Yeast metagen as well as preparation method and application thereof

Abstract

The invention relates to the technical field of microorganisms, and discloses a yeast metabolite which comprises saccharomyces cerevisiae EnhSa-13, a metabolite of the saccharomyces cerevisiae EnhSa-13 and a carrier, and the content of the metabolite in the yeast metabolite is larger than 1 wt%. The content of the saccharomyces cerevisiae EnhSa-13 and metabolites thereof in the yeast metabolite product is high, and the saccharomyces cerevisiae EnhSa-13 is rich in amino acid polypeptides and analogues, fatty acid and compounds thereof, linolenic acid and derivatives thereof, arachidic acid, carbohydrates and compounds thereof, fatty acid esters and the like. When being used as a feed additive, the feed additive can improve the activity and the quantity of beneficial microorganisms in rumen of ruminants, improve the utilization rate of feed, and improve the milk yield, the milk fat percentage and the DM apparent digestibility of dairy cows; the laying rate of laying hens and the quality of eggs are improved.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and in particular to a yeast postbiotic and a preparation method and application thereof. Background Art

[0002] Adding yeast agents to feed has certain beneficial effects on the growth of animal hosts, inflammatory diseases, and physiological abnormalities related to neurodevelopmental disorders, but the effects are unstable. On the one hand, the biological activity of yeast agents is affected by processing and storage; on the other hand, when exogenous yeast enters the animal intestine, the host needs to re-establish the microecological balance, and it is difficult to achieve results in a short period of time.

[0003] Yeast postbiotic products use inactivated yeast cells and their metabolites to nourish and stimulate the growth and reproduction of endogenous probiotics in the body. The aromatic substances and digestive enzymes contained in the metabolites can promote animal feeding and digestion, bactericidal substances can inhibit harmful bacteria, and cell wall polysaccharides can improve the body's immunity. They can also absorb mycotoxins and negative bacterial endotoxins in feed, reducing the harm caused to animals by adverse external factors. Therefore, improving the yield and quality of cells and their metabolites is an urgent problem to be solved. Summary of the invention

[0004] In view of this, the present application provides a yeast postbiotic to solve the problem of low content of bacteria and its metabolites and low content of effective ingredients in existing yeast postbiotic products.

[0005] In a first aspect, the present application provides a yeast postbiotic.

[0006] In an optional embodiment, the yeast postbiotics include Saccharomyces cerevisiae EnhSa-13 and its metabolites, and a carrier, wherein the content of the metabolites in the yeast postbiotics is greater than 1 wt %, and the carrier is a solid culture.

[0007] In an optional embodiment, among the metabolites, the proportion of amino acid polypeptides and analogs is more than 25wt%, the proportion of fatty acids and their derivatives is more than 15wt%, the proportion of linolenic acid and its derivatives is more than 5wt%, and the proportion of eicosanoids, fatty acid esters, diterpenoids or glycerophosphorylcholine is more than 1wt%.

[0008] In an optional embodiment, the crude protein content in the yeast postbiotics is 20.94wt% or more;

[0009] And / or, the content of acid-soluble protein in the yeast postbiotic is greater than 44.32 wt%.

[0010] The carrier comprises at least three of corn cob meal, corn germ meal, peanut meal, bran, palm meal and glutamic acid residue.

[0011] In a second aspect, the present application provides a method for preparing the yeast postbiotics.

[0012] In an optional embodiment, the preparation method comprises the following steps:

[0013] (1) After culturing Saccharomyces cerevisiae EnhSa-13, aerobic fermentation is performed using a low-concentration substrate sugar medium and a high-concentration glucose medium with feeding and feeding to obtain a yeast fermentation liquid; and molasses is added to perform a first anaerobic fermentation to form a first anaerobic fermentation liquid; the content of molasses in the low-concentration substrate sugar medium is less than 10wt%, and the content of glucose in the high-concentration glucose medium is more than 60wt%;

[0014] (2) The first anaerobic fermentation liquid obtained in step (1) is mixed with a carrier to perform a second anaerobic fermentation, and then the fermentation product is heated to autolyze the yeast cells to obtain a yeast postbiotic product.

[0015] In an optional embodiment, in step (1), the content of molasses in the low-concentration substrate sugar culture medium is 2wt%-10wt%;

[0016] And / or, the content of glucose in the high concentration glucose is 60wt%-80wt%;

[0017] and / or, based on the mass of the yeast fermentation liquid, the amount of molasses added is 2wt%-4wt%;

[0018] The temperatures of the expansion culture, aerobic fermentation and anaerobic fermentation are all 28°C-34°C.

[0019] In an optional embodiment, the low-concentration substrate sugar culture medium comprises: 2.0wt%-10.0wt% molasses, 0.5wt%-1.5wt% ammonium sulfate, 0.12wt%-0.20wt% yeast extract, 0.12wt%-0.18wt% magnesium sulfate, 0.006wt%-0.010wt% anhydrous calcium chloride, 0.4wt%-0.8wt% potassium dihydrogen phosphate, and 0.02wt%-0.04wt% defoaming agent;

[0020] And / or, the high concentration glucose comprises: 60wt%-80wt% glucose and 0.5wt%-1.5wt% yeast extract.

[0021] In an optional embodiment, in step (1), the cell concentration of Saccharomyces cerevisiae EnhSa-13 after expansion is 2×10 8 CFU / mL, after expansion, inoculated into the low-concentration substrate sugar medium at an inoculum size of 5vol%-15vol%;

[0022] And / or, when CER and OUR begin to decrease synchronously, start feeding high-concentration glucose medium, the ventilation ratio is 2-2.5VVM, the lower limit of dissolved oxygen is 30vol%, the RQ value is between 0.8-1, the initial flow rate is 300L / h-360L / h, when CER and OUR decrease synchronously again, the residual sugar is measured to be less than 1wt%, then the fermentation is ended;

[0023] And / or, the yeast fermentation liquid has a wet weight of 300 g / L or more and a viable bacterial count of 1.5-2 billion CFU / mL or more.

[0024] In an optional embodiment, in step (2), the volume ratio of the first anaerobic fermentation liquid to the added amount of the carrier is (1-1.5):1.

[0025] In a third aspect, the present application provides a use of the yeast postbiotic or the yeast postbiotic obtained by the preparation method in a feed additive.

[0026] In a fourth aspect, the present application provides a feed additive.

[0027] In an optional embodiment, the feed additive includes the yeast postbiotic or the yeast postbiotic obtained by the preparation method;

[0028] Optionally, the added amount of the yeast postbiotics is 0.1wt%-0.3wt% of the mass of the feed additive.

[0029] Compared with the prior art, the beneficial effects of this application are:

[0030] The present application provides a yeast postbiotic, including saccharomyces cerevisiae EnhSa-13 and its metabolites, and a carrier, wherein the content of the metabolites in the yeast postbiotic is greater than 1wt%, and the carrier is a solid culture. The saccharomyces cerevisiae strain EnhSa-13 and its metabolites in the yeast postbiotic product are high in content, and are rich in amino acid polypeptides and analogs, fatty acids and their complexes, linolenic acid and its derivatives, eicosanoids, carbohydrates and their complexes, fatty acid esters, and the like.

[0031] The present application also provides a method for preparing yeast postbiotics, wherein a fermentation broth with higher biomass is obtained in a low-sugar and high-sugar concentration flow-feeding feeding mode, and the product obtained after adding a carrier for fermentation contains more nutrients; the preparation method is simple, and the amount of glucose used is greatly reduced, thereby improving product quality while saving costs. As a feed additive, it can increase the activity and quantity of beneficial microorganisms in the rumen of ruminants, improve the utilization rate of feed, increase the milk production, milk fat rate and DM apparent digestibility of dairy cows, and improve the egg production rate and egg quality of laying hens. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 It is the wet weight of the different strains in Example 2 of the present application after 24 hours of shake flask fermentation.

[0034] Figure 2 This is the relative content ratio of the full spectrum metabolites of yeast postbiotics and fish meal in Experimental Example 1 of the present application.

[0035] Figure 3 The present invention shows the effects of adding the yeast postbiotic product obtained in Example 2 and the yeast culture product obtained in Comparative Example 1 to the basal diet on the rectal temperature (A) and respiratory rate (B) of dairy cows in Experimental Example 2 of the present application.

[0036] Figure 4 This is the effect on the milk production of dairy cows by adding the yeast postbiotic product obtained in Example 2 and the yeast culture product obtained in Comparative Example 1 to the basal diet in Experimental Example 2 of the present application.

[0037] Figure 5 The present invention is a test example 4 of the present application, in which the yeast postbiotics of the present invention are added, and the effects of no addition or the addition of an equal amount of common yeast culture on the milk yield (A), milk fat rate (B), DM apparent digestibility (C) and rumen microorganisms (D) of dairy cows. DETAILED DESCRIPTION

[0038] The following examples are provided for a better understanding of the present application, but are not limited to the best implementation mode described, and do not limit the content and protection scope of the present application. Any product identical or similar to the present application obtained by anyone under the inspiration of the present application or by combining the features of the present application with other prior arts shall fall within the protection scope of the present application.

[0039] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0040] In this application, the selection scope of "and / or" includes any one of two or more related listed items, and also includes any and all combinations of related listed items, and the said any and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. For example, "A and / or B" includes three parallel solutions of A, B and A+B.

[0041] In the present application, "plurality", "multiple", "multiple times", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0042] In this application, "optionally" refers to optional, that is, it refers to any one of the two parallel solutions of "yes" or "no". If there are multiple "options" in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "option" is independent.

[0043] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0044] In the present application, the percentage content, unless otherwise specified, refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.

[0045] In this application, when it comes to percentage concentration, unless otherwise specified, it refers to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of the component.

[0046] In the present application, wt% means weight percentage, and vol% means volume percentage.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0048] The present application is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application.

[0049] If no specific experimental steps or conditions are specified in the following experiments, the conventional experimental steps or conditions described in the literature in this field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0050] In the following experiments, the Saccharomyces cerevisiae strain Sa-10 was deposited in China General Microbiological Culture Collection Center with the deposit number: CGMCC NO.6120;

[0051] EnhSa-13 is deposited in China General Microbiological Culture Collection Center with the deposit number: CGMCCNO.24946.

[0052] In the following experiments, the YPD liquid medium includes the following components by mass percentage: 2% glucose, 2% tryptone, 1% yeast extract powder, and the balance is water; the YPD solid medium is based on the YPD liquid medium and adds 2wt% agar powder.

[0053] The molasses culture medium includes the following components by mass percentage: 14% sugarcane molasses, 1.0% ammonium sulfate, 0.16% yeast extract, 0.15% magnesium sulfate, 0.008% anhydrous calcium chloride, 0.15% potassium dihydrogen phosphate, 0.03% defoaming agent, and the balance is water.

[0054] Example 1 Strain Screening

[0055] This embodiment provides a strain screening method, which is as follows:

[0056] (1) Activation of strains: Five Saccharomyces cerevisiae strains Sa-10, EnhSa-13, Sa-16, Sa-17 and Sa-22 (Sa-16, Sa-17 and Sa-22 were all obtained from Saccharomyces cerevisiae strain Sa-10 by a combined mutagenesis screening method of diethyl sulfate and normal temperature and pressure plasma (ARTP)) preserved in glycerol tubes were streaked on YPD solid culture medium for activation at 30°C for 48 h. Then, a single activated colony was picked and inoculated into a shake flask containing 100 mL of YPD liquid culture medium for activation at 200 r / min, 30°C for 24 h.

[0057] (2) Shake flask fermentation: The liquid bacterial solution activated in step (1) (bacterial concentration of 2×10 8 CFU / mL) was inoculated into a shake flask containing 100 mL of molasses medium for fermentation, with an inoculation amount of 10 vol%, and culture conditions of 200 r / min, 30°C, and 24 h. Three parallels were made for each strain. In order to eliminate the instability of molasses itself, different batches of molasses were used for the experiments.

[0058] (3) 30L tank fermentation: The liquid bacterial solution activated in step (2) (bacterial concentration of 2×10 8 CFU / mL) was inoculated at an inoculum size of 10 vol.% into 500 mL YPD liquid culture medium for further expansion, the culture conditions were 200 r / min, 30°C, and cultured for 24 h. After that, the bacterial liquid was inoculated at an inoculum size of 10 vol% into a fermenter filled with 20 L molasses culture medium for fermentation, the culture conditions were 30°C, the initial rotation speed was 300 r / min, the ventilation ratio was 2VVM, and the fermentation was marked by the beginning of the rise of dissolved oxygen in the fermentation.

[0059] (4) Strain screening: Determine the wet weight of the bacteria after fermentation and record the relevant data, compare the differences between different strains, and screen out strains with better growth performance.

[0060] The wet weight determination method described therein is as follows: the fermentation broth is centrifuged to remove the upper liquid, leaving the bacterial cell sediment, and the excess water on the surface of the bacterial cell is gently absorbed using filter paper or absorbent paper until no water drips from the bacterial cell, and the mass of the bacterial cell measured in this way is used as the wet weight of the bacterial cell.

[0061] The results are as follows Figure 1 And as shown in Table 1.

[0062] Table 1 Parameters at the end of 30L tank fermentation

[0063]

[0064] Depend on Figure 1 The shake flask results show that under the same conditions, the wet weight of EnhSa-13 and Sa-16 increased significantly compared with the control strain Sa-10 after 24 hours of cultivation. From the results of the upper tank in Table 1, it can be seen that the fermentation of EnhSa-13 and Sa-22 ended in a shorter time (the dissolved oxygen suddenly began to rise sharply from below 10%) and the wet weight at the end of fermentation was higher. In summary, the growth performance of the cerevisiae yeast strain EnhSa-13 is strong, and this advantage has not changed in different batches of molasses and different fermentation equipment.

[0065] Example 2: Preparation of yeast postbiotics and detection of their metabolites

[0066] This embodiment provides a yeast postbiotic, which is prepared by the following steps:

[0067] (1) Seed culture: The cerevisiae strain EnhSa-13 preserved in a glycerol tube was streaked on a YPD solid culture medium for activation, and the culture condition was static culture at 30°C for 48 h. Then, a single activated colony was picked and inoculated into 100 mL of a primary YPD liquid culture medium, and the culture condition was 200 r / min, 30°C, and cultured for 24 h to obtain a primary seed solution. Then, the primary seed solution was inoculated into 500 mL of a secondary YPD liquid culture medium at an inoculum amount of 10 vol%, and the culture condition was 200 r / min, 30°C, and cultured for 24 h to obtain a secondary seed solution.

[0068] (2) Liquid fermentation: The secondary seed solution was inoculated in 300 L and 3 T fermentation tanks at an inoculum rate of 10 vol% for expansion. The culture conditions were 200 r / min, 30°C, 24 h, aeration ratio of 2VVM, tank pressure of 0.05 MPa, and the bacterial cell concentration was 2×10 8CFU / mL of bacterial liquid, and finally inoculated into a 30T fermenter filled with low-sugar medium at an inoculum of 10vol% for the sugar fermentation stage, the culture conditions were 150r / min, 30°C, tank pressure of 0.04MPa, ventilation ratio of 2VVM, and culture for 12h. During the culture process, an exhaust gas analyzer (Biological Institute of Shandong Academy of Sciences, FGA-3A) was connected to judge the start time of feeding by observing the changes in CER (carbon dioxide release rate) and OUR (oxygen uptake rate). When CER and OUR began to decrease synchronously, glucose feeding medium was started to be added, and the fed-batch culture stage was entered. The culture conditions were 180 r / min, 30°C, and a ventilation ratio of 2VVM. The rotation speed was set to be linked with the dissolved oxygen, the lower limit of dissolved oxygen was set to 30%, the addition time was 13 h, the initial flow rate was 330 L / h, and the feeding rate was gradually increased according to the RQ value (respiratory entropy) of the exhaust gas analyzer to stabilize the RQ value at 0.8-1, and the final flow rate was 600 L / h. When the feeding was completed and CER and OUR decreased synchronously again, the residual sugar was determined by the DNS method to be less than 1%, and the fermentation was completed, and a yeast fermentation broth with a wet weight of more than 300 g / L and a live yeast count of 1.5-2 billion CFU / mL was obtained. The yeast fermentation broth was then cooled to below 20°C, 3% (v / v) sterilized molasses was added thereto, the low speed was turned on at 50 r / min, sterile air was not passed, the fermentation tank pressure was maintained at 0.04 MPa, and fermented at 30°C for 24 hours to obtain yeast fermentation broth rich in metabolite precursors.

[0069] (3) Solid-state fermentation: The fermentation liquid containing the yeast EnhSa-13 cells and their metabolites obtained in step (2) is mixed evenly with the carrier at a mass ratio of 1.2:1, and the water content of the mixture is controlled at 40%, and solid-state anaerobic fermentation is carried out at 35°C for 72 hours. Then, the fermentation product is heated to 60°C and kept warm for 10 hours to allow the yeast cells to fully autolyze and break the cell wall. The plate live bacteria counting method is used to detect that there are basically no live bacteria. Then, the product is dried and crushed at 60°C to obtain a yeast postbiotic product with a significant fermentation flavor.

[0070] The low-sugar culture medium includes the following components by mass percentage: 2% molasses, 1.0% ammonium sulfate, 0.16% yeast extract, 0.15% magnesium sulfate, 0.008% anhydrous calcium chloride, 0.6% potassium dihydrogen phosphate, 0.03% defoaming agent, and the balance is water.

[0071] The glucose feed medium comprises the following components by mass percentage: 60% glucose, 1% yeast extract, and the balance water.

[0072] The carrier is 20% corn cob powder, 40% corn germ meal, 10% peanut meal, 17% bran, 10% palm meal and 3% glutamic acid residue.

[0073] (4) Full spectrum metabolism-LCMS metabolomics determination: Weigh 60 mg of yeast postbiotic product sample into a 1.5 mL centrifuge tube, add two small steel beads and 600 μL of methanol-water (V:V=7:3, containing mixed internal standard, 4 μg / mL); precool in a -40℃ refrigerator for 2 min, grind in a grinder (60 Hz, 2 min); extract in an ice-water bath for 30 min, stand overnight at -40℃; centrifuge at low temperature for 10 min (12000 r / min, 4℃), draw 150 μL of supernatant with a syringe, filter with a 0.22 μm organic phase pinhole filter, transfer to an LC injection vial, and store at -80℃ until LC-MS analysis. The analytical instrument for this experiment is a liquid chromatography-mass spectrometry system consisting of a Waters ACQUITY UPLC I-Class plus / Thermo QE ultra-high performance liquid phase tandem high-resolution mass spectrometer.

[0074] Chromatographic conditions: Chromatographic column: ACQUITY UPLC HSS T3 (100mm×2.1mm, 1.8um); Column temperature: 45°C; Mobile phase: A-0.1% formic acid aqueous solution, B-acetonitrile; A:B=1:1 isocratic elution; Flow rate: 0.35mL / min; Injection volume: 3μL. Three parallels were measured for each group of samples.

[0075] The above operation was performed simultaneously on the mixed fish meal formed by mixing the imported fish meal and the domestic fish meal in a mass ratio of 1:1. The data were then statistically analyzed and the two groups of samples were compared and analyzed.

[0076] The results are as follows Figure 2 As shown, the ordinate is the ratio of the component to the total metabolites (sequence number percent); the abscissa is the mixed fish meal and the yeast postbiotic product obtained in this example.

[0077] The results showed that the yeast postbiotic product of the present invention contained metabolites with a relative content greater than 1%, and the metabolites contained amino acid peptides and analogues (Amino acids peptides and analogues, 28.57%), fatty acids and their complexes (Fatty acids and conjugates, 18.95%), linolenic acid and its derivatives (Lineolic acids andderivatives, 7.32%), eicosanoids (Eicosanoids, 5.83%), carbohydrates and their complexes (Carbohydrates and carbohydrate conjugates, 3.98%), fatty acid esters (Fatty acidesters, 2.80%), diterpenoids (Diterpenoids, 2.30%), amines (Amines, 2.20%), pyrimidines and pyrimidine derivatives (Pyrimidines and pyrimidine derivatives, 1.83%), medium-chain hydroxy acids and their derivatives (Medium-chain hydroxy acids and The content of amino acid peptides and analogs is 1.14 times that of mixed fish meal. Peptides are important active substances in the nervous system, some of which have immunomodulatory activity. Small peptides can accelerate protein synthesis, promote mineral absorption, proliferate rumen microorganisms, and improve animal production performance. Fatty acids and their complexes are 2.64 times that of mixed fish meal, eicosanoids are 1.57 times that of mixed fish meal, linolenic acid and its derivatives are 13.78 times that of mixed fish meal, carbohydrates and their complexes are 2.18 times that of mixed fish meal, fatty acid esters are 4.49 times that of mixed fish meal, and medium-chain hydroxy acids and their derivatives are 9.27 times that of mixed fish meal. ω-6PUFAs such as linoleic acid and arachidonic acid, and ω-3PUFAs such as linolenic acid, eicosapentaenoic acid and docosahexaenoic acid are essential fatty acids for fish and shrimp. The fertility of mammals is closely related to the changes in fatty acids in follicles. Studies have found that high-quality oocytes often contain more linoleic acid and arachidonic acid. ω-3PUFAs have many physiological functions such as improving animal production and reproductive performance, immune regulation, and improving coat color and eggshell appearance.

[0078] Fish meal is widely used in animal production because of its rich nutrition and high biological value. According to the above results, the yeast postbiotic product of the present invention has certain advantages over fish meal, among which amino acid polypeptides and analogs, linolenic acid and its derivatives, fatty acid esters, medium-chain hydroxy acids and their derivatives are significantly higher than fish meal, and yeast postbiotics are also rich in yeast protein and unknown growth factors, which play core functions in the animal intestines, such as stimulating intestinal microbial activity, promoting nutrient digestion and absorption, and improving animal immunity.

[0079] Comparative Example 1

[0080] This embodiment provides a yeast postbiotic, which is prepared by the following steps:

[0081] (1) Seed culture: The brewer's yeast strain Sa-10 preserved in a glycerol tube was streaked and activated on a YPD solid culture medium, and the culture condition was static culture at 30°C for 48 h. Then, a single activated colony was picked and inoculated into 100 mL of a primary YPD liquid culture medium, and the culture condition was 200 r / min, 30°C, and cultured for 24 h to obtain a primary seed solution. Then, the primary seed solution was inoculated into 500 mL of a secondary YPD liquid culture medium at an inoculum amount of 10 vol%, and the culture condition was 200 r / min, 30°C, and cultured for 24 h to obtain a secondary seed solution.

[0082] (2) Liquid fermentation: The secondary seed solution was inoculated in 300 L and 3 T fermentation tanks at an inoculum rate of 10 vol% for expansion. The culture conditions were 200 r / min, 30°C, 24 h, aeration ratio of 2VVM, tank pressure of 0.05 MPa, and the bacterial cell concentration was 2×10 8 CFU / mL of bacterial liquid, and finally inoculated into a 30T fermenter filled with high base sugar medium at an inoculation rate of 10vol% for base sugar fermentation stage, the culture conditions are 150r / min, 30℃, tank pressure is 0.04MPa, ventilation ratio is 2VVM, and culture is 18h. When the dissolved oxygen begins to rise linearly, start to feed molasses feed medium, enter the feed culture stage, the culture conditions are 180r / min, 30℃, ventilation ratio is 2.5VVM, dissolved oxygen is controlled at 20%-40%, the flow time is 6h, the initial flow rate is 700L / h, when the dissolved oxygen rises linearly again, the residual sugar is determined by DNS method <1%, the fermentation is over, and the yeast fermentation liquid with a wet weight of more than 180g / L and a live yeast count of 800 million CFU / mL is obtained. The yeast fermentation liquid was then cooled to below 20°C, 8% (v / v) sterilized molasses medium was added thereto, the low speed was turned on at 50 r / min, sterile air was not passed, the fermentation tank pressure was maintained at 0.1 MPa, the fermentation time was 72 h, and liquid low-temperature anaerobic fermentation liquid was obtained.

[0083] The high-sugar molasses culture medium is composed of: 15% molasses, 1.0% ammonium sulfate, 0.16% yeast extract, 0.15% magnesium sulfate, 0.008% anhydrous calcium chloride, 0.15% potassium dihydrogen phosphate, and 0.03% defoaming agent.

[0084] The composition of the molasses feed medium is: 92% molasses, 4% ammonium sulfate, 0.2% yeast extract, 0.1% magnesium sulfate, 0.01% calcium chloride, 0.1% potassium dihydrogen phosphate, and 0.03% defoaming agent.

[0085] The composition of the molasses culture medium added in the liquid anaerobic fermentation process is: 12% molasses, 1.0% ammonium sulfate, 0.15% yeast extract, 0.04% magnesium sulfate, 0.006% anhydrous calcium chloride, and 0.05% potassium dihydrogen phosphate.

[0086] (3) Solid-state enzymatic hydrolysis aerobic fermentation: 10% (v / v) of sterilized molasses medium is added to the liquid low-temperature anaerobic fermentation bacterial liquid described in the previous step, mixed evenly, and then mixed with the solid medium at a mass ratio of 1:0.6, and hydrolase is added, the amount of low-temperature amylase is 6 U / g, the amount of neutral cellulase is 8 U / g, and the amount of acid protease is 12 U / g. The moisture content of the material is controlled at 40%, and the air humidity of the fermentation environment is 70%. The solid-state enzymatic hydrolysis process is carried out simultaneously with the aerobic fermentation. The aerobic fermentation is carried out at 32-35°C for 24 hours to obtain a solid-state aerobic fermentation product.

[0087] The molasses medium is composed of the following substances by mass percentage: 90% sugarcane molasses, 0.2% ammonium sulfate, 0.2% yeast extract, 0.03% magnesium sulfate, 0.002% calcium chloride, and 0.04% potassium dihydrogen phosphate. The solid medium is composed of the following substances by mass percentage: 20% corn flour, 30% bran, 25% corn germ meal, 15% corn cob powder, and 10% corn steep liquor powder.

[0088] (4) Solid-state anaerobic fermentation: The fermentation box containing the solid-state enzymatic aerobic fermentation product is sealed, the fermentation temperature is adjusted to 35°C, and the anaerobic fermentation is carried out for 72 hours. After the fermentation is completed, the solid-state anaerobic fermentation product is obtained. After high-temperature cell wall breaking and autolysis, low-temperature drying, crushing and packaging, the yeast culture product is obtained.

[0089] Test Example 1

[0090] The crude protein and acid-soluble protein contents of the yeast postbiotic product obtained in Example 2 of the present invention and the yeast culture product obtained in Comparative Example 1 were tested as follows:

[0091] The crude protein was determined according to the Kjeldahl method of GB / T 6432-2018, and the acid-soluble protein was determined according to the method of NY / T3801-2020. The results are shown in Table 2.

[0092] Table 2 Crude protein and acid-soluble protein test results

[0093]

[0094] High molecular weight proteins are easily precipitated under acidic conditions, and protein hydrolysates (acid-soluble proteins) with relatively small molecular weights are soluble in acidic solutions (including peptides and free amino acids). At the same time, after being treated with acidic solutions, the effects of organic nitrogen such as urea on protein content can be removed. As can be seen from Table 2, the yeast postbiotic product of the present invention not only has a high crude protein content, but also a high acid-soluble protein content, indicating that the product contains more small molecular peptides and free amino acids, which is more conducive to animal digestion and absorption, and thus has a stronger effect on animals.

[0095] Test Example 2

[0096] The effects of the yeast postbiotic product obtained in Example 2 of the present invention and the yeast culture product obtained in Comparative Example 1 on the adaptability and production performance of ruminants to heat stress were tested as follows:

[0097] Experimental method: 45 Holstein cows with similar milk production in mid-lactation, average lactation days of 150 days, two parities, and similar health conditions were selected. According to the principle of single-factor experimental design, the cows were randomly divided into 3 groups, with 15 cows in each group. One of the groups was the control group, which was fed with a basic diet; the remaining two groups were experimental groups, which were fed with a basic diet + yeast postbiotics in Example 2 (added at 30 g / head / d) and a basic diet + yeast culture of Comparative Example 1 (added at 100 g / head / d), respectively. The experimental period was 60 days. The basic diet is a total mixed diet (TMR) formulated according to the dairy cow feeding standards (NY / T34-2004). The measured indicators include rectal temperature, respiratory rate and milk production. The results are as follows Figure 3 and Figure 4 shown.

[0098] Figure 3 The effect of adding the yeast postbiotic product obtained in Example 2 of the present invention (30 g / head / d) and the yeast culture product obtained in Comparative Example 1 (100 g / head / d) to the basal diet on the rectal temperature (A) and respiratory rate (B) of dairy cows. The same lowercase letters indicate no statistically significant difference (P>0.05), and different lowercase letters indicate a statistically significant difference (P<0.05).

[0099] Figure 4The effect of adding the yeast postbiotic product obtained in Example 2 of the present invention (30 g / head / d) and the yeast culture product obtained in Comparative Example 1 (100 g / head / d) to the basal diet on the milk production of dairy cows. The same lowercase letters indicate no statistically significant difference (P>0.05), and different lowercase letters indicate a statistically significant difference (P<0.05).

[0100] Heat stress has a significant impact on the development of my country's dairy industry. In the past decade of heat stress research, THI = 72 has been widely adopted as the threshold for heat stress in dairy cows. In this experiment, THI was above 72 in almost all periods, so it was determined that these cows were in a heat stress environment. During heat stress, the most common physiological indicators used to assess heat stress in dairy cows are respiratory rate and rectal temperature. Animals expel excess heat from their bodies by increasing their respiratory rate. Some researchers have proposed that rectal temperature is a sensitive indicator of heat stress. Small changes in rectal temperature can profoundly affect the function of tissues, organs and endocrine system. Such changes may reduce lactation and cause various growth problems.

[0101] according to Figure 3 As shown, the rectal temperature and respiratory rate of the cows added with Example 2 (30g / head / d) and Comparative Example 1 (100g / head / d) in the basal diet were significantly lower than those of the control group (P<0.05), and there was no significant difference between the two groups (P>0.05). This indicates that the addition of the yeast postbiotics of the present invention can reduce the rectal temperature and respiratory rate of dairy cows during heat stress, and may improve the adaptability of dairy cows to heat stress. Moreover, the amount of Example 2 added is less than 1 / 3 of that of Comparative Example 1, but it has a similar effect to Comparative Example 1, indicating that the yeast postbiotics of the present invention have a stronger effect on animals. According to Figure 4 As shown, the milk production of Example 2 (30 g / head / d) and Comparative Example 1 (100 g / head / d) added to the basal diet was significantly higher than that of the control group (P<0.05), and the difference between the two groups was not significant (P>0.05). Similarly, it is shown that the yeast postbiotics of the present invention can increase the milk production of dairy cows, and the effect on animals is stronger than that of Comparative Example 1.

[0102] Test Example 3

[0103] The probiotic effects of the yeast postbiotic product obtained in Example 2 of the present invention on key rumen microorganisms in ruminants were tested as follows:

[0104] Selenomonas ruminantium is the dominant bacteria in the rumen, accounting for 21%-50% of the total bacteria in the rumen. It is one of the important rumen microorganisms involved in rumen fermentation and plays an important role in the production of propionic acid, a precursor of ruminant sugar production. Therefore, this test example explored the effect of the yeast postbiotics of the present invention on Selenomonas ruminantium in vitro.

[0105] (1) Experimental materials and strains: yeast postbiotics, Selenomonas ruminantium.

[0106] (2) Test method: Weigh 2 g of the yeast postbiotic product of Example 2, dilute to 20 mL with distilled water, sonicate for 1 h, centrifuge at 12000 r / min for 5 min, and take the supernatant to pass through a membrane (0.22 μm) for later use. Add the obtained yeast postbiotic extract to a special culture medium for ruminant Selenomonas at different ratios (5 vol%, 10 vol%, 20 vol%), test its OD600 value, place it in anaerobically at 37°C for 48 h, measure its OD600 value, and use the difference ΔOD600 between the two as an indicator for judging the growth-promoting effect.

[0107] The results are shown in Table 3.

[0108] Table 3 The probiotic effect of yeast postbiotic extract on Lunamonas ruminants

[0109]

[0110] The yeast postbiotic extract of the present invention added with 5%-20% has a probiotic effect on the ruminant Lunamonas, which plays an important role in regulating the rumen microorganisms of ruminants.

[0111] Test Example 4

[0112] The application of the yeast postbiotic product obtained in Example 2 of the present invention in ruminants was tested as follows:

[0113] (1) Experimental animals and materials: The experimental animals were 45 Holstein cows with an average lactation period of 20 days and 3 parities. The yeast postbiotics were the product of Example 2 of the present invention.

[0114] (2) Experimental design: According to the principle of single-factor experimental design, the cows were randomly divided into 3 groups, with 5 replicates in each group and 3 cows in each replicate. The control group of cows was fed a basal diet, the experimental group A added 30g / head / d of a certain brand of yeast culture to the basal diet, and the experimental group B added 30g / head / d of the yeast postbiotic product of Example 2 of the present invention to the basal diet. The experimental period was 90 days. The measured indicators included milk yield, milk fat rate, DM apparent digestibility and the effect on rumen microorganisms of dairy cows. The results are as follows Figure 5 shown.

[0115] Compared with the control group, the yeast postbiotics of the present invention increased milk production by 3%, milk fat rate by 13%, DM apparent digestibility by 4%, and the relative content of rumen microorganisms was also significantly increased. At the same time, compared with ordinary yeast culture, the yeast postbiotics of the present invention have certain advantages in various indicators, especially in terms of rumen microorganisms and DM apparent digestibility.

[0116] In summary, the yeast postbiotic product of the present invention can significantly improve the production performance of dairy cows, and all indicators have certain advantages compared with other yeast cultures.

[0117] Test Example 5

[0118] The application of the yeast postbiotic product obtained in Example 2 of the present invention in poultry was tested as follows:

[0119] (1) Experimental animals and materials: The experimental animals were 52-week-old healthy Hy-Line Brown laying hens with an average body weight of (2.04±0.11) kg, totaling 384. The yeast postbiotics used were the products of the present invention.

[0120] (2) Experimental design: According to the principle of single-factor experimental design, laying hens were randomly divided into 4 groups, with 8 replicates in each group, 12 hens in each replicate, and 3 hens in one cage. According to statistics, there were no significant differences in the body weight, egg production rate, average egg weight, etc. among the treatment groups (P>0.05). The pre-test period was 1 week and the formal test period was 12 weeks. With the basal diet as the control, the experimental groups were supplemented with 1000 mg / kg (experimental group 1, 0.1%), 2000 mg / kg (experimental group 2, 0.2%) and 3000 mg / kg (experimental group 3, 0.3%) of yeast postbiotics on the basis of the basal diet (Table 4). The measured indicators included egg quality, blood immune indicators, cecal microorganisms, and egg storage quality.

[0121] The results are shown in Table 5.

[0122] Table 4 Diet composition and nutritional level of laying hens (air-dried basis)

[0123]

[0124] 1 The nutritional content values ​​are calculated according to the 31st edition of NRC.

[0125] 2Premix (provided per kilogram of feed): VA, 12,500 IU; VD3, 4,125 IU; VE, 15 IU; VK, 2 mg; VB1, 1 mg; VB2, 8.5 mg; VB6, 8 mg; VB12, 5 mg; calcium pantothenate, 50 mg; niacin, 32.5 mg; biotin, 2 mg; folic acid, 5 mg; choline, 500 mg; Mn, 65 mg; I, 1 mg; Fe, 60 mg; Cu, 8 mg; Zn, 66 mg; Na2SO4, 4 g.

[0126] Table 5 Effects of adding yeast postbiotics to the diet on egg quality of laying hens (52-64wk) (n=8)

[0127]

[0128]

[0129] 1 P-values: A is one-way ANOVA with Duncan's multiple comparisons; L and Q are regression analyses of linear and quadratic effects of yeast culture.

[0130] As the addition level of yeast postbiotics increased, protein height (week 6, P = 0.05; week 9, P = 0.01; week 12, P = 0.04) and Haugh units (week 6, P = 0.05; week 9, P = 0.02; week 12, P = 0.04) showed a quadratic upward trend. Among them, compared with the control group, the addition of 0.1%-0.3% yeast postbiotics significantly increased the protein height and Haugh units in week 9 (P < 0.05) and significantly increased the Haugh units in week 12 (P < 0.05). As the addition level of yeast postbiotics increased, eggshell thickness (week 9, P = 0.02; week 12, P < 0.01) and eggshell strength (week 12, P = 0.01) showed a quadratic upward trend. Among them, compared with the control group, the addition of 0.2% and 0.3% yeast postbiotics significantly increased the eggshell thickness at week 9 (P = 0.02); the addition of 0.1% and 0.2% yeast postbiotics significantly increased the eggshell thickness (P < 0.01) and eggshell strength (P = 0.01) at week 12 of the experiment. In addition, the addition of 0.3% yeast postbiotics to the feed significantly increased the egg shape index of the eggs at week 9 of the experiment (P < 0.05). In summary, the addition of yeast postbiotics to the diet improves the egg white quality and eggshell quality of eggs.

[0131] The results are shown in Table 6.

[0132] Table 6 Effect of adding yeast postbiotics to the diet on serum immunoglobulin levels (n=8)

[0133]

[0134] According to the results of production performance and egg quality indexes, the control group and experimental group 1 were selected to determine the serum inflammatory factor content. At the end of the 12th week of the positive trial period, one chicken with a body weight close to the average was selected for each repetition, and 5 mL of blood was collected from the sterile wing vein of the fasting chicken. The blood was left to stand at room temperature for 2 hours, and centrifuged at 3 000r / min for 10 minutes to separate the serum. The ELISA kit was used to determine the content of immunoglobulin M (IgM), immunoglobulin A (IgA), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin 10 (IL-10) and tumor necrosis factor-α (TNF-α) in the serum. Compared with the control group, the addition of 0.1% yeast postbiotics to the diet significantly reduced the content of the proinflammatory factor IL-6 (P<0.05), and had no significant effect on the content of other immunoglobulins (P>0.05).

[0135] The results are shown in Table 7.

[0136] Table 7 Differential microorganisms in the cecum between the control group and the yeast postbiotic group (%, n = 8)

[0137]

[0138] Yeast postbiotics added to feed significantly reduced the relative abundance of Patescibacteria, Saccharimonadia and unclassified_p__Firmicutes in the cecal digesta of laying hens (P<0.05), but significantly increased the relative abundance of Fusobacteriota and Fusobacteriia in the cecal digesta of laying hens (P<0.05). At the family level, yeast postbiotics added to feed significantly increased the relative abundance of Peptococcaceae, Fusobacteriaceae, Clostridiaceae, Muribaculaceae and Anaerofustaceae in the cecal digesta of laying hens (P<0.05), and reduced the relative abundance of unclassified_p__Firmicutes under Firmicutes (P<0.05). Among them, Clostridiaceae can participate in the metabolism of various carbohydrates and produce short-chain fatty acids such as acetic acid, propionic acid and butyric acid after fermentation. At the genus level, the addition of yeast postbiotics to feed significantly increased the relative abundance of Anaerofustis, Fusobacterium and Eubacterium_brachy_group in the cecal chyme of laying hens (P<0.05).

[0139] The results are shown in Table 8.

[0140] Table 8 Effects of adding yeast postbiotics to the diet on short-chain fatty acids in cecal digesta (n=8)

[0141]

[0142] Compared with the control group, the addition of 0.1% yeast postbiotics to the diet significantly increased the content of butyric acid and isobutyric acid in the cecal digesta (P<0.05), and the content of other short-chain fatty acids showed an increasing trend (0.50≤P<0.10). This indicates that yeast postbiotics increase the content of short-chain fatty acids in the cecum by changing the composition of laying hens' cecal microorganisms, thereby regulating the nutritional metabolism and health of laying hens.

[0143] The results are shown in Table 9.

[0144] Table 9 Effects of adding yeast postbiotics to the diet on egg quality during storage of laying hens (64 weeks) (n = 8, 25 ° C)

[0145]

[0146]

[0147] Compared with the control group, the albumen height of eggs stored at 25℃ for 14 days in the group supplemented with 0.2% yeast postbiotics was significantly increased (P<0.05), and the yolk color of eggs stored at 25℃ for 14 days in the groups supplemented with 0.2% and 0.3% yeast postbiotics was significantly increased (P<0.05). The addition of yeast postbiotics to the diet had an increasing trend on the Haugh unit of eggs stored at 25℃ for 14 days (P=0.08), and an increasing trend on the pH value of egg white of eggs stored at 25℃ for 7 days (P=0.08). The addition of yeast postbiotics to the diet linearly reduced the eggshell ratio of eggs stored at 25℃ for 7 days (P>0.05), and linearly increased the yolk color of eggs stored at 25℃ for 14 days (P<0.05). The addition of yeast culture to the diet twice increased the albumen height and yolk color of eggs stored at 25℃ for 14 days (P<0.05), and twice reduced the pH value of egg white of eggs stored at 25℃ for 7 days (P<0.05).

[0148] In summary, adding yeast postbiotics to the diet can improve the production performance of laying hens, improve the quality of eggshells and egg whites, regulate the composition of the cecal microorganisms of laying hens, increase the content of short-chain fatty acids in the cecum by increasing the relative abundance of short-chain fatty acid-producing bacteria, increase the albumen height and yolk color of eggs stored at 25°C for 14 days, and extend the storage period of eggs.

[0149] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection created by this application.

Claims

1. A yeast postbiotic, characterized in that: The invention comprises saccharomyces cerevisiae EnhSa-13 and its metabolites and a carrier, wherein the content of the metabolites in the yeast postbiotics is greater than 1wt%.

2. The yeast postbiotic according to claim 1, characterized in that Among the metabolites, amino acid polypeptides and analogs account for more than 25wt%, fatty acids and their derivatives account for more than 15wt%, linolenic acid and its derivatives account for more than 5wt%, and eicosanoids, fatty acid esters, diterpenoid compounds or glycerolphosphorylcholine account for more than 1wt%.

3. The yeast postbiotic according to claim 1, characterized in that The crude protein content of the yeast postbiotics is greater than 20.94wt%; and / or, the content of acid-soluble protein in the yeast postbiotic is greater than 44.32 wt %; And / or, the carrier includes at least three of corn cob meal, corn germ meal, peanut meal, bran, palm meal, and glutamic acid residue.

4. A method for preparing the yeast postbiotic according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: (1) After culturing Saccharomyces cerevisiae EnhSa-13, aerobic fermentation is performed using a low-concentration substrate sugar medium and a high-concentration glucose medium with feeding and feeding to obtain a yeast fermentation liquid; and molasses is added to perform a first anaerobic fermentation to form a first anaerobic fermentation liquid; the content of molasses in the low-concentration substrate sugar medium is less than 10wt%, and the content of glucose in the high-concentration glucose medium is more than 60wt%; (2) The first anaerobic fermentation liquid obtained in step (1) is mixed with a carrier to perform a second anaerobic fermentation, and then the fermentation product is heated to autolyze the yeast cells to obtain a yeast postbiotic product.

5. The preparation method according to claim 4, characterized in that: In step (1), the content of molasses in the low-concentration substrate sugar culture medium is 2wt%-10wt%; And / or, the content of glucose in the high concentration glucose is 60wt%-80wt%; and / or, based on the mass of the yeast fermentation liquid, the amount of molasses added is 2wt%-4wt%; The temperatures of the expansion culture, aerobic fermentation and anaerobic fermentation are all 28°C-34°C.

6. The preparation method according to claim 5, characterized in that: The low-concentration substrate sugar culture medium comprises: 2.0wt%-10.0wt% molasses, 0.5wt%-1.5wt% ammonium sulfate, 0.12wt%-0.20wt% yeast extract, 0.12wt%-0.18wt% magnesium sulfate, 0.006wt%-0.010wt% anhydrous calcium chloride, 0.4wt%-0.8wt% potassium dihydrogen phosphate, and 0.02wt%-0.04wt% defoamer; And / or, the high concentration glucose comprises: 60wt%-80wt% glucose and 0.5wt%-1.5wt% yeast extract.

7. The preparation method according to claim 4, characterized in that: In step (1), the cell concentration of Saccharomyces cerevisiae EnhSa-13 after expansion is 2×10 8 CFU / mL, after expansion, inoculated into the low-concentration substrate sugar medium at an inoculum size of 5vol%-15vol%; And / or, when CER and OUR begin to decrease synchronously, start feeding high-concentration glucose medium, the ventilation ratio is 2-2.5VVM, the lower limit of dissolved oxygen is 30vol%, the RQ value is between 0.8-1, the initial flow rate is 300L / h-360L / h, when CER and OUR decrease synchronously again, the residual sugar is measured to be less than 1wt%, then the fermentation is ended; And / or, the yeast fermentation liquid has a wet weight of 300 g / L or more and a viable bacterial count of 1.5-2 billion CFU / mL or more.

8. The preparation method according to claim 4, characterized in that: In step (2), the volume ratio of the first anaerobic fermentation liquid to the added amount of the carrier is (1-1.5):

1.

9. Use of the yeast postbiotic according to any one of claims 1 to 3 or the yeast postbiotic obtained by the preparation method according to any one of claims 4 to 8 in feed additives.

10. A feed additive, characterized in that: The yeast postbiotic comprising any one of claims 1 to 3 or the yeast postbiotic obtained by the preparation method of any one of claims 4 to 8; Optionally, the added amount of the yeast postbiotics is 0.1wt%-0.3wt% of the mass of the feed additive.

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