WO2023015965A1 - 一种高免疫型酵母细胞壁及制备方法和应用 - Google Patents

一种高免疫型酵母细胞壁及制备方法和应用 Download PDF

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WO2023015965A1
WO2023015965A1 PCT/CN2022/089879 CN2022089879W WO2023015965A1 WO 2023015965 A1 WO2023015965 A1 WO 2023015965A1 CN 2022089879 W CN2022089879 W CN 2022089879W WO 2023015965 A1 WO2023015965 A1 WO 2023015965A1
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cell wall
yeast cell
preparation
hyperimmune
yeast
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English (en)
French (fr)
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覃先武
胡骏鹏
谢智文
戴晋军
黄鑫
龚发源
徐杰
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安琪酵母股份有限公司
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Priority to EP22854962.2A priority Critical patent/EP4368697A1/en
Publication of WO2023015965A1 publication Critical patent/WO2023015965A1/zh

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/14—Pretreatment of feeding-stuffs with enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/163—Sugars; Polysaccharides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • C12N1/063—Lysis of microorganisms of yeast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae

Definitions

  • the invention relates to the technical field of microorganism application, in particular to a hyperimmune yeast cell wall, its preparation method and application.
  • Yeast cell wall is a functional green feed additive or feed material derived from yeast. It is rich in glucan and mannan, and has the functions of enhancing immunity, promoting growth, relieving stress, adsorbing mycotoxins, adsorbing pathogenic bacteria, and anti-tumor. Biological efficacy. A large number of studies at home and abroad have found that the biological efficacy of yeast cell wall polysaccharides is closely related to its structure. Among them, the mannan in the yeast cell wall can be very similar to the receptors of pathogenic bacteria on the intestinal wall of animals. Pathogenic bacteria adhere and multiply in the intestinal tract. Molecular weight is another important factor affecting the biological activities of ⁇ -1,3-glucans such as anti-tumor and immune regulation.
  • Polysaccharides with a molecular weight of 100-200 kDa have strong biological activities, and glucans with a molecular weight greater than 90 kDa can form unique , Highly ordered triple helix structure, which is conducive to the combination with receptors and produces biological effects, which is very important for immune function.
  • the triple helix structure of yeast dextran is conducive to the adsorption of mycotoxins by glucan through intermolecular forces such as hydrogen bonds and van der Waals forces.
  • the common yeast cell wall products currently on the market are the by-products of yeast extract production.
  • the glucan molecular weight is greater than 200kDa and its water solubility is poor, which limits the use of yeast cell walls in many fields such as animal breeding, animal health care, plant protection, and human nutrition. application.
  • Methods to increase the dissolution rate of yeast cell wall and reduce the molecular weight of dextran include physical modification, chemical modification and biological enzymatic method.
  • the aggressive method uses a large amount of chemical reagents, some of which are highly toxic and likely to cause environmental pollution, and the chemical modification reaction is violent, which easily destroys the functional molecular structure of polysaccharides; the biological enzymatic method has a mild reaction, but the improvement of the dissolution rate is limited.
  • the technical problem to be solved by the present invention is that there is a lack of a highly immune yeast cell wall, which has a relatively high dissolution rate, and the dextran with a relative molecular weight of 80-200kDa accounts for more than 99% of the total mass of dextran.
  • one of the purposes of the present invention provides a kind of hyperimmune yeast cell wall;
  • the second object of the present invention is to provide the preparation method of the above hyperimmune yeast cell wall;
  • the third object of the present invention is to provide the above-mentioned The application of hyperimmune yeast cell wall or the hyperimmune yeast cell wall prepared by the above preparation method in feed;
  • the fourth object of the present invention is to provide feed containing the above hyperimmune yeast cell wall.
  • the invention provides a kind of hyperimmune yeast cell wall, the dextran whose relative molecular weight is 80-200kDa accounts for more than 99% of the total mass of dextran, and the dextran The content is 20-40%.
  • the lysis rate of the cell wall of the above-mentioned hyperimmune yeast is ⁇ 40%.
  • the manno-oligosaccharide content in the cell wall of the hyperimmune yeast is ⁇ 20% based on the mass of the hyperimmune yeast cell wall as 100%.
  • the present invention also provides a method for preparing the above-mentioned hyperimmune yeast cell wall, comprising the steps of:
  • step (2) subjecting the yeast cell wall milk obtained in step (1) to protease enzymolysis and secondary enzymolysis, wherein the enzymes used in the secondary enzymolysis are dextranase, mannanase, cellulase and amylase one or more of them;
  • step (3) acid hydrolyzing the enzymolysis solution obtained in step (2) to obtain the cell wall of hyperimmune yeast.
  • the autolytic wall breaking in the step (1) is carried out under the conditions of a salt concentration of 2.0-5.5%, a pH of 4.0-6.5, and a temperature of 45-75° C., preferably, the autolysis time is 15-30 hours.
  • the amount of protease added in step (2) is 1-10 ⁇ based on the dry matter of yeast cell wall milk.
  • the protease is papain, neutral protease, alkaline protease and bromelain.
  • the protease contains neutral protease and bromelain, further preferably, the added amount of the neutral protease is 2-5 ⁇ , and the added amount of bromelain is 2-5 ⁇ .
  • the protease is neutral protease and bromelain, or neutral protease, bromelain and papain, or papain, neutral protease, alkaline protease and bromelain.
  • the protease is added in an amount of 6-10 ⁇ .
  • the enzymatic hydrolysis temperature of the protease is 30-60° C.
  • the enzymatic hydrolysis pH is 4.5-7.0
  • the enzymatic hydrolysis time is 6-10 h.
  • the amount of enzyme used in the secondary enzymatic hydrolysis is 1-10 ⁇ based on the dry matter of yeast cell wall milk.
  • the temperature of the secondary enzymatic hydrolysis is 40-60°C, and the pH of the enzymatic hydrolysis is 4.0-7.0, the enzymatic hydrolysis time is 4-12h.
  • the enzyme used in the secondary enzymatic hydrolysis comprises dextranase and cellulase, preferably, the added amount of the dextranase is 2-3 ⁇ , and the added amount of the cellulase is 2 -3 ⁇ .
  • the enzymes used for the secondary enzymolysis are glucanase and cellulase, or glucanase, mannanase and cellulase, or glucanase, mannanase, cellulose enzymes and amylases.
  • the amount of enzyme used for the second enzymatic hydrolysis is 4-8 ⁇ .
  • the acid hydrolysis pH in step (3) is 1.5-3.5, the acid hydrolysis temperature is 50-100°C, and the acid hydrolysis time is 6-20h.
  • the acid used for acid hydrolysis is an inorganic acid and/or an organic acid , further preferably, the inorganic acid is one or more of hydrochloric acid, sulfuric acid and phosphoric acid, and more preferably, the organic acid is citric acid, malic acid, lactic acid, formic acid, acetic acid and propionic acid one or more of two.
  • the above preparation method further includes the step of drying the hyperimmune yeast cell wall: after the acid hydrolysis, adjust the pH to 4.0-7.0, and dry to obtain a powdery hyperimmune yeast cell wall.
  • the yeast-containing raw material is obtained by fermenting a Saccharomyces cerevisiae strain, preferably, the Saccharomyces cerevisiae strain is FX-2 (Saccharomyces cerevisiaed).
  • the above-mentioned fermentation pH is 4.0-7.0
  • the fermentation temperature is 28-35° C.
  • the fermentation time is 15-35 hours.
  • the above-mentioned fermentation adopts fed-batch feeding method to feed carbon source, nitrogen source and phosphorus source;
  • the carbon source is 6000-8000 parts, the nitrogen source is 400-700 parts, and the phosphorus source is 300-600 parts;
  • the carbon source is one or more of molasses, cornstarch, glucose, maltose, trehalose, trehalose and galactose, preferably molasses;
  • the nitrogen source is selected from one of urea, ammonia water or ammonium sulfate, preferably urea;
  • the phosphorus source is selected from phosphoric acid or potassium dihydrogen phosphate, preferably potassium dihydrogen phosphate.
  • the present invention also provides the application of the above hyperimmune yeast cell wall or the hyperimmune yeast cell wall prepared by the above preparation method in feed.
  • the present invention also provides a feed, which comprises the above-mentioned hyperimmune yeast cell wall or the hyperimmune yeast cell wall and basal ration obtained by the above preparation method, and the addition amount of the hyperimmune yeast cell wall in the feed is 0.5-10wt ⁇ .
  • the cell wall of the highly immune yeast of the present invention has a high dissolution rate, and the glucan with a relative molecular weight of 80-200kDa accounts for more than 99% of the total mass of glucan, and the immune effect is significantly improved; it is derived from yeast and is a green , environmentally friendly feed ingredients or additives;
  • the present invention adopts secondary enzymatic hydrolysis and acid hydrolysis process, and the preparation process is environmentally friendly. While improving the yeast cell wall solubility and degrading dextran, more functional molecular structures of polysaccharides are retained, and the yeast cell wall is improved. The immune function and its added value broaden the application field of yeast cell wall.
  • the invention provides a kind of hyperimmune yeast cell wall, the dextran whose relative molecular weight is 80-200kDa accounts for more than 99% of the total mass of dextran, and the dextran The content is 20-40%.
  • the dissolution rate of the cell wall of the hyperimmune yeast is ⁇ 40%.
  • the dissolution rate is increased, and the biological activity of the glucan molecule is improved, and the immune function of the yeast cell wall is improved.
  • the mannan oligosaccharide content in the cell wall of the hyperimmune yeast is ⁇ 20%.
  • the present invention also provides a method for preparing the above-mentioned hyperimmune yeast cell wall, comprising the steps of:
  • step (2) subjecting the yeast cell wall milk obtained in step (1) to protease enzymolysis and secondary enzymolysis, wherein the enzymes used in the secondary enzymolysis are dextranase, mannanase, cellulase and amylase one or more of them;
  • step (3) acid hydrolyzing the enzymolysis solution obtained in step (2) to obtain the cell wall of hyperimmune yeast.
  • the autolysis and wall breaking in step (1) is carried out at a salt concentration of 2.0-5.5%, a pH of 4.0-6.5, and a temperature of 45-75°C, preferably the autolysis time is 15 -30h.
  • the amount of protease added in step (2) is 1-10 ⁇ based on the dry matter of yeast cell wall milk.
  • the protease is papain, neutral protease, One or more of alkaline protease and bromelain; preferably, the protease contains neutral protease and bromelain; further preferably, the addition of the neutral protease is 2-5 ⁇ , the pineapple The amount of protease added is 2-5 ⁇ .
  • the protease is neutral protease and bromelain, or neutral protease, bromelain and papain, or papain, neutral protease, alkaline protease and bromelain.
  • the protease is added in an amount of 6-10 ⁇ .
  • the enzymatic hydrolysis temperature of the protease is 30-60° C.
  • the enzymatic hydrolysis pH is 4.5-7.0
  • the enzymatic hydrolysis time is 6-10 h.
  • the amount of enzyme used in the secondary enzymatic hydrolysis is 1-10 ⁇ ; preferably, the temperature of the secondary enzymatic hydrolysis is 40- At 60°C, the pH of the enzymatic hydrolysis is 4.0-7.0, and the enzymatic hydrolysis time is 4-12h.
  • the amount of enzyme added is insufficient, the efficiency of enzymatic hydrolysis is not enough. If the amount of enzyme added is too high, it will lead to excessive enzymolysis, high cost and failure to meet product quality requirements.
  • the enzymes used in the secondary enzymatic hydrolysis include dextranase and cellulase, preferably, the added amount of the dextranase is 2-3 ⁇ , the fiber Sulfase is added in an amount of 2-3 ⁇ .
  • the enzymes used in the secondary enzymatic hydrolysis are glucanase and cellulase, or glucanase, mannanase and cellulase, or glucanase, Mannanase, cellulase and amylase.
  • the amount of enzyme used in the secondary enzymolysis is 4-8 ⁇ .
  • the acid hydrolysis pH in step (3) is 1.5-3.5, the acid hydrolysis temperature is 50-100°C, and the acid hydrolysis time is 6-20h; preferably, the acid used for acid hydrolysis is Inorganic acid and/or organic acid; Further preferably, the inorganic acid is one or more of hydrochloric acid, sulfuric acid and phosphoric acid; More preferably, the organic acid is citric acid, malic acid, lactic acid, One or more of formic acid, acetic acid and propionic acid.
  • the above preparation method also includes the step of drying the cell wall of the hyperimmune yeast: after the acid hydrolysis, adjust the pH to 4.0-7.0, and dry to obtain powdered hyperimmune yeast cell wall.
  • the yeast-containing raw material is obtained by fermenting Saccharomyces cerevisiae strains, preferably, the Saccharomyces cerevisiae strains are Saccharomyces cerevisiaed strains FX-2 (Saccharomyces cerevisiaed), the The strain was deposited in the China Center for Type Culture Collection (CCTCC) on August 1, 2016.
  • the preservation number is CCTCC NO: M2016418.
  • the preservation address is: China. Wuhan. Wuhan University, postal code 430072, Tel: (027)-68754052;
  • the strain has been described in the patent publication with publication number CN108220175A.
  • the fermentation pH is 4.0-7.0
  • the fermentation temperature is 28-35° C.
  • the fermentation time is 15-35 hours.
  • the fermentation adopts fed-batch feeding method to feed carbon source, nitrogen source and phosphorus source;
  • the carbon source is 6000-8000 parts, the nitrogen source is 400-700 parts, and the phosphorus source is 300-600 parts;
  • the carbon source is one or more of molasses, cornstarch, glucose, maltose, trehalose, trehalose and galactose, preferably molasses;
  • the nitrogen source is selected from one of urea, ammonia water or ammonium sulfate, preferably urea;
  • the phosphorus source is selected from phosphoric acid or potassium dihydrogen phosphate, preferably potassium dihydrogen phosphate.
  • the present invention also provides the application of the above hyperimmune yeast cell wall or the hyperimmune yeast cell wall prepared by the above preparation method in feed.
  • the present invention also provides a feed, which comprises the above-mentioned hyperimmune yeast cell wall or the hyperimmune yeast cell wall and basal ration obtained by the above preparation method, and the addition amount of the hyperimmune yeast cell wall in the feed is 0.5-10wt ⁇ .
  • Table 1 embodiment of the present invention and comparative example use raw material and equipment source
  • the culture medium contains carbon source, nitrogen source and phosphorus source, wherein the carbon source is 7000g of molasses, the nitrogen source is 400g of urea, and the phosphorus source is 600g of potassium dihydrogen phosphate. Sterilize the culture medium at 121°C for 10 minutes, inoculate Saccharomyces cerevisiaed FX-2 (Saccharomyces cerevisiaed) for fermentation, the fermentation temperature is 28°C, the fermentation time is 15 hours, the fermentation pH is 4.0, and yeast milk is obtained;
  • step (3) Dilute the yeast cell wall milk obtained in step (2) with water to a dry matter concentration of 5%, add 1 ⁇ of papain (based on the dry matter mass of the yeast cell wall milk, the same below), control the temperature at 30°C, and pH4. 5. Enzymolysis for 6 hours;
  • step (4) Add 10wt% sulfuric acid to the enzymolysis solution obtained in step (4), adjust the pH to 1.5, heat up to 70°C, and keep warm for 6h;
  • the pH is adjusted to 4.0, and then spray-dried to obtain a high-immune yeast cell wall powder.
  • the hydrolyzed sample is injected into the liquid chromatography, and pure water is used as the mobile phase.
  • the Detection by differential detector and quantification by external standard method When dextran and mannan are hydrolyzed, because the sample may not be hydrolyzed completely, and the glucose and mannose produced by hydrolysis partly undergo other side reactions due to high temperature, the test results are higher than the actual dextran and mannan contained in the product. Glycans are low. In the detection, the dextran reference substance was used to correct the error caused by the above hydrolysis process.
  • Glucose and mannose mixed standard solution (2g/L) weigh 0.2000g each of glucose and mannose, and dilute to 100ml with pure water.
  • Dextran reference substance (curdlan from Alcaligenes faecalis): Sigma product, article number
  • ⁇ -glucan or mannan The content of ⁇ -glucan or mannan is calculated according to the following formula:
  • Dissolve the sample in water collect the precipitate by centrifugation, and calculate the ratio of the weight of the dissolved substance to the total weight.
  • m 1 weight of sediment after centrifugation, g
  • the calculation result is kept to 1 decimal place.
  • the hyperimmune yeast cell wall product prepared in step (1) is prepared into a solution, and the relative molecular weight of dextran is determined by high-phase liquid chromatography.
  • the culture medium contains carbon source, nitrogen source and phosphorus source, wherein the carbon source is 6000g of molasses, the nitrogen source is 700g of urea, and the phosphorus source is 300g of potassium dihydrogen phosphate. Sterilize the culture medium at 121°C for 10 minutes, inoculate Saccharomyces cerevisiaed FX-2 (Saccharomyces cerevisiaed) for fermentation, the fermentation temperature is 35°C, the fermentation time is 35h, and the fermentation pH is 7.0 to obtain yeast milk;
  • step (3) Dilute the yeast cell wall milk obtained in step (2) with water to a dry matter mass concentration of 20%, add 10 ⁇ of alkaline protease (based on the dry matter mass of the yeast cell wall milk, the same below), control the temperature at 60°C, and pH 7 .0, enzymatic hydrolysis for 10h;
  • step (4) Add citric acid to the enzymolysis solution obtained in step (4), adjust the pH to 3.5, heat up to 100° C., and keep warm for 20 hours;
  • the pH is adjusted to 7.0, and then spray-dried to obtain a high-immune yeast cell wall powder.
  • the culture medium contains carbon source, nitrogen source and phosphorus source, wherein the carbon source is 8000g of molasses, the nitrogen source is 550g of urea, and the phosphorus source is 450g of potassium dihydrogen phosphate. Sterilize the culture medium at 121°C for 10 minutes, inoculate Saccharomyces cerevisiaed FX-2 (Saccharomyces cerevisiaed) for fermentation and culture, the fermentation temperature is 30°C, the fermentation time is 20 hours, and the fermentation pH is 5.0 to obtain yeast milk;
  • step (3) adding water to dilute the yeast cell wall milk obtained in step (2) into a dry matter mass concentration of 10%, adding 5 ⁇ of neutral protease (based on yeast cell wall milk dry matter mass, the same below) and 5 ⁇ of bromelain ( Based on the mass of dry matter of yeast cell wall milk, the same below), control the temperature at 50°C, pH 5.0, and enzymatically hydrolyze for 8 hours;
  • step (4) Add phosphoric acid and acetic acid to the enzymolysis solution obtained in step (4), adjust the pH to 2.5, heat up to 80° C., and keep warm for 8 hours;
  • the pH is adjusted to 5.0, and then spray-dried to obtain a high-immune yeast cell wall powder.
  • the culture medium contains carbon source, nitrogen source and phosphorus source, wherein the carbon source is 7000g of molasses, the nitrogen source is 400g of urea, and the phosphorus source is 600g of potassium dihydrogen phosphate. Sterilize the culture medium at 121°C for 10 minutes, inoculate Saccharomyces cerevisiaed FX-2 (Saccharomyces cerevisiaed) for fermentation, the fermentation temperature is 28°C, the fermentation time is 15 hours, the fermentation pH is 4.0, and yeast milk is obtained;
  • step (3) Dilute the yeast cell wall milk obtained in step (2) with water to a dry matter concentration of 5%, add 2 ⁇ of neutral protease, control the temperature at 40°C, pH 6.0, and enzymatically hydrolyze for 7 hours;
  • step (4) Add 10wt% sulfuric acid to the enzymolysis solution obtained in step (4), adjust the pH to 2.0, heat up to 50° C., and keep warm for 13 hours;
  • the pH is adjusted to 5.0, and then spray-dried to obtain a high-immune yeast cell wall powder.
  • the culture medium contains carbon source, nitrogen source and phosphorus source, wherein the carbon source is 7000g of molasses, the nitrogen source is 400g of urea, and the phosphorus source is 600g of potassium dihydrogen phosphate. Sterilize the culture medium at 121°C for 10 minutes, inoculate Saccharomyces cerevisiaed FX-2 (Saccharomyces cerevisiaed) for fermentation, the fermentation temperature is 28°C, the fermentation time is 15 hours, the fermentation pH is 4.0, and yeast milk is obtained;
  • step (3) Dilute the yeast cell wall milk obtained in step (2) with water to a dry matter concentration of 5%, add 5 ⁇ of bromelain, control the temperature at 45°C, pH 5.5, and enzymatically hydrolyze for 9 hours;
  • step (4) Add citric acid to the enzymolysis solution obtained in step (4), adjust the pH to 3.0, heat up to 60° C., and keep warm for 16 hours;
  • the pH is adjusted to 6.0, and then spray-dried to obtain a high-immune yeast cell wall powder.
  • the culture medium contains carbon source, nitrogen source and phosphorus source, wherein the carbon source is 7000g of molasses, the nitrogen source is 400g of urea, and the phosphorus source is 600g of potassium dihydrogen phosphate. Sterilize the culture medium at 121°C for 10 minutes, inoculate Saccharomyces cerevisiaed FX-2 (Saccharomyces cerevisiaed) for fermentation, the fermentation temperature is 28°C, the fermentation time is 15 hours, the fermentation pH is 4.0, and yeast milk is obtained;
  • step (3) Dilute the yeast cell wall milk obtained in step (2) with water to a dry matter concentration of 5%, add 2 ⁇ of papain and 2 ⁇ of alkaline protease, control the temperature at 55° C., pH 6.5, and perform enzymatic hydrolysis for 8 hours;
  • step (4) Add 10wt% sulfuric acid to the enzymolysis solution obtained in step (4), adjust the pH to 1.5, heat up to 70°C, and keep warm for 6h;
  • the pH is adjusted to 4.0, and then spray-dried to obtain a high-immune yeast cell wall powder.
  • the culture medium contains carbon source, nitrogen source and phosphorus source, wherein the carbon source is 7000g of molasses, the nitrogen source is 400g of urea, and the phosphorus source is 600g of potassium dihydrogen phosphate. Sterilize the culture medium at 121°C for 10 minutes, inoculate Saccharomyces cerevisiaed FX-2 (Saccharomyces cerevisiaed) for fermentation, the fermentation temperature is 28°C, the fermentation time is 15 hours, the fermentation pH is 4.0, and yeast milk is obtained;
  • step (3) Dilute the yeast cell wall milk obtained in step (2) with water to a dry matter mass concentration of 5%, add 2 ⁇ of papain, 2 ⁇ of neutral protease and 0.2 ⁇ of bromelain, and control the temperature at 60°C and pH4. 5. Enzymolysis for 7 hours;
  • step (4) Add 10wt% sulfuric acid to the enzymolysis solution obtained in step (4), adjust the pH to 1.5, heat up to 70°C, and keep warm for 6h;
  • the culture medium contains carbon source, nitrogen source and phosphorus source, wherein the carbon source is 7000g of molasses, the nitrogen source is 400g of urea, and the phosphorus source is 600g of potassium dihydrogen phosphate. Sterilize the culture medium at 121°C for 10 minutes, inoculate Saccharomyces cerevisiaed FX-2 (Saccharomyces cerevisiaed) for fermentation, the fermentation temperature is 28°C, the fermentation time is 15 hours, the fermentation pH is 4.0, and yeast milk is obtained;
  • step (3) adding water to dilute the yeast cell wall milk obtained in step (2) into a dry matter mass concentration of 5%, adding 2 ⁇ of papain, 2 ⁇ of neutral protease, 2 ⁇ of bromelain and 2 ⁇ of alkaline protease, Control the temperature at 50°C, pH 5.5, and enzymatic hydrolysis for 6 hours;
  • step (4) Add 10wt% sulfuric acid to the enzymolysis solution obtained in step (4), adjust the pH to 1.5, heat up to 70°C, and keep warm for 6h;
  • the pH is adjusted to 4.0, and then spray-dried to obtain a high-immune yeast cell wall powder.
  • the culture medium contains carbon source, nitrogen source and phosphorus source, wherein the carbon source is 7000g of molasses, the nitrogen source is 400g of urea, and the phosphorus source is 600g of potassium dihydrogen phosphate. Sterilize the culture medium at 121°C for 10 minutes, inoculate Saccharomyces cerevisiaed FX-2 (Saccharomyces cerevisiaed) for fermentation, the fermentation temperature is 28°C, the fermentation time is 15 hours, the fermentation pH is 4.0, and yeast milk is obtained;
  • step (3) Dilute the yeast cell wall milk obtained in step (2) with water to a dry matter mass concentration of 5%, add 3 ⁇ of papain, 3 ⁇ of bromelain and 3 ⁇ of alkaline protease, control the temperature at 45°C, and pH5. 0, enzymatic hydrolysis for 8h;
  • step (4) Add 10wt% sulfuric acid to the enzymolysis solution obtained in step (4), adjust the pH to 1.5, heat up to 70°C, and keep warm for 6h;
  • the pH is adjusted to 4.0, and then spray-dried to obtain a high-immune yeast cell wall powder.
  • the culture medium contains carbon source, nitrogen source and phosphorus source, wherein the carbon source is 7000g of molasses, the nitrogen source is 400g of urea, and the phosphorus source is 600g of potassium dihydrogen phosphate. Sterilize the culture medium at 121°C for 10 minutes, inoculate Saccharomyces cerevisiaed FX-2 (Saccharomyces cerevisiaed) for fermentation, the fermentation temperature is 28°C, the fermentation time is 15 hours, the fermentation pH is 4.0, and yeast milk is obtained;
  • step (3) Dilute the yeast cell wall milk obtained in step (2) with water to a dry matter concentration of 5%, add 0.5 ⁇ of papain, control the temperature at 30°C, pH 4.5, and perform enzymatic hydrolysis for 6 hours;
  • step (4) Add 10wt% sulfuric acid to the enzymolysis solution obtained in step (4), adjust the pH to 1.5, heat up to 70°C, and keep warm for 6h;
  • the pH is adjusted to 4.0, and then spray-dried to obtain a high-immune yeast cell wall powder.
  • the culture medium contains carbon source, nitrogen source and phosphorus source, wherein the carbon source is 7000g of molasses, the nitrogen source is 400g of urea, and the phosphorus source is 600g of potassium dihydrogen phosphate. Sterilize the culture medium at 121°C for 10 minutes, inoculate Saccharomyces cerevisiaed FX-2 (Saccharomyces cerevisiaed) for fermentation, the fermentation temperature is 28°C, the fermentation time is 15 hours, the fermentation pH is 4.0, and yeast milk is obtained;
  • step (3) Dilute the yeast cell wall milk obtained in step (2) with water to a dry matter concentration of 5%, add 1 ⁇ of papain, control the temperature at 30° C., pH 4.5, and perform enzymatic hydrolysis for 6 hours;
  • step (4) Add 10wt% sulfuric acid to the enzymolysis solution obtained in step (4), adjust the pH to 1.5, heat up to 70°C, and keep warm for 6h;
  • the pH is adjusted to 4.0, and then spray-dried to obtain a high-immune yeast cell wall powder.
  • the culture medium contains carbon source, nitrogen source and phosphorus source, wherein the carbon source is 7000g of molasses, the nitrogen source is 400g of urea, and the phosphorus source is 600g of potassium dihydrogen phosphate. Sterilize the culture medium at 121°C for 10 minutes, inoculate Saccharomyces cerevisiaed FX-2 (Saccharomyces cerevisiaed) for fermentation, the fermentation temperature is 35°C, the fermentation time is 35h, and the fermentation pH is 7.0 to obtain yeast milk;
  • step (3) Dilute the yeast cell wall milk obtained in step (2) with water to a dry matter concentration of 20%, add 12 ⁇ of alkaline protease, control the temperature at 60° C., pH 7.0, and perform enzymatic hydrolysis for 10 hours;
  • step (4) Add citric acid to the enzymolysis solution obtained in step (4), adjust the pH to 3.5, heat up to 100° C., and keep warm for 20 hours;
  • the pH is adjusted to 7.0, and then spray-dried to obtain a high-immune yeast cell wall powder.
  • the culture medium contains carbon source, nitrogen source and phosphorus source, wherein the carbon source is 7000g of molasses, the nitrogen source is 400g of urea, and the phosphorus source is 600g of potassium dihydrogen phosphate. Sterilize the culture medium at 121°C for 10 minutes, inoculate Saccharomyces cerevisiaed FX-2 (Saccharomyces cerevisiaed) for fermentation, the fermentation temperature is 35°C, the fermentation time is 35h, and the fermentation pH is 7.0 to obtain yeast milk;
  • step (3) Dilute the yeast cell wall milk obtained in step (2) with water to a dry matter concentration of 20%, add 10 ⁇ of alkaline protease, control the temperature at 60° C., pH 7.0, and perform enzymatic hydrolysis for 10 hours;
  • step (4) Add citric acid to the enzymolysis solution obtained in step (4), adjust the pH to 3.5, heat up to 100° C., and keep warm for 20 hours;
  • the pH is adjusted to 7.0, and then spray-dried to obtain a high-immune yeast cell wall powder.
  • the culture medium contains carbon source, nitrogen source and phosphorus source, wherein the carbon source is 7000g of molasses, the nitrogen source is 400g of urea, and the phosphorus source is 600g of potassium dihydrogen phosphate. Sterilize the culture medium at 121°C for 10 minutes, inoculate Saccharomyces cerevisiaed FX-2 (Saccharomyces cerevisiaed) for fermentation, the fermentation temperature is 28°C, the fermentation time is 15 hours, the fermentation pH is 4.0, and yeast milk is obtained;
  • step (3) Dilute the yeast cell wall milk obtained in step (2) with water to a dry matter concentration of 5%, add 1 ⁇ of papain, control the temperature at 30° C., pH 4.5, and perform enzymatic hydrolysis for 6 hours;
  • step (4) The enzymolysis solution obtained in step (4) is heated to 90° C. for 1 hour, and then spray-dried to obtain a high-immune yeast cell wall powder.
  • the culture medium contains carbon source, nitrogen source and phosphorus source, wherein the carbon source is 7000g of molasses, the nitrogen source is 400g of urea, and the phosphorus source is 600g of potassium dihydrogen phosphate. Sterilize the culture medium at 121°C for 10 minutes, inoculate Saccharomyces cerevisiaed FX-2 (Saccharomyces cerevisiaed) for fermentation, the fermentation temperature is 28°C, the fermentation time is 15 hours, the fermentation pH is 4.0, and yeast milk is obtained;
  • step (3) Dilute the yeast cell wall milk obtained in step (2) with water to a dry matter concentration of 5%, add 1 ⁇ glucanase, control the temperature at 40°C, pH 4.0, and perform enzymatic hydrolysis for 4 hours;
  • step (4) Add 10wt% sulfuric acid to the enzymolysis solution obtained in step (4), adjust the pH to 1.5, heat up to 70°C, and keep warm for 6h;
  • the high-immune cell wall powders prepared in Examples 1-9 have a dextran content of 20%-40%, a manno-oligosaccharide content of 20% or more, and a dissolution rate higher than 40%.
  • the relative molecular weight is 80-200kDa dextran accounts for more than 99% of the total mass of dextran, the content of glucan and mannan in the cell wall samples prepared in Comparative Examples 1-6 has no big difference with the examples, but the comparative examples 1.
  • Comparative Example 2 The cell wall dissolution rates of Comparative Example 2, Comparative Example 5, and Comparative Example 6 were all lower than 40%, and dextran with a relative molecular weight of 200 kDa or more accounted for more than 99% of the total mass of dextran.
  • the dissolution rate of Comparative Example 3 and Comparative Example 4 was higher than 40%, but the dextran with a relative molecular weight below 80KDa accounted for more than 98% of the total mass of dextran.
  • a total of 72 Du ⁇ Chang ⁇ Da Sanyuan hybrid weaned piglets with similar body weight and good body condition at the age of 28 days were selected, and divided into 9 treatment groups according to the principle of similar average body weight and half male and half female. Each group had 4 replicates. Repeat a column of pigs, with one male and one female pig in each column.
  • the experimental basal diet was formulated according to the nutritional needs of weaned piglets in NRC 2012, as shown in Table 4.
  • the 9 experimental groups were fed with different yeast cell wall products on the basis of the basal diet. The specific grouping and diet design are shown in Table 5.
  • the daily management is carried out according to the feeding and management method of large-scale pig farms. After the piglets are purchased, they are fed with the transitional ration (basic ration) first, and then the experimental ration is fed after the test starts to ensure that the trough feed is sufficient for the piglets to eat freely. Water is supplied from drinking fountains, and the indoor temperature is maintained at room temperature. Immunization status of piglets after purchase: 28 days old against PRRS, 35 days old against pseudorabies (PR), 42 days old against swine fever (HC).
  • Test group 1 46.76 ⁇ 1.46 17.78 ⁇ 4.21 1.63 ⁇ 0.32 Test group 2 47.56 ⁇ 2.35 18.73 ⁇ 3.24 1.54 ⁇ 0.54 Test group 3 47.03 ⁇ 3.67 18.55 ⁇ 4.21 1.53 ⁇ 0.48 Test group 4 44.99 ⁇ 1.36 14.69 ⁇ 0.99 2.06 ⁇ 0.09 Test group 5 46.83 ⁇ 2.31 15.36 ⁇ 3.24 2.04 ⁇ 0.58 Test group 6 45.89 ⁇ 2.35 14.38 ⁇ 1.24 2.19 ⁇ 0.54 Test group 7 47.05 ⁇ 2.58 15.74 ⁇ 2.43 1.99 ⁇ 0.78 Test group 8 46.87 ⁇ 3.12 15.65 ⁇ 2.35 1.99 ⁇ 1.03 Test group 9 47.25 ⁇ 2.76 16.05 ⁇ 1.65 1.94 ⁇ 0.86
  • the serum globulin levels of each treatment group from high to low are Example 2, Example 3, Example 1, Comparative Example 6, Comparative Example 4, Comparative Example 5, Comparative Example 2, Comparative Example 1, Comparative Example 3; albumin/globulin from low to high are Example 3, Example 2, Example 1, Comparative Example 6, Comparative Example 4, Comparative Example 5, Comparative Example 2, Comparative Example 1 , Comparative Example 3; show that the highly immune yeast cell walls prepared in Example 1, Example 2, and Example 3 can significantly improve the immunity of piglets compared with the comparative examples.
  • the cell wall of the highly immune yeast of the present invention has a high dissolution rate, and in terms of mass percentage, more than 99% of the dextran molecular weight is 80-200kDa, and the immune effect is significantly improved; it is derived from yeast and is a green, environmentally friendly feed ingredients or additives.

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Abstract

提供了一种高免疫型酵母细胞壁及制备方法和应用。该高免疫型酵母细胞壁的溶解率≥40％，相对分子量为80-200kDa的葡聚糖占葡聚糖总质量的99％以上，以所述高免疫型酵母细胞壁质量为100％计，葡聚糖含量为20-40％。该高免疫型酵母细胞壁提高了溶解率和免疫功效，源于酵母，可用于饲料原料或添加剂。

Description

一种高免疫型酵母细胞壁及制备方法和应用 技术领域
本发明涉及微生物应用技术领域，具体涉及一种高免疫型酵母细胞壁及制备方法和应用。
背景技术
酵母细胞壁是源于酵母的一种功能性绿色饲料添加剂或饲料原料，含有丰富的葡聚糖和甘露聚糖，具有增强免疫、促生长、缓解应激、吸附霉菌毒素、吸附病原菌、抗肿瘤等生物功效。国内外大量研究发现，酵母细胞壁多糖的生物功效与其结构密切相关，其中酵母细胞壁中的甘露聚糖能与病原菌在动物肠壁上的受体非常相似，与病原菌的外源凝集素结合后，阻碍病原菌在肠道黏附和繁殖。分子量大小是影响β-1,3-葡聚糖抗肿瘤和免疫调节等生物活性的另一个重要因素，100-200kDa的多糖具有较强的生物活性，分子量大于90kDa的葡聚糖能够形成独特的、高度有序的三股螺旋结构，该结构有利于和受体结合，产生生物学效应，对起免疫作用至关重要。同时，酵母葡聚糖的三股螺旋结构，有利于葡聚糖通过氢键和范德华力等分子间作用力吸附霉菌毒素。
目前市售的常见的酵母细胞壁产品为生产酵母抽提物的副产物，其葡聚糖分子量大于200kDa，且水溶性差，限制了酵母细胞壁在动物养殖、动物保健、植物保护、人类营养等诸多领域应用。提高酵母细胞壁的溶解率及降低葡聚糖分子量的方法有物理改性法、化学改性法和生物酶法等，但是物理改性方法存在较大的不确定性，产品质量无法控制；化学改性方法使用大量的化学试剂，部分化学试剂有剧毒，容易引起环境污染，且化学改性反应剧烈，容易破坏多糖的功能性分子结构；生物酶法反应温和，但是对溶解率提升有限。
因此，需对酵母细胞壁产品进行酶解和酸水解等深加工处理，提高酵母细胞壁溶解率，聚焦特定分子量片段80kDa-200kDa，提高酵母细胞壁免疫功效，拓宽酵母细胞壁应用领域。
发明内容
本发明要解决的技术问题：缺乏一种高免疫型酵母细胞壁，其具有较高溶解率，相对分子量为80-200kDa的葡聚糖占葡聚糖总质量的99％以上。
针对现有技术存在的不足，本发明的目的之一提供一种高免疫型酵母细胞壁；本发明的目的之二是提供上述高免疫型酵母细胞壁的制备方法；本发明的目的之三是提供上述高免疫型酵母细胞壁或上述制备方法制得高免疫型酵母细胞壁在饲料中的应用；本发明的目的之四是提供含有上述高免疫型酵母细胞壁的饲料。
本发明的技术方案：
本发明提供一种高免疫型酵母细胞壁，其相对分子量为80-200kDa的葡聚糖占葡聚糖总质量的99％以上，以所述高免疫型酵母细胞壁质量为100％计，葡聚糖含量为20-40％。
优选的是，上述高免疫型酵母细胞壁的溶解率≥40％。
优选的是，以所述高免疫型酵母细胞壁质量为100％计，上述高免疫型酵母细胞壁中甘露寡糖含量≥20％。
本发明还提供一种上述高免疫型酵母细胞壁的制备方法，包括如下步骤：
(1)将含酵母的原料进行自溶破壁，分离得到酵母细胞壁乳；
(2)将步骤(1)得到酵母细胞壁乳依次经过蛋白酶酶解和二次酶解，其中，所述二次酶解所用酶为葡聚糖酶、甘露聚糖酶、纤维素酶和淀粉酶中的一种或两种以上；
(3)将步骤(2)得到酶解液进行酸水解，得到高免疫型酵母细胞壁。
优选的是，步骤(1)所述自溶破壁在盐浓度2.0-5.5％，pH为4.0-6.5，温度45-75℃条件下进行，优选自溶时间为15-30h。
优选的是，以酵母细胞壁乳干物质质量计，步骤(2)所述蛋白酶的添加量为1-10‰，优选地，所述蛋白酶为木瓜蛋白酶、中性蛋白酶、碱性蛋白酶和菠萝蛋白酶中的一种或两种以上，优选地，所述蛋白酶含有中性蛋白酶和菠萝蛋白酶，进一步优选地，所述中性蛋白酶的添加量为2-5‰，菠萝蛋白酶的添加量为2-5‰。
优选的是，所述蛋白酶为中性蛋白酶和菠萝蛋白酶，或中性蛋白酶、菠萝蛋白酶和木瓜蛋白酶，或木瓜蛋白酶、中性蛋白酶、碱性蛋白酶和菠萝蛋白酶。
优选的是，所述蛋白酶的添加量为6-10‰。
优选的是，所述蛋白酶酶解温度为30-60℃，酶解pH为4.5-7.0，酶解时间为6-10h。
优选的是，以酵母细胞壁乳干物质质量计，所述二次酶解所用酶的添加量为1-10‰，优选地，所述二次酶解温度为40-60℃，酶解pH为4.0-7.0，酶解时间为4-12h。
优选的是，所述二次酶解所用酶包含葡聚糖酶和纤维素酶，优选地，所述葡聚糖酶的添加量为2-3‰，所述纤维素酶的添加量为2-3‰。
优选的是，所述二次酶解所用酶为葡聚糖酶和纤维素酶，或葡聚糖酶、甘露聚糖酶和纤维素酶，或葡聚糖酶、甘露聚糖酶、纤维素酶和淀粉酶。
优选的是，所述二次酶解所用酶的添加量为4-8‰。
优选的是，步骤(3)所述酸解pH为1.5-3.5，酸解温度为50-100℃，酸解时间为6-20h，优选地，酸解所用酸为无机酸和/或有机酸，进一优选地，所述无机酸为盐酸、硫酸和磷酸中的一种或两种以上，更进一步优选地，所述有机酸为柠檬酸、苹果酸、乳酸、甲酸、乙酸和丙酸中的一种或两种以上。
优选的是，上述制备方法还包括将所述高免疫型酵母细胞壁进行干燥的步骤：酸解结束后，调pH＝4.0-7.0，干燥得到粉状高免疫型酵母细胞壁。
优选的是，在步骤(1)中，含酵母的原料通过酿酒酵母菌株发酵得到，优选地，所述酿酒酵母菌株为酿酒酵母菌株FX-2(Saccharomyces cerevisiaed)。
优选的是，上述发酵pH为4.0-7.0，发酵温度为28-35℃，发酵时间为15-35小时。
优选的是，上述发酵采用流加方式流加碳源、氮源和磷源；
所述碳源为6000-8000份，所述氮源为400-700份，所述磷源为300-600份；
所述碳源为糖蜜、玉米淀粉、葡萄糖、麦芽糖、海藻糖、海藻糖和半乳糖中的一种或两种以上，优选为糖蜜；
所述氮源选自于尿素、氨水或硫酸铵中的一种，优选为尿素；
所述磷源选自于磷酸或磷酸二氢钾中的一种，优选为磷酸二氢钾。
本发明还提供上述高免疫型酵母细胞壁或上述制备方法制得高免疫型酵母细胞壁在饲料中的应用。
本发明还提供一种饲料，其包含上述高免疫型酵母细胞壁或上述制备方法制得高免疫型酵母细胞壁和基础日粮，所述高免疫型酵母细胞壁在饲料中的添加量为0.5-10wt‰。
本发明的有益效果：
(1)本发明高免疫型酵母细胞壁具有高溶解率，相对分子量为80-200kDa的葡聚糖占葡聚糖总质量的99％以上，免疫功效显著提高；其源于酵母，是一种绿色、环保的饲料原料或添加剂；
(2)本发明采用二次酶解及酸解工艺，制备过程环境友好，在提高酵母细胞壁溶解性和降解葡聚糖的同时，更多保留了多糖的功能性分子结构，提高了酵母细胞壁的免疫功效及其附加值，拓宽酵母细胞壁的应用领域。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互结合。下面结合实施例来详细说明本发明。
本发明提供一种高免疫型酵母细胞壁，其相对分子量为80-200kDa的葡聚糖占葡聚糖总质量的99％以上，以所述高免疫型酵母细胞壁质量为100％计，葡聚糖含量为20-40％。
在本发明的一个优选实施方式中，上述高免疫型酵母细胞壁的溶解率≥40％。
相较于现有的酵母细胞壁，其溶解率增加，而且葡聚糖分子生物活性提高，酵母细胞壁的免疫功效提高。
在本发明的一个优选实施方式中，以所述高免疫型酵母细胞壁质量为100％计，上述高免疫型酵母细胞壁中甘露寡糖含量≥20％。
本发明还提供上述高免疫型酵母细胞壁的制备方法，包括如下步骤：
(1)将含酵母的原料进行自溶破壁，分离得到酵母细胞壁乳；
(2)将步骤(1)得到酵母细胞壁乳依次经过蛋白酶酶解和二次酶解，其中，所述二次酶解所用酶为葡聚糖酶、甘露聚糖酶、纤维素酶和淀粉酶中的一种或两种以上；
(3)将步骤(2)得到酶解液进行酸水解，得到高免疫型酵母细胞壁。
在本发明的又一个优选实施方式中，步骤(1)所述自溶破壁在盐浓度2.0-5.5％，pH为4.0-6.5，温度45-75℃条件下进行，优选自溶时间为15-30h。
在本发明的又一个优选实施方式中，以酵母细胞壁乳干物质质量计，步骤(2)所述蛋白酶的添加量为1-10‰，优选地，所述蛋白酶为木瓜蛋白酶、中性蛋白酶、碱性蛋白酶和菠萝蛋白酶中的一种或两种以上；优选地，所述蛋白酶含有中性蛋白酶和菠萝蛋白酶；进一步优选地，所述中性蛋白酶的添加量为2-5‰，所述菠萝蛋白酶的添加量为2-5‰。
在本发明的又一个优选实施方式中，所述蛋白酶为中性蛋白酶和菠萝蛋白酶，或中性蛋白酶、菠萝蛋白酶和木瓜蛋白酶，或木瓜蛋白酶、中性蛋白酶、碱性蛋白酶和菠萝蛋白酶。
在本发明的又一个优选实施方式中，所述蛋白酶的添加量为6-10‰。
在本发明的又一个优选实施方式中，所述蛋白酶酶解温度为30-60℃，酶解pH为4.5-7.0，酶解时间为6-10h。酶添加量不足时，酶解效率不够，酶添加过量会导致酶解过度，成本偏高且达不到产品质量要求。
在本发明的又一个优选实施方式中，以酵母细胞壁乳干物质质量计，所述二次酶解所用酶的添加量为1-10‰；优选地，所述二次酶解温度为40-60℃，酶解pH为4.0-7.0，酶解时间为4-12h。酶添加量不足时，酶解效率不够，酶添加过量会导致酶解过度，成本偏高，且达不到产品质量要求。
在本发明的又一个优选实施方式中，所述二次酶解所用酶包含葡聚糖酶和纤维素酶，优选地，所述葡聚糖酶的添加量为2-3‰，所述纤维素酶的添加量为2-3‰。
在本发明的又一个优选实施方式中，所述二次酶解所用酶为葡聚糖酶和纤维素酶，或葡聚糖酶、甘露聚糖酶和纤维素酶，或葡聚糖酶、甘露聚糖酶、纤维素酶和淀粉酶。
在本发明的又一个优选实施方式中，所述二次酶解所用酶的添加量为 4-8‰。
在本发明的又一个优选实施方式中，步骤(3)所述酸解pH为1.5-3.5，酸解温度为50-100℃，酸解时间为6-20h；优选地，酸解所用酸为无机酸和/或有机酸；进一优选地，所述无机酸为盐酸、硫酸和磷酸中的一种或两种以上；更进一步优选地，所述有机酸为柠檬酸、苹果酸、乳酸、甲酸、乙酸和丙酸中的一种或两种以上。
在本发明的又一个优选实施方式中，上述制备方法中还包括将所述高免疫型酵母细胞壁进行干燥的步骤：酸解结束后，调pH＝4.0-7.0，干燥得到粉状高免疫型酵母细胞壁。
在本发明的又一个优选实施方式中，在步骤(1)中，含酵母的原料通过酿酒酵母菌株发酵得到，优选地，所述酿酒酵母菌株为酿酒酵母菌株FX-2(Saccharomyces cerevisiaed)，该菌株于2016年8月1日保藏在中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:M2016418，保藏地址：中国.武汉.武汉大学，邮政编码430072，电话：(027)-68754052；该菌株在公开号为CN108220175A的专利公开文本中已有记载。
在本发明的又一个优选实施方式中，所述发酵pH为4.0-7.0，发酵温度为28-35℃，发酵时间为15-35小时。
在本发明的又一个优选实施方式中，所述发酵采用流加方式流加碳源、氮源和磷源；
所述碳源为6000-8000份，所述氮源为400-700份，所述磷源为300-600份；
所述碳源为糖蜜、玉米淀粉、葡萄糖、麦芽糖、海藻糖、海藻糖和半乳糖中的一种或两种以上，优选为糖蜜；
所述氮源选自于尿素、氨水或硫酸铵中的一种，优选为尿素；
所述磷源选自于磷酸或磷酸二氢钾中的一种，优选为磷酸二氢钾。
本发明还提供上述高免疫型酵母细胞壁或上述制备方法制得高免疫型酵母细胞壁在饲料中的应用。
本发明还提供一种饲料，其包含上述高免疫型酵母细胞壁或上述制备方法制得高免疫型酵母细胞壁和基础日粮，所述高免疫型酵母细胞壁在饲料中的添加量为0.5-10wt‰。
下面将通过具体的实施例进一步说明本发明的有益效果。
本发明实施例和对比例使用原料和设备来源见表1。
表1本发明实施例和对比例使用原料和设备来源
Figure PCTCN2022089879-appb-000001
实施例1
(一)高免疫型酵母细胞壁的制备
(1)培养液含有碳源、氮源、磷源，其中碳源为糖蜜7000g，氮源为尿素400g，磷源为磷酸二氢钾600g。将培养液在121℃灭菌10min，接种酿酒酵母菌FX-2(Saccharomyces cerevisiaed)进行发酵培养，发酵温度28℃，发酵时间15h，发酵pH4.0，获得酵母乳；
(2)将上述酵母乳进行自溶处理，加入氯化钠，使得氯化钠的质量浓度为2％，在pH4.0，温度45℃下自溶15h，5000rpm离心处理，得到上层的酵母自溶物和下层酵母细胞壁乳，收集酵母细胞壁乳进行酶解处理；
(3)将步骤(2)得到酵母细胞壁乳加水稀释成干物质质量浓度为5％，加入1‰的木瓜蛋白酶(以酵母细胞壁乳干物质质量计，下同)，控制温度30℃，pH4.5，酶解6h；
(4)继续加入1‰的葡聚糖酶(以酵母细胞壁乳干物质质量计，下同)，控制温度40℃，pH4.0，酶解4h；
(5)向步骤(4)得到的酶解液中加入10wt％的硫酸，调节pH为1.5，升温至70℃，保温6h；
(6)保温结束后，调pH为4.0，然后进行喷雾干燥处理，得到高免疫型酵母细胞壁粉末。
(二)高免疫型酵母细胞壁的产品测定
1.甘露聚糖和葡聚糖的测定
1.1检测原理
根据葡聚糖和甘露糖在流动相和液相色谱柱的固定相之间具有不同的分配系数，将水解后的样品注入液相色谱，用纯水做流动相，糖类分子流出后，经示差检测器检测，用外标法定量。在葡聚糖和甘露聚糖水解时，由于样品可能存在水解不彻底，以及水解产生的葡萄糖和甘露糖由于高温造成部分发生其它副反应，导致检测结果比产品中实际含有的葡聚糖和甘露聚糖偏低。检测中采用葡聚糖对照品对上述水解过程引起的误差进行修正。
1.2仪器
a)水浴锅；
b)旋涡混合器；
c)电炉；
d)压力蒸汽灭菌器；
e)高效液相色谱仪：带示差检测器和糖柱(6.5mm×300mm waters sugar pak-1)。
1.3试剂
a)纯水；
b)盐酸：37％左右；
c)葡萄糖：AR；
d)甘露糖：AR；
e)氢氧化钠：AR；
f)葡萄糖和甘露糖混合标液(2g/L)：分别称取葡萄糖和甘露糖各0.2000g，用纯水定容至100ml。
g)葡聚糖对照品(curdlan from Alcaligenes faecalis)：Sigma产品，货号
h)氢氧化钠溶液：300g/L。
1.4样品处理
准确称取400mg(精确至0.1mg)样品放入一个20ml的耐热玻璃制的带螺帽的小试管中，加入6.0ml盐酸(37％)，小心的将小瓶盖紧后用旋涡混合器混合，得到均一的悬浮液。将小瓶放入30℃水浴中处理45min，每15min用旋涡混合器振荡混合一次。然后将悬浮物定量的转移到200ml杜氏瓶中，用约100ml～120ml的水，分几次洗涤小试管，洗涤液并入杜氏瓶中。将杜氏瓶放入高压灭菌锅，121℃下处理60min。取出后冷却，用氢氧化钠溶液将溶液调pH到6～7，然后定容至200ml。使用0.45微米孔径的醋酸纤维素膜过滤备用。同时准确称取葡聚糖对照品(见1.3中g))200mg，按样品处理方法进行同样的处理。
1.5色谱条件
采用纯水做为流动相，流速为0.5ml/min，柱温80℃，待仪器基线平稳后再进样。
1.6标准曲线的绘制
分别吸取甘露糖/葡萄糖标液(见1.3中f))1、2、3、4、5ml到10ml容量瓶中，用高纯水定容到刻度，得到甘露糖、葡萄糖各为200、400、600、800、1000mg/l的混合标样。在上述色谱条件下准确进样20ul，得到色谱峰面积和标准物质量浓度之间的回归方程，绘制标准曲线。
1.7样品及对照品的测定
在同样的色谱条件下，将处理好的样品和葡聚糖对照品分别注入色谱仪中，记录各色谱峰的保留时间和峰面积。用糖标样色谱峰的保留时间定性，用糖标样色谱峰的峰面积来定量。
1.8结果计算
β-葡聚糖或甘露聚糖的含量按下式计算：
X＝(A1×0.2×100)÷(m1×1000)×0.9×F……………(1)
F＝P×(100-W)÷[(A2×0.2×100)÷(m2×1000)×0.9]…(2)
式中：
X---样品中葡聚糖或甘露聚糖的含量，％；
A1---根据样品溶液的峰面积，在标准曲线上查得的样品溶液的葡萄糖或甘露糖的含量，mg/L；
A2---根据葡聚糖对照品溶液的峰面积，在标准曲线上查得的样品溶液的葡萄糖的含量，mg/L；
m1---称取样品的质量，g；
m2---称取葡聚糖对照品的质量，g；
0.2---样品/葡聚糖对照品处理后定容的体积，L；
0.9---将葡萄糖或甘露糖换算成葡聚糖或甘露聚糖的系数；
F---样品酸水解中葡萄糖和甘露糖被破坏造成结果偏低的经验补偿系数；
P---葡聚糖对照品的纯度(依据试剂厂家提供的检测报告)；
W---葡聚糖对照品的水分(依据试剂厂家提供的检测报告)。
备注：在同一个实验室内，一般1～2个月检测F值即可。F值在1.14左右，实验室定期对F值进行修正。
1.9允许误差
在重复性检测条件下获得的两次独立测定结果的相对误差不得超过表2中所规定的数值：
表2重复性检测相对误差
含量 ≤10％ 10-30％(不含10％和30％) ≥30％
甘露聚糖 10％ 5％ 3％
葡聚糖 10％ 5％ 3％
2.溶解率的测定方法
2.1测定原理
使样品溶解于水，通过离心收集沉淀，计算出溶出物质重量占总重的比例。
2.2试剂与仪器
a)蒸馏水；
b)离心机(5000g)；
c)水分测定仪；
d)分析天平；
2.3测定步骤
准确称取样品10g(精确到0.1mg),记为m 0，溶解于200mL蒸馏水中，使其充分溶解后全部转入离心杯中，5000g离心5min，准确测定沉淀重量，记为m 1，并测定沉淀干物质含量(按照GB 5009.3-2010第一法检测)，沉淀干物质含量记为D。
2.4结果计算
样品溶解率为：X＝(m 0-m 1×D)÷m 0×100％
X——样品溶解率，％；
m 0——称取样品重量，g；
m 1——离心后沉淀重量，g；
D——离心后沉淀干物质含量，％。
计算结果保留至小数点后1位。
3.按照如下方法检测葡聚糖的相对分子量：
测定方法：将步骤(一)制备得到的高免疫型酵母细胞壁产品配制成溶液，利用高相液相色谱法测定葡聚糖的相对分子量。
分析条件：Shodex OHpak SB-805HQ凝胶柱(8mm×300mm)；检测器：示差检测器(Optilab rEX)，十八角静态激光散射仪(DAWN HELLOS)，紫外检测器；检测波长：658nm(葡聚糖)；流动相：0.5mo1/L NaCl溶液；柱温：25℃；流速：0.5mL/min；进样量：20μL。
高效液相色谱仪：型号GPC/RI/MALLS，Waters 515泵，美国Waters公司制。
相关检测结果见表3所示。
实施例2
(一)高免疫型酵母细胞壁的制备
(1)培养液含有碳源、氮源、磷源，其中碳源为糖蜜6000g，氮源为尿素700g，磷源为磷酸二氢钾300g。将培养液在121℃灭菌10min，接种酿酒酵母菌FX-2(Saccharomyces cerevisiaed)进行发酵培养，发酵温度35℃，发酵时间35h，发酵pH7.0，获得酵母乳；
(2)将上述酵母乳进行自溶处理，加入氯化钠，使得氯化钠的质量浓度为5.5％，在pH6.5，温度75℃下自溶30h，5000rpm离心处理，得到上层的酵母自溶物和下层酵母细胞壁乳，收集酵母细胞壁乳进行酶解处理；
(3)将步骤(2)得到酵母细胞壁乳加水稀释成干物质质量浓度为20％，加入10‰的碱性蛋白酶(以酵母细胞壁乳干物质质量计，下同)，控制温度60℃，pH7.0，酶解10h；
(4)继续加入10‰的甘露聚糖酶(以酵母细胞壁乳干物质质量计，下同)，控制温度60℃，pH7.0，酶解12h；
(5)向步骤(4)得到的酶解液中加入柠檬酸，调节pH为3.5，升温至100℃，保温20h；
(6)保温结束后，调pH为7.0，然后进行喷雾干燥处理，得到高免疫型酵母细胞壁粉末。
按照实施例1中产品测定方法测定，相关检测结果如表3所示。
实施例3
(一)高免疫型酵母细胞壁的制备
(1)培养液含有碳源、氮源、磷源，其中碳源为糖蜜8000g，氮源为尿素550g，磷源为磷酸二氢钾450g。将培养液在121℃灭菌10min，接种酿酒酵 母菌FX-2(Saccharomyces cerevisiaed)进行发酵培养，发酵温度30℃，发酵时间20h，发酵pH5.0，获得酵母乳；
(2)将上述酵母乳进行自溶处理，加入氯化钠，使得氯化钠的质量浓度为3％，在pH5.0，温度55℃下自溶20h，5000rpm离心处理，得到上层的酵母自溶物和下层酵母细胞壁乳，收集酵母细胞壁乳进行酶解处理；
(3)将步骤(2)得到酵母细胞壁乳加水稀释成干物质质量浓度为10％，加入5‰的中性蛋白酶(以酵母细胞壁乳干物质质量计，下同)和5‰的菠萝蛋白酶(以酵母细胞壁乳干物质质量计，下同)，控制温度50℃，pH5.0，酶解8h；
(4)继续加入5‰的纤维素酶(以酵母细胞壁乳干物质质量计，下同)和5‰的淀粉酶(以酵母细胞壁乳干物质质量计，下同)，控制温度50℃，pH5.0，酶解8h；
(5)向步骤(4)得到的酶解液中加入磷酸和乙酸，调节pH为2.5，升温至80℃，保温8h；
(6)保温结束后，调pH为5.0，然后进行喷雾干燥处理，得到高免疫型酵母细胞壁粉末。
按照实施例1中产品测定方法测定，相关检测结果如表3所示。
实施例4
(一)高免疫型酵母细胞壁的制备
(1)培养液含有碳源、氮源、磷源，其中碳源为糖蜜7000g，氮源为尿素400g，磷源为磷酸二氢钾600g。将培养液在121℃灭菌10min，接种酿酒酵母菌FX-2(Saccharomyces cerevisiaed)进行发酵培养，发酵温度28℃，发酵时间15h，发酵pH4.0，获得酵母乳；
(2)将上述酵母乳进行自溶处理，加入氯化钠，使得氯化钠的质量浓度为2％，在pH4.0，温度45℃下自溶15h，5000rpm离心处理，得到上层的酵母自溶物和下层酵母细胞壁乳，收集酵母细胞壁乳进行酶解处理；
(3)将步骤(2)得到酵母细胞壁乳加水稀释成干物质质量浓度为5％，加入2‰的中性蛋白酶，控制温度40℃，pH6.0，酶解7h；
(4)继续加入2‰的纤维素酶，控制温度40℃，pH6.0，酶解6h；
(5)向步骤(4)得到的酶解液中加入10wt％的硫酸，调节pH为2.0，升温至50℃，保温13h；
(6)保温结束后，调pH为5.0，然后进行喷雾干燥处理，得到高免疫型酵母细胞壁粉末。
实施例5
(一)高免疫型酵母细胞壁的制备
(1)培养液含有碳源、氮源、磷源，其中碳源为糖蜜7000g，氮源为尿素400g，磷源为磷酸二氢钾600g。将培养液在121℃灭菌10min，接种酿酒酵母菌FX-2(Saccharomyces cerevisiaed)进行发酵培养，发酵温度28℃，发酵时间15h，发酵pH4.0，获得酵母乳；
(2)将上述酵母乳进行自溶处理，加入氯化钠，使得氯化钠的质量浓度为2％，在pH4.0，温度45℃下自溶15h，5000rpm离心处理，得到上层的酵母自溶物和下层酵母细胞壁乳，收集酵母细胞壁乳进行酶解处理；
(3)将步骤(2)得到酵母细胞壁乳加水稀释成干物质质量浓度为5％，加入5‰的菠萝蛋白酶，控制温度45℃，pH5.5，酶解9h；
(4)继续加入5‰的淀粉酶，控制温度50℃，pH4.0，酶解5h；
(5)向步骤(4)得到的酶解液中加入柠檬酸，调节pH为3.0，升温至60℃，保温16h；
(6)保温结束后，调pH为6.0，然后进行喷雾干燥处理，得到高免疫型酵母细胞壁粉末。
实施例6
(一)高免疫型酵母细胞壁的制备
(1)培养液含有碳源、氮源、磷源，其中碳源为糖蜜7000g，氮源为尿素400g，磷源为磷酸二氢钾600g。将培养液在121℃灭菌10min，接种酿酒酵母菌FX-2(Saccharomyces cerevisiaed)进行发酵培养，发酵温度28℃，发酵时间15h，发酵pH4.0，获得酵母乳；
(2)将上述酵母乳进行自溶处理，加入氯化钠，使得氯化钠的质量浓度为2％，在pH4.0，温度45℃下自溶15h，5000rpm离心处理，得到上层的酵母自溶物和下层酵母细胞壁乳，收集酵母细胞壁乳进行酶解处理；
(3)将步骤(2)得到酵母细胞壁乳加水稀释成干物质质量浓度为5％，加入2‰的木瓜蛋白酶和2‰的碱性蛋白酶，控制温度55℃，pH6.5，酶解8h；
(4)继续加入2‰的葡聚糖酶和2‰的纤维素酶，控制温度60℃，pH5.0，酶解7h；
(5)向步骤(4)得到的酶解液中加入10wt％的硫酸，调节pH为1.5，升温至70℃，保温6h；
(6)保温结束后，调pH为4.0，然后进行喷雾干燥处理，得到高免疫型酵母细胞壁粉末。
实施例7
(一)高免疫型酵母细胞壁的制备
(1)培养液含有碳源、氮源、磷源，其中碳源为糖蜜7000g，氮源为尿素400g，磷源为磷酸二氢钾600g。将培养液在121℃灭菌10min，接种酿酒酵母菌FX-2(Saccharomyces cerevisiaed)进行发酵培养，发酵温度28℃，发酵时间15h，发酵pH4.0，获得酵母乳；
(2)将上述酵母乳进行自溶处理，加入氯化钠，使得氯化钠的质量浓度为2％，在pH4.0，温度45℃下自溶15h，5000rpm离心处理，得到上层的酵母自溶物和下层酵母细胞壁乳，收集酵母细胞壁乳进行酶解处理；
(3)将步骤(2)得到酵母细胞壁乳加水稀释成干物质质量浓度为5％，加入2‰的木瓜蛋白酶、2‰的中性蛋白酶和0.2‰的菠萝蛋白酶，控制温度60℃，pH4.5，酶解7h；
(4)继续加入3‰的甘露聚糖酶和3‰的淀粉酶，控制温度40℃，pH7.0，酶解10h；
(5)向步骤(4)得到的酶解液中加入10wt％的硫酸，调节pH为1.5，升温至70℃，保温6h；
(6)保温结束后，调pH为4.0，然后进行喷雾干燥处理，得到高免疫 型酵母细胞壁粉末。
实施例8
(一)高免疫型酵母细胞壁的制备
(1)培养液含有碳源、氮源、磷源，其中碳源为糖蜜7000g，氮源为尿素400g，磷源为磷酸二氢钾600g。将培养液在121℃灭菌10min，接种酿酒酵母菌FX-2(Saccharomyces cerevisiaed)进行发酵培养，发酵温度28℃，发酵时间15h，发酵pH4.0，获得酵母乳；
(2)将上述酵母乳进行自溶处理，加入氯化钠，使得氯化钠的质量浓度为2％，在pH4.0，温度45℃下自溶15h，5000rpm离心处理，得到上层的酵母自溶物和下层酵母细胞壁乳，收集酵母细胞壁乳进行酶解处理；
(3)将步骤(2)得到酵母细胞壁乳加水稀释成干物质质量浓度为5％，加入2‰的木瓜蛋白酶、2‰的中性蛋白酶、2‰的菠萝蛋白酶和2‰的碱性蛋白酶，控制温度50℃，pH5.5，酶解6h；
(4)继续加入3‰的葡聚糖酶、3‰的甘露聚糖酶和3‰的纤维素酶，控制温度50℃，pH9.0，酶解9h；
(5)向步骤(4)得到的酶解液中加入10wt％的硫酸，调节pH为1.5，升温至70℃，保温6h；
(6)保温结束后，调pH为4.0，然后进行喷雾干燥处理，得到高免疫型酵母细胞壁粉末。
实施例9
(一)高免疫型酵母细胞壁的制备
(1)培养液含有碳源、氮源、磷源，其中碳源为糖蜜7000g，氮源为尿素400g，磷源为磷酸二氢钾600g。将培养液在121℃灭菌10min，接种酿酒酵母菌FX-2(Saccharomyces cerevisiaed)进行发酵培养，发酵温度28℃，发酵时间15h，发酵pH4.0，获得酵母乳；
(2)将上述酵母乳进行自溶处理，加入氯化钠，使得氯化钠的质量浓度为2％，在pH4.0，温度45℃下自溶15h，5000rpm离心处理，得到上层的酵母自溶物和下层酵母细胞壁乳，收集酵母细胞壁乳进行酶解处理；
(3)将步骤(2)得到酵母细胞壁乳加水稀释成干物质质量浓度为5％，加入3‰的木瓜蛋白酶、3‰的菠萝蛋白酶和3‰的碱性蛋白酶，控制温度45℃，pH5.0，酶解8h；
(4)继续加入2‰的葡聚糖酶、2‰的甘露聚糖酶、2‰的纤维素酶和2‰的淀粉酶，控制温度60℃，pH5.0，酶解11h；
(5)向步骤(4)得到的酶解液中加入10wt％的硫酸，调节pH为1.5，升温至70℃，保温6h；
(6)保温结束后，调pH为4.0，然后进行喷雾干燥处理，得到高免疫型酵母细胞壁粉末。
对比例1
(一)高免疫型酵母细胞壁的制备
(1)培养液含有碳源、氮源、磷源，其中碳源为糖蜜7000g，氮源为尿素400g，磷源为磷酸二氢钾600g。将培养液在121℃灭菌10min，接种酿酒酵母菌FX-2(Saccharomyces cerevisiaed)进行发酵培养，发酵温度28℃，发酵时间15h，发酵pH4.0，获得酵母乳；
(2)将上述酵母乳进行自溶处理，加入氯化钠，使得氯化钠的质量浓度为2％，在pH4.0，温度45℃下自溶15h，5000rpm离心处理，得到上层的酵母自溶物和下层酵母细胞壁乳，收集酵母细胞壁乳进行酶解处理；
(3)将步骤(2)得到酵母细胞壁乳加水稀释成干物质质量浓度为5％，加入0.5‰的木瓜蛋白酶，控制温度30℃，pH4.5，酶解6h；
(4)继续加入1‰的葡聚糖酶，控制温度40℃，pH4.0，酶解4h；
(5)向步骤(4)得到的酶解液中加入10wt％的硫酸，调节pH为1.5，升温至70℃，保温6h；
(6)保温结束后，调pH为4.0，然后进行喷雾干燥处理，得到高免疫型酵母细胞壁粉末。
按照实施例1中产品测定方法测定，相关检测结果如表3所示。
对比例2
(一)高免疫型酵母细胞壁的制备
(1)培养液含有碳源、氮源、磷源，其中碳源为糖蜜7000g，氮源为尿素400g，磷源为磷酸二氢钾600g。将培养液在121℃灭菌10min，接种酿酒酵母菌FX-2(Saccharomyces cerevisiaed)进行发酵培养，发酵温度28℃，发酵时间15h，发酵pH4.0，获得酵母乳；
(2)将上述酵母乳进行自溶处理，加入氯化钠，使得氯化钠的质量浓度为2％，在pH4.0，温度45℃下自溶15h，5000rpm离心处理，得到上层的酵母自溶物和下层酵母细胞壁乳，收集酵母细胞壁乳进行酶解处理；
(3)将步骤(2)得到酵母细胞壁乳加水稀释成干物质质量浓度为5％，加入1‰的木瓜蛋白酶，控制温度30℃，pH4.5，酶解6h；
(4)继续加入0.5‰的葡聚糖酶，控制温度40℃，pH4.0，酶解4h；
(5)向步骤(4)得到的酶解液中加入10wt％的硫酸，调节pH为1.5，升温至70℃，保温6h；
(6)保温结束后，调pH为4.0，然后进行喷雾干燥处理，得到高免疫型酵母细胞壁粉末。
按照实施例1中产品测定方法测定，相关检测结果如表3所示。
对比例3
(一)高免疫型酵母细胞壁的制备
(1)培养液含有碳源、氮源、磷源，其中碳源为糖蜜7000g，氮源为尿素400g，磷源为磷酸二氢钾600g。将培养液在121℃灭菌10min，接种酿酒酵母菌FX-2(Saccharomyces cerevisiaed)进行发酵培养，发酵温度35℃，发酵时间35h，发酵pH7.0，获得酵母乳；
(2)将上述酵母乳进行自溶处理，加入氯化钠，使得氯化钠的质量浓度为5.5％，在pH6.5，温度75℃下自溶30h，5000rpm离心处理，得到上层的酵母自溶物和下层酵母细胞壁乳，收集酵母细胞壁乳进行酶解处理；
(3)将步骤(2)得到酵母细胞壁乳加水稀释成干物质质量浓度为20％，加入12‰的碱性蛋白酶，控制温度60℃，pH7.0，酶解10h；
(4)继续加入10‰的甘露聚糖酶，控制温度60℃，pH7.0，酶解12h；
(5)向步骤(4)得到的酶解液中加入柠檬酸，调节pH为3.5，升温至100℃，保温20h；
(6)保温结束后，调pH为7.0，然后进行喷雾干燥处理，得到高免疫型酵母细胞壁粉末。
按照实施例1中产品测定方法测定，相关检测结果如表3所示。
对比例4
(一)高免疫型酵母细胞壁的制备
(1)培养液含有碳源、氮源、磷源，其中碳源为糖蜜7000g，氮源为尿素400g，磷源为磷酸二氢钾600g。将培养液在121℃灭菌10min，接种酿酒酵母菌FX-2(Saccharomyces cerevisiaed)进行发酵培养，发酵温度35℃，发酵时间35h，发酵pH7.0，获得酵母乳；
(2)将上述酵母乳进行自溶处理，加入氯化钠，使得氯化钠的质量浓度为5.5％，在pH6.5，温度75℃下自溶30h，5000rpm离心处理，得到上层的酵母自溶物和下层酵母细胞壁乳，收集酵母细胞壁乳进行酶解处理；
(3)将步骤(2)得到酵母细胞壁乳加水稀释成干物质质量浓度为20％，加入10‰的碱性蛋白酶，控制温度60℃，pH7.0，酶解10h；
(4)继续加入12‰的甘露聚糖酶，控制温度60℃，pH7.0，酶解12h；
(5)向步骤(4)得到的酶解液中加入柠檬酸，调节pH为3.5，升温至100℃，保温20h；
(6)保温结束后，调pH为7.0，然后进行喷雾干燥处理，得到高免疫型酵母细胞壁粉末。
按照实施例1中产品测定方法测定，相关检测结果如表3所示。
对比例5
(一)高免疫型酵母细胞壁的制备
(1)培养液含有碳源、氮源、磷源，其中碳源为糖蜜7000g，氮源为尿素400g，磷源为磷酸二氢钾600g。将培养液在121℃灭菌10min，接种酿酒酵母菌FX-2(Saccharomyces cerevisiaed)进行发酵培养，发酵温度28℃，发酵时间15h，发酵pH4.0，获得酵母乳；
(2)将上述酵母乳进行自溶处理，加入氯化钠，使得氯化钠的质量浓度为2％，在pH4.0，温度45℃下自溶15h，5000rpm离心处理，得到上层的酵母自溶物和下层酵母细胞壁乳，收集酵母细胞壁乳进行酶解处理；
(3)将步骤(2)得到酵母细胞壁乳加水稀释成干物质质量浓度为5％，加入1‰的木瓜蛋白酶，控制温度30℃，pH4.5，酶解6h；
(4)继续加入1‰的葡聚糖酶，控制温度40℃，pH4.0，酶解4h；
(5)将步骤(4)得到的酶解液升温至90℃保温1h，然后进行喷雾干燥处理，得到高免疫型酵母细胞壁粉末。
按照实施例1中产品测定方法测定，相关检测结果如表3所示。
对比例6
(一)高免疫型酵母细胞壁的制备
(1)培养液含有碳源、氮源、磷源，其中碳源为糖蜜7000g，氮源为尿素400g，磷源为磷酸二氢钾600g。将培养液在121℃灭菌10min，接种酿酒酵母菌FX-2(Saccharomyces cerevisiaed)进行发酵培养，发酵温度28℃，发酵时间15h，发酵pH4.0，获得酵母乳；
(2)将上述酵母乳进行自溶处理，加入氯化钠，使得氯化钠的质量浓度为2％，在pH4.0，温度45℃下自溶15h，5000rpm离心处理，得到上层的酵母自溶物和下层酵母细胞壁乳，收集酵母细胞壁乳进行酶解处理；
(3)将步骤(2)得到酵母细胞壁乳加水稀释成干物质质量浓度为5％，加入1‰的葡聚糖酶，控制温度40℃，pH4.0，酶解4h；
(4)继续加入1‰的木瓜蛋白酶，控制温度30℃，pH4.5，酶解6h；
(5)向步骤(4)得到的酶解液中加入10wt％的硫酸，调节pH为1.5，升温至70℃，保温6h；
(6)保温结束后，调pH为4.0，然后进行喷雾干燥处理，得到高免疫 型酵母细胞壁粉末。
按照实施例1中产品测定方法测定，相关检测结果如表3所示。
表3实施例1-9和对比例1-6检测结果
Figure PCTCN2022089879-appb-000002
由表3可知，实施例1-9制备的高免疫细胞壁粉末，其葡聚糖含量均在20％-40％之间，甘露寡糖含量均大于等于20％，溶解率均高于40％，且相对分子量为80-200kDa的葡聚糖占葡聚糖总质量的99％以上，对比例1-6制备的细胞壁样品葡聚糖、甘露聚糖含量与实施例无较大差异，但对比例1、对比例2、对比例5、对比例6的细胞壁溶解率均低于40％，且相对分子量为200kDa以上的葡聚糖占葡聚糖总质量的99％以上。对比例3和对比例4溶解率高于40％，但是相对分子量为80KDa以下的葡聚糖占葡聚糖总质量的98％以上。
应用试验例
选取28日龄体重接近、体况良好的杜×长×大三元杂交断奶仔猪72头，按平均体重相近似、公母各半的原则分为9个处理组，每组4个重复，每个重复一栏猪，每栏公母各1头猪。试验基础日粮按NRC 2012断奶仔猪营养需要进行配制，见表4，9个试验组分别在基础日粮的基础上饲喂不同的酵母细胞壁产品。具体分组及日粮设计见表5。
日常管理按规模化猪场饲养管理方法进行，仔猪购入后先饲喂过渡日粮(基础日粮)，试验开始后饲喂试验日粮，保证料槽饲料充足以供仔猪自由采食，自动饮水器供水，室内温度维持常温。仔猪购买进场后免疫情况：28日龄免疫蓝耳(PRRS)，35日龄免疫伪狂犬(PR)，42日龄免疫猪瘟(HC)。
试验结束时，全部空腹仔猪前腔静脉采血5ml，置于离心管中，静置30min，3500r/min离心10min，收集血清，置于-20℃保存待测。检测总蛋白(TP)、白蛋白(ALB)、球蛋白(GLO)，试验结果如表6所示。
表4试验基础日粮及营养水平
日粮组成 含量 营养指标 含量
玉米/％ 67.00 净能(kcal/kg) 2900.50
豆粕(46％蛋白质)/％ 23.30 粗蛋白质/％ 19.06
发酵豆粕/％ 5.88 粗灰分/％ 5.00
大豆油/％ 1.05 干物质/％ 86.5
细石粉/％ 1.00 赖氨酸/％ 1.41
磷酸二氢钙/％ 0.065 蛋氨酸+胱氨酸/％ 0.79
4％仔猪预混料/％ 0.50 苏氨酸/％ 0.87
食盐/％ 0.37 可消化磷/％ 0.18
98.5％赖氨酸/％ 0.2    
氯化胆碱/％ 0.05    
表5试验设计
Figure PCTCN2022089879-appb-000003
表6试验结果
处理组 总蛋白 球蛋白 白蛋白/球蛋白
试验组1 46.76±1.46 17.78±4.21 1.63±0.32
试验组2 47.56±2.35 18.73±3.24 1.54±0.54
试验组3 47.03±3.67 18.55±4.21 1.53±0.48
试验组4 44.99±1.36 14.69±0.99 2.06±0.09
试验组5 46.83±2.31 15.36±3.24 2.04±0.58
试验组6 45.89±2.35 14.38±1.24 2.19±0.54
试验组7 47.05±2.58 15.74±2.43 1.99±0.78
试验组8 46.87±3.12 15.65±2.35 1.99±1.03
试验组9 47.25±2.76 16.05±1.65 1.94±0.86
由表6可知，试验14d后，各处理组血清球蛋白水平从高到低依次为实施例2、实施例3、实施例1、对比例6、对比例4、对比例5、对比例2、对比例1、对比例3；白蛋白/球蛋白从低到高依次为实施例3、实施例2、实施例1、对比例6、对比例4、对比例5、对比例2、对比例1、对比例3；表明实施例1、实施例2、 实施例3制备的高免疫型酵母细胞壁较对比例制备高免疫酵母细胞壁可明显提高仔猪机体免疫力。
综上所述，本发明高免疫型酵母细胞壁具有高溶解率，以质量百分比计，99％以上葡聚糖分子量为80-200kDa，免疫功效显著提高；其源于酵母，是一种绿色、环保的饲料原料或添加剂。
以上所述，仅是本发明实施的较佳实施例，并非对本发明做任何形式上的限制，凡在本发明的精神和原则之内所做的修改、等同替换和改进等，均需要包含在本发明的保护范围之内。

Claims (20)

  1. 一种高免疫型酵母细胞壁，其特征在于，其相对分子量为80-200kDa的葡聚糖占葡聚糖总质量的99％以上，以所述高免疫型酵母细胞壁质量为100％计，葡聚糖含量为20-40％。
  2. 根据权利要求1所述的高免疫型酵母细胞壁，其特征在于，其溶解率≥40％。
  3. 根据权利要求1或2所述的高免疫型酵母细胞壁，其特征在于，以所述高免疫型酵母细胞壁质量为100％计，甘露寡糖含量≥20％。
  4. 权利要求1-3任一项所述的高免疫型酵母细胞壁的制备方法，其特征在于，包括如下步骤：
    (1)将含酵母的原料进行自溶破壁，分离得到酵母细胞壁乳；
    (2)将步骤(1)得到酵母细胞壁乳依次经过蛋白酶酶解和二次酶解，其中，所述二次酶解所用酶为葡聚糖酶、甘露聚糖酶、纤维素酶和淀粉酶中的一种或两种以上；
    (3)将步骤(2)得到酶解液进行酸水解，得到高免疫型酵母细胞壁。
  5. 根据权利要求4所述的制备方法，其特征在于，步骤(1)所述自溶破壁在盐浓度2.0-5.5％，pH为4.0-6.5，温度45-75℃条件下进行，优选自溶时间为15-30h。
  6. 根据权利要求4或5所述的制备方法，其特征在于，以酵母细胞壁乳干物质质量计，步骤(2)所述蛋白酶的添加量为1-10‰；优选地，所述蛋白酶为木瓜蛋白酶、中性蛋白酶、碱性蛋白酶和菠萝蛋白酶中的一种或两种以上，优选地，所述蛋白酶含有中性蛋白酶和菠萝蛋白酶；进一步优选地，所述中性蛋白酶的添加量为2-5‰，所述菠萝蛋白酶的添加量为2-5‰。
  7. 根据权利要求6所述的制备方法，其特征在于，所述蛋白酶为中性蛋白酶和菠萝蛋白酶，或中性蛋白酶、菠萝蛋白酶和木瓜蛋白酶，或木瓜蛋白酶、中性蛋白酶、碱性蛋白酶和菠萝蛋白酶。
  8. 根据权利要求7所述的制备方法，其特征在于，所述蛋白酶的添加量为6-10‰。
  9. 根据权利要求4-8任一项所述的制备方法，其特征在于，所述蛋白酶酶解温度为30-60℃，酶解pH为4.5-7.0，酶解时间为6-10h。
  10. 根据权利要求4-9任一项所述的制备方法，其特征在于，以酵母细胞壁乳干物质质量计，所述二次酶解所用酶的添加量为1-10‰；优选地，所述二次酶解温度为40-60℃，酶解pH为4.0-7.0，酶解时间为4-12h。
  11. 根据权利要求4-10任一项所述的制备方法，其特征在于，所述二次酶解所用酶包含葡聚糖酶和纤维素酶；优选地，所述葡聚糖酶的添加量为2-3‰,所述纤维素酶的添加量为2-3‰。
  12. 根据权利要求11所述的制备方法，其特征在于，所述二次酶解所用酶为葡聚糖酶和纤维素酶，或葡聚糖酶、甘露聚糖酶和纤维素酶，或葡聚糖酶、甘露聚糖酶、纤维素酶和淀粉酶。
  13. 根据权利要求12所述的制备方法，其特征在于，所述二次酶解所用酶的添加量为4-8‰。
  14. 根据权利要求4-13任一项所述的制备方法，其特征在于，步骤(3)所述酸解pH为1.5-3.5，酸解温度为50-100℃，酸解时间为6-20h；优选地，酸解所用酸为无机酸和/或有机酸；进一优选地，所述无机酸为盐酸、硫酸和磷酸中的一种或两种以上；更进一步优选地，所述有机酸为柠檬酸、苹果酸、乳酸、甲酸、乙酸和丙酸中的一种或两种以上。
  15. 根据权利要求4-14任一项所述的制备方法，其特征在于，还包括将所述高免疫型酵母细胞壁进行干燥步骤：酸解结束后，调pH＝4.0-7.0，干燥得到粉状高免疫型酵母细胞壁。
  16. 根据权利要求4-15任一项所述的制备方法，其特征在于，步骤(1)含酵母的原料通过酿酒酵母菌株发酵得到；优选地，所述酿酒酵母为酿酒酵母FX-2(Saccharomyces cerevisiaed)，保藏编号为CCTCC NO：M20016418。
  17. 根据权利要求16所述制备方法，其特征在于，所述发酵pH为4.0-7.0，发酵温度为28-35℃，发酵时间为15-35小时。
  18. 根据权利要求16或17所述的制备方法，其特征在于，所述发酵采用流加方式流加碳源、氮源和磷源；
    所述碳源为6000-8000份，所述氮源为400-700份，所述磷源为300-600 份；所述碳源为糖蜜、玉米淀粉、葡萄糖、麦芽糖、海藻糖、海藻糖和半乳糖中的一种或两种以上，优选为糖蜜；
    所述氮源选自于尿素、氨水或硫酸铵中的一种，优选为尿素；
    所述磷源选自于磷酸或磷酸二氢钾中的一种，优选为磷酸二氢钾。
  19. 根据权利要求1-3任一项所述的高免疫型酵母细胞壁或权利要求4-18任一项所述的制备方法制得高免疫型酵母细胞壁在饲料中的应用。
  20. 一种饲料，其特征在于，其包含权利要求1-3任一项所述高免疫型酵母细胞壁或权利要求4-18任一项所述制备方法制得高免疫型酵母细胞壁和基础日粮，所述高免疫型酵母细胞壁在饲料中的添加量为0.5-10wt‰。
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