CN107937292B - Saccharomyces cerevisiae culture and fermentation process thereof - Google Patents
Saccharomyces cerevisiae culture and fermentation process thereof Download PDFInfo
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Abstract
The invention discloses a saccharomyces cerevisiae culture and a fermentation process thereof. The fermentation process of the saccharomyces cerevisiae culture comprises the following steps: 1) seed culture in a shake flask; 2) liquid state deep aerobic fermentation; 3) liquid state low temperature anaerobic fermentation; 4) solid state enzymolysis aerobic fermentation; 5) solid anaerobic fermentation; 6) and (5) performing solid high-temperature wall breaking autolysis to obtain the saccharomyces cerevisiae culture. The invention makes the yeast ferment fully in the liquid fermentation stage and the solid fermentation stage, and produces more metabolites, thereby improving the product effect. The saccharomyces cerevisiae culture prepared by the method provided by the invention has unique aromatic odor, and after high-temperature wall breaking, probiotic components such as yeast cell walls and contents can exert the effects of insufficient food calling, stress resistance, intestinal probiotic and the like which are not possessed by live bacteria.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a saccharomyces cerevisiae culture and a fermentation process thereof.
Background
The Saccharomyces cerevisiae culture is a microecological preparation, and is formed by fully fermenting specific yeast strains according to a specific process and a specific culture medium. The microecological product is different from a product containing active yeast cells, and mainly comprises extracellular metabolites generated by saccharomyces cerevisiae in the fermentation process, in addition, the microecological product also comprises a denatured solid matrix after fermentation, cell contents after wall breaking of the saccharomyces cerevisiae, yeast cell walls and the like, and is rich in B-group vitamins, minerals, small peptides, organic acids, vitamins, nucleotides, mannooligosaccharides, beta-glucan, unknown growth factors and the like, and the substances are used as nutrient substrates of probiotics in animal gastrointestinal tracts, so that the growth of the probiotics can be effectively stimulated, intestinal flora is adjusted, the utilization rate of the animal to feed is effectively improved, and further the production performance of the animal is improved. It is a micro-ecological feed additive integrating multiple effects of nutrition, health care and the like.
The general production process of yeast culture mainly comprises a liquid fermentation method and a solid fermentation method, the existing technology only adopts one fermentation method, but the liquid submerged fermentation method is simply and singly utilized, more metabolic active products of yeast cells rather than the yeast are obtained, only the solid fermentation method is adopted, the inoculation amount is often insufficient, the yeast activity is low, the solid fermentation is insufficient, liquid-solid two-phase fermentation combining the two fermentation methods is adopted, but the liquid phase is only used as the propagation stage of the yeast, the metabolic products of the liquid anaerobic fermentation are not carried out, and the low-temperature anaerobic fermentation can promote the synthesis of aromatic substances such as organic acids, esters, alcohols, ketones, terpenes and the like.
Disclosure of Invention
In view of the above, the present invention provides a saccharomyces cerevisiae culture and a fermentation process thereof, wherein the saccharomyces cerevisiae is fully fermented in both a liquid fermentation stage and a solid fermentation stage to produce more metabolites, thereby improving the product effect.
Based on the above purpose, the fermentation process of the saccharomyces cerevisiae culture provided by the invention comprises the following steps:
1) and (3) seed culture in a shaking flask: firstly, inoculating saccharomyces cerevisiae into a liquid culture medium for primary and secondary amplification culture to obtain a shake flask seed culture solution;
2) liquid-state deep aerobic fermentation: inoculating the obtained shake flask seed culture solution into a base liquid culture medium, aerating for aerobic fermentation, and then adding a feed supplement culture medium for continuous aerobic fermentation to obtain a yeast liquid aerobic fermentation bacterium solution;
3) liquid low-temperature anaerobic fermentation: adding molasses culture medium into the obtained yeast liquid aerobic fermentation bacterial liquid, and carrying out anaerobic fermentation to obtain low-temperature anaerobic fermentation bacterial liquid;
4) solid-state enzymolysis aerobic fermentation: supplementing the molasses culture medium into the obtained low-temperature anaerobic fermentation bacterial liquid, then mixing the molasses culture medium with a solid culture medium, adding hydrolase, and carrying out solid aerobic fermentation to obtain a solid aerobic fermentation product;
5) solid anaerobic fermentation: carrying out solid anaerobic fermentation on the obtained solid aerobic fermentation product to obtain a solid anaerobic fermentation product;
6) solid-state high-temperature wall breaking autolysis: and (3) carrying out heat preservation treatment on the obtained solid anaerobic fermentation product to ensure that yeast cells are fully autolyzed to obtain the saccharomyces cerevisiae culture.
As an embodiment of the invention, the method also comprises a step of low-temperature drying the saccharomyces cerevisiae culture after the step 6).
Optionally, the saccharomyces cerevisiae culture is subjected to short-time airflow drying by an airflow dryer to obtain a dried yeast culture, and the dried yeast culture is crushed, packaged into bags and stored in shade.
As an embodiment of the present invention, in step 1), the liquid medium is a yeast extract peptone glucose medium (also called YPD liquid medium) having a composition of: each 1L of water contains 10-20 g of glucose, 10-30 g of tryptone and 5-15 g of yeast extract powder.
As an embodiment of the invention, in the step 2), the obtained shake flask seed culture solution is inoculated into the base liquid culture medium in an inoculum size of 4-10% by volume percentage for propagation;
the aerobic fermentation conditions in aeration are as follows: the temperature is 28-30 ℃, the rotating speed is 150-200 rpm, the tank pressure is 0.03-0.04 Mpa, the aeration ratio (vvm for short: the ratio of aeration per minute to the actual feed liquid volume of the tank body) is 2:1, and the fermentation time is 18-24 hours;
the conditions for continuously carrying out aerobic fermentation by adding the supplemented medium are as follows: 20-40% of dissolved oxygen, 30-34 ℃ of fermentation temperature, 200-250 rpm of rotation speed, 0.03-0.04 Mpa of tank pressure, 2-2.5: 1 of ventilation volume (tank volume v/sterile air volume v), and 5-6 hours of fermentation time;
the base sugar liquid culture medium comprises the following substances in percentage by mass: 10-16% of cane molasses, 0.8-1.5% of ammonium sulfate, 0.08-0.16% of yeast extract, 0.1-0.2% of magnesium sulfate, 0.005-0.02% of anhydrous calcium chloride, 0.05-0.2% of potassium dihydrogen phosphate, 0.03-0.06% of defoaming agent and the balance of water; adjusting the initial pH value of the base liquid culture medium to 6.0-6.5;
the feed medium consists of the following substances in percentage by mass: 90-95% of cane molasses, 2-5% of ammonium sulfate, 0.1-0.3% of yeast extract, 0.05-0.20% of magnesium sulfate, 0.008-0.015% of calcium chloride, 0.05-0.15% of potassium dihydrogen phosphate, 0.02-0.04% of defoaming agent and the balance of water;
the number of viable bacteria in the liquid aerobic zymophyte liquid of the microzyme is 6-10 hundred million CFU/ml.
The defoaming agent used in the invention comprises four types of natural oil ester, higher alcohol, fatty acid, polyether, organic silicon and the like, and the polyether and the organic silicon have superior performance and wide application. Among these, tetraol polyether (PPE) is the most effective.
As an embodiment of the invention, in the step 3), 8-15% by volume of molasses culture medium is added into the obtained yeast liquid aerobic fermentation broth;
the anaerobic fermentation conditions are as follows: the fermentation temperature is 15-20 ℃, the rotating speed is 50-60 rpm, the pressure of the fermentation tank is kept to be not higher than 0.1Mpa in a closed manner, and the fermentation time is 48-72 hours;
the molasses culture medium comprises the following substances in percentage by mass: 8-15% of cane molasses, 0.8-1.5% of ammonium sulfate, 0.08-0.16% of yeast extract, 0.01-0.06% of magnesium sulfate, 0.005-0.02% of anhydrous calcium chloride, 0.01-0.05% of potassium dihydrogen phosphate and the balance of water.
As an embodiment of the invention, in the step 4), 6-12% by volume of the molasses culture medium is added into the obtained low-temperature anaerobic fermentation bacterial liquid; the molasses culture medium comprises the following substances in percentage by mass: 75-90% of cane molasses, 0.1-0.6% of ammonium sulfate, 0.1-0.3% of yeast extract, 0.01-0.05% of magnesium sulfate, 0.001-0.005% of calcium chloride, 0.01-0.05% of monopotassium phosphate and the balance of water, and the pH is adjusted to be 5.8;
the yeast liquid low-temperature anaerobic fermentation bacterial liquid supplemented with the molasses culture medium and the solid culture medium are mixed according to the mass ratio of 1: 0.5-0.8, the stacking height after mixing is 0.2-0.6 m, the water content is 35-45%, the air humidity of a fermentation environment is 70-80%, the temperature of solid aerobic fermentation is 32-38 ℃, and the fermentation time is 12-24 hours;
the number of live microzyme in the solid aerobic fermentation product is 4-10 hundred million CFU/g;
the hydrolase is at least one selected from low-temperature amylase, neutral cellulase and acid protease, the enzyme activity of the low-temperature amylase is more than or equal to 2000U/ml, the enzyme activity of the neutral cellulase is more than or equal to 2000U/ml, and the enzyme activity of the acid protease is more than or equal to 50000U/ml.
Definition of amylase activity units: the amount of enzyme required to hydrolyze starch to 1mg of reducing sugar per minute at 37 ℃ and pH 6.8. During amylase activity determination, starch is converted into reducing sugar within certain initial action time at 37 ℃ and pH6.8, and then the reducing sugar is subjected to colorimetric determination to obtain the generation amount of the reducing sugar through the action of the starch and a DNS reagent, so that the initial speed of enzyme reaction, namely the activity of the enzyme is calculated. 1U-1 mg reducing sugar/min ml enzyme.
Definition of activity unit of neutral cellulase: 1ml of liquid enzyme, at (50. + -. 0.1) ℃ and pH6.0, 1h hydrolyses the sodium carboxymethyl cellulose substrate to give an amount of reducing sugar equivalent to 1mg of glucose, expressed as 1 enzyme activity unit in U/ml.
Acid protease activity unit location: hydrolysis of casein at pH3.6 and a temperature of 40 ℃ per minute to yield 1. mu.g tyrosine per minute is defined as one unit of protease activity.
As an embodiment of the present invention, in step 4), the solid medium is composed of the following substances by mass percent:
preparing a solid culture medium according to 20-30% of corn flour, 30-40% of bran, 20-30% of corn germ meal, 10-20% of corn steep liquor dry powder and 10-20% of corncob powder;
or preparing a solid culture medium according to 20-40% of corn flour, 20-30% of wheat middling, 20-30% of DDGS (dried distillers grains and soluble substances thereof) and 20-40% of corncob powder;
or preparing a solid culture medium according to 10-20% of corn flour, 25-40% of wheat middling, 10-20% of corn bran and 20-35% of corn protein feed;
or preparing a solid culture medium according to 20-35% of corn flour, 20-40% of wheat middling, 20-30% of corn germ meal, 10-20% of corn steep liquor dry powder and 10-20% of corn cob powder.
As an embodiment of the invention, in step 5), the solid anaerobic fermentation is performed by using a closed fermentation box;
the fermentation temperature of the solid anaerobic fermentation is 32-38 ℃, and the fermentation time is 48-72 h;
the number of live microzyme in the solid anaerobic fermentation product is 2-6 hundred million CFU/ml, and the content of acid soluble protein is 35-40%.
In step 6), the temperature of the heat preservation treatment is 45-60 ℃ for 6-12 hours, the temperature of the solid anaerobic fermentation product is raised to the temperature, the heat preservation treatment is carried out to ensure that the yeast is completely autolyzed, and the dilution coating flat plate detection basically has no viable bacteria.
In the present invention, each of the culture media used needs to be sterilized.
In the invention, the saccharomyces cerevisiae is saccharomyces cerevisiae Sa-10 which is preserved in China general microbiological culture collection center with the preservation number of CGMCC No. 6120.
The invention also provides a saccharomyces cerevisiae culture which is prepared according to the fermentation process of the saccharomyces cerevisiae culture.
From the above, the fermentation process of the saccharomyces cerevisiae culture provided by the invention adopts five-stage fermentation modes of liquid deep aerobic fermentation, liquid low-temperature anaerobic fermentation, solid enzymatic aerobic fermentation, solid anaerobic fermentation and high-temperature autolysis, fully utilizes the advantages of the liquid aerobic fermentation and the solid fermentation, wherein the liquid deep aerobic fermentation is high-density culture, provides enough saccharomycete cell number for the subsequent stage, and then adopts the liquid low-temperature anaerobic fermentation, which is favorable for the full anaerobic fermentation of saccharomycete in the liquid state, so that the liquid stage can generate more abundant metabolites, and then the solid enzymatic aerobic fermentation is carried out, the aerobic proliferation of the saccharomycete is carried out while the raw material is subjected to enzymatic hydrolysis, the enzymolysis can fully degrade the substances which can not be utilized by the saccharomycete in the solid raw material, and is more favorable for the propagation and metabolism of the saccharomycete in the solid fermentation stage, then solid anaerobic fermentation is carried out, yeast further fully carries out anaerobic fermentation to obtain rich metabolic components, the smell of solid raw materials is effectively improved, the solid raw materials generate strong acid flavor and have excellent food calling performance, high-temperature autolysis is carried out at the last stage, yeast cells are crushed, yeast cell contents are fully released, and finally, the functional yeast culture with high nutritional value and unique activity is prepared.
Drawings
FIG. 1 is a flow chart of the Saccharomyces cerevisiae culture production process of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to specific embodiments and the accompanying drawings.
In the following embodiments, the saccharomyces cerevisiae is saccharomyces cerevisiae Sa-10, which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 6120.
Example 1
As an embodiment of the invention, the fermentation process of the yeast culture comprises the following steps:
(1) shake flask seed culture (primary, secondary seed liquid): activating a saccharomyces cerevisiae strain Sa-10 preserved at an ultralow temperature (for example, liquid nitrogen at-196 ℃), inoculating 1-2 rings of the activated saccharomyces cerevisiae into a primary shake flask culture medium, performing shake culture on the activated saccharomyces cerevisiae strain in a shaking table at 180-200 rpm at 28-30 ℃ for 17-24 hours to obtain a primary seed solution, then inoculating the primary seed solution into a secondary shake flask culture medium, and performing shake culture on the primary seed solution in the shaking table at 180-200 rpm at 28-30 ℃ for 20-24 hours to obtain a shake flask seed culture solution.
Wherein, the primary shake flask culture medium and the secondary shake flask culture medium are YPD liquid culture media, and the YPD liquid culture media comprise the following components: 20g of glucose, 20g of tryptone, 10g of yeast extract powder, 1000ml of water and natural pH; the culture medium is sterilized at 116 deg.C for 30 min.
(2) Liquid-state deep aerobic fermentation: inoculating the obtained shake flask seed culture solution into a base sugar liquid culture medium according to the inoculation amount of 10% (v/v) for a base sugar fermentation culture stage, adjusting the culture condition to 30 ℃, setting the rotation speed to 150 rpm, setting the tank pressure to 0.04Mpa, setting the aeration ratio to be 2:1 (vvm: the ratio of aeration per minute to the actual feed liquid volume of the tank body), culturing for about 18 hours, transferring to a fed-batch culture medium when dissolved oxygen starts to rise linearly, entering the fed-batch culture stage, setting the rotation speed to 200rpm, setting the aeration ratio to be 2.5:1 (tank volume v/sterile air volume v), setting the dissolved oxygen to be 20-40%, feeding for 6 hours, setting the flow rate to be 700L/h, and finishing fermentation when the dissolved oxygen rises linearly again, wherein the residual sugar is less than 1%, thereby obtaining the yeast liquid aerobic fermentation broth.
The sugar solution culture medium comprises the following substances in percentage by mass: 15% of cane molasses, 1.0% of ammonium sulfate, 0.16% of yeast extract, 0.15% of magnesium sulfate, 0.008% of anhydrous calcium chloride, 0.15% of monopotassium phosphate, 0.03% of defoaming agent and the balance of water, and adjusting the initial pH of the culture medium to 6.2; the feed supplement culture medium is a molasses liquid culture medium and comprises the following substances in percentage by mass: cane molasses 92%, ammonium sulfate 4%, yeast extract 0.2%, magnesium sulfate 0.1%, calcium chloride 0.01%, potassium dihydrogen phosphate 0.1%, defoaming agent 0.03%, and water in balance, and the initial pH of the culture medium is adjusted to 6.0. The pH is not regulated in the fermentation process, the biomass wet weight reaches 180g/L, the number of live yeast bacteria reaches 8 hundred million CFU/ml, and the liquid submerged aerobic zymocyte liquid is obtained.
(3) Liquid low-temperature anaerobic fermentation: cooling the liquid deep aerobic zymogen liquid of the microzyme to 18 ℃, then adding 8% (v/v) of sterilized molasses culture medium into the liquid deep aerobic zymogen liquid, starting low-speed stirring at 50rpm, stopping sterile air, keeping the pressure of a fermentation tank at 0.1Mpa, and fermenting for 72h to obtain the liquid low-temperature anaerobic zymogen liquid.
The supplemented molasses culture medium comprises the following substances in percentage by mass: 12% of cane molasses, 1.0% of ammonium sulfate, 0.15% of yeast extract, 0.04% of magnesium sulfate, 0.006% of anhydrous calcium chloride, 0.05% of monopotassium phosphate and the balance of water, and adjusting the pH to 5.8;
(4) solid-state enzymolysis aerobic fermentation: adding 10% (V/V) of sterilized molasses culture medium into the liquid low-temperature anaerobic fermentation bacterial liquid obtained in the previous step, immediately mixing the liquid low-temperature anaerobic fermentation bacterial liquid and the solid culture medium according to the mass ratio of 1:0.6, adding hydrolase with the dosage of 6U/g low-temperature amylase, 8U/g neutral cellulase and 12U/g acid protease per gram of solid culture medium, controlling the water content of the materials at 40%, controlling the air humidity of the fermentation environment at 70%, synchronously performing the solid enzymolysis process and aerobic fermentation, the live bacteria number of the initial saccharomycetes in the solid aerobic fermentation product is 2-4 hundred million CFU/g, the solid aerobic fermentation product is put into a solid fermentation box, the thickness of the material placed in the fermentation box is 0.4m, and carrying out aerobic fermentation for 24 hours at the temperature of 32-35 ℃ to obtain a solid aerobic fermentation product, wherein the number of the final live saccharomycetes in the solid aerobic fermentation product is 8 hundred million CFU/g.
The molasses culture medium comprises the following substances in percentage by mass: 90% of cane molasses, 0.2% of ammonium sulfate, 0.2% of yeast extract, 0.03% of magnesium sulfate, 0.002% of calcium chloride, 0.04% of monopotassium phosphate and the balance of water, and the pH value is adjusted to 5.8;
the solid culture medium comprises the following substances in percentage by mass: the solid culture medium is prepared from 20% of corn flour, 30% of bran, 25% of corn germ meal, 15% of corncob meal and 10% of corn steep liquor dry powder.
In the step, corn flour and bran mainly provide a carbon source, corn germ meal and corn steep liquor dry powder mainly provide a nitrogen source, and corncob powder mainly serves as an adsorption component and effectively adsorbs bacteria liquid.
5) Solid anaerobic fermentation: and (3) sealing the fermentation box filled with the solid enzymatic aerobic fermentation product, adjusting the fermentation temperature to 35 ℃, carrying out anaerobic fermentation for 72 hours until the acid soluble protein content reaches 38.28%, and obtaining the solid anaerobic fermentation product after the fermentation is finished, wherein the number of live microzyme in the solid anaerobic fermentation product is 4 hundred million CFU/ml.
6) Solid-state high-temperature wall breaking autolysis: and heating the product after anaerobic fermentation to 60 ℃ for heat preservation treatment for 10 hours to ensure that yeast cells are fully autolyzed and wall-broken, diluting and coating a flat plate to detect that no yeast exists after the yeast autolyzes completely, and obtaining the autolyzed yeast culture, wherein the content of the yeast cell wall polysaccharide mannan reaches 1.5 percent.
7) Low-temperature drying, crushing and packaging: and (3) drying the autolyzed yeast culture by using an airflow drying agent at low temperature in a short time to obtain a dried yeast culture, crushing the dried yeast culture, packaging the dried yeast culture into bags, and storing the bags in the shade to obtain a finished yeast culture product which has the fermentation fragrance and is acidic.
Example 2
As an embodiment of the invention, the fermentation process of the yeast culture comprises the following steps:
(1) shake flask seed culture (primary, secondary seed liquid): activating a saccharomyces cerevisiae strain Sa-10 preserved at an ultralow temperature (for example, liquid nitrogen at-196 ℃), inoculating 1-2 rings of the activated saccharomyces cerevisiae into a primary shake flask culture medium, performing shake culture on the activated saccharomyces cerevisiae strain in a shaking table at 180-200 rpm at 28-30 ℃ for 17-24 hours to obtain a primary seed solution, then inoculating the primary seed solution into a secondary shake flask culture medium, and performing shake culture on the primary seed solution in the shaking table at 180-200 rpm at 28-30 ℃ for 20-24 hours to obtain a shake flask seed culture solution.
Wherein, the primary shake flask culture medium and the secondary shake flask culture medium are YPD liquid culture media, and the YPD liquid culture media comprise the following components: 10g of glucose, 10g of tryptone, 5g of yeast extract powder, 1000ml of water and natural pH; the culture medium is sterilized at 116 deg.C for 30 min. .
(2) Liquid-state deep aerobic fermentation: and inoculating the obtained shake flask seed culture solution into a base sugar liquid culture medium by 8% (v/v) inoculation amount to perform a base sugar fermentation culture stage, wherein the culture condition temperature is 30 ℃, the rotation speed is set to be 180 r/min, the tank pressure is 0.03Mpa, the aeration ratio is 2:1 (vvm: the ratio of aeration per minute to the actual feed liquid volume of the tank body), the culture time is 18 hours, when the dissolved oxygen rises linearly, the residual sugar is less than 1%, the feeding of the feed medium is started, the temperature is 30 ℃, the aeration ratio is 2.5:1 (tank volume v/sterile air volume v), the dissolved oxygen is 20-40%, the feeding time is 5 hours, the flow rate is 840L/h, when the dissolved oxygen starts to rise linearly again, and the fermentation is finished, so that the yeast liquid aerobic fermentation liquid is prepared.
The sugar solution culture medium comprises the following substances in percentage by mass: 13% of cane molasses, 0.8% of ammonium sulfate, 0.1% of yeast extract, 0.15% of magnesium sulfate, 0.01% of anhydrous calcium chloride, 0.15% of monopotassium phosphate, 0.03% of defoaming agent and the balance of water, and adjusting the initial pH of the culture medium to be 6.2; the feed medium consists of the following substances in percentage by mass: 90% of cane molasses, 3.5% of ammonium sulfate, 0.15% of yeast extract, 0.2% of magnesium sulfate, 0.01% of calcium chloride, 0.2% of monopotassium phosphate, 0.03% of defoaming agent and the balance of water, and the initial pH of the culture medium is adjusted to be 6.2. The pH is not regulated in the fermentation process, the dissolved oxygen is controlled to be 20-40 percent in the fermentation process, the biomass wet weight reaches 170g/L, the viable count of yeast reaches 7 hundred million CFU/ml, and the liquid-state deep aerobic zymocyte liquid is obtained.
(3) Liquid low-temperature anaerobic fermentation: cooling the liquid deep aerobic zymocyte liquid of the microzyme to 20 ℃, then adding 9% (v/v) of sterilized molasses culture medium into the liquid deep aerobic zymocyte liquid, starting low-speed stirring at 30rpm, stopping sterile air, keeping the pressure of a fermentation tank at 0.1Mpa, and fermenting for 48 hours to obtain the liquid low-temperature anaerobic zymocyte liquid.
The supplemented molasses culture medium comprises the following substances in percentage by mass: 9% of cane molasses, 0.9% of ammonium sulfate, 0.12% of yeast extract, 0.04% of magnesium sulfate, 0.008% of anhydrous calcium chloride, 0.04% of potassium dihydrogen phosphate and the balance of water, and the pH value is adjusted to 6.0;
(4) solid-state enzymolysis aerobic fermentation: adding 7% (V/V) of sterilized molasses culture medium into the liquid low-temperature anaerobic fermentation bacterial liquid obtained in the previous step, immediately mixing the yeast liquid aerobic fermentation bacterial liquid and the solid culture medium according to the mass ratio of 1:0.5, adding hydrolase, wherein the dosage of the hydrolase is that a certain amount of enzyme activity is added into each gram of solid culture medium, the dosage of low-temperature amylase is 8U/g, the dosage of neutral cellulase is 8U/g, the water content of the material is controlled to be 35%, the air humidity of the fermentation environment is 70-80%, the initial viable count of yeast in the solid aerobic fermentation product is 2 hundred million CFU/g, the solid aerobic fermentation product is put into a solid fermentation box, the thickness of the material placed in the fermentation box is not more than 0.6m, and carrying out aerobic fermentation for 16 hours at the temperature of 32-35 ℃ to obtain a solid aerobic fermentation product, wherein the number of the final live saccharomycetes in the solid aerobic fermentation product is 6 hundred million CFU/g.
The molasses supplementing culture medium comprises the following substances in percentage by mass: 82% of cane molasses, 0.5% of ammonium sulfate, 0.2% of yeast extract, 0.01% of magnesium sulfate, 0.001% of calcium chloride, 0.01% of monopotassium phosphate and the balance of water, and the pH value is adjusted to 5.8;
the solid culture medium comprises the following substances in percentage by mass: preparing a solid culture medium by using 30% of corn flour, 30% of wheat middling, 20% of DDGS (distillers dried grains with solubles) and 20% of corncob powder;
in the step, the corn flour and the wheat middling mainly provide a carbon source, the DDGS mainly provides a nitrogen source, and the corncob flour mainly serves as an adsorption component and effectively adsorbs bacteria liquid.
5) Solid anaerobic fermentation: and (3) sealing the fermentation box filled with the solid enzymatic aerobic fermentation product, keeping an anaerobic state, adjusting the fermentation temperature to about 35 ℃, carrying out anaerobic fermentation for 60 hours, and finishing fermentation to obtain the solid anaerobic fermentation product, wherein the content of acid soluble protein reaches 36.46%.
6) Solid-state high-temperature wall breaking autolysis: and heating the product after anaerobic fermentation to 55 ℃ for heat preservation treatment for 12h to ensure that yeast cells are fully autolyzed and wall-broken, diluting and coating a flat plate to detect that no yeast exists after the yeast autolyzes completely, and obtaining the autolyzed yeast culture, wherein the content of the yeast cell wall polysaccharide mannan reaches 1%.
7) Low-temperature drying, crushing and packaging: drying the autolyzed yeast culture at low temperature to obtain dried yeast culture, pulverizing, packaging into bags, and storing in shade to obtain final product with fermentation fragrance and acidity.
Example 3
As another embodiment of the present invention, the fermentation process of the yeast culture comprises the following steps:
(1) shake flask seed culture (primary, secondary seed liquid): activating a saccharomyces cerevisiae strain Sa-10 preserved at an ultralow temperature (for example, liquid nitrogen at-196 ℃), inoculating 1-2 rings of the activated saccharomyces cerevisiae into a primary shake flask culture medium, performing shake culture on the activated saccharomyces cerevisiae strain in a shaking table at 180-200 rpm at 28-30 ℃ for 17-24 hours to obtain a primary seed solution, then inoculating the primary seed solution into a secondary shake flask culture medium, and performing shake culture on the primary seed solution in the shaking table at 180-200 rpm at 28-30 ℃ for 20-24 hours to obtain a shake flask seed culture solution.
Wherein, the primary shake flask culture medium and the secondary shake flask culture medium are YPD liquid culture media, and the YPD liquid culture media comprise the following components: 15g of glucose, 30g of tryptone, 15g of yeast extract powder, 1000ml of water and natural pH; the culture medium is sterilized at 116 deg.C for 30 min. .
(2) Liquid-state deep aerobic fermentation: inoculating the obtained shake flask seed culture solution into a base sugar liquid culture medium by an inoculation amount of 10% (v/v) for a base sugar fermentation culture stage, wherein the culture condition temperature is 30 ℃, the rotating speed is set to be 200 r/min, the aeration ratio is 2:1 (vvm: the ratio of aeration per minute to the actual feed liquid volume of the tank body), the tank pressure is 0.04Mpa, the culture time is 16 hours, when the dissolved oxygen rises linearly, the residual sugar is less than 1%, the feeding of the feed medium is started, the temperature is 30 ℃, the aeration ratio is 2:1 (tank volume v/sterile air volume v), the dissolved oxygen is 20-40%, the feeding time is 5.5 hours, the flow rate is 765L/h, when the dissolved oxygen starts to rise linearly again, and the fermentation is finished, so that the yeast liquid aerobic fermentation liquid is prepared.
The sugar solution culture medium comprises the following substances in percentage by mass: 11% of cane molasses, 1.0% of ammonium sulfate, 0.12% of yeast extract, 0.15% of magnesium sulfate, 0.02% of anhydrous calcium chloride, 0.15% of monopotassium phosphate, 0.04% of defoaming agent and the balance of water, and adjusting the initial pH of the culture medium to 6.3; the feed medium consists of the following substances in percentage by mass: cane molasses 90%, ammonium sulfate 3.5%, yeast extract 0.15%, magnesium sulfate 0.2%, calcium chloride 0.012%, potassium dihydrogen phosphate 0.2%, defoaming agent 0.03%, and water in balance, and the initial pH of the culture medium is adjusted to 6.2. The pH is not regulated in the fermentation process, the biomass wet weight reaches 180g/L, the number of live yeast bacteria reaches 8 hundred million CFU/ml, and the liquid submerged aerobic zymocyte liquid is obtained.
(3) Liquid low-temperature anaerobic fermentation: cooling the liquid deep aerobic zymogen liquid of the microzyme to 22 ℃, then adding 6% (v/v) of the sterilized molasses culture medium into the liquid deep aerobic zymogen liquid, starting low-speed stirring at 60rpm, stopping sterile air, keeping the pressure of a fermentation tank at 0.1Mpa, and fermenting for 60 hours to obtain the liquid low-temperature anaerobic zymogen liquid.
The supplemented molasses culture medium comprises the following substances in percentage by mass: 10% of cane molasses, 1.2% of ammonium sulfate, 0.14% of yeast extract, 0.03% of magnesium sulfate, 0.008% of calcium chloride, 0.03% of monopotassium phosphate and the balance of water, and the pH value is adjusted to 6.0;
(4) solid-state enzymolysis aerobic fermentation: 8 percent (V/V) of molasses culture medium which is sterilized is added into the liquid low-temperature anaerobic fermentation bacterial liquid in the last step, the liquid aerobic fermentation bacterial liquid of the microzyme and the solid culture medium are immediately mixed according to the mass ratio of 1:0.5, adding hydrolase, wherein the dosage of the hydrolase is that a certain amount of enzyme activity is added into each gram of solid culture medium, the dosage of low-temperature amylase is 6U/g, the dosage of acid protease is 10U/g, the water content of the material is controlled to be 35%, the air humidity of the fermentation environment is 70-80%, the initial viable count of yeast in the solid aerobic fermentation product is 2 hundred million CFU/g, the solid aerobic fermentation product is put into a solid fermentation box, the thickness of the material placed in the fermentation box is not more than 0.6m, and carrying out aerobic fermentation for 16 hours at the temperature of 32-35 ℃ to obtain a solid aerobic fermentation product, wherein the number of the final live saccharomycetes in the solid aerobic fermentation product is 6 hundred million CFU/g.
The molasses supplementing culture medium comprises the following substances in percentage by mass: 86% of cane molasses, 0.5% of ammonium sulfate, 0.2% of yeast extract, 0.03% of magnesium sulfate, 0.002% of calcium chloride, 0.04% of monopotassium phosphate and the balance of water, and the pH value is adjusted to 5.8;
the solid culture medium comprises the following substances in percentage by mass: preparing a solid culture medium according to 20% of corn flour, 30% of wheat middling, 20% of corn bran and 30% of corn protein feed;
in the step, corn flour and wheat middling mainly provide a carbon source, corn protein feed mainly provides a nitrogen source, and corn husks mainly serve as an adsorption component and effectively adsorb bacteria liquid.
5) Solid anaerobic fermentation: and (3) sealing the fermentation box filled with the solid enzymatic aerobic fermentation product, adjusting the fermentation temperature to about 32 ℃, carrying out anaerobic fermentation for 48 hours, and finishing the fermentation to obtain the solid anaerobic fermentation product, wherein the content of acid soluble protein reaches 35.86%.
6) Solid-state high-temperature wall breaking autolysis: and heating the product after anaerobic fermentation to 50 ℃ for heat preservation treatment for 15h to ensure that yeast cells are fully autolyzed and wall-broken, diluting and coating a flat plate to detect that no yeast exists after the yeast autolyzes completely, and obtaining the autolyzed yeast culture, wherein the content of the yeast cell wall polysaccharide mannan reaches 1.2%.
7) Low-temperature drying, crushing and packaging: drying the autolyzed yeast culture at low temperature to obtain dried yeast culture, pulverizing, packaging into bags, and storing in shade to obtain final product with fermentation fragrance and acidity.
Comparative example 1
The liquid low-temperature anaerobic fermentation step in the embodiment 1 of the invention is omitted, and the rest is the same as the embodiment 1, and the steps are as follows:
(1) shake flask seed culture (primary, secondary seed liquid): activating a saccharomyces cerevisiae strain Sa-10 preserved at an ultralow temperature (for example, liquid nitrogen at-196 ℃), inoculating 1-2 rings of the activated saccharomyces cerevisiae into a primary shake flask culture medium, performing shake culture on the activated saccharomyces cerevisiae strain in a shaking table at 180-200 rpm at 28-30 ℃ for 17-24 hours to obtain a primary seed solution, then inoculating the primary seed solution into a secondary shake flask culture medium, and performing shake culture on the primary seed solution in the shaking table at 180-200 rpm at 28-30 ℃ for 20-24 hours to obtain a shake flask seed culture solution.
Wherein, the primary shake flask culture medium and the secondary shake flask culture medium are YPD liquid culture media, and the YPD liquid culture media comprise the following components: 20g of glucose, 20g of tryptone, 10g of yeast extract powder, 1000ml of water and natural pH; the culture medium is sterilized at 116 deg.C for 30 min.
(2) Liquid-state deep aerobic fermentation: inoculating the obtained shake flask seed culture solution into a base sugar liquid culture medium by 8% (v/v) inoculation amount to perform a base sugar fermentation culture stage, adjusting the culture condition to 30 ℃, setting the rotation speed to 150 rpm, setting the tank pressure to 0.04Mpa, setting the aeration ratio to be 2:1 (vvm: the ratio of aeration per minute to the actual feed liquid volume of the tank body), culturing for about 18 hours, transferring to a fed-batch culture medium when dissolved oxygen starts to rise linearly, entering the fed-batch culture stage, setting the rotation speed to 200rpm, setting the aeration ratio to be 2.5:1 (tank volume v/sterile air volume v), setting the dissolved oxygen to be 20-40%, feeding for 6 hours, setting the flow rate to be 700L/h, and finishing fermentation when the dissolved oxygen rises linearly again, wherein the residual sugar is less than 1%, thereby obtaining the yeast liquid submerged aerobic fermentation bacterial liquid.
The sugar solution culture medium comprises the following substances in percentage by mass: 13% of cane molasses, 0.8% of ammonium sulfate, 0.15% of yeast extract, 0.1% of magnesium sulfate, 0.01% of anhydrous calcium chloride, 0.1% of monopotassium phosphate, 0.03% of defoaming agent and the balance of water; the feed medium consists of the following substances in percentage by mass: 90% of cane molasses, 3.5% of ammonium sulfate, 0.15% of yeast extract, 0.1% of magnesium sulfate, 0.01% of calcium chloride, 0.1% of monopotassium phosphate, 0.03% of defoaming agent and the balance of water; the initial pH of the medium was adjusted to 6.2. The pH is not regulated in the fermentation process, the dissolved oxygen in the fermentation process is controlled to be 20-40%, the biomass wet weight reaches 160g/L, and the number of the live yeast bacteria reaches 6 hundred million CFU/ml.
(3) Solid anaerobic fermentation: mixing the solid culture medium and the obtained yeast liquid submerged aerobic fermentation bacteria liquid according to the mass ratio of 1:0.5, putting into a fermentation box for sealing treatment, adjusting the fermentation temperature to about 35 ℃, carrying out anaerobic fermentation for 72 hours, and finishing the fermentation to obtain a solid anaerobic fermentation product.
The solid culture medium comprises the following substances in percentage by mass: preparing a solid culture medium by using 30% of corn flour, 30% of wheat middling, 20% of corn germ meal, 10% of corn steep liquor dry powder and 10% of corncob powder;
in the step, corn flour and wheat middling mainly provide a carbon source, corn germ meal and corn steep liquor dry powder mainly provide a nitrogen source, and corncob powder is mainly used as an adsorption component and effectively adsorbs bacteria liquid.
4) Low-temperature drying, crushing and packaging: drying the obtained solid anaerobic fermentation product at low temperature to obtain dry yeast culture, pulverizing, packaging into bags, and storing in shade to obtain final product of yeast culture.
Comparative example 2
The solid-state enzymatic aerobic fermentation in the embodiment 1 of the invention is omitted, and the other steps are the same as the embodiment 1 of the invention, specifically as follows:
(1) shake flask seed culture (primary, secondary seed liquid): activating a saccharomyces cerevisiae strain Sa-10 preserved at an ultralow temperature (for example, liquid nitrogen at-196 ℃), inoculating 1-2 rings of the activated saccharomyces cerevisiae into a primary shake flask culture medium, performing shake culture on the activated saccharomyces cerevisiae strain in a shaking table at 180-200 rpm at 28-30 ℃ for 17-24 hours to obtain a primary seed solution, then inoculating the primary seed solution into a secondary shake flask culture medium, and performing shake culture on the primary seed solution in the shaking table at 180-200 rpm at 28-30 ℃ for 20-24 hours to obtain a shake flask seed culture solution.
Wherein, the primary shake flask culture medium and the secondary shake flask culture medium are YPD liquid culture media, and the YPD liquid culture media comprise the following components: 20g of glucose, 20g of tryptone, 10g of yeast extract powder, 1000ml of water and natural pH; the culture medium is sterilized at 116 deg.C for 30 min.
(2) Liquid-state deep aerobic fermentation: and inoculating the yeast seed culture solution into a base sugar liquid culture medium by an inoculation amount of 8% (v/v) for a base sugar fermentation culture stage, wherein the culture condition temperature is 30 ℃, the rotation speed is set to be 200rpm, the tank pressure is 0.03MPa, the culture time is 18 hours, when dissolved oxygen rises linearly, residual sugar is less than 1%, feeding of a supplemented medium is started, the temperature is 30 ℃, the dissolved oxygen is 20-40%, the feeding time is 6 hours, the flow rate is 700L/h, when the dissolved oxygen rises linearly again, the fermentation is finished, and the yeast liquid deep aerobic fermentation broth is prepared.
The sugar solution culture medium comprises the following substances in percentage by mass: 13% of cane molasses, 0.8% of ammonium sulfate, 0.15% of yeast extract, 0.1% of magnesium sulfate, 0.01% of anhydrous calcium chloride, 0.1% of monopotassium phosphate, 0.03% of defoaming agent and the balance of water; the feed medium consists of the following substances in percentage by mass: 90% of cane molasses, 3.5% of ammonium sulfate, 0.15% of yeast extract, 0.1% of magnesium sulfate, 0.01% of calcium chloride, 0.1% of monopotassium phosphate, 0.03% of defoaming agent and the balance of water; the initial pH of the medium was adjusted to 6.2. The pH is not regulated in the fermentation process, the dissolved oxygen in the fermentation process is controlled to be 20-40%, the biomass wet weight reaches 160g/L, and the number of the live yeast bacteria reaches 6 hundred million CFU/ml.
(3) Liquid low-temperature anaerobic fermentation: cooling the liquid aerobic zymogen liquid of the microzyme to 18 ℃, then supplementing 8% (v/v) of sterilized molasses culture medium into the liquid aerobic zymogen liquid, starting low-speed stirring at 50rpm, stopping sterile air, keeping the pressure of a fermentation tank at 0.1Mpa, and fermenting for 72 hours to obtain the liquid low-temperature anaerobic zymogen liquid.
The supplemented molasses culture medium comprises the following substances in percentage by mass: 7 percent of cane molasses, 0.5 percent of ammonium sulfate, 0.15 percent of yeast extract, 0.02 percent of magnesium sulfate, 0.002 percent of calcium chloride, 0.02 percent of monopotassium phosphate and the balance of water, and the pH value is adjusted to 5.8;
(4) solid anaerobic fermentation: mixing the solid culture medium and the obtained liquid low-temperature anaerobic fermentation bacteria liquid according to the mass ratio of 1:0.6, putting the mixture into a fermentation box for sealing treatment, adjusting the fermentation temperature to about 35 ℃, carrying out anaerobic fermentation for 48 hours, and finishing the fermentation to obtain a solid anaerobic fermentation product, wherein the content of acid soluble protein is 28.42%.
The solid culture medium comprises the following substances in percentage by mass: preparing a solid culture medium by using 30% of corn flour, 30% of wheat middling, 20% of corn germ meal, 10% of corn steep liquor dry powder and 10% of corncob powder;
in the step, corn flour and wheat middling mainly provide a carbon source, corn germ meal and corn steep liquor dry powder mainly provide a nitrogen source, and corncob powder is mainly used as an adsorption component and effectively adsorbs bacteria liquid.
5) Low-temperature drying, crushing and packaging: drying the obtained solid anaerobic fermentation product at low temperature to obtain dry yeast culture, pulverizing, packaging into bags, and storing in shade to obtain final product of yeast culture.
The organic acid content of the liquid fermentation broth obtained by the liquid low-temperature anaerobic fermentation in step (3) in examples 1-3 of the present invention and the fermentation broth obtained by the liquid deep aerobic fermentation in step (2) in comparative examples 1-2 were tested, and the results are shown in the following table.
The organic acid determination method comprises the following steps: the instrument was shimadzu LC-2030, column: c18 column, 4.6mm X250 mm, 5 μm, or equivalent performance column, analytical conditions: elution is carried out isocratically for 20min with a 0.1% phosphoric acid solution-methanol 97.5+2.5 (vol.%) mobile phase, column temperature: 40 ℃, sample introduction: 10 mu L, the detection wavelength is 210nm, and the content of organic acid in the liquid low-temperature anaerobic fermentation product is measured. The results are shown in Table 1.
TABLE 1 organic acid test results
Note: the organic acid can effectively reduce the pH value of the digestate, inhibit the propagation of harmful bacteria and improve the palatability of the feed.
The yeast cultures obtained in examples 1-3 and comparative example 1 were tested for nucleotide content.
The nucleotide determination method comprises the following steps: shimadzu LC-2030 and UV detector, analysis conditions: mobile phase: phosphate buffer solution: methanol (0.01mol/L) 1000:40, flow rate 1.0ml/min, column oven 25 ℃, detection wavelength: 254nm, sample size: 10ul, the nucleotide content in the yeast culture obtained in each example and comparative example was determined. The results are shown in Table 2.
TABLE 2 detection of nucleotides in finished Yeast culture products
Note: the nucleotide is used as an essential substance for cell synthesis, and has physiological functions of protecting intestinal mucosa, enhancing immunity of organisms and the like.
The yeast cultures obtained in examples 1-3 and comparative example 2 were tested for polypeptide content. The results are shown in Table 3.
TABLE 3 polypeptide detection results
| Test items | Example 1 | Example 2 | Example 3 | Comparative example 2 |
| Polypeptide content (%) | 38.28 | 36.46 | 35.86 | 28.42 |
Note: the polypeptide content is the percentage of the polypeptide in the total protein, and the polypeptide is an active substance, so that the polypeptide is more favorable for digestion and absorption of animals.
Therefore, compared with the prior art, the fermentation process of the yeast culture provided by the invention has the following advantages:
(1) the invention adopts the saccharomyces cerevisiae with excellent performance selected by the prior knowledge as the strain, the strain is safe and reliable, and the raw materials of cane molasses, corn flour, bran, wheat middling, corn germ meal, corn bran, DDGS, corn cob meal and the like are all bulk raw materials, and are mostly agricultural product processing byproducts, and the price is low.
(2) The invention adopts a new method combining two fermentation modes of yeast liquid fermentation, solid state fermentation and aerobic fermentation and anaerobic fermentation, and the organic acid content, the nucleotide content and the polypeptide content of the product obtained by fermentation are obviously higher than those of a comparative example through five-stage fermentation modes of liquid deep aerobic fermentation, liquid low-temperature anaerobic fermentation, solid state enzymolysis aerobic fermentation, solid state anaerobic fermentation and high-temperature autolysis.
(3) The method adopts the enzymolysis treatment of the solid culture medium and the synchronous aerobic fermentation and propagation of the saccharomycetes, utilizes high-activity enzyme substances to decompose substances which are difficult to utilize by the saccharomycetes in the solid culture medium, and simultaneously carries out the aerobic fermentation of the saccharomycetes, and compared with the comparative proportion 2, the nucleotide content in the saccharomyces cerevisiae cultures obtained by the treatment method is obviously higher than that in the comparative proportion in the saccharomyces cerevisiae cultures of the examples 1 to 3, so that the quality of the saccharomyces cerevisiae cultures is effectively improved.
(4) The invention is subjected to liquid anaerobic fermentation, the organic acid content is rich, compared with comparative example 1, the organic acid content is obviously improved, the pH value of the initial stage is 4.6-5.0 during the solid fermentation of the inoculated saccharomycetes, the fermentation environment is acidic, the growth of harmful bacteria can be effectively inhibited, and the product quality is effectively ensured.
(5) All zymophyte liquid is mixed into a solid culture medium, and is continuously fermented to prepare a finished product of the saccharomyces cerevisiae culture, a fermentation product can be directly used without separation and purification, no waste liquid and waste residue are discharged, and the environmental protection requirement is met.
The saccharomyces cerevisiae culture prepared by the method provided by the invention has unique aromatic odor, and after high-temperature wall breaking, probiotic components such as yeast cell walls and contents can exert the effects of insufficient food calling, stress resistance, intestinal probiotic and the like which are not possessed by live bacteria.
Those of ordinary skill in the art will understand that: the invention is not to be considered as limited to the specific embodiments thereof, but is to be understood as being modified in all respects, all changes and equivalents that come within the spirit and scope of the invention.
Claims (4)
1. A fermentation process of a saccharomyces cerevisiae culture is characterized by comprising the following steps:
1) and (3) seed culture in a shaking flask: firstly, inoculating saccharomyces cerevisiae into a liquid culture medium for primary and secondary amplification culture to obtain a shake flask seed culture solution;
2) liquid-state deep aerobic fermentation: inoculating the obtained shake flask seed culture solution into a base liquid culture medium, aerating for aerobic fermentation, and then adding a feed supplement culture medium for continuous aerobic fermentation to obtain a yeast liquid aerobic fermentation bacterium solution;
3) liquid low-temperature anaerobic fermentation: adding molasses culture medium into the obtained yeast liquid aerobic fermentation bacterial liquid, and carrying out anaerobic fermentation to obtain low-temperature anaerobic fermentation bacterial liquid;
4) solid-state enzymolysis aerobic fermentation: supplementing the molasses culture medium into the obtained low-temperature anaerobic fermentation bacterial liquid, then mixing the molasses culture medium with a solid culture medium, adding hydrolase, and carrying out solid aerobic fermentation to obtain a solid aerobic fermentation product;
5) solid anaerobic fermentation: carrying out solid anaerobic fermentation on the obtained solid aerobic fermentation product to obtain a solid anaerobic fermentation product;
6) solid-state high-temperature wall breaking autolysis: carrying out heat preservation treatment on the obtained solid anaerobic fermentation product to ensure that yeast cells are fully autolyzed to obtain a saccharomyces cerevisiae culture;
in the step 2), inoculating the obtained shake flask seed culture solution into the base liquid culture medium by an inoculation amount of 4-10% in volume percentage for propagation;
the aerobic fermentation conditions in aeration are as follows: the temperature is 28-30 ℃, the rotating speed is 150-200 rpm, the tank pressure is 0.03-0.04 Mpa, the aeration ratio is 2:1, the fermentation time is 18-24 hours, and the aeration ratio is the ratio of the aeration volume per minute to the actual feed liquid volume of the tank body;
the conditions for continuously carrying out aerobic fermentation by adding the supplemented medium are as follows: the dissolved oxygen is 20-40%, the fermentation temperature is 30-34 ℃, the rotating speed is 200-250 rpm, the tank pressure is 0.03-0.04 Mpa, the aeration ratio is 2-2.5: 1, the fermentation time is 5-6 hours, and the aeration ratio is the ratio of the tank volume to the sterile air volume;
the base sugar liquid culture medium comprises the following substances in percentage by mass: 10-16% of cane molasses, 0.8-1.5% of ammonium sulfate, 0.08-0.16% of yeast extract, 0.1-0.2% of magnesium sulfate, 0.005-0.02% of anhydrous calcium chloride, 0.05-0.2% of potassium dihydrogen phosphate, 0.03-0.06% of defoaming agent and the balance of water; adjusting the initial pH value of the base liquid culture medium to 6.0-6.5;
the feed medium consists of the following substances in percentage by mass: 90-95% of cane molasses, 2-5% of ammonium sulfate, 0.1-0.3% of yeast extract, 0.05-0.20% of magnesium sulfate, 0.008-0.015% of calcium chloride, 0.05-0.15% of potassium dihydrogen phosphate, 0.02-0.04% of defoaming agent and the balance of water;
the number of viable bacteria in the liquid aerobic zymophyte liquid of the microzyme is 6-10 hundred million CFU/ml;
in the step 3), 8-15% by volume of molasses culture medium is added into the obtained yeast liquid aerobic fermentation bacterial liquid;
the anaerobic fermentation conditions are as follows: the fermentation temperature is 15-20 ℃, the rotating speed is 50-60 rpm, the pressure of the fermentation tank is kept to be not higher than 0.1Mpa in a closed manner, and the fermentation time is 48-72 hours;
the molasses culture medium comprises the following substances in percentage by mass: 8-15% of cane molasses, 0.8-1.5% of ammonium sulfate, 0.08-0.16% of yeast extract, 0.01-0.06% of magnesium sulfate, 0.005-0.02% of anhydrous calcium chloride, 0.01-0.05% of potassium dihydrogen phosphate and the balance of water;
in the step 4), 6 to 12 volume percent of the molasses culture medium is supplemented into the obtained low-temperature anaerobic fermentation bacterial liquid;
the yeast liquid low-temperature anaerobic fermentation bacterial liquid supplemented with the molasses culture medium and the solid culture medium are mixed according to the mass ratio of 1: 0.5-0.8, the stacking height after mixing is 0.2-0.6 m, the water content is 35-45%, the air humidity of a fermentation environment is 70-80%, the temperature of solid aerobic fermentation is 32-38 ℃, and the fermentation time is 12-24 hours;
the number of live microzyme in the solid aerobic fermentation product is 4-10 hundred million CFU/g;
the hydrolase is selected from low-temperature amylase, neutral cellulase and acid protease or low-temperature amylase and neutral cellulase or low-temperature amylase and acid protease, the enzyme activity of the low-temperature amylase is more than or equal to 2000U/ml, the enzyme activity of the neutral cellulase is more than or equal to 2000U/ml, and the enzyme activity of the acid protease is more than or equal to 50000U/ml;
in the step 4), the solid culture medium consists of the following substances in percentage by mass:
preparing a solid culture medium according to 20-30% of corn flour, 30-40% of bran, 20-30% of corn germ meal, 10-20% of corn steep liquor dry powder and 10-20% of corncob powder;
in the step 5), a closed fermentation box is adopted for carrying out the solid anaerobic fermentation;
the fermentation temperature of the solid anaerobic fermentation is 32-38 ℃, and the fermentation time is 48-72 h;
the number of live microzyme in the solid anaerobic fermentation product is 2-6 hundred million CFU/ml, and the content of acid soluble protein is 35-40%.
2. The fermentation process of saccharomyces cerevisiae culture according to claim 1, further comprising a step of low temperature drying of said saccharomyces cerevisiae culture after step 6).
3. The fermentation process of a saccharomyces cerevisiae culture according to claim 1, wherein in step 1), the liquid medium is a yeast extract peptone glucose medium consisting of: each 1L of water contains 10-20 g of glucose, 10-30 g of tryptone and 5-15 g of yeast extract powder.
4. The fermentation process of the saccharomyces cerevisiae culture according to claim 1, wherein in the step 6), the temperature of the heat preservation treatment is 45-60 ℃ and the time is 6-12 h.
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