time.

The invention provides a novel preparation method of the yeast cell wall, so that the solubility of the yeast cell wall is increased, and the yeast cell wall has a bacteriostatic effect. The yeast cell wall provided by the invention contains 25-50% of effective component beta-glucan, more than or equal to 20% of mannan, has a dissolution rate of 30-50%, and can specifically inhibit the growth of escherichia coli and staphylococcus aureus. Compared with the existing yeast cell wall, the solubility of the cell wall is improved, and the antibacterial effect is achieved, so that the property of the conventional yeast cell wall is modified, and a modified product which is more suitable for being used as a feed raw material is obtained.

Specifically, the present invention proposes the following technical solutions.

In a first aspect, the invention provides a yeast cell wall having a beta-glucan content of 25-50%, wherein more than 99% of the beta-glucan contentRelative molecular weight of 3.0X 10⁵-1.0×10⁶(ii) a The content of mannan in the yeast cell wall is more than or equal to 20%, wherein the relative molecular weight of more than 99% of mannan is 8.0 x 10⁵-2.0×10⁶。

In a second aspect, the invention provides a yeast cell wall with increased solubility, wherein the content of beta-glucan in the yeast cell wall is 25-50%, and wherein more than 99% of beta-glucan has a relative molecular weight of 3.0 x 10⁵-1.0×10⁶(ii) a The content of mannan in the yeast cell wall is more than or equal to 20%, wherein the relative molecular weight of more than 99% of mannan is 8.0 x 10⁵-2.0×10⁶The lysis rate of the yeast cell wall is 30% to 50%, preferably 35% to 50%.

In a third aspect, the invention provides a yeast cell wall with bacteriostatic effect, wherein the content of beta-glucan in the yeast cell wall is 25-50%, and the relative molecular weight of more than 99% of beta-glucan is 3.0 multiplied by 10⁵-1.0×10⁶(ii) a The content of mannan in the yeast cell wall is more than or equal to 20%, wherein the relative molecular weight of more than 99% of mannan is 8.0 x 10⁵-2.0×10⁶The yeast cell wall exhibits bacteriostatic effects against escherichia coli and staphylococcus aureus.

Preferably, the minimum inhibitory concentration of the yeast cell wall for escherichia coli and staphylococcus aureus is 5% yeast cell wall dry matter mass concentration according to the yeast cell wall described above.

Preferably, the yeast cell wall is obtained by enzymatic hydrolysis of mannanase, alkaline protease, papain, cellulase and beta-glucanase according to the above.

Preferably, the yeast cell wall is obtained by sequentially carrying out enzymolysis on mannanase, alkaline protease, papain, cellulase and beta-glucanase according to the yeast cell wall.

More preferably, the yeast cell wall is obtained by sequentially carrying out enzymolysis on the yeast cell wall by mannanase, alkaline protease, papain, cellulase and beta-glucanase.

In a fourth aspect, the invention also provides a method for preparing yeast cell walls, which comprises the following steps:

(1) carrying out autolysis wall breaking on a raw material containing yeast, and separating to obtain yeast cell wall milk;

(2) carrying out enzymolysis on yeast cell wall milk by mannase, alkaline protease, papain, cellulase and beta-glucanase to obtain a yeast cell wall; preferably sequentially carrying out enzymolysis on mannanase, alkaline protease, papain, cellulase and beta-glucanase; further preferably, the enzyme is obtained by sequentially carrying out enzymolysis on mannanase, alkaline protease, papain, cellulase and beta-glucanase.

Preferably, according to the preparation method, the autolytic wall breaking is performed at a salt concentration of 3-5%, a pH value of 5.5-6.5, and a temperature of 55-75 ℃; the time of the autolysis treatment is preferably 20 to 30 hours.

Preferably, according to the preparation method, the addition mass of the mannanase, the alkaline protease, the papain, the cellulase and the beta-glucanase is 3-5 per thousand, 2-6 per thousand, 0.1-0.7 per thousand, 0.2-1 per thousand and 0.2-1 per thousand respectively based on the mass of yeast cell wall dry matter.

Preferably, according to the preparation method, the addition amounts of the mannanase, the alkaline protease, the papain, the cellulase and the beta-glucanase are respectively (0.06U-0.1U), (0.1U-0.3U), (0.005U-0.035U), (0.001U-0.005U) and (0.0016U-0.008U) in each gram of yeast cell wall dry matter.

Preferably, according to the above-described production method,

the enzymolysis temperature of the mannase is 35-60 ℃, and the pH value is 7.0-11.0;

the enzymolysis temperature of the alkaline protease is 40-70 ℃, and the pH value is 6.5-8.5;

the enzymolysis temperature of the papain is 40-52 ℃, and the enzymolysis pH is 4.0-6.0;

the enzymolysis temperature of the cellulase is 50-68 ℃, and the pH value is 4.0-5.5;

the enzymolysis temperature of the beta-glucanase is 40-67 ℃, and the pH value is 4.5-6.5.

Preferably, according to the preparation method,

the enzymolysis time of the mannase is 10-15 h;

the enzymolysis time of the alkaline protease is 7-10 h;

the enzymolysis time of the papain is 3-8 h;

the enzymolysis time of the cellulase is 3-8 h;

the enzymolysis time of the beta-glucanase is 6-12 h.

Preferably, according to the preparation method, in the step (2), the yeast cell wall milk is prepared into a suspension solution with the concentration of 8-20% (w/v) based on the dry matter of the yeast cell wall, then mannanase, alkaline protease and papain are respectively adopted for enzymolysis, and then cellulase and beta-glucanase are adopted for enzymolysis.

Preferably, according to the preparation method, in the step (2), before enzymolysis by cellulase and beta-glucanase, enzyme deactivation operation is carried out, and reactants are preferably treated at the temperature of 85-100 ℃ for 0.5-1.5 hours.

Preferably, according to the preparation method, before autolysis wall breaking in the step (1), fermentation treatment is carried out on yeast by taking molasses as a carbon source and ammonium sulfate as a nitrogen source, and preferably, molasses with 25-35% of total sugar concentration is taken as the carbon source.

Preferably, according to the above-described preparation method, the temperature of the fermentation treatment is 25 to 35 ℃, preferably 28 to 30 ℃, and the pH value is 4.0 to 6.0.

Preferably, according to the preparation method, the yeast is Saccharomyces cerevisiae Z2.2(Saccharomyces cerevisiae Hansen Z2.2) with the preservation number of CCTCC NO: M205128; saccharomyces cerevisiae FX-2(Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418; saccharomyces cerevisiae Z2.4(Saccharomyces cerevisiae Hansen Z2.4) with a preservation number of CCTCC NO: M205130; are all preserved in China center for type culture Collection; preferably Saccharomyces cerevisiae FX-2(Saccharomyces cerevisiae FX-2) with the accession number CCTCC NO: M2016418.

In a fifth aspect, the invention also provides the yeast cell wall prepared by the preparation method, the dissolution rate of the yeast cell wall is improved, and the yeast cell wall has a bacteriostatic effect.

Preferably, according to the yeast cell wall, the dissolving rate of the yeast cell wall is 30-50%, the content of beta-glucan is 25-50%, the content of mannan is more than or equal to 20%, and the minimum inhibitory concentration of the yeast cell wall to escherichia coli and staphylococcus aureus is 5% dry matter concentration.

In a sixth aspect, the invention also provides a feed comprising a yeast cell wall as described in any one of the above.

In a seventh aspect, the invention also provides the use of the yeast cell wall of any of the above in the field of feed additives.

The beneficial effects obtained by the invention are as follows: compared with the existing common unmodified yeast cell wall product, the modified yeast cell wall product prepared by the invention can improve the solubility of the yeast cell wall and enhance the bacteriostatic effect, and can be fed to any animal, including livestock, poultry, aquatic products, ruminants and the like. When mixed with feed or used as additive for feeding, the product has effects of improving animal immunity, and effectively inhibiting Escherichia coli and Staphylococcus aureus, thereby improving animal productivity and health status, and reducing use of feed antibiotics.

Information on the preservation of the strains

The strain used by the invention, namely the Saccharomyces cerevisiae FX-2(Saccharomyces cerevisiae FX-2), is preserved in China Center for Type Culture Collection (CCTCC) in 2016, 8, 1 and 8, with the preservation number of CCTCC NO: M2016418, and the preservation address is as follows: china, wuhan university, zip code: 430072; telephone: (027) -68754052.

The strain Saccharomyces cerevisiae Z2.2(Saccharomyces cerevisiae Hansen Z2.2) used by the invention is preserved in China Center for Type Culture Collection (CCTCC) at 25/10/2005, with the preservation number of CCTCC NO: M205128 and the preservation address: china, wuhan university, zip code: 430072; telephone: (027) -68752319.

The strain Saccharomyces cerevisiae Z2.4(Saccharomyces cerevisiae Hansen Z2.4) used by the invention is preserved in China Center for Type Culture Collection (CCTCC) at 25/10/2005, the preservation number is CCTCC NO: M205130, the preservation address is as follows: china, wuhan university, zip code: 430072; telephone: (027) -68752319.

Drawings

FIG. 1 is a graph showing the bacteriostatic effect of gentamicin and physiological saline on Escherichia coli, and yeast cell walls prepared in example one, example two, comparative example one and comparative example two, wherein reference numeral 1 is the yeast cell wall prepared in example one, reference numeral 2 is the yeast cell wall prepared in example two, reference numeral 3 is physiological saline, reference numeral 4 is the yeast cell wall prepared in comparative example one, reference numeral 5 is the yeast cell wall prepared in comparative example two, and reference numeral 6 is gentamicin.

FIG. 2 is a graph showing the bacteriostatic effects of gentamicin and physiological saline on Escherichia coli, and yeast cell walls prepared in example III, comparative example IV, and comparative example V, wherein reference numeral 7 is the yeast cell wall prepared in comparative example III, reference numeral 8 is the yeast cell wall prepared in example III, reference numeral 9 is physiological saline, reference numeral 10 is the yeast cell wall prepared in comparative example IV, reference numeral 11 is the yeast cell wall prepared in comparative example V, and reference numeral 12 is gentamicin.

Fig. 3 is a graph showing the bacteriostatic effect of gentamicin and physiological saline on staphylococcus aureus, wherein the yeast cell wall prepared in the first embodiment is shown in the reference numeral 1, the yeast cell wall prepared in the second embodiment is shown in the reference numeral 2, the physiological saline is shown in the reference numeral 3, the yeast cell wall prepared in the first embodiment is shown in the reference numeral 4, the yeast cell wall prepared in the second embodiment is shown in the reference numeral 5, and gentamicin is shown in the reference numeral 6.

Fig. 4 is a graph showing the bacteriostatic effect of gentamicin and physiological saline on staphylococcus aureus, and the yeast cell walls prepared in example three, comparative example four and comparative example five, wherein reference numeral 7 is the yeast cell wall prepared in comparative example three, reference numeral 8 is the yeast cell wall prepared in example three, reference numeral 9 is physiological saline, reference numeral 10 is the yeast cell wall prepared in comparative example four, reference numeral 11 is the yeast cell wall prepared in comparative example five, and reference numeral 12 is gentamicin.

Detailed Description

As described above, the present invention provides a method for preparing a modified yeast cell wall, which can increase the lysis rate and enhance the bacteriostatic effect compared to the conventional yeast cell wall. The invention provides a modified yeast cell wall with improved solubility and bacteriostatic effect, wherein the content of beta-glucan in the yeast cell wall is 25-50%, and the relative molecular weight of more than 99% of beta-glucan is 3.0 multiplied by 10⁵-1.0×10⁶Preferably 3.3X 10⁵-1.0×10⁶(ii) a Mannan is more than or equal to 20%, wherein the relative molecular weight of mannan of more than 99% is 8.0 × 10⁵-2.0×10⁶Preferably 8.6X 10⁵-2.0×10⁶. The yeast cell walls can be used as feed additives, thereby reducing the use of antibiotics in the feed. Without being bound by theory, the yeast cell wall provided by the present invention exhibits enhanced solubility and bacteriostatic effects, which are related to the molecular weight of β -glucan and the molecular weight and content of mannan, etc. in the yeast cell wall. The modified yeast cell wall has small molecular weight of beta-glucan and mannan, greatly improves the solubility and shows excellent bacteriostatic effect.

The preparation method of the yeast cell wall breaks the wall by autolysis, and then carries out compound enzymolysis treatment on a plurality of enzymes. By means of an autolysed salt, preferably sodium chloride and/or ethyl acetate. The treatment under the slightly acidic environment can maintain the sterile environment, stimulate the endogenous enzyme activity of the yeast, promote the lysis of the yeast cells, and achieve the purposes of removing the contents in the yeast cells and leading the residual yeast cell wall milk to contain higher polysaccharide components.

And then separating the autolyzed substances, removing yeast autolysate to obtain yeast cell wall milk, and performing composite enzymolysis on the yeast cell wall milk by adopting various enzymes, wherein the enzymolysis treatment is performed on the yeast cell wall milk by adopting mannanase, alkaline protease, papain, cellulase and beta-glucanase. The structure of the yeast cell wall is phosphorylated mannan, protein and glucan from the outer layer to the inner layer in sequence, so that the inventor finds out through experimental investigation that mannase is firstly used for treating to destroy the structure of the yeast cell wall so as to separate mannan components; then, alkaline protease and papain are adopted for treatment, so that the structure of the yeast cell wall can be destroyed, and protein components are separated; and finally, treating by adopting cellulase and beta-glucanase, degrading glucan in the cell wall to change the glucan into a small molecular fragment, thereby exposing the functional site. The alkaline protease and the papain have the function of further destroying the cell wall structure in the cell wall milk treated by the mannanase so as to separate protein components, so that the alkaline protease and the papain can be added simultaneously for enzymolysis treatment or can be added respectively for enzymolysis treatment. The cellulase and the beta-glucanase have the functions of further converting glucan in the yeast cell wall treated by the mannanase and the protease into small molecular fragments so as to expose functional sites, so that the cellulase and the beta-glucanase can be added simultaneously for enzymolysis treatment or can be added respectively for enzymolysis treatment.

Meanwhile, the present invention provides a method for preparing a modified yeast cell wall, wherein in a preferred embodiment of the present invention, the method comprises the steps of:

(1) autolytic wall breaking

Autolyzing the yeast-containing material at salt concentration of 3-5%, pH of 5.5-6.5 and temperature of 55-75 deg.C, and separating to obtain yeast cell wall milk;

(2) enzymolysis treatment

a. Diluting yeast cell wall milk with water to dry matter mass concentration of 8-20%, adding mannase with mass concentration of 3-5 ‰ (the enzyme content is calculated by mass of yeast cell wall dry matter, the same below), controlling temperature at 35-60 deg.C, pH to 7.0-11.0, and hydrolyzing for 10-15 hr; then adding alkaline protease with the mass concentration of 2-6 per mill, controlling the temperature at 40-70 ℃, controlling the pH to 6.5-8.5, and hydrolyzing for 7-10 hours; then regulating the temperature to 40-52 ℃, adding 0.1-0.7 per mill (dry matter content) of papain, and hydrolyzing for 3-8 hours;

b. enzyme deactivation treatment is carried out at 85-100 ℃ for 0.5-1.5 hours;

(3) secondary enzymolysis treatment

c. Preparing enzymolysis reactant subjected to enzyme deactivation treatment obtained in the last step into a solution with the dry matter mass concentration of 8-20%, adding cellulase with the mass concentration of 0.2-1 per mill, controlling the temperature to be 40-60 ℃, controlling the pH to be 4.0-5.5, and hydrolyzing for 3-8 hours; then adding beta-glucanase with the mass concentration of 0.2-1 per mill, controlling the temperature to be 40-67 ℃, the pH to be 4.5-6.5, and hydrolyzing for 6-12 hours to obtain an enzymolysis product;

(4) centrifugal drying

And (4) heating the enzymolysis product obtained in the step (3) to 90 ℃, preserving the temperature for 30min-1h, and drying to obtain the modified yeast cell wall.

In another preferred embodiment of the invention, before autolysis treatment, Saccharomyces cerevisiae strain is adopted, molasses is used as carbon source, ammonium sulfate is used as nitrogen source, fermentation pH value is 4.0-6.0, temperature is 28-30 ℃, fermentation time is 16-24 hours, and yeast material with optimal yeast cell wall polysaccharide content is obtained. In a preferred embodiment, the Saccharomyces cerevisiae strain is Saccharomyces cerevisiae Z2.2(Saccharomyces cerevisiae Hansen Z2.2) with a accession number of CCTCC NO: M205128; saccharomyces cerevisiae FX-2(Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418; saccharomyces cerevisiae Z2.4(Saccharomyces cerevisiae Hansen Z2.4) with the preservation number of CCTCC NO: M205130, which are all preserved in the China center for type culture Collection. More preferably, the Saccharomyces cerevisiae strain is Saccharomyces cerevisiae FX-2(Saccharomyces cerevisiae FX-2) with the collection number of CCTCC NO: M2016418.

The manufacturers of the raw materials and equipment used in the present example, and the equipment and analysis method used in the product analysis are described below, wherein the chemical substances are not indicated as being chemically pure grades of conventional reagents. Information on the raw materials used in examples and comparative examples is shown in the following table.

TABLE 1 information on the raw materials used in the present invention

Raw materials	Manufacturer of the product
		Mannase (20 ten thousand U/g)	Shanxi Tang Biotech Co., Ltd
Alkaline protease (50 ten thousand U/g)	NANNING PANGBO BIOLOGICAL ENGINEERING Co.,Ltd.
		Papain (50 ten thousand U/g)	NANNING DONG HIGHER BIO TECH Co.,Ltd.
Cellulase (5 ten thousand U/g)	NANNING PANGBO BIOLOGICAL ENGINEERING Co.,Ltd.
		Beta-glucanase (8 ten thousand U/g)	German Union enzyme preparations
Nuclease (10 ten thousand U/g)	Angel Yeast Co.,Ltd.
		Bacterial protease (10 ten thousand U/g)	Novoxin Biotechnology Co., Ltd (Tianjin)
Peptone (good order FP101)	Angel Yeast Co.,Ltd.
		Yeast extract powder (cargo number FM888)	Angel Yeast Co.,Ltd.
Molasses (total sugar content 45% -80%)	Chifeng blue sky sugar industry Co Ltd
		MH broth	BEIJING LAND BRIDGE TECHNOLOGY Co.,Ltd.
MH agar medium	BEIJING LAND BRIDGE TECHNOLOGY Co.,Ltd.
		Escherichia coli (ATCC25922)	American Type Culture Collection (ATCC)
Staphylococcus aureus (ATCC29213)	American Type Culture Collection (ATCC)
		Oxford cup	Yichang Sitong chemical Co Ltd
Gentamicin	Beijing Wanjia first Biotechnology Co Ltd

Example one

(I) preparation of modified Yeast cell walls

The modified yeast cell wall is prepared by the following method:

(1) saccharomyces cerevisiae Z2.2(Saccharomyces cerevisiae Hansen Z2.2) with the preservation number of CCTCC NO: M205128 is adopted, and the source and the preparation method of the strain are described in Chinese patent application with the patent application number of 200710135740.6 and the publication number of CN 101361524A. Observing by an optical microscope, the cell diameter of the saccharomyces cerevisiae strain is about 4-6 mu m, the saccharomyces cerevisiae strain is in an ellipsoid shape and is propagated asexually in a budding mode; after being cultured on a solid medium plate at 28 ℃ for 24 hours, the medium is milk white, opaque and round colony.

The strain slant is inoculated into a shake flask liquid culture medium of 100mL by the inoculum size of 2 rings, and is placed for shake culture at the set rotating speed of 250r/min and the temperature of 30 ℃ for 18 h. Wherein the 100mL liquid shake flask culture medium has the formula: 100mL of water, 2g of peptone, 1g of yeast extract powder and 2g of glucose, and adjusting the pH value to (5.0 +/-0.2).

Inoculating the strain subjected to shake flask activation culture into a 50L fermentation tank, setting the rotation speed at 400r/min, the fermentation temperature at 30 ℃ and the fermentation time at 16h to obtain the primary yeast raw material. Wherein, the formula of the fermentation medium is as follows: molasses with total sugar concentration of 30% (diluted with water, 30% total sugar concentration determined by referring to light industry standard QBT2684-2005 of the people's republic of China, and also determined by the same method in the following examples), 10L; (NH)₄)₂SO₄，500g；NH₄H₂PO₄，80g，MgSO₄，56g；ZnSO₄，28g；H₂O, 20L, pH 4.0.

(2) Carrying out autolysis treatment on the yeast primary raw material, adding sodium chloride to ensure that the mass concentration of the sodium chloride is 3%, the pH value is 6.5, the temperature is 55 ℃, carrying out autolysis for 20 hours, then carrying out centrifugal treatment at 5000rpm to obtain yeast autolysate on the upper layer and yeast cell wall milk on the lower layer, and collecting the yeast cell wall milk for carrying out enzymolysis treatment.

(3) Enzymolysis:

a. diluting the yeast cell wall milk obtained in the step (2) with water to obtain 15% concentration, adding mannase (calculated by yeast cell wall dry matter, the same below) with 3 ‰ mass concentration, controlling temperature at 55 deg.C and pH value at 8.0, and hydrolyzing for 15 hr;

b. continuously adding alkaline protease with the mass concentration of 3 per thousand and papain with the mass concentration of 0.5 per thousand, controlling the temperature at 55 ℃, the pH value at 8.5 and the hydrolysis time at 7 hours;

c. c, preserving the temperature of the reactant obtained in the step b for 1h at 90 ℃, and inactivating to obtain an enzyme-inactivated solution;

d. then adjusting the temperature of the solution after enzyme deactivation to 60 ℃, adjusting the pH value to 5.5, adding 0.5 per mill mass concentration of cellulase and 0.5 per mill mass concentration of beta-glucanase, and hydrolyzing for 8 hours;

e. and d, heating the solution obtained in the step d to 90 ℃, preserving heat for 1h, and then carrying out spray drying treatment to obtain the modified yeast cell wall powder.

(II) product determination of modified Yeast cell walls

1. Determination of beta-glucans and mannans in yeast cell walls

1.1 principle of detection

According to different distribution coefficients of glucan and mannose between a mobile phase and a stationary phase of a liquid chromatographic column, injecting a hydrolyzed sample into the liquid chromatogram, using pure water as the mobile phase, detecting saccharide molecules after flowing out by a differential detector, and quantifying by an external standard method.

In the hydrolysis of the beta-glucan and the mannan, the detection result is lower than that of the beta-glucan and the mannan actually contained in the product due to the possible incomplete hydrolysis of the sample and the partial occurrence of other side reactions of glucose and mannose generated by the hydrolysis caused by high temperature. And correcting errors caused by the hydrolysis process by using a glucan reference substance in the detection.

1.2 instruments

a) A water bath kettle;

b) a vortex mixer;

c) an electric furnace;

d) a pressure steam sterilizer;

e) high performance liquid chromatograph: with a differential detector and a sugar column (6.5 mm. times.300 mm waters sugar pak-1).

1.3 reagents

a) Pure water;

b) hydrochloric acid: about 37 percent;

c) glucose: AR;

d) mannose: AR;

e) sodium hydroxide: AR;

f) glucose and mannose mixed standard solution (2 g/L): 0.2000g of glucose and 0.2000g of mannose are respectively weighed and are made into 100ml by pure water.

g) Dextran control (curdlan from Alcaligenes faecalalis): sigma, cat # C7821.

h) Sodium hydroxide solution: 300 g/L.

1.4 sample treatment

A sample of 400mg (to the nearest 0.1mg) was weighed accurately into a 20ml glass-lined small test tube, 6.0ml hydrochloric acid (37%) was added, and the vial was carefully capped and mixed with a vortex mixer to obtain a uniform suspension. The vials were placed in a 30 ℃ water bath for 45min and mixed with a vortex mixer with shaking every 15 min. The suspension is then quantitatively transferred to a 200ml dewar and the cuvette is washed several times with about 100ml to 120ml of water, the wash being incorporated into the dewar. Placing the Du's bottle into an autoclave, and treating at 121 deg.C for 60 min. Taking out and cooling, adjusting the pH of the solution to 6-7 by using a sodium hydroxide solution, and then fixing the volume to 200 ml. Filter through a 0.45 micron pore size cellulose acetate membrane for use.

At the same time, 200mg of a dextran control (see 1.3(g)) was accurately weighed and subjected to the same treatment as the sample treatment method.

1.5 chromatographic conditions

Pure water is used as a mobile phase, the flow rate is 0.5ml/min, the column temperature is 80 ℃, and the sample is injected after the baseline of the instrument is stable.

1.6 drawing of Standard Curve

Respectively sucking 1ml, 2 ml, 3 ml, 4 ml and 5ml of mannose/glucose standard solution (see A.3(f)) into 10ml volumetric flasks, and fixing the volume to the scale with high-purity water to obtain mixed standard samples of 200 mg/l, 400 mg/l, 600 mg/l, 800 mg/l and 1000mg/l of mannose and glucose respectively. And (3) accurately injecting 20ul of sample under the chromatographic conditions to obtain a regression equation between the chromatographic peak area and the mass concentration of the standard substance, and drawing a standard curve.

1.7 measurement of samples and controls

Under the same chromatographic conditions, the treated sample and the dextran control were injected into the chromatograph separately, and the retention time and peak area of each chromatographic peak were recorded. The retention time of the chromatographic peak of the sugar standard sample is used for qualitative determination, and the peak area of the chromatographic peak of the sugar standard sample is used for quantitative determination.

1.8 calculation of results

The content of beta-glucan or mannan is calculated as follows:

X＝(A₁×0.2×100)÷(m₁×1000)×0.9×F…………………………………(1)

F＝P×(100-W)÷[(A₂×0.2×100)÷(m₂×1000)×0.9]………………………(2)

in the formula:

x- - -the content of beta-glucan or mannan in the sample,%;

A₁- -the glucose or mannose content of the sample solution, mg/L, is found on the standard curve according to the peak area of the sample solution;

A₂-the glucose content of the sample solution, mg/L, is found on the standard curve according to the peak area of the dextran control solution;

m₁-weighing the mass of the sample, g;

m₂weighing the mass of the dextran control, g;

0.2- -volume of the treated sample/dextran control, L;

0.9- - -coefficient for conversion of glucose or mannose to β -glucan or mannan;

f- -empirical compensation factor for low results due to destruction of glucose and mannose in acid hydrolysis of samples;

p- - -the purity of the dextran control (according to the test report provided by the reagent manufacturer);

w- -moisture of the dextran control (according to the test report provided by the reagent manufacturer).

Remarking: and (4) detecting the F value in the same laboratory for 1-2 months generally. The F value is about 1.25, and the F value is periodically corrected by a laboratory.

1.9 tolerance errors

The relative difference between the results of two independent measurements obtained under repeated measurement conditions must not exceed the values specified in the following table:

table 2 allowable error values

2. Method for measuring dissolution rate

2.1 principle of measurement

The sample was dissolved in water, and the precipitate was collected by centrifugation, and the weight ratio of the dissolved substance to the total weight was calculated.

2.2 reagents and instruments

a) Distilled water;

b) a centrifuge (5000 g);

c) a moisture meter;

d) an analytical balance;

2.3 measurement procedure

Accurately weigh 10g of sample (accurate to 0.1mg) and record as m₀Dissolving in 200mL of distilled water, transferring into a centrifuge cup after fully dissolving, centrifuging for 5min at 5000g, and accurately measuring the precipitate weight as m₁And the sediment dry matter content (measured according to the first method of GB 5009.3-2010) was determined and recorded as D.

2.4 calculation of results

The sample dissolution rate was:

X＝(m₀-m₁×D)÷m₀×100％

x-sample dissolution,%;

m₀-weighing the sample weight, g;

m₁-centrifugation followed by weight of the pellet, g;

d-the content of dry matter of the precipitate after centrifugation,%.

The calculation result is retained to 1 bit after the decimal point.

3. The molecular weight of glucan and the molecular weight of mannan were measured as follows:

the determination method comprises the following steps: preparing the modified yeast cell wall product prepared in the step (I) into a solution, and measuring the molecular weight of glucan and mannan by using a high-phase liquid chromatography.

Analysis conditions were as follows: shodex Ohpak SB-805HQ gel column (8 mm. times.300 mm); a detector: differential detector (Optilab reex), eighteen-angle static laser scattering instrument (DAWN HELLOS), uv detector; detection wavelength: 658nm (dextran), 280nm (mannan); mobile phase: 0.5mo1/L NaCl solution; column temperature: 25 ℃; flow rate: 0.5 mL/min; sample introduction amount: 20 μ L.

High performance liquid chromatograph: model GPC/RI/MALLS, Waters 515 Pump, manufactured by Waters corporation, USA.

The yeast cell wall product prepared in the first example is detected to have a dissolution rate of 48.5%, a mass content of beta-glucan of 35% and a mass content of mannan of 30%. The molecular weight of glucan detected in the yeast cell wall was 8.45X 10⁵98.12% of total glucan, and the molecular weight of mannan is 1.98 × 10⁶And accounts for 99.45 percent of the total amount of mannan._.

(III) bacteriostatic effect of modified yeast cell wall

Preparing the modified yeast cell wall sample prepared in the step (one) into a solution, taking out 500mL to 1000mL, centrifuging at 6000g of 5000-. The specific experimental procedures and experimental results are as follows:

1. preparation of bacteriostatic test sample

Preparing the modified yeast cell wall sample prepared in the step (I) into a solution with the dry matter concentration of 10%, taking 500mL, centrifuging at 5000g for 20min, taking the supernatant, and evaporating and concentrating at 80 ℃ by using a rotary evaporator until the dry matter concentration is 40% for a subsequent antibacterial activity test.

2. Protocol for testing bacteriostatic activity

The difference of the bacteriostatic effect of the modified yeast cell wall prepared by the invention and the bacteriostatic effect of the common yeast cell wall are respectively tested from the size of the bacteriostatic zone and the MIC experiment. The experimental process comprises the preparation of a culture medium, the culture of strains, a zone of inhibition test and an MIC test.

2.1 preparation of the culture medium.

MH broth and MH agar medium were autoclaved at 121 ℃ for 20min, and MH agar plates were prepared for use.

2.2 Strain culture

Inoculating the preserved Escherichia coli and Staphylococcus aureus to MH broth, and incubating in constant temperature shaking table at 37 deg.C for 12-14 h. And inoculating the recovered escherichia coli and staphylococcus aureus to MH broth again, and culturing for about 16-20 h. The OD value is between 0.4 and 0.7 by the detection of a spectrophotometer. The number of viable bacteria is about 10¹⁰CFU/mL (CFU/mL indicates the total number of bacterial colonies contained per mL of sample).

2.3 zone of inhibition test

An Oxford cup method is adopted to carry out a bacteriostatic circle experiment, the determination principle is to sterilize a culture medium, then an Oxford cup (a small circular tube with the inner diameter of 6mm, the outer diameter of 8mm and the height of 10mm, the two ends of the tube are smooth, a glass tube and a porcelain tube can also be used) is directly and vertically placed on the surface of the culture medium by aseptic operation, the pressure is lightly applied to ensure that the Oxford cup is in contact with the culture medium without a gap, a sample to be detected is added into the cup, and the Oxford cup can be generally filled with 240 microliters so as not to overflow. After the mixture is filled, the mixture is cultured for a certain time at a certain temperature, and a transparent circle with the oxford cup as the center can be observed. This is because, during the culturing process, the test bacteria start to grow on the one hand, and the sample to be measured spreads spherically on the other hand, and the closer to the cup, the higher the concentration of the sample to be measured, and the farther from the cup, the lower the concentration of the sample to be measured. As the concentration of the sample to be detected is reduced, a minimum inhibitory concentration zone exists, and bacteria can not grow in the zone range and are in a transparent circle, namely an 'inhibitory zone'. The higher the concentration of the sample to be detected is, the larger the inhibition zone is. The size of the inhibition zone can be directly measured by a ruler.

2.3.1 preparation of plates containing bacteria

Diluting the pure culture bacterial liquid obtained in the step 2.2 by 1000 times by using normal saline (sterilized at 121 ℃ under high pressure for 20min) to prepare bacterial suspension;

pouring the sterilized MH agar into plates, and dripping 200 mu L of bacterial suspension into each plate after the MH agar is solidified;

and (3) quickly and uniformly coating the bacteria liquid on the whole dish by using the sterilized glass rod to manufacture the bacteria-containing dish.

2.3.2 the prepared yeast cell wall samples were adjusted to the same concentration.

2.3.3 put the oxford cup into the prepared plate containing the bacteria.

2.3.4 Yeast cell wall samples were added to Oxford cups (250. mu.L each) to level the surface with the Oxford cups.

2.3.5 the plate of 2.3.4 was placed in a constant temperature incubator and incubated at 37 ℃ for about 14 hours, and the results were observed.

In the experimental process, the modified yeast cell wall prepared in the first embodiment is used as a sample to be tested, gentamicin is used as a positive control, and physiological saline is used as a negative control. Wherein gentamicin is an aminoglycoside antibiotic and has good bacteriostatic action on escherichia coli and staphylococcus aureus.

The results are shown in FIGS. 1 and 3. Wherein, figure 1 is a bacteriostatic effect diagram of yeast cell walls on escherichia coli, and figure 3 is a bacteriostatic effect diagram of yeast cell walls on staphylococcus aureus. As can be seen from FIGS. 1 and 3, the yeast cell wall prepared in the first example has a significant bacteriostatic effect on Escherichia coli and Staphylococcus aureus. The size of the inhibition zone of gentamicin on escherichia coli is 35mm, the size of the inhibition zone of staphylococcus aureus is 30mm, the size of the inhibition zone of modified yeast cell wall on escherichia coli prepared in the first embodiment is 28mm, and the size of the inhibition zone of staphylococcus aureus is 21mm, and experimental results show that the inhibition effect of the yeast cell wall on escherichia coli and staphylococcus aureus prepared in the first embodiment is second to gentamicin.

MIC assay

The minimum inhibitory concentration is generally used to refer to the lowest concentration of drug that inhibits a significant increase in a microorganism when incubated for a certain period of time in a particular environment. In the present invention, the lowest concentration of yeast cell wall that inhibits the significant growth of E.coli and S.aureus was used when incubated at 37 ℃ for 12-14 h. The specific experimental process and experimental results are as follows:

determination of the MIC experiment of E.coli:

2.4.1 preparation of bacterial suspension: diluting the pure cultured Escherichia coli liquid in step 2.2 with MH broth 100 times, shaking to make viable count of Escherichia coli suspension 10⁸CFU/mL。

2.4.2 Place 10 tubes in tube racks, numbered 1-10, respectively, wherein tubes numbered 1 have nothing added and tubes numbered 2-10 have 1ml MH broth added, respectively.

2.4.3 Add 1mL of yeast cell wall sample (40% dry matter concentration) to test tube number 1, add 1mL of yeast cell wall sample to test tube number 2, shake well, remove 1mL of solution from test tube number 2 and add to test tube number 3, shake well, remove 1mL of solution from test tube number 3 and add to test tube number 4, and so on to test tube number 10. 1mL of the solution was removed from the test tube designated by the reference numeral 10 and discarded, and the concentration of the dry substance of the yeast cell wall in test tubes numbered 1 to 10 was 40%, 40%. times.2, respectively, by stepwise dilution^-1，40％×2^-2，40％×2^-3，40％×2^-4，40％×2^-5，40％×2^-6，40％×2^-7，40％×2^-8And 40% x 2^-9。

2.4.4 Add 1mL of prepared E.coli suspension to each of the 10 tubes and mix well.

2.4.5 after finishing, placing the mixture in a constant-temperature incubator at 37 ℃, incubating for about 12-14h, and observing the result.

The results show that the yeast cell wall prepared in example one can effectively inhibit the growth of Escherichia coli, and the minimum inhibitory concentration against Escherichia coli is 5% dry matter concentration (i.e., 0.05 g/mL).

Determination of the MIC test of Staphylococcus aureus:

according to the same determination method of the above Escherichia coli MIC experiment, the yeast cell wall prepared in the first example can effectively inhibit the growth of Staphylococcus aureus, and the minimum inhibitory concentration to Staphylococcus aureus is 5% dry matter concentration (i.e., 0.05 g/mL).

Example two

The yeast cell wall is prepared by the following method, the content of the prepared yeast cell wall is measured, and the bacteriostatic effect and the solubility of the yeast cell wall are measured.

(I) preparation of modified Yeast cell walls

The modified yeast cell wall is prepared by the following method:

(1) the Saccharomyces cerevisiae strain FX-2(Saccharomyces cerevisiae FX-2) is adopted, and the preservation number is CCTCC NO: M2016418. The strain is from a fermented dough, the fermented dough contains various wild bacteria, a dough leaching solution is prepared by taking the fermented dough as a sample, a pure strain is obtained by separation through a dilution coating flat plate separation method, and the strain is identified to belong to saccharomyces cerevisiae, wherein the identification method of the strain comprises the following steps: the identification is carried out by sequencing 5.8S rRNA, and the result shows that the sequence homology is more than 99 percent, so that the strain obtained by separation is determined to belong to saccharomyces cerevisiae, and the biological classification is saccharomyces cerevisiae FX-2. Observing by an optical microscope, the cell diameter of the saccharomyces cerevisiae strain is about 4-6 mu m, the saccharomyces cerevisiae strain is in an ellipsoid shape and is propagated asexually in a budding mode; after being cultured on a solid medium plate at 28 ℃ for 24 hours, the medium is milk white, opaque and round colony.

The strain slant is inoculated into a shake flask liquid culture medium of 100mL by the inoculum size of 2 rings, and is placed for shake culture at the set rotating speed of 250r/min and the temperature of 30 ℃ for 18 h. Wherein the formula of the liquid shake flask culture medium is as follows: 100mL of water, 2g of peptone, 1g of yeast extract powder and 2g of glucose, and adjusting the pH value to (5.0 +/-0.2).

Inoculating the strain subjected to shake flask activation culture into a 50L fermentation tank, setting the rotation speed at 400r/min, the fermentation temperature at 28 ℃ and the fermentation time for 24h to obtain the primary yeast raw material. Wherein the formula of the fermentation medium is: molasses with total sugar concentration of 28%, 10L; (NH)₄)₂SO₄，500g；NH₄H₂PO₄，80g，MgSO₄，56g；ZnSO₄，28g；H₂O, 20L and pH value of 6.0.

(2) Carrying out autolysis treatment on the yeast primary raw material, adding sodium chloride to ensure that the mass concentration of the sodium chloride is 5%, the pH value is 5.5, the temperature is 75 ℃, carrying out autolysis for 25 hours, then carrying out centrifugal treatment at 5000rpm to obtain yeast autolysate on the upper layer and yeast cell wall milk on the lower layer, and collecting the yeast cell wall milk for carrying out enzymolysis treatment.

(3) Enzymolysis:

a. diluting the yeast cell wall milk obtained in the step (2) with water to obtain 20% concentration, adding mannase (calculated by yeast cell wall dry matter, the same below) with 5 ‰ mass concentration, controlling temperature at 45 deg.C and pH value at 10.0, and hydrolyzing for 10 hr;

b. continuously adding alkaline protease with the mass concentration of 6 per mill, controlling the temperature at 50 ℃, the pH value at 7.5, and hydrolyzing for 10 hours;

c. b, adjusting the temperature of the solution treated in the step b to be 58 ℃ and the pH value to be 7, then continuously adding papain with the mass concentration of 0.3 per thousand, and carrying out enzymolysis for 5 hours;

d. c, adjusting the temperature of the solution treated in the step c to 85 ℃, and performing heat preservation treatment for 1.5 hours to obtain an inactivated solution;

e. adjusting the temperature of the solution after enzyme deactivation to 50 ℃, adjusting the pH value to 4.5, then adding 1 per mill mass concentration of cellulase, and carrying out enzymolysis for 5 hours;

f. adding beta-glucanase with the mass concentration of 1 per mill into the solution treated in the step e, and hydrolyzing for 12 hours;

g. and f, heating the solution obtained in the step f to 85 ℃, preserving heat for 1.5h, and then carrying out spray drying treatment to obtain the modified yeast cell wall powder.

(II) product determination of modified Yeast cell walls

The solubility and content of the modified yeast cell wall prepared in example two were measured in the same manner as in example one.

Detection ofThe yeast cell wall product prepared in example two was obtained with a lysis rate of 50.0%, a β -glucan content of 48.6% by mass, and a mannan content of 35.2% by mass. Wherein the glucan has a molecular weight of 3.35X 10⁵The mannan has a molecular weight of 8.64 × 10, and accounts for 99.91% of total glucan⁵And accounts for 99.25% of the total amount of mannan.^.

(III) bacteriostatic effect of modified yeast cell wall

The bacteriostatic effect of the modified yeast cell wall was determined in the same manner as in example one.

The measurement results are shown in fig. 1 and 3. Wherein, figure 1 is a bacteriostatic effect diagram of yeast cell walls on escherichia coli, and figure 3 is a bacteriostatic effect diagram of yeast cell walls on staphylococcus aureus. As can be seen from FIGS. 1 and 3, the yeast cell wall prepared in example two has a significant bacteriostatic effect on Escherichia coli and Staphylococcus aureus. The size of the inhibition zone of gentamicin on escherichia coli is 35mm, the size of the inhibition zone of staphylococcus aureus is 30mm, the size of the inhibition zone of modified yeast cell wall prepared in the second embodiment on escherichia coli is 28mm, and the size of the inhibition zone of staphylococcus aureus is 35 mm.

MIC experiment results show that the yeast cell wall prepared in the second example can effectively inhibit the growth of Escherichia coli and Staphylococcus aureus, and the minimum inhibitory concentration to Escherichia coli and Staphylococcus aureus is 5% dry matter concentration (namely 0.05 g/mL).

EXAMPLE III

(I) preparation of modified Yeast cell walls

The modified yeast cell wall is prepared by the following method:

(1) saccharomyces cerevisiae Z2.4(Saccharomyces cerevisiae Hansen Z2.4) with the preservation number of CCTCC NO: M205130 is adopted, and the source and the preparation method of the strain are described in Chinese patent application with the patent application number of 200810105972.1 and the publication number of CN 101575578A. Observing by an optical microscope, the cell diameter of the saccharomyces cerevisiae strain is about 4-6 mu m, the saccharomyces cerevisiae strain is in an ellipsoid shape and is propagated asexually in a budding mode; after being cultured on a solid medium plate at 28 ℃ for 24 hours, the medium is milk white, opaque and round colony.

The strain slant is inoculated into a shake flask liquid culture medium of 100mL by the inoculation amount of 2 rings, and is placed for shake culture at the set rotating speed of 250r/min and the temperature of 25 ℃ for 25 h. Wherein the formula of the liquid shake flask culture medium is as follows: 100mL of water, 2g of peptone, 1g of yeast extract powder and 2g of glucose, and adjusting the pH value to (5.0 +/-0.2) to obtain the activated culture bacterial liquid.

Inoculating the bacterial liquid subjected to shake flask activation culture into a 50L fermentation tank, setting the rotating speed at 400r/min, the fermentation temperature at 30 ℃ and the fermentation time for 20h to obtain the primary yeast raw material. Wherein, the formula of the fermentation medium is as follows: molasses with total sugar concentration of 35%, 10L; (NH)₄)₂SO₄，500g；NH₄H₂PO₄，80g，MgSO₄，56g；ZnSO₄，28g；H₂O, 20L, pH 5.0.

(2) Carrying out autolysis treatment on the yeast primary raw material, adding sodium chloride to ensure that the mass concentration of the sodium chloride is 4%, the pH value is 6, the temperature is 60 ℃, carrying out autolysis for 30 hours, then carrying out centrifugal treatment at 5000rpm to obtain yeast autolysate on the upper layer and yeast cell wall milk on the lower layer, and collecting the yeast cell wall milk for enzymolysis treatment.

(3) Enzymolysis:

a. diluting the yeast cell wall milk obtained in the step (2) with water to 8% concentration, adding mannase (calculated by yeast cell wall dry matter, the same below) with 4 ‰ mass concentration, controlling temperature at 60 deg.C and pH value at 7.0, and hydrolyzing for 10 hr;

b. continuously adding alkaline protease with the mass concentration of 2 per mill, controlling the temperature at 70 ℃, the pH value at 9.5, and hydrolyzing for 8 hours;

c. b, adjusting the temperature of the solution treated in the step b to 50 ℃ and the pH value to 6, then continuously adding papain with the mass concentration of 0.7 per thousand, and carrying out enzymolysis for 3 hours;

d. c, adjusting the temperature of the solution treated in the step c to be 100 ℃, and performing heat preservation treatment for 0.5h to obtain an inactivated solution;

e. adjusting the temperature of the solution after enzyme deactivation to 40 ℃, adjusting the pH value to 4.0, then adding 0.2 per mill mass concentration of cellulase, and carrying out enzymolysis for 3 hours;

f. adjusting the temperature of the solution treated in the step e to 67 ℃, adjusting the pH value to 6.5, then adding beta-glucanase with the mass concentration of 0.2 per mill, and hydrolyzing for 10 hours;

g. and f, heating the solution obtained in the step f to 100 ℃, preserving heat for 0.5h, and then carrying out spray drying treatment to obtain the modified yeast cell wall powder.

(II) product determination of modified Yeast cell walls

The solubility and content of the modified yeast cell wall prepared in example three were measured in the same manner as in example one.

The yeast cell wall product prepared in the third example was tested to have a lysis rate of 35.0%, a β -glucan content of 35.5%, and a mannan content of 28.5%. Wherein the molecular weight of dextran is 9.66 × 10⁵The mannan has a molecular weight of 1.06 × 10, and accounts for 99.9% of total glucan⁶And accounts for 99.57 percent of the total amount of mannan.^.

(III) bacteriostatic effect of modified yeast cell wall

The measurement results are shown in fig. 2 and 4. Wherein, FIG. 2 is a diagram of the bacteriostatic effect of the yeast cell wall on Escherichia coli, and FIG. 4 is a diagram of the bacteriostatic effect of the yeast cell wall on Staphylococcus aureus. As can be seen from FIGS. 2 and 4, the yeast cell wall prepared in example III has a significant bacteriostatic effect on Escherichia coli and Staphylococcus aureus. The size of the inhibition zone of gentamicin on escherichia coli is 35mm, the size of the inhibition zone of staphylococcus aureus is 30mm, the size of the inhibition zone of modified yeast cell wall prepared in the third embodiment on escherichia coli is 24mm, and the size of the inhibition zone of staphylococcus aureus is 15 mm.

MIC experiment results show that the yeast cell wall prepared in the third example can effectively inhibit the growth of Escherichia coli and Staphylococcus aureus, and the minimum inhibitory concentration to Escherichia coli and Staphylococcus aureus is 5% dry matter concentration (namely 0.05 g/mL).

Comparative example 1

(ii) preparation of Yeast cell wall

The modified yeast cell wall is prepared by the following method:

(1) the Saccharomyces cerevisiae strain FX-2(Saccharomyces cerevisiae FX-2) is adopted, and the preservation number is CCTCC NO: M2016418.

And (3) putting the strain slant into a 100mL shake flask liquid culture medium by the inoculation amount of 2 rings, placing the shake flask liquid culture medium for shake culture, setting the rotation speed to be 250r/min, setting the temperature to be 30 ℃, and culturing for 18h to obtain the activated culture bacterial liquid. Wherein, the formula of the liquid shake flask culture medium is as follows: 100mL of water, 2g of peptone, 1g of yeast extract powder and 2g of glucose, and adjusting the pH value to (5.0 +/-0.2).

Inoculating the bacterial liquid subjected to shake flask activation culture into a 50L fermentation tank, setting the rotating speed at 400r/min, the fermentation temperature at 30 ℃ and the fermentation time at 16h to obtain the primary yeast raw material. Wherein, the formula of the fermentation medium is as follows: molasses with total sugar concentration of 30%, 10L; (NH)₄)₂SO₄，500g；NH₄H₂PO₄，80g，MgSO₄，56g；ZnSO₄，28g；H₂O, 20L, pH 4.0.

(3) Enzymolysis:

b. continuously adding alkaline protease with the mass concentration of 3 per mill, controlling the temperature to be 55 ℃, the pH value to be 8.5 and the hydrolysis time to be 7 hours;

c. c, adjusting the temperature of the solution treated in the step b to be 90 ℃, and performing heat preservation treatment for 1h to obtain an inactivated solution;

d. adjusting the temperature of the solution after enzyme deactivation to 60 ℃, adjusting the pH value to 5.5, then adding 0.5 per mill mass concentration of cellulase, and carrying out enzymolysis for 8 hours;

e. d, adjusting the pH value of the solution treated in the step d to 6.0 and the temperature to 55 ℃, then adding beta-glucanase with the mass concentration of 0.5 per thousand into the solution, and hydrolyzing for 5 hours;

f. and e, heating the solution obtained in the step e to 90 ℃, carrying out heat preservation treatment for 1h, and then carrying out spray drying treatment to obtain yeast cell wall powder.

(II) product determination of Yeast cell walls

The solubility and content of the yeast cell wall obtained in comparative example A were measured in the same manner as in example A.

The yeast cell wall product prepared in the comparative example one was tested to have a dissolution rate of 32.0%, a beta-glucan content of 30.0% and a mannan content of 25.0%. Wherein the molecular weight of the beta-glucan is 4.45 x 10⁶The mannan has a molecular weight of 8.5 × 10, and accounts for 99.92% of total glucan⁵And accounts for 99.25% of the total amount of mannan.^.

(III) bacteriostatic effect of modified yeast cell wall

The bacteriostatic effect of the yeast cell wall prepared in comparative example one was measured in the same manner as in example one.

The measurement results are shown in fig. 1 and 3. Wherein, figure 1 is a bacteriostatic effect diagram of yeast cell walls on escherichia coli, and figure 3 is a bacteriostatic effect diagram of yeast cell walls on staphylococcus aureus. As can be seen from FIGS. 1 and 3, the yeast cell wall prepared in comparative example one has no significant bacteriostatic effect on Escherichia coli and Staphylococcus aureus. The MIC experimental results also show that the yeast cell wall prepared in the comparative example I can not effectively inhibit the growth of escherichia coli and staphylococcus aureus.

Comparative example No. two

(ii) preparation of Yeast cell wall

The yeast cell wall is prepared by the following method:

And (3) putting the strain slant into a 100mL shake flask liquid culture medium by the inoculation amount of 2 rings, placing the shake flask liquid culture medium for shake culture, setting the rotation speed to be 250r/min, setting the temperature to be 30 ℃, and culturing for 18h to obtain the activated culture bacterial liquid. Wherein the formula of the liquid shake flask culture medium is as follows: 100mL of water, 2g of peptone, 1g of yeast extract powder and 2g of glucose, and adjusting the pH value to (5.0 +/-0.2).

(3) Enzymolysis:

b. b, adjusting the pH value of the solution treated in the step a to 6, controlling the temperature to be 55 ℃, then continuously adding papain with the mass concentration of 0.5 per thousand, and carrying out enzymolysis for 7 hours;

c. c, adjusting the temperature of the solution treated in the step c to be 90 ℃, and performing heat preservation treatment for 1h to obtain an inactivated solution;

e. d, adjusting the temperature of the solution treated in the step d to 55 ℃, adjusting the pH value to 6.0, adding beta-glucanase with the mass concentration of 0.5 per thousand, and hydrolyzing for 5 hours;

(II) product determination of Yeast cell walls

The solubility and content of the yeast cell wall prepared in comparative example two were measured in the same manner as in example one.

The yeast cell wall product prepared in the comparative example was tested to have a dissolution rate of 32.0%, a beta-glucan content of 28.0% and a mannan content of 32.0%. Wherein the molecular weight of dextran is 1.15 × 10⁶The mannan has a molecular weight of 1.0 × 10, and accounts for 99.05% of total glucan⁶And accounts for 99.45 percent of the total amount of mannan.^.

(III) bacteriostatic Effect of Yeast cell wall

The bacteriostatic effect of the yeast cell wall obtained in comparative example two was measured in the same manner as in example one.

The measurement results are shown in fig. 1 and 3. Wherein, figure 1 is a bacteriostatic effect diagram of yeast cell walls on escherichia coli, and figure 3 is a bacteriostatic effect diagram of yeast cell walls on staphylococcus aureus. As can be seen from FIGS. 1 and 3, the yeast cell wall prepared in comparative example No. exhibits no significant bacteriostatic effect against E.coli and S.aureus. The results of the MIC experiments show that the yeast cell wall prepared in the comparative example II can not effectively inhibit the growth of Escherichia coli and Staphylococcus aureus.

Comparative example No. three

(ii) preparation of Yeast cell wall

The modified yeast cell wall is prepared by the following method:

(3) Enzymolysis:

c. adjusting the pH value of the solution treated in the step b to 6, controlling the temperature to be 55 ℃, then continuously adding papain with the mass concentration of 0.5 per thousand for enzymolysis for 7 hours;

d. c, adjusting the temperature of the solution treated in the step c to be 90 ℃, and performing heat preservation treatment for 1h to obtain an inactivated solution;

e. adjusting the temperature of the solution after enzyme deactivation to 60 ℃, adjusting the pH value to 5.5, then adding 0.5 per mill mass concentration of cellulase, and carrying out enzymolysis for 8 hours;

f, heating the solution obtained in the step e to 90 ℃, carrying out heat preservation treatment for 1h, and then carrying out spray drying treatment to obtain yeast cell wall powder.

(II) product determination of modified Yeast cell walls

The solubility and content of the yeast cell wall obtained in comparative example III were measured in the same manner as in example I.

The yeast cell wall product prepared in the third comparative example was tested to have a dissolution rate of 30.4%, a beta-glucan content of 25.5% and a mannan content of 30.2%. Wherein the molecular weight of dextran is 4.7 × 10⁶The mannan has a molecular weight of 8.72 × 10, and accounts for 99.90% of total glucan⁵And accounts for 99.57 percent of the total amount of mannan.^.

(III) bacteriostatic Effect of Yeast cell wall

And (3) determining the bacteriostatic effect of the yeast cell wall prepared in the third comparative example according to the same method as the first example.

The measurement results are shown in fig. 2 and 4. Wherein, FIG. 2 is a diagram of the bacteriostatic effect of the yeast cell wall on Escherichia coli, and FIG. 4 is a diagram of the bacteriostatic effect of the yeast cell wall on Staphylococcus aureus. As can be seen from FIGS. 2 and 4, the yeast cell wall prepared in comparative example III has no significant bacteriostatic effect on Escherichia coli and Staphylococcus aureus. The results of the MIC experiments show that the yeast cell wall prepared in the third comparative example can not effectively inhibit the growth of Escherichia coli and Staphylococcus aureus.

Comparative example No. four

(ii) preparation of Yeast cell wall

The modified yeast cell wall is prepared by the following method:

And (3) putting the strain slant into a 100mL shake flask liquid culture medium by the inoculation amount of 2 rings, placing the shake flask liquid culture medium for shake culture, setting the rotation speed to be 250r/min, setting the temperature to be 30 ℃, and culturing for 18h to obtain the activated culture bacterial liquid. Wherein the formula of the liquid shake flask culture medium is as follows: 100mL of water, 2g of peptone, 1g of yeast extract powder and 2g of glucose, wherein the pH value is (5.0 +/-0.2).

(3) Enzymolysis:

c. adjusting the pH value of the solution treated in the step b to 6, controlling the temperature to be 55 ℃, then continuously adding papain with the mass concentration of 0.5 per thousand, and carrying out enzymolysis for 7 hours;

e. adjusting the temperature of the solution after enzyme deactivation to 55 ℃, adjusting the pH value to 6.0, then adding beta-glucanase with 0.5 per mill mass concentration, and carrying out enzymolysis for 16 hours;

(II) product determination of Yeast cell walls

The solubility and content of the yeast cell wall obtained in comparative example four were measured in the same manner as in example one.

The yeast cell wall product prepared in the comparative example four is detected to have the dissolution rate of 32.0 percent, the mass content of beta-glucan of 32.8 percent and the mass content of mannan of 29.7 percent. Wherein the molecular weight of dextran is 4.47 × 10⁶The mannan has a molecular weight of 9.65 × 10, and accounts for 99.50% of total glucan⁵And accounts for 99.65% of the total amount of mannan.^.

(III) bacteriostatic Effect of Yeast cell wall

And (3) determining the bacteriostatic effect of the yeast cell wall prepared by the fourth comparative example according to the same method as the first example.

The measurement results are shown in fig. 2 and 4. Wherein, FIG. 2 is a diagram of the bacteriostatic effect of the yeast cell wall on Escherichia coli, and FIG. 4 is a diagram of the bacteriostatic effect of the yeast cell wall on Staphylococcus aureus. As can be seen from FIGS. 2 and 4, the yeast cell wall prepared in comparative example four has no significant bacteriostatic effect on Escherichia coli and Staphylococcus aureus. The MIC experiment result shows that the yeast cell walls prepared in the comparative example four can not effectively inhibit the growth of escherichia coli and staphylococcus aureus.

Comparative example five

(ii) preparation of Yeast cell wall

Firstly, utilizing a Saccharomyces cerevisiae strain, namely Saccharomyces cerevisiae FX-2(Saccharomyces cerevisiae FX-2) with the preservation number of CCTCC NO: M2016418, referring to the method disclosed in the embodiment of Chinese invention patent application with the application number of 201210025279.X and the publication number of CN103243028A, preparing 20kg of yeast milk according to the proportion of 10 mass percent of dry matter (the mass proportion of yeast dry matter to water is 1: 9), then cooking for 1 hour at 89 ℃, cooling to 65 ℃, adding papain according to the proportion of 2 per mill of the addition amount of the yeast dry matter, adjusting the pH to 5.5, and acting for 6 hours; then adjusting the pH value to 5.0, adjusting the temperature to 68 ℃, adding nuclease according to the proportion of 1.0 per mill of the addition amount of the yeast dry matter, after 8 hours of action, cooling to 50 ℃, adjusting the pH value to 5.6, adding bacterial protease according to the proportion of 0.2 per mill of the addition amount of the yeast dry matter, and acting for 5 hours; then inactivating enzyme at 85 ℃ for 30 minutes, finally centrifuging, taking heavy phase after centrifugation, spraying powder and drying to obtain the yeast cell wall.

(II) product determination of Yeast cell walls

The solubility and content of the modified yeast cell wall prepared in comparative example five were measured in the same manner as in example one.

The yeast cell wall product prepared in the fifth comparative example was tested to have a dissolution rate of 22.50%, a beta-glucan content of 20.0%, and a mannan content of 60%. Wherein the molecular weight of dextran is 1.07 × 10⁷The mannan has a molecular weight of 99.8% of the total glucanIs 4.6X 10⁶And accounts for 99.50 percent of the total amount of mannan.^.

(III) bacteriostatic Effect of Yeast cell wall

The bacteriostatic effect of the yeast cell wall prepared in the fifth comparative example was measured in the same manner as in the first example.

The measurement results are shown in fig. 2 and 4. Wherein, FIG. 2 is a diagram of the bacteriostatic effect of the yeast cell wall on Escherichia coli, and FIG. 4 is a diagram of the bacteriostatic effect of the yeast cell wall on Staphylococcus aureus. As can be seen from FIGS. 2 and 4, the yeast cell wall prepared in the fifth comparative example has no significant bacteriostatic effect on Escherichia coli and Staphylococcus aureus. MIC experimental results show that the yeast cell walls prepared in the fifth comparative example can not effectively inhibit the growth of escherichia coli and staphylococcus aureus.

It can be seen from the above examples that autolysis treatment is performed by using yeast strains, and then enzymolysis treatment is performed by using mannanase, alkaline protease, papain, cellulase and beta-glucanase to obtain modified yeast cell walls, which is mainly characterized in that the modified yeast cell walls show obvious bacteriostatic effects compared with the yeast cell walls obtained in the comparative examples, are second to gentamicin, and even have superior inhibitory effects on staphylococcus aureus to gentamicin. In addition, in the beta-glucan prepared in the first to third examples, the relative molecular mass of nearly 99% or more of the beta-glucan is 3.3X 10⁵-1.0×10⁶And the relative molecular mass of more than 99 percent of the mannan in the prepared mannan is 8.6 multiplied by 10⁵-2.0×10⁶The molecular weights of the beta-glucan and mannan were smaller than those of comparative examples one to five, and at the same time, the dissolution rates were higher. Comparative examples one to four enzymes are adopted for enzymolysis in the process of preparing the yeast cell wall, and the bacteriostatic effect is not shown. In the fifth comparative example, papain, nuclease and bacterial protease are used for enzymolysis, and the obtained yeast cell wall does not show a bacteriostatic effect.

In the livestock breeding industry, a large number of normal bacteria are inhabited in the intestinal tracts of livestock and poultry raised in a common environment, and the bacteria mainly comprise bacilli, cocci, lactic acid bacteria, bifidobacteria, clostridia and the like, and form a microbial barrier of the intestinal tracts. When the resistance of the cultured animals is reduced, the balance between the number of intestinal microbial flora and the flora is broken, pathogenic microorganisms can make the animals sick, if the pathogenic microorganisms are not treated in time, the disease is easy to spread in the cultured animals, and the health of the animals is seriously influenced. For a long time, the antibiotics for animals play an important role in preventing and treating animal epidemic diseases and guaranteeing public health safety. Antibiotics added in the livestock breeding process interfere the growth and reproduction of bacteria by selectively acting on specific parts (target positions) of the microorganisms, and kill or inhibit the microorganisms. The mechanisms of action of antibiotics can be divided into 4 classes: interfere with bacterial cell wall synthesis, damage bacterial cell membrane permeability, inhibit bacterial protein synthesis, and inhibit bacterial nucleic acid synthesis. However, antibiotic residues also pose a serious problem in terms of food safety, environmental pollution, risk of superbacteria, and the like. No. 2428, issued by the ministry of agriculture in 2016, has decided to stop colistin sulfate as a feed additive for animal growth globally 30 days 4-7 in 2017. In general, antibiotic replacement products are becoming a market hotspot and a trend towards industry revolution. The yeast cell wall beta-glucan and mannan prepared by the method has smaller molecular weight range and higher dissolution rate, and has obvious bacteriostatic effect on intestinal typical pathogenic bacteria such as escherichia coli and staphylococcus aureus. The feed additive is applied to the field of feed addition, can improve the immunity of animals and enhance the body health of the animals, and can be used as an ideal substitute of feed antibiotics.

The foregoing is considered as illustrative and not restrictive in character, and that various modifications, equivalents, and improvements made within the spirit and principles of the invention are intended to be included within the scope of the invention.

Claims

1. A yeast cell wall, wherein the mass content of beta-glucan in the yeast cell wall is 35-50%, wherein more than 99% of the beta-glucan phase is presentHas a molecular weight of 3.0X 10⁵-1.0×10⁶(ii) a The mass content of mannan in the yeast cell wall is 20-35%, wherein the relative molecular weight of mannan of more than 99% is 8.0 × 10⁵-2.0×10⁶。

2. A yeast cell wall with improved solubility, wherein the mass content of beta-glucan in the yeast cell wall is 35-50%, and wherein the relative molecular weight of more than 99% of beta-glucan is 3.0 x 10⁵-1.0×10⁶(ii) a The mass content of mannan in the yeast cell wall is 20-35%, wherein the relative molecular weight of mannan of more than 99% is 8.0 × 10⁵-2.0×10⁶The dissolving rate of the yeast cell wall is 30-50%.

3. Yeast cell wall according to claim 2, wherein the lysis rate of the yeast cell wall is between 35% and 50%.

4. A yeast cell wall with antibacterial effect, wherein the mass content of beta-glucan in the yeast cell wall is 35-50%, wherein the relative molecular weight of more than 99% of beta-glucan is 3.0 x 10⁵-1.0×10⁶(ii) a The mass content of mannan in the yeast cell wall is 20-35%, wherein the relative molecular weight of mannan of more than 99% is 8.0 × 10⁵-2.0×10⁶The yeast cell wall exhibits bacteriostatic effects against escherichia coli and staphylococcus aureus.

5. Yeast cell wall according to any of claims 1 to 4, wherein the minimum inhibitory concentration of the yeast cell wall for E.coli and S.aureus is 5% yeast cell wall dry matter mass concentration.

6. A yeast cell wall according to any of claims 1 to 4, wherein the yeast cell wall is enzymatically hydrolyzed using mannanase, alkaline protease, papain, cellulase and β -glucanase;

the addition mass of the mannase, the alkaline protease, the papain, the cellulase and the beta-glucanase is 3-5 per thousand, 2-6 per thousand, 0.1-0.7 per thousand, 0.2-1 per thousand and 0.2-1 per thousand respectively based on the mass of yeast cell wall dry matter; adding mannase for enzymolysis, adding alkaline protease and papain simultaneously or respectively for enzymolysis, and adding cellulase and beta-glucanase simultaneously or respectively for enzymolysis;

the enzymolysis temperature of the beta-glucanase is 40-67 ℃, and the pH value is 4.5-6.5;

the enzymolysis time of the mannase is 10-15 h;

the enzymolysis time of the alkaline protease is 7-10 h;

the enzymolysis time of the papain is 3-8 h;

the enzymolysis time of the cellulase is 3-8 h;

the enzymolysis time of the beta-glucanase is 6-12 h.

7. The yeast cell wall of claim 6, wherein the yeast cell wall is enzymatically hydrolyzed by mannanase, followed by alkaline protease and papain, and then by cellulase and beta-glucanase.

8. Yeast cell wall according to claim 6, wherein the yeast cell wall is obtained by enzymatic hydrolysis with mannanase, followed by enzymatic hydrolysis with alkaline protease, followed by enzymatic hydrolysis with papain, followed by enzymatic hydrolysis with cellulase and then enzymatic hydrolysis with β -glucanase.

9. Yeast cell wall according to claim 5, wherein the yeast cell wall is obtained by enzymatic hydrolysis of mannanase, alkaline protease, papain, cellulase and β -glucanase;

the enzymolysis time of the mannase is 10-15 h;

the enzymolysis time of the alkaline protease is 7-10 h;

the enzymolysis time of the papain is 3-8 h;

the enzymolysis time of the cellulase is 3-8 h;

the enzymolysis time of the beta-glucanase is 6-12 h.

10. Yeast cell wall according to claim 9, wherein the yeast cell wall is obtained by enzymatic hydrolysis first with mannanase, followed by enzymatic hydrolysis with alkaline protease and papain, followed by enzymatic hydrolysis with cellulase and β -glucanase.

11. Yeast cell wall according to claim 9, wherein the yeast cell wall is obtained by enzymatic hydrolysis first with mannanase, followed by enzymatic hydrolysis with alkaline protease, followed by enzymatic hydrolysis with papain, followed by enzymatic hydrolysis with cellulase and then enzymatic hydrolysis with β -glucanase.

12. A method for preparing yeast cell walls, which is characterized by comprising the following steps:

(2) carrying out enzymolysis on yeast cell wall milk by mannase, alkaline protease, papain, cellulase and beta-glucanase to obtain a yeast cell wall; adding mannase for enzymolysis, adding alkaline protease and papain simultaneously or respectively for enzymolysis, and adding cellulase and beta-glucanase simultaneously or respectively for enzymolysis;

the addition quality, the enzymolysis temperature, the enzymolysis pH and the enzymolysis time of the mannase, the alkaline protease, the papain, the cellulase and the beta-glucanase are as follows:

the addition mass of the mannase, the alkaline protease, the papain, the cellulase and the beta-glucanase is 3-5 per thousand, 2-6 per thousand, 0.1-0.7 per thousand, 0.2-1 per thousand and 0.2-1 per thousand respectively based on the mass of yeast cell wall dry matter;

the enzymolysis time of the mannase is 10-15 h;

the enzymolysis time of the alkaline protease is 7-10 h;

the enzymolysis time of the papain is 3-8 h;

the enzymolysis time of the cellulase is 3-8 h;

the enzymolysis time of the beta-glucanase is 6-12 h.

13. The method according to claim 12, wherein in the step (2), the yeast cell wall milk is first enzymatically hydrolyzed by mannanase, then by alkaline protease and papain, and then enzymatically hydrolyzed by cellulase and β -glucanase to obtain the yeast cell wall.

14. The method according to claim 12, wherein in the step (2), the yeast cell wall milk is first enzymatically hydrolyzed by mannanase, then by alkaline protease, then by papain, then by cellulase, and then by β -glucanase to obtain the yeast cell wall.

15. The method according to claim 12, wherein the autolytic wall-breaking is performed at a salt concentration of 3-5%, a pH of 5.5-6.5, and a temperature of 55-75 ℃.

16. The method according to claim 15, wherein the time for autolytic wall-breaking is 20-30 h.

17. The method according to any one of claims 12 to 16, wherein the mannanase, the alkaline protease, the papain, the cellulase and the beta-glucanase are added in amounts of (0.06U-0.1U), (0.1U-0.3U), (0.005U-0.035U), (0.001U-0.005U) and (0.0016U-0.008U), respectively, per gram of yeast cell wall dry matter.

18. The method according to any one of claims 12 to 16, wherein in the step (2), the yeast cell wall milk is prepared into a suspension solution with a mass concentration of 8 to 20% of dry matter of the yeast cell wall, and then the enzymatic hydrolysis is performed by mannanase, alkaline protease and papain, and then the enzymatic hydrolysis is performed by cellulase and beta-glucanase.

19. The method according to claim 17, wherein in the step (2), the yeast cell wall milk is prepared into a suspension solution with a mass concentration of 8-20% based on dry matter of the yeast cell wall, and then the enzymatic hydrolysis is performed by mannanase, alkaline protease and papain, and then the enzymatic hydrolysis is performed by cellulase and beta-glucanase.

20. The process according to claim 18, wherein in the step (2), the enzyme deactivation is carried out before the enzymatic hydrolysis with the cellulase and the β -glucanase.

21. The method according to claim 20, wherein the reaction product obtained by enzymolysis with mannanase and then with alkaline protease and papain is subjected to enzyme inactivation at 85 to 100 ℃ for 0.5 to 1.5 hours before the enzymolysis with cellulase and β -glucanase.

22. The process according to claim 19, wherein in the step (2), the enzyme deactivation is carried out before the enzymatic hydrolysis with the cellulase and the β -glucanase.

23. The method according to claim 22, wherein the reaction product obtained by enzymolysis with mannanase and then with alkaline protease and papain is subjected to enzyme inactivation at 85 to 100 ℃ for 0.5 to 1.5 hours before the enzymolysis with cellulase and β -glucanase.

24. The method according to any one of claims 12 to 16, wherein the yeast is subjected to a fermentation treatment using molasses as a carbon source and ammonium sulfate as a nitrogen source before the autolytic wall breaking in step (1).

25. The method of claim 24, wherein molasses having a total sugar concentration of 25% to 35% is used as the carbon source.

26. The method according to claim 24, wherein the fermentation treatment is carried out at a temperature of 25 to 35 ℃ and a pH of 4.0 to 6.0.

27. The method of claim 26, wherein the temperature of the fermentation treatment is 28-30 ℃.

28. The method according to claim 17, wherein the yeast is subjected to fermentation treatment using molasses as a carbon source and ammonium sulfate as a nitrogen source before autolytic wall breaking in step (1).

29. The method of claim 28, wherein molasses having a total sugar concentration of 25% -35% is used as the carbon source.

30. The method according to claim 28, wherein the fermentation treatment is carried out at a temperature of 25 to 35 ℃ and a pH of 4.0 to 6.0.

31. The method of claim 30, wherein the temperature of the fermentation treatment is 28 to 30 ℃.

32. The method according to claim 18, wherein the yeast is subjected to fermentation treatment using molasses as a carbon source and ammonium sulfate as a nitrogen source before autolytic wall breaking in step (1).

33. The method of claim 32, wherein molasses having a total sugar concentration of 25% -35% is used as the carbon source.

34. The method according to claim 32, wherein the fermentation treatment is carried out at a temperature of 25 to 35 ℃ and a pH of 4.0 to 6.0.

35. The method of claim 34, wherein the temperature of the fermentation treatment is 28-30 ℃.

36. The method according to claim 19, wherein the yeast is subjected to fermentation treatment using molasses as a carbon source and ammonium sulfate as a nitrogen source before autolytic wall breaking in step (1).

37. The method of claim 36, wherein molasses having a total sugar concentration of 25% -35% is used as the carbon source.

38. The method according to claim 36, wherein the fermentation treatment is carried out at a temperature of 25 to 35 ℃ and a pH of 4.0 to 6.0.

39. The method of claim 38, wherein the temperature of the fermentation treatment is 28-30 ℃.

40. The method according to claim 20, wherein the yeast is subjected to fermentation treatment using molasses as a carbon source and ammonium sulfate as a nitrogen source before autolytic wall breaking in step (1).

41. The method of claim 40, wherein molasses having a total sugar concentration of 25% -35% is used as the carbon source.

42. The method according to claim 40, wherein the fermentation treatment is carried out at a temperature of 25 to 35 ℃ and a pH of 4.0 to 6.0.

43. The method of claim 42, wherein the temperature of the fermentation treatment is 28-30 ℃.

44. The method according to claim 22, wherein the yeast is subjected to fermentation treatment using molasses as a carbon source and ammonium sulfate as a nitrogen source before autolytic wall breaking in step (1).

45. The method of claim 44, wherein molasses having a total sugar concentration of 25% -35% is used as the carbon source.

46. The method according to claim 44, wherein the fermentation treatment is carried out at a temperature of 25 to 35 ℃ and a pH of 4.0 to 6.0.

47. The method of claim 46, wherein the temperature of the fermentation treatment is 28-30 ℃.

48. The method according to any one of claims 12 to 16, wherein the yeast is saccharomyces cerevisiae Z2.2 (2: (c)Saccharomyces cerevisiae Hansen Z2.2), and the preservation number is CCTCC NO: M205128; saccharomyces cerevisiae FX-2Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418; saccharomyces cerevisiae Z2.4: (Saccharomyces cerevisiae Hansen Z2.4), and the preservation number is CCTCC NO: M205130; are all preserved in China center for type culture Collection.

49. The method of claim 48, wherein the yeast is Saccharomyces cerevisiae FX-2 (C.)Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418.

50. The method according to claim 17, wherein the yeast is Saccharomyces cerevisiae Z2.2 (C.) (Saccharomyces cerevisiae Hansen Z2.2), and the preservation number is CCTCC NO: M205128; saccharomyces cerevisiae FX-2Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418; saccharomyces cerevisiae Z2.4: (Saccharomyces cerevisiae Hansen Z2.4), and the preservation number is CCTCC NO: M205130; are all preserved in China center for type culture Collection.

51. The method according to claim 50, wherein the yeast is Saccharomyces cerevisiae FX-2 (FX-2: (S.cerevisiae)Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418.

52. The method according to claim 18, wherein the yeast is Saccharomyces cerevisiae Z2.2 (C.) (Saccharomyces cerevisiae Hansen Z2.2), and the preservation number is CCTCC NO: M205128; saccharomyces cerevisiae FX-2Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418; saccharomyces cerevisiae Z2.4: (Saccharomyces cerevisiae Hansen Z2.4), and the preservation number is CCTCC NO: M205130; are all preserved in China center for type culture Collection.

53. The method according to claim 52, wherein the yeast is Saccharomyces cerevisiae FX-2 (C.) (Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418.

54. The method of claim 19, wherein the yeast is Saccharomyces cerevisiae Z2.2 (2: (S.cerevisiae)Saccharomyces cerevisiae Hansen Z2.2), and the preservation number is CCTCC NO: M205128; saccharomyces cerevisiae FX-2Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418; saccharomyces cerevisiae Z2.4: (Saccharomyces cerevisiae Hansen Z2.4), and the preservation number is CCTCC NO: M205130; are all preserved in China center for type culture Collection.

55. The method according to claim 54, wherein the yeast is used as the yeastIs Saccharomyces cerevisiae FX-2: (Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418.

56. The method of claim 20, wherein the yeast is Saccharomyces cerevisiae Z2.2 (2: (S.cerevisiae)Saccharomyces cerevisiae Hansen Z2.2), and the preservation number is CCTCC NO: M205128; saccharomyces cerevisiae FX-2Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418; saccharomyces cerevisiae Z2.4: (Saccharomyces cerevisiae Hansen Z2.4), and the preservation number is CCTCC NO: M205130; are all preserved in China center for type culture Collection.

57. The method according to claim 56, wherein the yeast is Saccharomyces cerevisiae FX-2 (FX-2: (S.cerevisiae)Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418.

58. The method of claim 22, wherein the yeast is Saccharomyces cerevisiae Z2.2 (2: (S.cerevisiae)Saccharomyces cerevisiae Hansen Z2.2), and the preservation number is CCTCC NO: M205128; saccharomyces cerevisiae FX-2Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418; saccharomyces cerevisiae Z2.4: (Saccharomyces cerevisiae Hansen Z2.4), and the preservation number is CCTCC NO: M205130; are all preserved in China center for type culture Collection.

59. The method of claim 58, wherein the yeast is Saccharomyces cerevisiae FX-2 (C.) (Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418.

60. The method of claim 24, wherein the yeast is Saccharomyces cerevisiae Z2.2 (C.) (Saccharomyces cerevisiae Hansen Z2.2), and the preservation number is CCTCC NO: M205128; saccharomyces cerevisiae FX-2Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418; saccharomyces cerevisiae Z2.4（Saccharomyces cerevisiae Hansen Z2.4), and the preservation number is CCTCC NO: M205130; are all preserved in China center for type culture Collection.

61. The method according to claim 60, wherein the yeast is Saccharomyces cerevisiae FX-2 (C.) (Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418.

62. The method of claim 26, wherein the yeast is Saccharomyces cerevisiae Z2.2 (2: (S.cerevisiae)Saccharomyces cerevisiae Hansen Z2.2), and the preservation number is CCTCC NO: M205128; saccharomyces cerevisiae FX-2Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418; saccharomyces cerevisiae Z2.4: (Saccharomyces cerevisiae Hansen Z2.4), and the preservation number is CCTCC NO: M205130; are all preserved in China center for type culture Collection.

63. The method according to claim 62, wherein the yeast is Saccharomyces cerevisiae FX-2 (FX-2: (S.cerevisiae)Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418.

64. The method of claim 28, wherein the yeast is Saccharomyces cerevisiae Z2.2 (2: (S.cerevisiae)Saccharomyces cerevisiae Hansen Z2.2), and the preservation number is CCTCC NO: M205128; saccharomyces cerevisiae FX-2Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418; saccharomyces cerevisiae Z2.4: (Saccharomyces cerevisiae Hansen Z2.4), and the preservation number is CCTCC NO: M205130; are all preserved in China center for type culture Collection.

65. The method according to claim 64, wherein the yeast is Saccharomyces cerevisiae FX-2 (FX-2: (S.cerevisiae)Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418.

66. The method of claim 32, wherein the yeast is Saccharomyces cerevisiae Z2.2 (2: (S.cerevisiae)Saccharomyces cerevisiae Hansen Z2.2), and the preservation number is CCTCC NO: M205128; saccharomyces cerevisiae FX-2Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418; saccharomyces cerevisiae Z2.4: (Saccharomyces cerevisiae Hansen Z2.4), and the preservation number is CCTCC NO: M205130; are all preserved in China center for type culture Collection.

67. The method according to claim 66, wherein the yeast is Saccharomyces cerevisiae FX-2 (C.) (Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418.

68. The method of claim 36, wherein the yeast is Saccharomyces cerevisiae Z2.2 (2: (S.cerevisiae)Saccharomyces cerevisiae Hansen Z2.2), and the preservation number is CCTCC NO: M205128; saccharomyces cerevisiae FX-2Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418; saccharomyces cerevisiae Z2.4: (Saccharomyces cerevisiae Hansen Z2.4), and the preservation number is CCTCC NO: M205130; are all preserved in China center for type culture Collection.

69. The method according to claim 68, wherein the yeast is Saccharomyces cerevisiae FX-2 (FX-2: (FX-2)Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418.

70. The method of claim 40, wherein the yeast is Saccharomyces cerevisiae Z2.2 (C.) (Saccharomyces cerevisiae Hansen Z2.2), and the preservation number is CCTCC NO: M205128; saccharomyces cerevisiae FX-2Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418; saccharomyces cerevisiae Z2.4: (Saccharomyces cerevisiae Hansen Z2.4), and the preservation number is CCTCC NO: M205130; are all preserved in China center for type culture Collection.

71. The method according to claim 70, wherein the yeast is Saccharomyces cerevisiae FX-2 (FX-2)Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418.

72. The method of claim 44, wherein the yeast is Saccharomyces cerevisiae Z2.2 (C.) (Saccharomyces cerevisiae Hansen Z2.2), and the preservation number is CCTCC NO: M205128; saccharomyces cerevisiae FX-2Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418; saccharomyces cerevisiae Z2.4: (Saccharomyces cerevisiae Hansen Z2.4), and the preservation number is CCTCC NO: M205130; are all preserved in China center for type culture Collection.

73. The method of claim 72, wherein the yeast is Saccharomyces cerevisiae FX-2 (FX-2: (FX-2)Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418.

74. The yeast cell wall produced by the production method according to any one of claims 12 to 73, wherein the lysis rate of the yeast cell wall is increased and the yeast cell wall has a bacteriostatic effect.

75. Yeast cell wall according to claim 74, wherein the yeast cell wall has a lysis rate of 30-50%, a β -glucan content of 35-50% by mass and a mannan content of 20-35% by mass, the minimum inhibitory concentration of the yeast cell wall against E.coli and S.aureus is 5% dry matter mass concentration.

76. A feed comprising a yeast cell wall according to any of claims 1 to 11 or a yeast cell wall according to claim 74 or 75.

77. Use of a yeast cell wall according to any of claims 1 to 11 and a yeast cell wall according to claim 74 or 75 in the field of feed additives.