CN111826293A - Saccharomyces cerevisiae micro-ecological preparation and application thereof - Google Patents
Saccharomyces cerevisiae micro-ecological preparation and application thereof Download PDFInfo
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- CN111826293A CN111826293A CN202010929407.8A CN202010929407A CN111826293A CN 111826293 A CN111826293 A CN 111826293A CN 202010929407 A CN202010929407 A CN 202010929407A CN 111826293 A CN111826293 A CN 111826293A
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/18—Baker's yeast; Brewer's yeast
- C12N1/185—Saccharomyces isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/85—Saccharomyces
- C12R2001/865—Saccharomyces cerevisiae
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/60—Feeding-stuffs specially adapted for particular animals for weanlings
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/18—Baker's yeast; Brewer's yeast
Abstract
The invention provides a saccharomyces cerevisiae micro-ecological preparation and application thereof. The preservation number of the saccharomyces cerevisiae is GDMCC No. 70011; the liquid saccharomyces cerevisiae microbial inoculum obtained by fully culturing and fermenting the saccharomyces cerevisiae in a culture medium is mixed with isomaltose hypgather, glucose and molasses, a microecological preparation is obtained by post treatment, and the microecological preparation is applied to the preparation of animal feed, can exert the functions of efficiently enhancing the immunity and disease resistance of the organism, has excellent and stable activity in the organism, shows the balance of stimulating the microecological environment in the intestinal tract of the organism, and has the advantages of few components, low price of raw materials and remarkable effect when being applied to the preparation of the animal feed. In addition, in the scheme, the saccharomyces cerevisiae can easily absorb selenium components in the liquid culture medium, the selenium expression capacity is excellent, and the organism immunity effect of the follow-up preparation of the microecological preparation is further promoted.
Description
Technical Field
The invention relates to the field of preparation of microecologics, and particularly relates to a saccharomyces cerevisiae microecologic preparation and application thereof.
Background
Saccharomyces cerevisiae is also known as baker's yeast or budding yeast. Saccharomyces cerevisiae is the most widely related yeast to human, not only because it is traditionally used for making bread, steamed bread and other food and brewing wine, but also as a eukaryotic model organism in modern molecular and cellular biology, and its function is equivalent to prokaryotic model organism Escherichia coli. Saccharomyces cerevisiae is the most commonly used biological species in fermentation. The cells of Saccharomyces cerevisiae are spherical or ovoid, 5-10 μm in diameter. The reproduction method is budding reproduction. Because of the use of a large amount of antibiotics, pathogenic microorganisms generate drug resistance and drugs are remained in animal products, potential harm is caused to the health of human beings, and because the saccharomyces cerevisiae is added into livestock and poultry feed products, the digestion capacity, the immunity and the disease resistance of organisms are improved, so that the application of the saccharomyces cerevisiae to antibiotic substitute products also becomes a hot point.
However, impurities are easily introduced in the culture process of the saccharomyces cerevisiae in the prior art, and the impurity removal process can cause the problem of high production cost. In addition, the saccharomyces cerevisiae cultured by the prior art is unstable in activity, shows activity imbalance in different temperature environments, and has poor anti-mixed bacteria capability and poor reproductive capacity, so that the saccharomyces cerevisiae has poor effect in the application process.
Disclosure of Invention
Based on the above, in order to solve the problems that impurities are easily introduced in the saccharomyces cerevisiae culture process, the activity of the saccharomyces cerevisiae is unstable, the activity is unbalanced in different temperature environments, and the application effect is poor due to poor anti-infectious bacteria capability and poor reproductive capacity in the application process of the saccharomyces cerevisiae in the prior art, the invention provides a micro-ecological preparation containing the saccharomyces cerevisiae, a preparation method and application thereof, wherein the specific technical scheme is as follows:
a Saccharomyces cerevisiae (Saccharomyces cerevisiae) with a preservation time of: number 04/29 in 2020; the preservation unit is as follows: guangdong province culture Collection of microorganisms (GDMCC) with the address: building No. 59, building No. 5 of the first-furious Zhonglu 100 yard in Guangzhou city; the preservation number is: GDMCC No. 70011; the preservation form is as follows: vacuum freeze drying tube.
A micro-ecological preparation containing saccharomyces cerevisiae is prepared by adopting the saccharomyces cerevisiae, mixing a liquid saccharomyces cerevisiae microbial inoculum obtained by fully culturing and fermenting the saccharomyces cerevisiae in a culture medium with a carrier, and performing post-treatment.
Further, the carrier is isomaltose hypgather, glucose and molasses.
Further, the ratio of the isomaltooligosaccharide to the glucose to the molasses is 1-5:6-15:1-3 by volume ratio.
Further, the culture medium comprises a liquid culture medium, a primary fermentation culture medium, a secondary fermentation culture medium and a tertiary fermentation culture medium.
Further, the liquid medium is used for forming shake flask liquid strains.
Further, the primary fermentation medium is used for inoculating the shake flask liquid strain, and the primary seed solution is prepared at the fermentation temperature of 26-29 ℃, the stirring speed of 250-260rpm/min, the ventilation volume of 1.0-1.2V/V.min, the dissolved oxygen concentration of not less than 40% and the fermentation time of 16-20 h.
Further, the secondary fermentation culture medium is used for inoculating the primary seed liquid, and the secondary seed liquid is prepared at the fermentation temperature of 25-29 ℃, the stirring speed of 150-170rpm/min, the ventilation volume of 1.0-1.2V/V.min, the dissolved oxygen concentration of not less than 40 percent and the fermentation time of 20-25 h.
Further, the third-level fermentation medium is used for inoculating the second-level seed liquid, the stirring speed is 200-250rpm/min, the ventilation volume is 0.8-1.0V/V.min, the dissolved oxygen concentration is not lower than 40%, and the fermentation time is 10-15h at the fermentation temperature of 25-29 ℃, so that the liquid saccharomyces cerevisiae agent is prepared.
In addition, the invention also provides application of the saccharomyces cerevisiae-containing microecological preparation in preparation of animal feed.
The saccharomyces cerevisiae is obtained by screening, has excellent activity stability, strong anti-infectious bacteria capability and strong reproductive capacity. The micro-ecological preparation containing the saccharomyces cerevisiae can replace antibiotics in the existing animal daily ration, can exert the functions of efficiently enhancing the immunity and disease resistance of the organism, has excellent and stable activity in the organism, shows the balance of stimulating micro-ecology in the intestinal tract of the organism, and has the advantages of few components, low price of raw materials and remarkable effect when being applied to the preparation of animal feed. In addition, in the scheme, the saccharomyces cerevisiae can easily absorb selenium components in the liquid culture medium, the selenium expression capacity is excellent, and the organism immunity effect of the follow-up preparation of the microecological preparation is further promoted.
Drawings
FIG. 1 is a growth curve of primary seeds of Saccharomyces cerevisiae in example 2;
FIG. 2 is a growth curve of secondary seeds of Saccharomyces cerevisiae in example 2;
FIG. 3 is a growth curve of tertiary seeds of Saccharomyces cerevisiae in example 2.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to embodiments thereof. It should be understood that the detailed description and specific examples, while indicating the scope of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
In one embodiment of the invention, the saccharomyces cerevisiae has the preservation number of GDMCC No.70011, and the preservation form is vacuum freeze-drying tube preservation.
The Saccharomyces cerevisiae cell is oval, circular or cylindrical, has no flagellum and no movement, and has cell wall containing no cellulose, polysaccharides such as dextran, mannan, etc., and components such as protein, lipid and chitin, etc., and has rapid propagation, strong metabolism and many metabolites. In animal husbandry, the development and utilization of saccharomyces cerevisiae are also deepened, and the characteristics and distribution of saccharomyces cerevisiae contain a large amount of saccharomyces cerevisiae in the animal digestive tract, so that the saccharomyces cerevisiae can be separated from animal excrement.
A microecological preparation containing saccharomyces cerevisiae is prepared by mixing a liquid saccharomyces cerevisiae microbial inoculum obtained by fully culturing and fermenting the saccharomyces cerevisiae in a culture medium with a carrier and carrying out post-treatment.
In one embodiment, the carrier is isomaltooligosaccharide, glucose, and molasses.
In one embodiment, the ratio of isomaltooligosaccharide, glucose and molasses is 1-5:6-15:1-3 by volume.
In one embodiment, the culture medium comprises a liquid culture medium, a primary fermentation medium, a secondary fermentation medium, and a tertiary fermentation medium.
In one embodiment, the liquid medium is used to form shake flask liquid seed cultures.
In one embodiment, the formula of the liquid medium is as follows: 10-20g/L, L g of glucose powder-0.01-0.05 g/L of selenium-methyl selenocysteine, and the balance of water.
In one embodiment, the liquid culture medium is prepared by mixing the components uniformly according to the content, and sterilizing at 120 deg.C for 20-30 min.
In one embodiment, the primary fermentation medium is used for inoculating the shake flask liquid strain, and the primary seed solution is prepared at the fermentation temperature of 26-29 ℃, the stirring speed of 250-260rpm/min, the ventilation volume of 1.0-1.2V/V.min, the dissolved oxygen concentration of not less than 40% and the fermentation time of 16-20 h.
In one embodiment, the amount of liquid seed in the shake flask in the primary fermentation medium is 6-10%. The inoculation amount is the ratio of the volume of the liquid strain transferred into the shake flask to the volume of the first fermentation medium after inoculation.
In one embodiment, the primary fermentation medium comprises the following components: 10-20g/L of agar, 2-8g/L of peptone, 6-10g/L of urea, 1-5g/L of sodium acetate, 6-8g/L of dipotassium hydrogen phosphate and the balance of water.
In one embodiment, the primary fermentation medium is prepared by the following method: the components of the formula are uniformly mixed according to the content, and the mixture is sterilized for 20-30min at the temperature of 100-120 ℃.
In one embodiment, the secondary fermentation medium is used for inoculating the primary seed liquid, and the secondary seed liquid is prepared at the fermentation temperature of 25-29 ℃, the stirring speed of 150-170rpm/min, the ventilation quantity of 1.0-1.2V/V.min, the dissolved oxygen concentration of not less than 40% and the fermentation time of 20-25 h.
In one embodiment, the amount of inoculum in the secondary fermentation medium and in the primary seed liquid is 3-6%. The inoculum size as used herein refers to the ratio of the volume of the primary seed solution transferred to the volume of the secondary fermentation medium after inoculation.
In one embodiment, the formula of the secondary fermentation medium is as follows: 10-15g/L of molasses, 1-3g/L of urea, 1-6g/L of cane sugar, 1-2g/L of citric acid hydrogen diamine, 1-3g/L of dipotassium hydrogen phosphate and the balance of water.
In one embodiment, the secondary fermentation medium is prepared by the following method: the components of the formula are uniformly mixed according to the content, and the mixture is sterilized for 20-30min at the temperature of 100-120 ℃.
In one embodiment, the third-level fermentation medium is used for inoculating the second-level seed liquid, and the liquid saccharomyces cerevisiae agent is prepared at the fermentation temperature of 25-29 ℃, the stirring speed of 200-250rpm/min, the ventilation quantity of 0.8-1.0V/V.min, the dissolved oxygen concentration of not less than 40% and the fermentation time of 10-15 h.
In one embodiment, the three-stage fermentation medium is inoculated in an amount of 5-10%. The inoculation amount as used herein refers to the ratio of the volume of the secondary seed solution transferred to the volume of the tertiary fermentation medium after inoculation.
In one embodiment, the formula of the tertiary fermentation medium is as follows: 10-15g/L of soybean meal, 0.1-0.5g/L of cane sugar, 8-15g/L of molasses, 1-4g/L of ammonium chloride, 1-3g/L of dipotassium phosphate, 0.5-0.9g/L of cysteine hydrochloride and the balance of water.
In one embodiment, the primary fermentation medium is prepared by the following method: the components of the formula are uniformly mixed according to the content, the pH value is adjusted to 7.0-7.2, and the sterilization is carried out for 20-30min at the temperature of 100 ℃ and 120 ℃.
In one embodiment, the method for mixing the liquid saccharomyces cerevisiae agent with the carrier comprises the following steps: under the condition that the temperature is 24-28 ℃, the weight percentage of the mixture is 0.5-1.7: 5-10 of the liquid saccharomyces cerevisiae microbial inoculum and the carrier are added into a stirring container, the stirring speed of the stirring container is started to be 100-300rpm/min, and the mixture is obtained after stirring for 20-60 min.
In one embodiment, the post-processing is: freeze drying the mixture, and pulverizing to 60-120 mesh to obtain micro-ecological preparation containing Saccharomyces cerevisiae.
In addition, the saccharomyces cerevisiae-containing micro-ecological preparation is applied to the preparation of animal feed.
In one embodiment, the saccharomyces cerevisiae containing micro-ecological preparation is added in the following amount in the preparation of animal feed: 0.5-3.5 g/Kg.
Embodiments of the present invention will be described in detail below with reference to specific examples.
Example 1:
in the embodiment, the saccharomyces cerevisiae is obtained by extracting, separating, screening and purifying pig manure.
The pig manure is the manure of a healthy pig, the manure needs to be frozen for 1d in liquid nitrogen after being selected, then the manure is placed at the temperature of 75 ℃ for storage, and a saccharomyces cerevisiae mixture is extracted and separated for later use.
Separation of yeasts 500 Saccharomyces cerevisiae strains, i.e.the above-mentioned Saccharomyces cerevisiae mixture, were selected from 100 fecal samples in this test.
Inoculating 10% of saccharomyces cerevisiae in the following formula components: culturing beef extract 10g, glucose powder 20g, L-selenium-methyl selenocysteine 0.05g, and water 1L in culture medium at 26 deg.C for 24h, if the culture medium becomes turbid, microscopic checking whether it is infected with bacteria, screening Saccharomyces cerevisiae capable of growing for the first time to obtain selenium-rich Saccharomyces cerevisiae, and standing at 75 deg.C for use.
Inoculating the selenium-enriched saccharomyces cerevisiae with the inoculation amount of 1% (volume ratio) obtained by the first screening into the following formula components: 0.01g of anhydrous copper sulfate, 10g of beef extract, 20g of glucose powder, 0.05g of L-selenium-methyl selenocysteine and 1L of water, and observing after culturing at 26 ℃ for 24h, if the culture medium becomes turbid, the strain is proved to be high-copper resistant. Then, whether the bacteria are infected or not is checked under a microscope. And screening the saccharomyces cerevisiae capable of growing for the second time, and placing at 75 ℃ for later use.
Inoculating the saccharomyces cerevisiae with the inoculation amount of 1% (volume ratio) obtained by the second screening to the components of the formula: 0.01g of zinc oxide, 10g of beef extract, 20g of glucose powder, 0.05g of L-selenium-methyl selenocysteine and 1L of water, and observing after culturing at 26 ℃ for 24h, if the culture medium becomes turbid, the strain is proved to be high-zinc resistant. Then, whether the bacteria are infected or not is checked under a microscope. And thirdly, screening the saccharomyces cerevisiae capable of growing, and placing the saccharomyces cerevisiae at 75 ℃ for later use.
Inoculating the saccharomyces cerevisiae with the inoculation amount of 1% (volume ratio) obtained by the third screening to the components of the formula: after 8g of compound vitamin, 10g of beef extract, 20g of glucose powder, 0.05g of L-selenium-methyl selenocysteine and 1L of water are cultured in a culture medium at 26 ℃ for 24h, if the culture medium becomes turbid, the strain is suitable for survival in a high-vitamin environment. Then, whether the bacteria are infected or not is checked under a microscope. And screening the saccharomyces cerevisiae capable of growing for the fourth time, and placing at 75 ℃ for later use.
The Saccharomyces cerevisiae selected by the method is preserved in Guangdong province microorganism culture collection center at No. 29, 04/2020, with the preservation number of GDMCC No. 70011.
Example 2:
preparing a liquid culture medium: the formula comprises the following components: 10g of glucose powder, 0.01g of L-selenium-methyl selenocysteine and 1L of water are evenly mixed and sterilized for 20min at the temperature of 120 ℃.
Preparing a first-stage fermentation culture medium: the formula of the primary fermentation medium comprises the following components: agar 10g, peptone 2g, urea 10g, sodium acetate 5g, dipotassium hydrogen phosphate 8g, and water 1L, mixing well, sterilizing at 120 deg.C for 30 min.
Preparing a secondary fermentation medium: the formula of the secondary fermentation medium is as follows: 15g of molasses, 1-3g/L of urea, 1g of sucrose, 2g of diammonium hydrogen citrate, 1g of dipotassium hydrogen phosphate and 1L of water, uniformly mixing, and sterilizing at 100 ℃ for 20 min.
Preparing a three-stage fermentation medium: according to the formula of the three-stage fermentation medium: 15g of soybean meal powder, 0.1g of cane sugar, 8g of molasses, 4g of ammonium chloride, 1g of dipotassium hydrogen phosphate, 0.5g of cysteine hydrochloride and 1L of water, uniformly mixing, adjusting the pH value to 7.0, and sterilizing at 100 ℃ for 20 min.
The preparation method of the micro-ecological preparation containing the saccharomyces cerevisiae comprises the following steps:
A1. taking the saccharomyces cerevisiae with the screened preservation number of GDMCC No.70011 in the example 1 as a strain, inoculating the saccharomyces cerevisiae with the inoculation amount of 7 percent (volume ratio) into a liquid culture medium, and carrying out shake culture at 28 ℃ for 18h to obtain a shake flask liquid strain; the Saccharomyces cerevisiae is 0.5 × 109cfu/mL。
A2. Transferring the shake flask liquid strain into the primary fermentation medium at an inoculation amount of 6% (volume ratio), and preparing into primary seed liquid at a fermentation temperature of 26 ℃, a stirring speed of 250rpm/min, a ventilation amount of 1.0V/V.min, a dissolved oxygen concentration of not less than 40% and a fermentation time of 16 h;
A3. transferring the primary seed liquid into the secondary fermentation culture medium by 3 percent (volume ratio), and preparing a secondary seed liquid at the fermentation temperature of 25 ℃, the stirring speed of 150rpm/min, the ventilation volume of 1.2V/V.min, the dissolved oxygen concentration of not less than 40 percent and the fermentation time of 20 hours;
A4. transferring the secondary seed liquid into the tertiary fermentation medium in an inoculation amount of 5% (volume ratio), and preparing the liquid saccharomyces cerevisiae agent at a fermentation temperature of 25 ℃, a stirring speed of 200rpm/min, a ventilation volume of 0.8V/V.min, a dissolved oxygen concentration of not less than 40% and a fermentation time of 10 h;
A5. mixing isomaltose hypgather, glucose and molasses in a volume ratio of 1:6:1 to obtain a carrier;
A6. under the condition of 24 ℃, mixing the components in percentage by mass as follows: 5, adding the liquid saccharomyces cerevisiae microbial inoculum and the carrier into a stirring container, starting the stirring speed of the stirring container to be 100rpm/min, and stirring for 20min to obtain a mixture;
A7. freeze drying the mixture, and pulverizing to 60 mesh to obtain micro-ecological preparation containing Saccharomyces cerevisiae.
As shown in fig. 1-3, the growth curves of the first seed, the second seed and the third seed in the first seed liquid, the second seed liquid and the third seed liquid are measured by a spectrophotometer, the wavelength of the spectrophotometer is set at 560nm, the absorbance values at different time points are recorded, and the absorbance values and the time are plotted to obtain the growth curve charts shown in fig. 1-3. As can be seen from the analysis of FIGS. 1-3, after the activation of Saccharomyces cerevisiae, the activity is higher, and after the activation of Saccharomyces cerevisiae by the first fermentation medium, the second fermentation medium, and the third fermentation medium, Saccharomyces cerevisiae with better activity is easily obtained, and it shows higher stability and strong reproductive ability. In addition, the saccharomyces cerevisiae is not easy to generate mixed bacteria in the culture process, and the saccharomyces cerevisiae with the purity of 99.5 percent can be obtained.
Example 3:
preparing a liquid culture medium: the formula comprises the following components: 10g of glucose powder, 0.01g of L-selenium-methyl selenocysteine and 1L of water are evenly mixed and sterilized for 20min at the temperature of 120 ℃.
Preparing a first-stage fermentation culture medium: the formula of the primary fermentation medium comprises the following components: agar 10g, peptone 2g, urea 10g, sodium acetate 5g, dipotassium hydrogen phosphate 8g, and water 1L, mixing well, sterilizing at 120 deg.C for 30 min.
Preparing a secondary fermentation medium: the formula of the secondary fermentation medium is as follows: 15g of molasses, 3g of urea, 6g of cane sugar, 2g of diamine hydrogen citrate, 3g of dipotassium hydrogen phosphate and 1L of water are uniformly mixed and sterilized for 30min at 120 ℃.
Preparing a three-stage fermentation medium: according to the formula of the three-stage fermentation medium: 15g of soybean meal powder, 0.5g of cane sugar, 15g of molasses, 4g of ammonium chloride, 3g of dipotassium hydrogen phosphate, 0.9g of cysteine hydrochloride and the balance of water, uniformly mixing, adjusting the pH value to 7.2, and sterilizing at 120 ℃ for 30 min.
The preparation method of the micro-ecological preparation containing the saccharomyces cerevisiae comprises the following steps:
A1. taking the saccharomyces cerevisiae with the screened preservation number of GDMCC No.70011 in the example 1 as a strain, inoculating the saccharomyces cerevisiae with the inoculation amount of 5 percent (volume ratio) into a liquid culture medium, and carrying out shake culture at 28 ℃ for 18h to obtain a shake flask liquid strain; the Saccharomyces cerevisiae is 0.5 × 109cfu/mL。
A2. Transferring the shake flask liquid strain to the primary fermentation medium at an inoculation amount of 10% (volume ratio), and preparing a primary seed solution at a fermentation temperature of 29 ℃, a stirring speed of 260rpm/min, a ventilation amount of 1.2V/V.min, a dissolved oxygen concentration of not less than 40% and a fermentation time of 20 h;
A3. transferring the primary seed liquid into the secondary fermentation culture medium according to the inoculation amount of 6 percent (volume ratio), and preparing a secondary seed liquid at the fermentation temperature of 29 ℃, the stirring speed of 170rpm/min, the ventilation volume of 1.2V/V.min, the dissolved oxygen concentration of not less than 40 percent and the fermentation time of 25 hours;
A4. transferring the secondary seed liquid into the tertiary fermentation medium in an inoculation amount of 10% (volume ratio), and preparing the liquid saccharomyces cerevisiae agent at a fermentation temperature of 29 ℃, a stirring speed of 250rpm/min, a ventilation volume of 1.0V/V.min, a dissolved oxygen concentration of not less than 40% and a fermentation time of 15 h;
A5. mixing isomaltose hypgather, glucose and molasses in a volume ratio of 1:6:1 to obtain a carrier;
A6. under the condition of 28 ℃, mixing the components in percentage by mass as 1.7: 10, adding the liquid saccharomyces cerevisiae microbial inoculum and the carrier into a stirring container, starting the stirring container at a stirring speed of 300rpm/min, and stirring for 60min to obtain a mixture;
A7. freeze drying the mixture, and pulverizing to 120 mesh to obtain micro-ecological preparation containing Saccharomyces cerevisiae.
Example 4:
preparing a liquid culture medium: the formula comprises the following components: 15g of glucose powder, 0.03g of L-selenium-methyl selenocysteine and 1L of water are uniformly mixed and sterilized for 30min at the temperature of 120 ℃.
Preparing a first-stage fermentation culture medium: the formula of the primary fermentation medium comprises the following components: agar 15g, peptone 5g, urea 8g, sodium acetate 3g, dipotassium hydrogen phosphate 7g, and water 1L, mixing well, sterilizing at 110 deg.C for 25 min.
Preparing a secondary fermentation medium: the formula of the secondary fermentation medium is as follows: 12g of molasses, 2g of urea, 4g of cane sugar, 1g of diamine hydrogen citrate, 2g of dipotassium hydrogen phosphate and water are uniformly mixed and sterilized for 25min at 110 ℃.
Preparing a three-stage fermentation medium: according to the formula of the three-stage fermentation medium: 12g of soybean meal powder, 0.3g of cane sugar, 12g of molasses, 2g of ammonium chloride, 2g of dipotassium hydrogen phosphate, 0.8g of cysteine hydrochloride and 1L of water, uniformly mixing, adjusting the pH value to 7.0, and sterilizing at 110 ℃ for 25 min.
The preparation method of the micro-ecological preparation containing the saccharomyces cerevisiae comprises the following steps:
A1. taking the saccharomyces cerevisiae with the screened preservation number of GDMCC No 70011 in the example 1 as a strain, inoculating the saccharomyces cerevisiae with the inoculation amount of 7 percent (volume ratio) into a liquid culture medium, and carrying out shake culture at 28 ℃ for 18h to obtain a shake flask liquid strain; the Saccharomyces cerevisiae is 1.0 × 109cfu/mL。
A2. Transferring the shake flask liquid strain to the primary fermentation medium at an inoculation amount of 8% (volume ratio), and preparing a primary seed solution at a fermentation temperature of 28 ℃, a stirring speed of 255rpm/min, a ventilation amount of 1.0V/V.min, a dissolved oxygen concentration of not less than 40% and a fermentation time of 18 h;
A3. transferring the primary seed liquid into the secondary fermentation culture medium by an inoculation amount of 5 percent (volume ratio), and preparing a secondary seed liquid at a fermentation temperature of 28 ℃, a stirring speed of 160rpm/min, a ventilation rate of 1.1V/V.min, a dissolved oxygen concentration of not less than 40 percent and a fermentation time of 23 hours;
A4. transferring the secondary seed liquid into the tertiary fermentation medium in an inoculation amount of 8% (volume ratio), and preparing the liquid saccharomyces cerevisiae agent at a fermentation temperature of 28 ℃, a stirring speed of 230rpm/min, a ventilation volume of 0.9V/V.min, a dissolved oxygen concentration of not less than 40% and a fermentation time of 12 h;
A5. mixing isomaltose hypgather, glucose and molasses in a volume ratio of 3:12:2 to obtain a carrier;
A6. under the condition of 25 ℃, mixing the components in percentage by mass as follows: adding the liquid saccharomyces cerevisiae microbial inoculum of 8 and the carrier into a stirring container, starting the stirring container at a stirring speed of 200rpm/min, and stirring for 40min to obtain a mixture;
A7. freeze drying the mixture, and pulverizing to 100 mesh to obtain micro-ecological preparation containing Saccharomyces cerevisiae.
Application test example 1:
and (3) detecting the liquid saccharomyces cerevisiae microbial inoculum in the embodiment 3 and detecting the prepared saccharomyces cerevisiae-containing microecologics.
Reference document GB5009.93-2010 determination of selenium in food for a method for detecting the content of selenium in a microecological preparation.
In the liquid saccharomyces cerevisiae bacterial agent in the embodiment 3, 2.5g of dry bacteria can be obtained in each liter of the liquid saccharomyces cerevisiae bacterial agent, and the content of the total selenium obtained by detection in the obtained dry bacteria is 50271 mu g/g. The culture method in the scheme can ensure higher activity and reproductive capacity of the saccharomyces cerevisiae, and simultaneously can obtain and apply the saccharomyces cerevisiae with high selenium content.
The liquid saccharomyces cerevisiae microbial inoculum is applied to the preparation of the microecological preparation to obtain 15.5g of dry microecological preparation, and the content of the total selenium obtained by detection is 50219 mu g/g, which shows that the content of the total selenium is not greatly different when the liquid saccharomyces cerevisiae microbial inoculum is applied to the preparation of the microecological preparation, so that the content of the selenium in the microecological preparation can be ensured.
Application test example 2:
the probiotic prepared in example 4 was applied to the preparation of animal feed, weaned piglets were selected as test subjects in this application test, 3 treatment groups were selected, and labeled treatment group 1, treatment group 2 and treatment group 3, each treatment group having 5 replicates, each having 10 piglets, and a control group was set. The test period is 60 days, the whole process is free to feed and drink water, and after the test is finished, 5 butchering heads are selected for each group to take the longissimus dorsi to measure the meat quality index.
The addition amount of the microecologics in the basal ration of the piglets is as follows: 0.5-3.5 g/Kg.
The formulation table of the piglet diet in application test example 1 is shown in table 1 below.
Table 1:
the preparation method of the piglet diet in the table 1 comprises the following steps: adding the components into a stirring kettle according to a formula, adding a certain amount of water, stirring and decocting for 20-120 min at the temperature of 60-100 ℃ and under the pressure of 1.2-5 MPa, concentrating to be viscous, and cooling to room temperature.
The average daily gain, average daily feed intake and feed-weight ratio were calculated by feeding the piglet diets in table 1 to the corresponding treatment groups, respectively, and the results are shown in table 2 below.
Table 2:
as can be seen from the data analysis in Table 2, the addition of a certain amount of the microecologics to the ration of the piglets in the treatment group is helpful for improving the weight gain of the piglets, but the average daily feed intake is reduced, and the weight gain of the piglets can still be ensured on the premise of less daily ration feeding of the piglets.
Slaughtering and taking the longissimus dorsi to determine the meat quality index, wherein the crude protein is determined by adopting a Dumas combustion method; muscle fat was measured by soxhlet extraction; l (lightness of flesh), a (redness) and b (yellowness) were measured using a colorimeter and the recordings are shown in table 3 below.
Table 3:
as can be seen from the analysis in table 3, the piglets in the treated group were superior to the control group in the values of crude protein, muscle fat, and flesh color brightness. The microecological preparation added in the scheme is beneficial to improving the pork quality, does not need to increase the growth period and change the existing feeding mode, and achieves better production performance.
Relevant morbidity rates were recorded throughout the test period observed for piglets in application test 1 and the structural record is shown in table 4 below.
Table 4:
| group of | Control group | Treatment group 1 | Treatment group 2 | Treatment group 3 |
| Incidence rate/%) | 9 | 1 | 0 | 0 |
As can be seen from the data analysis in table 4, the incidence of the piglets in the treatment group is low during the feeding process, wherein the incidence of the piglets in the treatment group 1 is 1%, and the incidence of the piglets in the treatment groups 2 and 3 is 0, but the incidence of the piglets in the control group is 9%, which indicates that the addition of a certain amount of the probiotics to the ration can effectively improve the body immunity of the piglets and reduce the incidence of the piglets.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. Saccharomyces cerevisiae with the preservation number of GDMCC No. 70011.
2. A microecological preparation containing saccharomyces cerevisiae is characterized in that the microecological preparation is prepared by adopting the saccharomyces cerevisiae of claim 1, mixing a liquid saccharomyces cerevisiae microbial inoculum obtained by fully culturing and fermenting the saccharomyces cerevisiae in a culture medium with a carrier, and carrying out post-treatment.
3. The saccharomyces cerevisiae containing microecological preparation according to claim 2, wherein the carrier is isomaltooligosaccharide, glucose and molasses.
4. The saccharomyces cerevisiae-containing microecological preparation according to claim 3, wherein the ratio of the isomaltooligosaccharide, the glucose and the molasses is 1-5:6-15:1-3 by volume.
5. The saccharomyces cerevisiae containing microecological preparation according to claim 2, wherein said culture medium comprises a liquid culture medium, a primary fermentation medium, a secondary fermentation medium and a tertiary fermentation medium.
6. The saccharomyces cerevisiae containing micro-ecological formulation according to claim 5, wherein said liquid culture medium is used for forming shake flask liquid spawn.
7. The micro-ecological preparation containing saccharomyces cerevisiae as claimed in claim 6, wherein the primary fermentation medium is used for inoculating the shake flask liquid strain, and is prepared into the primary seed solution at the fermentation temperature of 26-29 ℃, the stirring rate of 250-260rpm/min, the aeration rate of 1.0-1.2V/V-min, the dissolved oxygen concentration of not less than 40% and the fermentation time of 16-20 h.
8. The saccharomyces cerevisiae-containing microecological preparation according to claim 7, wherein the secondary fermentation medium is used for inoculating the primary seed solution, and the secondary seed solution is prepared at a fermentation temperature of 25-29 ℃, a stirring rate of 150-170rpm/min, an aeration rate of 1.0-1.2V/V-min, a dissolved oxygen concentration of not less than 40%, and a fermentation time of 20-25 h.
9. The micro-ecological preparation containing saccharomyces cerevisiae as claimed in claim 8, wherein the tertiary fermentation medium is used for inoculating the secondary seed liquid, and the liquid saccharomyces cerevisiae agent is prepared at a fermentation temperature of 25-29 ℃, a stirring speed of 200-250rpm/min, an aeration rate of 0.8-1.0V/V-min, a dissolved oxygen concentration of not less than 40%, and a fermentation time of 10-15 h.
10. Use of a saccharomyces cerevisiae containing micro-ecological preparation according to any of the claims 2-9 for the preparation of animal feed.
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| CN101463329A (en) * | 2009-01-07 | 2009-06-24 | 北京龙科方舟生物工程技术中心 | Saccharomyces cerevisiae, yeast preparation including the same, feed, and preparation and use thereof |
| CN107988089A (en) * | 2017-11-09 | 2018-05-04 | 广东海纳川生物科技股份有限公司 | The preparation method and Bu Ladi yeast preparations of a kind of Bu Ladi yeast preparations |
| CN109423451A (en) * | 2017-08-24 | 2019-03-05 | 康盛中科(北京)技术有限公司 | A kind of Wine brewing yeast strain KSA01 and application thereof |
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| CN101463329A (en) * | 2009-01-07 | 2009-06-24 | 北京龙科方舟生物工程技术中心 | Saccharomyces cerevisiae, yeast preparation including the same, feed, and preparation and use thereof |
| CN109423451A (en) * | 2017-08-24 | 2019-03-05 | 康盛中科(北京)技术有限公司 | A kind of Wine brewing yeast strain KSA01 and application thereof |
| CN107988089A (en) * | 2017-11-09 | 2018-05-04 | 广东海纳川生物科技股份有限公司 | The preparation method and Bu Ladi yeast preparations of a kind of Bu Ladi yeast preparations |
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Application publication date: 20201027 |