CN115678789A - Novel method for improving activity of beer yeast and preparation of composite microbial inoculum - Google Patents
Novel method for improving activity of beer yeast and preparation of composite microbial inoculum Download PDFInfo
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Abstract
The invention provides a new method for improving the activity of beer yeast, which comprises the following steps: (1) preparing astragalus extract; (2) preparing a hawthorn extract; (3) preparing a licorice extract; (4) preparing an extract of eucommia ulmoides leaves; (5) Mixing radix astragali extract, fructus crataegi extract, glycyrrhrizae radix extract and folium Eucommiae extract at a certain proportion to obtain extract mixed solution; (6) activating the beer yeast to obtain activated seed liquid; (7) Adding activated seed liquid into YPD liquid culture medium, and adding 3-20% mixed extract solution for co-culture. The invention also provides a preparation method of the composite microbial inoculum. The invention obviously improves the activity of the beer yeast by adding the astragalus, the hawthorn, the liquorice and the eucommia leaves into the beer yeast for co-culture, and prepares the composite microbial inoculum by drying the mixed solution of the extracts of the astragalus, the hawthorn, the liquorice and the eucommia leaves into powder and mixing the powder with freeze-dried beer yeast powder, and the composite microbial inoculum is used as a green feed additive for the breeding industry.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a novel method for improving the activity of beer yeast and preparation of a composite microbial inoculum.
Background
The brewer's yeast is a yeast used for brewing beer, and the cell form thereof is an ellipsoid having a nearly spherical shape. The beer yeast contains almost no sugar, fat and starch, and contains high-quality complete protein, high-quality mineral substances, high-quality functional dietary fiber and the like. Therefore, the beer yeast can be used as a probiotic feed additive in the breeding industry.
However, after feeding as a feed, brewers yeast needs to be effectively resistant to the effects of the animal's digestive system, particularly gastric acid and bile salts, on its activity. Therefore, how to improve the activity of the existing beer yeast feed becomes a difficult problem to be solved urgently in the current field.
Disclosure of Invention
The invention aims to provide a novel method for improving the activity of beer yeast and a preparation method of a composite microbial inoculum, wherein the activity (namely, the growth reproductive capacity and the acid and bile salt resistance) of the beer yeast is remarkably improved by adding astragalus, hawthorn, liquorice and folium cortex eucommiae into the beer yeast for co-culture, and the composite microbial inoculum is prepared by drying a mixed solution of extracts of the astragalus, the hawthorn, the liquorice and the folium cortex eucommiae into powder and mixing the powder with freeze-dried beer yeast powder and is used as a green feed additive for the breeding industry.
The invention adopts the following technical scheme to solve the technical problems:
a novel method for improving the activity of beer yeast comprises the following steps:
(1) Putting the astragalus slices into clear water, and soaking overnight; performing ultrasonic extraction with ultrasonic power of 800W for 30min at 50 deg.C the next day; filtering, and centrifuging to obtain radix astragali extract;
(2) Putting hawthorn into clear water, boiling for 1h, and filtering to obtain a first leaching solution; mixing the filtered residue with clear water, boiling for 30min, and filtering to obtain a second leaching solution; mixing the two leaching solutions, adding 60% anhydrous ethanol, and precipitating with ethanol overnight; centrifuging the next day, collecting supernatant, and recovering ethanol to obtain fructus crataegi extract;
(3) Putting liquorice into clear water, soaking for 1h, and decocting for 2h; after filtering, decocting the residue for 2 times under the same conditions for 1.5h and 1h respectively; mixing the three decoctions, adding 70% anhydrous ethanol, and precipitating with ethanol overnight; centrifuging the next day, collecting supernatant, and recovering ethanol to obtain Glycyrrhrizae radix extract;
(4) Adding ethanol into folium Eucommiae, and soaking overnight; performing ultrasonic treatment at 800W and 60 deg.C for 40min; filtering, and performing rotary evaporation to obtain folium Eucommiae extract;
(5) Mixing the obtained radix astragali extract, fructus crataegi extract, glycyrrhrizae radix extract and folium Eucommiae extract according to a ratio of 2:3:1:2 to obtain an extract mixed solution;
(6) Inoculating beer yeast into YPD liquid culture medium, and culturing overnight in 37 deg.C incubator to obtain activated seed liquid;
(7) Adding the activated seed solution into a fresh YPD liquid culture medium, adding a 3-20% extract mixed solution, and co-culturing at 37 ℃.
In a preferred embodiment of the present invention, in the step (1), the astragalus membranaceus tablets are mixed according to a mass ratio of 1:5, putting the mixture into clear water, and soaking the mixture overnight; after ultrasonic extraction, filtering with four layers of gauze, and centrifuging the filtrate at 20 deg.C at 10000rpm/min for 5min; centrifuging, and collecting supernatant to 200mL to obtain radix astragali extract.
In a preferred embodiment of the present invention, in the step (2), the hawthorn is added to the mixture of the hawthorn and the water in a mass ratio of 1:10 putting the mixture into clear water, boiling the mixture for 1 hour at 100 ℃, and filtering the mixture to obtain a first leaching solution; mixing the filtered residue with clear water according to the mass ratio of 1:5 mixing and boiling for 30min again, and filtering to obtain a second leaching solution; mixing the two leaching solutions, adding absolute ethyl alcohol, preparing 60% alcohol precipitation solution, and standing overnight at 4 ℃; centrifuging at 11000rpm/min for 5min at 4 deg.C the next day; and (4) recovering ethanol after taking the supernatant to obtain an extracting solution, and fixing the volume to 200mL to obtain the hawthorn extract.
In a preferred embodiment of the present invention, in the step (3), the liquorice is added in a mass ratio of 1:10 putting into clear water, soaking and decocting; filtering with four layers of gauze, and decocting the residue for 2 times under the same condition; mixing the three decoctions, adding anhydrous ethanol, preparing 70% ethanol precipitation solution, and standing at 4 deg.C overnight; centrifuging at 10000rpm/min at 20 deg.C for 5min the next day; and (4) recovering ethanol after taking the supernatant to obtain an extracting solution, and fixing the volume to 200mL to obtain the liquorice extract.
In a preferred embodiment of the present invention, in the step (4), after soaking the leaves of eucommia ulmoides in 70% ethanol overnight, performing ultrasonic treatment; filtering with four layers of gauze, rotary evaporating to remove ethanol to obtain water solution, and metering to volume of 200mL to obtain folium Eucommiae extract.
In a preferred embodiment of the present invention, in the step (6), the lager brewing yeast preserved in the single loop slant is extracted with an inoculating loop, inoculated into 100mL of YPD liquid medium, and cultured overnight in an incubator at 37 ℃ to obtain an activated seed solution.
In a preferred embodiment of the present invention, in the step (7), the specific amount of the mixed extract solution is 10%.
A preparation method of a complex microbial inoculum comprises the following steps:
(1) Putting the astragalus slices into clear water, and soaking overnight; performing ultrasonic extraction with ultrasonic power of 800W for 30min at 50 deg.C the next day; filtering, and centrifuging to obtain radix astragali extract;
(2) Putting hawthorn into clear water, boiling for 1h, and filtering to obtain a first leaching solution; mixing the filtered residue with clear water, boiling for 30min, and filtering to obtain second extractive solution; mixing the two leaching solutions, adding 60% anhydrous ethanol, and precipitating with ethanol overnight; centrifuging the next day, collecting supernatant, and recovering ethanol to obtain fructus crataegi extract;
(3) Putting liquorice into clear water, soaking for 1h, and decocting for 2h; after filtering, decocting the residue for 2 times under the same conditions for 1.5h and 1h respectively; mixing the three decoctions, adding 70% anhydrous ethanol, and precipitating with ethanol overnight; centrifuging the next day, collecting supernatant, and recovering ethanol to obtain Glycyrrhrizae radix extract;
(4) Adding ethanol into folium Eucommiae, and soaking overnight; performing ultrasonic treatment at 800W and 60 deg.C for 40min; filtering, and performing rotary evaporation to obtain folium Eucommiae extract;
(5) Mixing the obtained astragalus extract, hawthorn extract, liquorice extract and eucommia ulmoides leaf extract according to a ratio of 2:3:1:2 to obtain an extract mixed solution; then, spray drying the extract mixed solution to obtain extract mixed powder;
(6) Inoculating beer yeast into YPD liquid culture medium, and culturing overnight in 37 deg.C incubator to obtain activated seed liquid;
(7) After overnight culture, centrifugally collecting thalli, and washing the thalli for a plurality of times by using sterile normal saline to obtain beer yeast mud; adding a protective agent into the beer yeast strain mud, pre-freezing, and freeze-drying in a freeze dryer; after the freeze drying is finished, grinding into powder;
(8) And (4) mixing the extract mixed powder obtained in the step (5) with the beer yeast freeze-dried powder obtained in the step (7) to obtain the compound microbial inoculum required by the target.
In a preferred embodiment of the present invention, in the step (4), the spray drying is performed under conditions of an inlet air temperature of 170 ℃ and a peristaltic pump speed of 7rpm/min.
In a preferred embodiment of the present invention, in the step (6), after the overnight culture is completed, the cells are centrifuged for 6min at 4 ℃ and 5000rpm/min by using a high-speed refrigerated centrifuge, and the cells are collected and washed with sterile physiological saline for 3 times to obtain beer yeast sludge; adding a protective agent with 20 percent of the volume of the fermentation liquor into the beer yeast strain mud, pre-freezing for 2 hours at the temperature of minus 70 ℃, and then placing the mixture into a freeze dryer for freeze drying for 36 to 48 hours; after the freeze drying is finished, grinding into powder; wherein, the protective agent comprises the following components: 10% of skim milk, 10% of trehalose and 5% of sucrose.
In the present invention, the YPD liquid medium used is a medium commonly used in the art, and its composition is not described in detail. The beer yeast acted with the extract mixed solution can be any beer yeast available in the field.
The principle of the invention is as follows:
the astragalus contains more than 20 trace elements, more than 50 saponins and triterpenoids and more than 20 flavonoids. The astragalus polysaccharide is a polysaccharide extract of astragalus roots, has the highest content in astragalus, has strong immunocompetence and is an important part for enhancing the immunity of organisms.
The hawthorn mainly contains inorganic salts, amino acids, organic acids and flavonoid compounds. Triterpenes are important components in organic acid compounds in hawthorn, and have important functions of tonifying heart, improving circulation, helping digestion and the like. The flavonoids compounds in the hawthorn have the effects of reducing blood pressure, resisting oxidation and the like, and are main bioactive components of the pharmacological action of the hawthorn.
The main active ingredients of Glycyrrhrizae radix are saponin, brass and polysaccharide compound etc. Wherein the saponin material is mainly glycyrrhizic acid, the flavonoid material is mainly glycyrrhizin and isoliquiritigenin, and the polysaccharide material is mainly glycyrrhiza polysaccharide. The glycyrrhiza polysaccharide has biological activities of immunoregulation, anti-tumor, antioxidation, antivirus, antibiosis and the like, and can improve the immunity of animal organisms and promote the production function of the animals.
The active ingredients of folium Eucommiae include flavonoids, iridoids, phenylpropanoids, lignanoids, polysaccharides, and gutta Percha. Wherein, the lignin and iridoid compounds are essential components of eucommia She Nazhu, and the flavonoid compounds are the compounds with the largest content in eucommia leaves.
Compared with the prior art, the invention has the advantages that:
(1) The mixed solution of the extract can improve the growth and reproduction capacity of the beer yeast; the research shows that: astragalus polysaccharide in the astragalus extract, organic acid and flavonoid compounds in the hawthorn extract, glycyrrhizic acid in the licorice extract and flavonoid and lignin compounds in the eucommia bark leaf extract influence the growth and reproduction capacity of beer yeast; when they act together, the influence on the growth and reproduction ability of the lager brewing yeast is most favorable;
(2) The extract mixed solution can improve the acid resistance of the beer yeast; the research shows that: astragalus polysaccharide in the astragalus extract, flavonoid compounds in the hawthorn extract, glycyrrhiza polysaccharide in the liquorice extract and flavonoid and lignin compounds in the eucommia ulmoides leaf extract contribute to the acid resistance of beer yeast; when the components play a role together, the acid resistance of the beer yeast is improved most obviously;
(3) The mixed solution of the extract can improve the bile salt resistance of the beer yeast; the research shows that: astragalus polysaccharide in the astragalus extract, organic acid and flavonoid compounds in the hawthorn extract, glycyrrhizic acid in the licorice extract and flavonoid and lignin compounds in the eucommia ulmoides leaf extract are beneficial to improving the bile salt resistance of the beer yeast; when the two materials play a role together, the improvement on the bile salt resistance of the beer yeast is most remarkable;
(4) The astragalus, hawthorn, liquorice and eucommia leaves belong to edible and medicinal plants for feeding, the beer yeast belongs to a microorganism for feeding, and the composite microbial inoculum prepared by compounding the astragalus, hawthorn, liquorice and eucommia leaves can be used as a novel green feed additive to be developed and applied to the breeding industry.
Drawings
FIG. 1 is a graph showing the effect of mixed solutions of different concentrations of extracts on the growth and reproduction ability of Saccharomyces cerevisiae in example 5;
FIG. 2 is a graph showing the effect of different concentrations of the Astragalus membranaceus extract and the mixed solution on the growth and reproduction ability of Saccharomyces cerevisiae in example 5;
FIG. 3 is a graph comparing the effect of different concentrations of hawthorn extract and mixed solution on the growth and reproduction ability of brewers' yeast in example 5;
FIG. 4 is a graph showing the effect of different concentrations of licorice extract and mixed solution on the growth and reproduction ability of Saccharomyces cerevisiae in example 5;
FIG. 5 is a graph showing the effect of different concentrations of the eucommia ulmoides leaf extract and the mixed solution on the growth and reproduction ability of brewer's yeast in example 5;
FIG. 6 is a graph showing the effect of the 10% extract mixed solution on the acid resistance of lager brewing yeast in example 6;
FIG. 7 is a graph showing the effect of the single solution and the mixed solution on the acid resistance of beer yeast in example 6;
FIG. 8 is a graph showing the effect of the 10% mixed solution on the bile salt resistance of lager brewing yeast in example 7;
FIG. 9 is a graph showing the effect of a single solution and a mixed solution on the bile salt resistance of lager brewing yeast in example 7;
FIG. 10 is a schematic diagram of a lager brewing yeast lyophilized powder and an extract mixed powder obtained in example 8 (in the figure, A shows the lager brewing yeast lyophilized powder, and B shows the extract mixed powder).
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
Example 1
The new method for improving the activity of the beer yeast comprises the following steps:
(1) Mixing the astragalus membranaceus tablets according to a mass ratio of 1:5, putting the mixture into clear water, and soaking the mixture overnight; performing ultrasonic extraction with ultrasonic power of 800W for 30min at 50 deg.C the next day; filtering with four layers of gauze, and centrifuging at 20 deg.C at 10000rpm/min for 5min; taking the supernatant and fixing the volume to 200mL to obtain the astragalus extract.
(2) Mixing the hawthorn with the following raw materials in a mass ratio of 1:10 putting into clear water, boiling for 1h at 100 ℃, and filtering to obtain a first leaching solution; mixing the residue filtered by the four layers of gauze with clear water (the mass ratio of the residue to the clear water is 1:5), boiling for 30min, and filtering to obtain a second leaching solution; mixing the two leaching solutions, adding anhydrous ethanol, preparing 60% ethanol precipitation solution, and standing overnight at 4 deg.C; centrifuging at 11000rpm/min at 4 ℃ for 5min the next day; and (4) recovering ethanol after taking the supernatant to obtain an extracting solution, and fixing the volume to 200mL to obtain the hawthorn extract.
(3) Liquorice is added according to the mass ratio of 1:10, putting the mixture into clear water, soaking for 1 hour, and decocting for 2 hours; filtering with four layers of gauze, decocting the residue for 2 times under the same conditions for 1.5 hr and 1 hr respectively; mixing the three decoctions, adding anhydrous ethanol, preparing 70% ethanol precipitation solution, and standing at 4 deg.C overnight; centrifuging at 10000rpm/min at 20 deg.C for 5min the next day; and (4) recovering ethanol after taking the supernatant to obtain an extracting solution, and fixing the volume to 200mL to obtain the liquorice extract.
(4) Adding 70% ethanol into folium Eucommiae, and soaking overnight; performing ultrasonic treatment at 800W and 60 deg.C for 40min; filtering with four layers of gauze, rotary evaporating to remove ethanol to obtain water solution, and metering to volume of 200mL to obtain folium Eucommiae extract.
(5) Mixing the obtained astragalus extract, hawthorn extract, liquorice extract and eucommia ulmoides leaf extract according to a mass ratio of 2:3:1:2 mixing to obtain an extract mixed solution.
(6) The beer yeast preserved in the slant of one loop was streaked with an inoculating loop, inoculated into 100mL YPD liquid medium, and cultured overnight in an incubator at 37 ℃ to obtain activated seed liquid.
(7) Adding 1% of the above activated seed solution to fresh YPD liquid medium, adding 3% of the mixed solution of the extracts, and co-culturing at 37 deg.C.
It should be noted that in this example, the YPD liquid medium used is a medium commonly used in the art, and the components thereof are not described in detail. The beer yeast acted with the extract mixed solution can be any beer yeast available in the field.
Example 2
The procedure of the new method for improving the activity of Saccharomyces cerevisiae in this example is substantially the same as that of example 1, and the main difference is that: in step (7), 1% of the above activated seed solution and 5% of the mixed extract solution were added to a fresh YPD liquid medium, and co-cultured at 37 ℃.
Example 3
The procedure of the new method for improving the activity of Saccharomyces cerevisiae in this example is substantially the same as that of example 1, and the main difference is that: in step (7), 1% of the above activated seed solution and 10% of the mixed extract solution were added to a fresh YPD liquid medium, and co-cultured at 37 ℃.
Example 4
The procedure of the new method for improving the activity of Saccharomyces cerevisiae in this example is substantially the same as that of example 1, and the main difference is that: in step (7), 1% of the above activated seed solution and 20% of the mixed extract solution were added to a fresh YPD liquid medium, and co-cultured at 37 ℃.
Example 5
This example illustrates the effect of the extract mixture solution of the present invention on the growth and reproduction ability of brewers' yeast.
1. Effect experiment of mixed solution of extracts of different concentrations:
1. the experimental steps are as follows:
(1) Mixing the astragalus root tablets according to a mass ratio of 1:5, putting the mixture into clear water, and soaking the mixture overnight; performing ultrasonic extraction with ultrasonic power of 800W for 30min at 50 deg.C the next day; filtering with four layers of gauze, and centrifuging at 20 deg.C at 10000rpm/min for 5min; taking the supernatant and fixing the volume to 200mL to obtain the astragalus extract.
(2) Mixing the hawthorn with the following raw materials in a mass ratio of 1:10 putting the mixture into clear water, boiling the mixture for 1 hour at 100 ℃, and filtering the mixture to obtain a first leaching solution; mixing the residue filtered by the four layers of gauze with clear water (the mass ratio of the residue to the clear water is 1:5), boiling for 30min, and filtering to obtain a second leaching solution; mixing the two leaching solutions, adding anhydrous ethanol, preparing 60% ethanol precipitation solution, and standing overnight at 4 deg.C; centrifuging at 11000rpm/min for 5min at 4 deg.C the next day; and (4) recovering ethanol after taking the supernatant to obtain an extracting solution, and fixing the volume to 200mL to obtain the hawthorn extract.
(3) Liquorice is added according to the mass ratio of 1:10, putting the mixture into clear water, soaking for 1 hour, and decocting for 2 hours; filtering with four layers of gauze, decocting the residue for 2 times under the same conditions for 1.5 hr and 1 hr respectively; mixing the three decoctions, adding anhydrous ethanol, preparing 70% ethanol precipitation solution, and standing at 4 deg.C overnight; centrifuging at 10000rpm/min at 20 deg.C for 5min the next day; and (4) recovering ethanol after taking the supernatant to obtain an extracting solution, and fixing the volume to 200mL to obtain the liquorice extract.
(4) Adding 70% ethanol into folium Eucommiae, and soaking overnight; performing ultrasonic treatment at 800W and 60 deg.C for 40min; filtering with four layers of gauze, rotary evaporating to remove ethanol to obtain water solution, and metering to volume of 200mL to obtain folium Eucommiae extract.
(5) Mixing the obtained astragalus extract, hawthorn extract, liquorice extract and eucommia ulmoides leaf extract according to a mass ratio of 2:3:1:2 mixing to obtain an extract mixed solution.
(6) A loop of the slant-deposited Saccharomyces cerevisiae (BY 4742, purchased from BNCC) was streaked with an inoculating loop, inoculated into 100mL of YPD liquid medium, and cultured overnight in an incubator at 37 ℃ to obtain an activated seed solution.
(7) Adding 1% of the above activated seed solution into fresh YPD liquid culture medium, adding 0,3%,5%,10%, and 20% of mixed extract solution, co-culturing at 37 deg.C, and measuring OD every 2h 600 The value is obtained.
2. The experimental results are as follows: as shown in fig. 1.
FIG. 1 shows the effect of adding mixed solutions of extracts of different concentrations on the growth and reproduction ability of Saccharomyces cerevisiae in this example. As can be seen from FIG. 1, the growth and reproduction ability of Saccharomyces cerevisiae could be improved by adding 3%,5%,10% and 20%, but the improvement of growth and reproduction ability of Saccharomyces cerevisiae was most obvious by adding 10%.
Therefore, the beer yeast growth and reproduction capacity can be effectively improved by adding the 3-20% extract mixed solution, and the best concentration is 10%.
2. Control experiment for "single solution":
1. the experimental steps are as follows:
(1) Mixing the astragalus root tablets according to a mass ratio of 1:5, putting the mixture into clear water, and soaking the mixture overnight; performing ultrasonic extraction with ultrasonic power of 800W for 30min at 50 deg.C the next day; filtering with four layers of gauze, and centrifuging at 20 deg.C at 10000rpm/min for 5min; taking the supernatant and fixing the volume to 200mL to obtain the astragalus extract.
(2) Mixing the hawthorn with the following raw materials in a mass ratio of 1:10 putting the mixture into clear water, boiling the mixture for 1 hour at 100 ℃, and filtering the mixture to obtain a first leaching solution; mixing the residue filtered by the four layers of gauze with clear water (the mass ratio of the residue to the clear water is 1:5), boiling for 30min, and filtering to obtain a second leaching solution; mixing the two leaching solutions, adding anhydrous ethanol, preparing 60% ethanol precipitation solution, and standing overnight at 4 deg.C; centrifuging at 11000rpm/min for 5min at 4 deg.C the next day; and (4) recovering ethanol after taking the supernatant to obtain an extracting solution, and fixing the volume to 200mL to obtain the hawthorn extract.
(3) Liquorice is added according to the mass ratio of 1:10, putting the mixture into clear water, soaking for 1 hour, and decocting for 2 hours; filtering with four layers of gauze, decocting the residue for 2 times under the same conditions for 1.5 hr and 1 hr respectively; mixing the three decoctions, adding anhydrous ethanol, preparing 70% ethanol precipitation solution, and standing at 4 deg.C overnight; centrifuging at 10000rpm/min at 20 deg.C for 5min the next day; and (4) recovering ethanol after taking the supernatant to obtain an extracting solution, and fixing the volume to 200mL to obtain the liquorice extract.
(4) Adding 70% ethanol into folium Eucommiae, and soaking overnight; performing ultrasonic treatment at 800W and 60 deg.C for 40min; filtering with four layers of gauze, rotary evaporating to remove ethanol to obtain water solution, and metering to volume of 200mL to obtain folium Eucommiae extract.
(5) The beer yeast preserved in the slant of one loop was streaked with an inoculating loop, inoculated into 100mL YPD liquid medium, and cultured overnight in an incubator at 37 ℃ to obtain activated seed liquid.
(6) Adding 1% of the above activated seed solution of step (4) into fresh YPD liquid culture medium, adding 0,3%,5%,10%, and 20% of radix astragali extract respectively, co-culturing at 37 deg.C, and measuring OD once every 2h 600 The value is obtained.
(7) Adding 1% of the above activated seed solution of step (4) into fresh YPD liquid culture medium, adding 0,3%,5%, and 10% of fructus crataegi extract, co-culturing at 37 deg.C, and measuring OD every 2h 600 The value is obtained.
(8) Adding 1% of the above activated seed solution of step (4) into fresh YPD liquid culture medium, adding 0,3%,5%, and 10% of Glycyrrhrizae radix extract, co-culturing at 37 deg.C, and measuring OD every 2h 600 The value is obtained.
(9) Adding 1% of the above activated seed solution of step (4) into fresh YPD liquid culture medium, adding 0,3%,5%,10%, and 20% of folium Eucommiae extract respectively, co-culturing at 37 deg.C, and measuring OD once every 2h 600 The value is obtained.
2. The experimental results are as follows: as shown in fig. 2-5.
FIG. 2 shows the effect of adding different concentrations of Astragalus membranaceus extract and mixed solution on the growth and reproduction ability of Saccharomyces cerevisiae, wherein the OD value of the mixed solution is significantly higher than that of Astragalus membranaceus extract. FIG. 3 shows the effect of adding different concentrations of fructus crataegi extract and mixed solution on the growth and reproduction ability of cerevisiae Fermentum, wherein the mixed solution has higher growth and reproduction ability than fructus crataegi extract. FIG. 4 is a graph showing the effect of adding different concentrations of licorice extract and mixed solution on the growth and reproduction ability of brewers' yeast, wherein the increase of the mixed solution is significantly higher than that of the Atractylodis rhizoma extract. FIG. 5 shows the effect of adding different concentrations of folium Eucommiae extract and mixed solution on the growth and reproduction ability of brewer's yeast, wherein the improvement of the mixed solution is significantly higher than that of the folium Eucommiae extract.
Therefore, the mixed solution of the extract can improve the growth and reproduction capacity of the beer yeast more than a single solution.
Experimental example 6
This example is intended to demonstrate the effect of the extract mixed solution of the present invention on the acid resistance of beer yeast.
1. Effect experiment of extract mixed solution:
1. the experimental steps are as follows:
(1) Mixing the astragalus membranaceus tablets according to a mass ratio of 1:5, putting the mixture into clear water, and soaking the mixture overnight; performing ultrasonic extraction with ultrasonic power of 800W for 30min at 50 deg.C the next day; filtering with four layers of gauze, and centrifuging at 20 deg.C at 10000rpm/min for 5min; taking the supernatant and fixing the volume to 200mL to obtain the astragalus extract.
(2) Mixing the hawthorn in a mass ratio of 1:10 putting the mixture into clear water, boiling the mixture for 1 hour at 100 ℃, and filtering the mixture to obtain a first leaching solution; mixing the residue filtered by the four layers of gauze with clear water (the mass ratio of the residue to the clear water is 1:5), boiling for 30min, and filtering to obtain a second leaching solution; mixing the two leaching solutions, adding anhydrous ethanol, preparing 60% ethanol precipitation solution, and standing overnight at 4 deg.C; centrifuging at 11000rpm/min for 5min at 4 deg.C the next day; and (4) recovering ethanol after taking the supernatant to obtain an extracting solution, and fixing the volume to 200mL to obtain the hawthorn extract.
(3) Liquorice is added according to the mass ratio of 1:10 putting the mixture into clear water, soaking for 1 hour, and decocting for 2 hours; filtering with four layers of gauze, decocting the residue for 2 times under the same conditions for 1.5 hr and 1 hr respectively; mixing the three decoctions, adding anhydrous ethanol, preparing 70% ethanol precipitation solution, and standing overnight at 4 deg.C; centrifuging at 10000rpm/min at 20 deg.C for 5min the next day; and (4) recovering ethanol after taking the supernatant to obtain an extracting solution, and fixing the volume to 200mL to obtain the liquorice extract.
(4) Adding 70% ethanol into folium Eucommiae, and soaking overnight; performing ultrasonic treatment at 800W and 60 deg.C for 40min; filtering with four layers of gauze, rotary evaporating to remove ethanol to obtain water solution, and metering to volume of 200mL to obtain folium Eucommiae extract.
(5) Mixing the obtained astragalus extract, hawthorn extract, liquorice extract and eucommia ulmoides leaf extract according to a mass ratio of 2:3:1:2, mixing to obtain an extract mixed solution.
(6) A loop of the slant-deposited Saccharomyces cerevisiae (BY 4742, purchased from BNCC) was streaked with an inoculating loop, inoculated into 100mL of YPD liquid medium, and cultured overnight in an incubator at 37 ℃ to obtain an activated seed solution.
(7) Adding 1% inoculum of activated seed solution into fresh YPD liquid culture medium with pH of 2, 3, and 7, respectively, culturing at 37 deg.C for 4 hr according to food retention time in stomach, and measuring OD 600 Values, as a control. Meanwhile, 1% of inoculation amount of activated seed solution is added into fresh YPD liquid culture media with pH values of 2, 3 and 7 respectively, and 10% of extract mixed solution is added; the OD was measured by culturing for 4 hours at 37 ℃ in an incubator according to the residence time of food in the stomach 600 Values were used as experimental groups.
2. The experimental results are as follows: as shown in fig. 6.
FIG. 6 is a graph showing the effect of adding a 10% extract mixed solution on the acid resistance of beer yeast in this example. As can be seen from fig. 6, when pH =2, pH =3, and pH =7, the OD value of the lager brewing yeast was significantly increased after the 10% mixed solution was added, indicating that the acid resistance of the lager brewing yeast could be improved by the 10% mixed solution.
Therefore, the acid resistance of the beer yeast can be effectively improved by adding the mixed solution of the extract.
2. Control experiment for "single solution":
1. the experimental steps are as follows:
(1) Mixing the astragalus membranaceus tablets according to a mass ratio of 1:5, putting the mixture into clear water, and soaking the mixture overnight; the next day, ultrasonic extraction is carried out with the ultrasonic power of 800W for 30min and the temperature of 50 ℃; filtering with four layers of gauze, and centrifuging at 20 deg.C at 10000rpm/min for 5min; taking the supernatant, and diluting to 200mL to obtain the astragalus extract.
(2) Mixing the hawthorn in a mass ratio of 1:10 putting the mixture into clear water, boiling the mixture for 1 hour at 100 ℃, and filtering the mixture to obtain a first leaching solution; mixing the residue filtered by the four layers of gauze with clear water (the mass ratio of the residue to the clear water is 1:5), boiling for 30min, and filtering to obtain a second leaching solution; mixing the two leaching solutions, adding absolute ethyl alcohol, preparing 60% alcohol precipitation solution, and standing overnight at 4 ℃; centrifuging at 11000rpm/min for 5min at 4 deg.C the next day; and (4) recovering ethanol after taking the supernatant to obtain an extracting solution, and fixing the volume to 200mL to obtain the hawthorn extract.
(3) And (2) mixing the liquorice in a mass ratio of 1:10, putting the mixture into clear water, soaking for 1 hour, and decocting for 2 hours; filtering with four layers of gauze, decocting the residue for 2 times under the same conditions for 1.5 hr and 1 hr respectively; mixing the three decoctions, adding anhydrous ethanol, preparing 70% ethanol precipitation solution, and standing overnight at 4 deg.C; centrifuging at 10000rpm/min at 20 deg.C for 5min the next day; and (4) recovering ethanol after taking the supernatant to obtain an extracting solution, and fixing the volume to 200mL to obtain the liquorice extract.
(4) Adding 70% ethanol into folium Eucommiae, and soaking overnight; performing ultrasonic treatment at 800W and 60 deg.C for 40min; filtering with four layers of gauze, rotationally evaporating ethanol to obtain an aqueous solution, and metering to 200mL to obtain the eucommia ulmoides leaf extract.
(5) The beer yeast preserved in the slant of one loop was streaked with an inoculating loop, inoculated into 100mL YPD liquid medium, and cultured overnight in an incubator at 37 ℃ to obtain activated seed liquid.
(6) Adding 1% of the inoculation amount of activated seed solution into fresh YPD liquid culture medium with pH values of 2, 3 and 7 respectively, and adding 10% of radix astragali extract; the OD was measured by incubating the food in an incubator at 37 ℃ for 4 hours according to the residence time in the stomach 600 Numerical values.
(7) Adding 1% of the inoculation amount of activated seed solution into fresh YPD liquid culture medium with pH values of 2, 3 and 7 respectively, and adding 5% of fructus crataegi extract; the OD was measured by incubating the food in an incubator at 37 ℃ for 4 hours according to the residence time in the stomach 600 Numerical values.
(8) Adding 1% of inoculation amount of activated seed solution into fresh YPD liquid culture medium with pH values of 2, 3 and 7 respectively, and adding 3% of Glycyrrhrizae radix extract; the OD was measured by incubating the food in an incubator at 37 ℃ for 4 hours according to the residence time in the stomach 600 Numerical values.
(9) Fresh YPD liquids at pH values of 2, 3 and 7, respectivelyAdding 1% of the inoculation amount of the activated seed solution into the culture medium, and then adding 10% of the eucommia ulmoides leaf extract; the OD was measured by culturing for 4 hours at 37 ℃ in an incubator according to the residence time of food in the stomach 600 Numerical values.
2. The experimental results are as follows: as shown in fig. 7.
FIG. 7 shows the effect of adding single solution and mixed solution on acid resistance of beer yeast. At pH =2, pH =3 and pH =7, the addition of the mixed solution has a greater influence on the increase in acid resistance of the lager brewing yeast than the addition of the single solution.
Therefore, the acid resistance of the beer yeast can be improved by adding the mixed extract solution in the invention compared with that of a single solution.
Experimental example 7
This example illustrates the effect of the extract mixture solution of the present invention on the bile salt resistance of brewers yeast.
1. Effect experiment of extract mixed solution:
1. the experimental steps are as follows:
(1) Mixing the astragalus root tablets according to a mass ratio of 1:5, putting the mixture into clear water, and soaking the mixture overnight; the next day, ultrasonic extraction is carried out with the ultrasonic power of 800W for 30min and the temperature of 50 ℃; filtering with four layers of gauze, and centrifuging at 20 deg.C at 10000rpm/min for 5min; taking the supernatant, and diluting to 200mL to obtain the astragalus extract.
(2) Mixing the hawthorn with the following raw materials in a mass ratio of 1:10 putting the mixture into clear water, boiling the mixture for 1 hour at 100 ℃, and filtering the mixture to obtain a first leaching solution; mixing the residue filtered by the four layers of gauze with clear water (the mass ratio of the residue to the clear water is 1:5), boiling for 30min, and filtering to obtain a second leaching solution; mixing the two leaching solutions, adding anhydrous ethanol, preparing 60% ethanol precipitation solution, and standing overnight at 4 deg.C; centrifuging at 11000rpm/min for 5min at 4 deg.C the next day; and (4) recovering ethanol after taking the supernatant to obtain an extracting solution, and fixing the volume to 200mL to obtain the hawthorn extract.
(3) Liquorice is added according to the mass ratio of 1:10, putting the mixture into clear water, soaking for 1 hour, and decocting for 2 hours; filtering with four layers of gauze, decocting the residue for 2 times under the same conditions for 1.5 hr and 1 hr respectively; mixing the three decoctions, adding anhydrous ethanol, preparing 70% ethanol precipitation solution, and standing overnight at 4 deg.C; centrifuging at 10000rpm/min at 20 deg.C for 5min the next day; and (4) recovering ethanol after taking the supernatant to obtain an extracting solution, and fixing the volume to 200mL to obtain the liquorice extract.
(4) Adding 70% ethanol into folium Eucommiae, and soaking overnight; performing ultrasonic treatment at 800W and 60 deg.C for 40min; filtering with four layers of gauze, rotary evaporating to remove ethanol to obtain water solution, and metering to volume of 200mL to obtain folium Eucommiae extract.
(5) Mixing the obtained astragalus extract, hawthorn extract, liquorice extract and eucommia ulmoides leaf extract according to a mass ratio of 2:3:1:2 mixing to obtain an extract mixed solution.
(6) A ring slant of the deposited Saccharomyces cerevisiae (BY 4742, purchased from BNCC) was streaked with an inoculating ring, inoculated into 100mL of YPD liquid medium, and cultured overnight in an incubator at 37 ℃ to obtain an activated seed solution.
(7) 1% inoculum of activated seed solution was added to fresh YPD liquid medium, and 1mL of the mixed culture solution was collected as a blank. Meanwhile, 1% inoculum of activated seed solution was added to fresh YPD liquid medium containing 0.3% and 0.5% bile salts, 1mL was collected as a control group, 10% extract mixed solution was added, and 1mL of mixed culture solution was collected as an experimental group; the CFU and survival in bile salts were calculated for each sample by incubation at 37 ℃ for 4h, based on the food residence time in the stomach, dilution plating, and then incubation at 37 ℃ for 24 h.
2. The experimental results are as follows: as shown in fig. 8.
FIG. 8 is a graph showing the effect of the addition of the 10% mixed solution on the bile salt resistance of lager brewing yeast in this example. As can be seen from FIG. 8, when 0.3% and 0.5% of bile salt was added, 10% of the mixed solution was added. The survival rate of the beer yeast is obviously increased, which shows that the 10 percent mixed solution can improve the bile salt resistance of the beer yeast.
Therefore, the extract mixed solution can effectively improve the bile salt resistance of the beer yeast.
2. Control experiment of "single solution":
1. the experimental steps are as follows:
(1) Mixing the astragalus root tablets according to a mass ratio of 1:5, putting the mixture into clear water, and soaking the mixture overnight; performing ultrasonic extraction with ultrasonic power of 800W for 30min at 50 deg.C the next day; filtering with four layers of gauze, and centrifuging at 20 deg.C at 10000rpm/min for 5min; taking the supernatant, and diluting to 200mL to obtain the astragalus extract.
(2) Mixing the hawthorn with the following raw materials in a mass ratio of 1:10 putting the mixture into clear water, boiling the mixture for 1 hour at 100 ℃, and filtering the mixture to obtain a first leaching solution; mixing the residue filtered by the four layers of gauze with clear water (the mass ratio of the residue to the clear water is 1:5), boiling for 30min, and filtering to obtain a second leaching solution; mixing the two leaching solutions, adding anhydrous ethanol, preparing 60% ethanol precipitation solution, and standing overnight at 4 deg.C; centrifuging at 11000rpm/min for 5min at 4 deg.C the next day; and (4) recovering ethanol after taking the supernatant to obtain an extracting solution, and fixing the volume to 200mL to obtain the hawthorn extract.
(3) Liquorice is added according to the mass ratio of 1:10, putting the mixture into clear water, soaking for 1 hour, and decocting for 2 hours; filtering with four layers of gauze, decocting the residue for 2 times under the same conditions for 1.5h and 1h respectively; mixing the three decoctions, adding anhydrous ethanol, preparing 70% ethanol precipitation solution, and standing at 4 deg.C overnight; centrifuging at 10000rpm/min at 20 deg.C for 5min the next day; and (4) recovering ethanol after taking the supernatant to obtain an extracting solution, and fixing the volume to 200mL to obtain the liquorice extract.
(4) Adding 70% ethanol into folium Eucommiae, and soaking overnight; performing ultrasonic treatment at 800W and 60 deg.C for 40min; filtering with four layers of gauze, rotationally evaporating ethanol to obtain an aqueous solution, and metering to 200mL to obtain the eucommia ulmoides leaf extract.
(5) The beer yeast preserved in the slant of one loop was streaked with an inoculating loop, inoculated into 100mL YPD liquid medium, and cultured overnight in an incubator at 37 ℃ to obtain activated seed liquid.
(6) 1% inoculum of activated seed solution was added to fresh YPD liquid medium, and 1mL of the mixed culture solution was collected as a blank. Meanwhile, 1% of inoculum size of activated seed solution is added into fresh YPD liquid culture medium containing 0.3% and 0.5% of bile salt, 1mL is respectively collected to be used as a control group, 10% of astragalus extract is added, and 1mL of mixed culture solution is collected to be used as an experimental group; the CFU and survival in bile salts were calculated for each sample by incubation at 37 ℃ for 4h, based on the food residence time in the stomach, dilution plating, and then incubation at 37 ℃ for 24 h.
(7) 1% inoculum of activated seed solution was added to fresh YPD liquid medium, and 1mL of the mixed culture solution was collected as a blank. Meanwhile, 1% of inoculum size of activated seed liquid is added into fresh YPD liquid culture medium containing 0.3% and 0.5% of bile salt, 1mL is respectively collected to be used as a control group, 5% of hawthorn extract is added, and 1mL of mixed culture liquid is collected to be used as an experimental group; the CFU and survival in bile salts were calculated for each sample by incubation at 37 ℃ for 4h, based on the food residence time in the stomach, dilution plating, and then incubation at 37 ℃ for 24 h.
(8) 1% inoculum of activated seed solution was added to fresh YPD liquid medium, and 1mL of the mixed culture solution was collected as a blank. Meanwhile, 1% inoculum of activated seed solution was added to fresh YPD liquid medium containing 0.3% and 0.5% bile salts, 1mL was collected as a control group, 3% licorice extract was added, and 1mL of mixed culture solution was collected as an experimental group; the CFU and survival in bile salts were calculated for each sample by incubation at 37 ℃ for 4h, based on the food residence time in the stomach, dilution plating, and then incubation at 37 ℃ for 24 h.
(9) 1% inoculum of activated seed solution was added to fresh YPD liquid medium, and 1mL of the mixed culture solution was collected as a blank. Meanwhile, 1% of inoculum size of activated seed liquid is added into fresh YPD liquid culture medium containing 0.3% and 0.5% of bile salt, 1mL is respectively collected to be used as a control group, 10% of eucommia ulmoides leaf extract is added, and 1mL of mixed culture liquid is collected to be used as an experimental group; the CFU and survival in bile salts were calculated for each sample by incubation at 37 ℃ for 4h, based on the food residence time in the stomach, dilution plating, and then incubation at 37 ℃ for 24 h.
2. The experimental results are as follows: as shown in fig. 9.
FIG. 9 is a graph showing the effect of adding a single solution and a mixed solution on the bile salt resistance of lager brewing yeast in this example. As can be seen from FIG. 9, when 0.3% and 0.5% bile salts were added, the survival rate of lager brewing yeast was slightly improved by adding the mixed solution compared to the single solution.
It is found that the mixed solution can improve the bile salt resistance of the lager brewing yeast more than the single solution.
Example 8
The preparation method of the complex microbial inoculum of the embodiment comprises the following steps:
(1) Mixing the astragalus membranaceus tablets according to a mass ratio of 1:5, putting the mixture into clear water, and soaking the mixture overnight; the next day, ultrasonic extraction is carried out with the ultrasonic power of 800W for 30min and the temperature of 50 ℃; filtering with four layers of gauze, and centrifuging at 20 deg.C at 10000rpm/min for 5min; taking the supernatant and fixing the volume to 200mL to obtain the astragalus extract.
(2) Mixing the hawthorn with the following raw materials in a mass ratio of 1:10 putting into clear water, boiling for 1h at 100 ℃, and filtering to obtain a first leaching solution; mixing the residue filtered by the four layers of gauze with clear water (the mass ratio of the residue to the clear water is 1:5), boiling for 30min, and filtering to obtain a second leaching solution; mixing the two leaching solutions, adding anhydrous ethanol, preparing 60% ethanol precipitation solution, and standing overnight at 4 deg.C; centrifuging at 11000rpm/min at 4 ℃ for 5min the next day; and (4) recovering ethanol after taking the supernatant to obtain an extracting solution, and fixing the volume to 200mL to obtain the hawthorn extract.
(3) Liquorice is added according to the mass ratio of 1:10 putting the mixture into clear water, soaking for 1 hour, and decocting for 2 hours; filtering with four layers of gauze, decocting the residue for 2 times under the same conditions for 1.5h and 1h respectively; mixing the three decoctions, adding anhydrous ethanol, preparing 70% ethanol precipitation solution, and standing at 4 deg.C overnight; centrifuging at 10000rpm/min at 20 deg.C for 5min the next day; and (4) recovering ethanol after taking the supernatant to obtain an extracting solution, and fixing the volume to 200mL to obtain the liquorice extract.
(4) Adding 70% ethanol into folium Eucommiae, and soaking overnight; performing ultrasonic treatment at 800W and 60 deg.C for 40min; filtering with four layers of gauze, rotationally evaporating ethanol to obtain an aqueous solution, and metering to 200mL to obtain the eucommia ulmoides leaf extract.
(5) Mixing the obtained astragalus extract, hawthorn extract, liquorice extract and eucommia ulmoides leaf extract according to a mass ratio of 2:3:1:2 mixing to obtain an extract mixed solution; then, the extract mixed solution was spray-dried under conditions of an inlet air temperature of 170 ℃ and a peristaltic pump speed of 7rpm/min to obtain extract mixed powder.
(6) A ring slant of the deposited Saccharomyces cerevisiae (BY 4742, purchased from BNCC) was streaked with an inoculating ring, inoculated into 100mL of YPD liquid medium, and cultured overnight in an incubator at 37 ℃.
(7) After the overnight culture is finished, centrifuging for 6min at 4 ℃ and 5000rpm/min by using a high-speed refrigerated centrifuge, collecting thalli, and washing for 3 times by using sterile normal saline to obtain beer yeast mud; adding a protective agent with 20 percent of the volume of the fermentation liquor into the beer yeast strain mud, pre-freezing for 2 hours at the temperature of minus 70 ℃, and then placing the mixture into a freeze dryer for freeze drying for 36 to 48 hours; after the freeze drying is finished, grinding into powder; wherein, the protective agent comprises the following components: 10% of skim milk, 10% of trehalose and 5% of sucrose.
(8) Mixing the extract mixed powder obtained in the step (5) with the beer yeast freeze-dried powder obtained in the step (7) according to the ratio of 2:1 to obtain the compound bacterial agent required by the target.
FIG. 10 shows the lager brewing yeast lyophilized powder (A) and the extract mixed powder (B) obtained in the present example.
In the embodiment, the composite microbial inoculum of the mixed powder of the astragalus, the hawthorn, the liquorice and the eucommia leaves and the beer yeast freeze-dried powder can be obtained.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A novel method for improving the activity of beer yeast is characterized by comprising the following steps:
(1) Putting the astragalus slices into clear water, and soaking overnight; performing ultrasonic extraction with ultrasonic power of 800W for 30min at 50 deg.C the next day; filtering, and centrifuging to obtain radix astragali extract;
(2) Putting hawthorn into clear water, boiling for 1h, and filtering to obtain a first leaching solution; mixing the filtered residue with clear water, boiling for 30min, and filtering to obtain second extractive solution; mixing the two leaching solutions, adding 60% anhydrous ethanol, and precipitating with ethanol overnight; centrifuging the next day, collecting supernatant, and recovering ethanol to obtain fructus crataegi extract;
(3) Putting liquorice into clear water, soaking for 1h, and decocting for 2h; after filtering, decocting the residue for 2 times under the same conditions for 1.5h and 1h respectively; mixing the three decoctions, adding 70% anhydrous ethanol, and precipitating with ethanol overnight; centrifuging the next day, collecting supernatant, and recovering ethanol to obtain Glycyrrhrizae radix extract;
(4) Adding ethanol into folium Eucommiae, and soaking overnight; performing ultrasonic treatment at 800W and 60 deg.C for 40min; filtering, and performing rotary evaporation to obtain folium Eucommiae extract;
(5) Mixing the obtained radix astragali extract, fructus crataegi extract, glycyrrhrizae radix extract and folium Eucommiae extract according to a ratio of 2:3:1:2 to obtain an extract mixed solution;
(6) Inoculating beer yeast into YPD liquid culture medium, and culturing overnight in 37 deg.C incubator to obtain activated seed liquid;
(7) Adding the activated seed solution into a fresh YPD liquid culture medium, adding a mixed solution of 3-20% of the extract, and co-culturing at 37 ℃.
2. The novel method for improving the activity of brewer's yeast according to claim 1, wherein in step (1), the astragalus membranaceus slices are mixed according to a mass ratio of 1:5, putting the mixture into clear water, and soaking the mixture overnight; after ultrasonic extraction, filtering with four layers of gauze, and centrifuging the filtrate at 20 ℃ at 10000rpm/min for 5min; after centrifugation, taking the supernatant and fixing the volume to 200mL to obtain the astragalus extract.
3. The novel method for improving the activity of brewer's yeast according to claim 1, wherein in the step (2), the hawthorn is added into the beer yeast according to the mass ratio of 1:10 putting the mixture into clear water, boiling the mixture for 1 hour at 100 ℃, and filtering the mixture to obtain a first leaching solution; mixing the filtered residue with clear water according to a mass ratio of 1:5 mixing and boiling for 30min again, and filtering to obtain a second leaching solution; mixing the two leaching solutions, adding absolute ethyl alcohol, preparing 60% alcohol precipitation solution, and standing overnight at 4 ℃; centrifuging at 11000rpm/min at 4 ℃ for 5min the next day; and (4) recovering ethanol after taking the supernatant to obtain an extracting solution, and fixing the volume to 200mL to obtain the hawthorn extract.
4. The novel method for improving the activity of brewer's yeast according to claim 1, wherein in step (3), licorice is added in a mass ratio of 1:10 soaking in clear water, and decocting; filtering with four layers of gauze, decocting the residue for 2 times under the same conditions; mixing the three decoctions, adding anhydrous ethanol, preparing 70% ethanol precipitation solution, and standing at 4 deg.C overnight; centrifuging at 10000rpm/min at 20 deg.C for 5min the next day; and (4) recovering ethanol after taking the supernatant to obtain an extracting solution, and fixing the volume to 200mL to obtain the liquorice extract.
5. The novel method for improving the activity of brewer's yeast according to claim 1, wherein in step (4), after 70% ethanol soaking eucommia ulmoides leaves overnight, sonication is carried out; filtering with four layers of gauze, rotary evaporating to remove ethanol to obtain water solution, and metering to volume of 200mL to obtain folium Eucommiae extract.
6. The novel method for improving the activity of a lager brewing yeast according to claim 1, wherein in the step (6), the lager brewing yeast preserved in the slant of the loop is extracted by inoculating with an inoculating loop, inoculated into 100mL YPD liquid medium, and cultured overnight in an incubator at 37 ℃ to obtain an activated seed solution.
7. The novel method for improving the activity of brewer's yeast according to claim 1, wherein in step (7), the specific addition amount of the extract mixed solution is 10%.
8. The preparation method of the composite microbial inoculum is characterized by comprising the following steps:
(1) Putting the astragalus slices into clear water, and soaking overnight; performing ultrasonic extraction with ultrasonic power of 800W for 30min at 50 deg.C the next day; filtering, and centrifuging to obtain radix astragali extract;
(2) Putting hawthorn into clear water, boiling for 1h, and filtering to obtain a first leaching solution; mixing the filtered residue with clear water, boiling for 30min, and filtering to obtain a second leaching solution; mixing the two leaching solutions, adding 60% anhydrous ethanol, and precipitating with ethanol overnight; centrifuging the next day, collecting supernatant, and recovering ethanol to obtain fructus crataegi extract;
(3) Putting liquorice into clear water, soaking for 1h, and decocting for 2h; after filtration, the residue is decocted for 2 times under the same conditions, and the time is 1.5h and 1h respectively; mixing the three decoctions, adding 70% anhydrous ethanol, and precipitating with ethanol overnight; centrifuging the next day, collecting supernatant, and recovering ethanol to obtain Glycyrrhrizae radix extract;
(4) Adding ethanol into folium Eucommiae, and soaking overnight; performing ultrasonic treatment at 800W and 60 deg.C for 40min; filtering, and performing rotary evaporation to obtain folium Eucommiae extract;
(5) Mixing the obtained astragalus extract, hawthorn extract, liquorice extract and eucommia ulmoides leaf extract according to a ratio of 2:3:1:2 to obtain an extract mixed solution; then, spray drying the extract mixed solution to obtain extract mixed powder;
(6) Inoculating beer yeast into YPD liquid culture medium, and culturing overnight in 37 deg.C incubator to obtain activated seed liquid;
(7) After overnight culture, centrifugally collecting thalli, and washing for a plurality of times by using sterile normal saline to obtain beer yeast mud; adding a protective agent into the beer yeast strain mud, pre-freezing, and freeze-drying in a freeze dryer; after freeze-drying, grinding into powder;
(8) And (4) mixing the extract mixed powder obtained in the step (5) with the beer yeast freeze-dried powder obtained in the step (7) to obtain the compound microbial inoculum required by the target.
9. The preparation method of the complex microbial inoculum according to claim 8, wherein in the step (4), the spray drying condition is that the inlet air temperature is 170 ℃ and the peristaltic pump speed is 7rpm/min.
10. The method for preparing a composite microbial inoculum according to claim 8, wherein in the step (6), after overnight culture is finished, a high-speed refrigerated centrifuge is used for centrifuging for 6min at 4 ℃ and 5000rpm/min, thalli are collected and washed for 3 times by using sterile normal saline to obtain beer yeast sludge; adding a protective agent with 20 percent of the volume of the fermentation liquor into the beer yeast strain mud, pre-freezing for 2 hours at the temperature of minus 70 ℃, and then placing the mixture into a freeze dryer for freeze drying for 36 to 48 hours; after the freeze drying is finished, grinding into powder; wherein, the protective agent comprises the following components: 10% of skim milk, 10% of trehalose and 5% of sucrose.
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| JP2008099682A (en) * | 2006-09-22 | 2008-05-01 | Sanei Gen Ffi Inc | Composition containing functional raw material and/or extract |
| CN102366024A (en) * | 2011-09-16 | 2012-03-07 | 汪松林 | Production method of pig feed additive containing traditional Chinese medicine and active probiotics |
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