CN110628648B - Rhizomucor fungi producing liquefied amylase and application - Google Patents
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Abstract
The invention provides rhizomucor for producing liquefied amylase and application thereof, and relates to the technical field of white spirit brewing production. The Rhizomucor sp TSM10.4 for producing the liquefied amylase is preserved in China general microbiological culture collection center with the preservation number of CGMCC No. 17200. The rhizomucor in the invention can grow well at the temperature of 30-50 ℃ and produce liquefied amylase; the solid fermentation powder can be prepared into solid fermentation bacteria powder for solid fermentation, and is applied to the production of the brewing yeast for making wine according to a proportion, so that the enhanced brewing yeast for making wine with high activity of liquefied amylase can be prepared; the enhanced brewing yeast is applied to brewing of white spirit, the liquor yield can be improved, and the liquor quality is improved, namely the top-grade liquor yield is improved.
Description
Technical Field
The invention relates to the technical field of white spirit brewing production, in particular to rhizomucor for producing liquefied amylase and application thereof.
Background
The yeast is the bone of the liquor, the yeast is an extremely important element in the brewing production of the liquor, provides biological enzyme, flavor substance and flavor precursor substance for the brewing production of the liquor, and has important influence on the yield and the quality of the liquor.
The liquefied amylase is an important enzyme system in the yeast for making hard liquor, and is an important factor influencing the yield and quality of the white liquor. In the brewing of white spirit, the liquefied amylase can firstly decompose starch in raw materials into dextrin, a small amount of maltose and the like, and further decompose the dextrin into reducing sugar, so that ethanol and other fragrant and flavor substances can be generated.
Rhizomucor fungi widely exist in the process of brewing white spirit, Ri\21232, Keetc. (2017) separate rhizomucor pustulatus from Maotai region Maotai-flavor vinasse; the Mucor miehei is separated from Maotai Daqu by the family and the company (2015); zhang Xiang et al (2013) found that Rhizomucor fungi are widely present in the distiller's yeast by culturable method, and the separation rate reaches 9.9%. At present, researches show that rhizomucor has activities of saccharifying enzyme, cellulase and the like, but reports of producing liquefied amylase and applications of rhizomucor in liquor production are not shown.
Disclosure of Invention
The invention aims to provide rhizomucor for producing liquefied amylase and application thereof, which can improve the liquor yield and the top-grade liquor yield of liquor brewing. The technical effects that can be produced by the preferred technical scheme in the technical schemes provided by the invention are described in detail in the following.
In order to achieve the purpose, the invention provides the following technical scheme:
the Rhizomucor sp TSM10.4 for producing the liquefied amylase is preserved in the China general microbiological culture collection management center in 2019, 4 months and 1 days, and the preservation number is CGMCC No. 17200.
The invention provides solid fermentation fungus powder prepared from the rhizomucor for producing the liquefied amylase.
The preparation method of the solid zymophyte powder provided by the invention comprises the following steps:
(1) using the root mucor producing the liquefied amylase as a strain to prepare spore suspension;
(2) inoculating the spore suspension prepared in the step (1) by using the sterilized solid fermentation culture medium, wherein the temperature of the solid fermentation culture medium is 30-50 ℃ during inoculation; culturing at 30-50 deg.C after inoculation;
(3) and (3) drying the culture obtained in the step (2) at the temperature of 30-50 ℃, and crushing to obtain the solid zymophyte powder.
Furthermore, in the step (1), the strains need to be selected from strains which are fresh and activated, grow vigorously and have rich spores.
Further, in the step (2), after the spore suspension prepared in the step (1) is inoculated, standing culture is carried out for 2.5 days to 3.5 days;
in the step (3), after the culture is dried, the water content is controlled to be 10-12%.
The invention provides an enhanced brewing yeast containing the solid fermentation bacteria powder.
Furthermore, the viable count of rhizomucor in the enhanced brewing yeast is 1 multiplied by 104CFU/g-1×107CFU/g。
Further, the solid fermentation bacteria powder is added according to the weight percentage
Further, the solid fermentation bacteria powder is added according to the weight percentage
The method for increasing the wine yield provided by the invention is used for brewing wine by using the enhanced brewing yeast for making wine.
Based on the technical scheme, the embodiment of the invention can at least produce the following technical effects:
the rhizomucor of the liquefied amylase, the solid fermentation fungus powder, the preparation method of the rhizomucor of the liquefied amylase and the method for strengthening brewing yeast and increasing the liquor yield are provided by the invention, and the rhizomucor can well grow at the temperature of 30-50 ℃ and generate the liquefied amylase; the solid fermentation powder can be prepared into solid fermentation bacteria powder for solid fermentation, and is applied to the production of the brewing yeast for making wine according to a proportion, so that the enhanced brewing yeast for making wine with high activity of liquefied amylase can be prepared; the enhanced brewing yeast is applied to brewing of white spirit, the liquor yield can be improved, and the liquor quality is improved, namely the top-grade liquor yield is improved.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
In one embodiment, the culture medium is:
1. primary screening of culture medium:
the components and the weight percentages of the components are respectively as follows: 2% of soluble starch, 0.4% of yeast extract, 0.1% of sodium dihydrogen phosphate, 0.1% of disodium hydrogen phosphate, 0.003% of amber red, 0.002% of kanamycin, 2.0% of agar and the balance of water.
The pH of the primary screening medium was 5.0.
2. Re-screening the culture medium:
the components and the weight percentages of the components are respectively as follows: 2% of soluble starch, 0.4% of yeast extract, 0.1% of sodium dihydrogen phosphate, 0.1% of disodium hydrogen phosphate, 2.0% of agar and the balance of water.
The pH of the rescreened medium was 4.5.
3. PDA culture medium:
200 g of potato, 1000 ml of water is added, the mixture is boiled for 20 minutes, four layers of gauze are filtered, the volume of filtrate is up to 1000 ml, 20 g of glucose and 15 g of agar are added.
The pH of the PDA medium was 6.0.
4. Solid fermentation medium:
water and bran in a weight ratio of 0.7-1: 1, and uniformly mixing.
Secondly, the standard of the activity determination of the liquefied amylase and the viable count determination of the rhizomucor is as follows:
1. determination of the Activity of a liquefying amylase
The method is carried out according to the method for measuring the liquefaction capacity in QB/T4257-2011 general analytical method for brewing Daqu.
2. Determination of viable count of Rhizomucor
The counting was carried out according to GB4789 (mould counting method).
Third, embodiment:
the first embodiment is as follows:
1. strain screening:
(1) preliminary screening
Grinding Daqu into powder, adding 25g into sterile physiological saline containing 225mL, and shaking at 37 deg.C for 30min (180r/min) to obtain 10-1Bacterial suspension; standing for 30min, collecting supernatant 1mL, adding into sterile physiological saline 9mL, and mixing to obtain 10-2Bacterial suspension; and so on to obtain 10-3、10-4、10-5、10-6Bacterial suspension; respectively take 10-4、10-5、10-61mL of each diluted bacterial suspension is put into a sterile culture dish, a prescreening culture medium which is melted and cooled to 50 ℃ is added to be uniformly mixed, the mixture is placed into a constant-temperature incubator at 37 ℃ to be cultured for 4 days after solidification, a proper amount of 0.02mol/L iodine solution is dripped around a growing bacterial colony, if the bacterial colony is a colorless transparent ring, the bacterial strain is a bacterial strain which generates liquefying amylase, and the bacterial strain which generates the colorless transparent ring is selected to be subjected to streak separation until a pure bacterial strain is obtained.
(2) Double sieve
Melting a re-screened culture medium, pouring the melted re-screened culture medium into a sterile culture dish (15-20 mL of culture medium is poured into a culture dish with the diameter of 9 cm), solidifying, then dotting the pure strains obtained by primary screening to the center of the culture dish, respectively placing the pure strains in a constant-temperature culture box with the temperature of 30 ℃, 35 ℃, 40 ℃, 45 ℃ and 50 ℃ for culturing for 48 hours, dripping a proper amount of 0.02mol/L iodine solution around the grown bacterial colonies to observe whether colorless transparent circles are generated, if the colorless transparent circles are formed, the bacterial strains are the bacterial strains generating the liquefied amylase, the larger the transparent circles are, the stronger the enzyme activity is, and finally obtaining the bacterial strain TSM10.4 which can grow in the temperature range of 30-50 ℃ and has the strongest liquefied amylase activity.
2. Identification of Strain TSM10.4
The strain TSM10.4 is cultured on a PDA culture medium plate at 30 ℃ for 4 days, the diameter reaches 8.6cm, the hyphae are initially white, then the hyphae become dark gray, and the reverse side is colorless. No exudate and no soluble pigment are produced. Hyphae are not septate, branch, possess cyst axis, have no cyst support, and have a spherical cyst spore shape. Heat resistance, good growth at 50 ℃.
The molecular biological characteristics of the strain TSM10.4 are as follows: the ITS rDNA sequence was aligned with the GenBank database and had greater than 99% homology to the ITS rDNA sequence of Mucor radiculosa (Rhizomucor sp.).
By combining the morphological characteristics and the hereditary characteristics, the strain for producing the liquefied amylase can be identified as Rhizomucor sp, which is named as Rhizomucor TSM10.4 and is preserved in China general microbiological culture collection center in 2019, 4 and 1, with the preservation number of CGMCC No. 17200.
Example two:
the preparation method of the solid fermentation bacterium powder comprises the following specific steps:
(1) weighing 25g-30g of solid fermentation medium in a 500mL triangular flask, and sterilizing at 121 ℃ for 40 minutes;
(2) taking a fresh activated, vigorous-growing and rich-spore rhizomucor TSM10.4 strain, adding 5mL of sterile normal saline, oscillating to disperse spores to prepare spore suspension, inoculating the spore suspension after a solid fermentation culture medium is cooled to below 50 ℃, and standing and culturing for 3 days at 37 ℃;
(3) drying the culture at 45 ℃ for 12-24 h, controlling the water content to 10-12%, crushing, and packaging in a self-sealing bag to obtain the solid zymophyte powder.
Weighing the above bacteria powder product, and counting according to GB4789 (mould counting method), wherein the viable count of Rhizomucor in the solid fermentation bacteria powder is 1.3 × 107More than CFU/g, 0.2 percent of mixed bacteria.
According to a liquefying capacity measuring method in QB/T4257-2011 general analytical method for brewing Daqu, the activity of the liquefying amylase in the solid fermentation powder is 14.63 g/g.h.
Example three:
preparing reinforced medium-temperature Daqu:
weighing 5 kg of the solid fermentation powder prepared in the embodiment 2, adding 1000 kg of the crushed starter propagation raw material (the starter propagation raw material is the conventional medium-temperature starter propagation raw material), uniformly mixing, and preparing the enhanced medium-temperature starter propagation according to the conventional medium-temperature starter propagation preparation process.
The viable count of the intensified intermediate-temperature rhizomucor daquus prepared by the embodiment is 8.5 multiplied by 106CFU/g, thin skin, regular section, dense hypha, grey white color, pure yeast aroma and no peculiar smell, and the activity of the liquefied amylase is increased by 49.91% (the activity of the unartified contrast Daqu liquefied amylase is 5.53g/g.h, and the activity of the intensified medium-temperature Daqu liquefied amylase is 8.29 g/g.h).
Example four:
preparing reinforced medium-high temperature Daqu:
weighing 1 kg of the solid fermentation powder prepared in the embodiment 2, adding 1000 kg of the crushed starter propagation raw material (the starter propagation raw material is a conventional medium-high temperature starter propagation raw material), uniformly mixing, and preparing the enhanced medium-high temperature starter propagation according to a conventional medium-high temperature starter propagation preparation process.
The viable count of the enhanced medium-high temperature rhizomucor daqu prepared by the embodiment is 1.7 multiplied by 106CFU/g, thin skin, regular section, dense hypha, grey white color, strong fragrance, pure fragrance and no peculiar smell. The activity of the liquefying amylase is increased by 38.32 percent (the activity of the non-strengthened contrast Daqu liquefying amylase is 3.81g/g.h, and the activity of the strengthened medium-high temperature Daqu liquefying amylase is 5.27 g/g.h).
Example five:
preparing the reinforced high-temperature Daqu:
0.5 kg of the solid fermentation powder prepared in the embodiment 2 is weighed, added into 1000 kg of the crushed starter propagation raw materials (the starter propagation raw materials are the conventional high-temperature starter propagation raw materials), uniformly mixed, and prepared into the enhanced high-temperature starter propagation according to the conventional high-temperature starter propagation preparation process.
The viable count of the enhanced high-temperature rhizomucor daquus prepared by the embodiment is 1.8 multiplied by 105CFU/g, brown appearance, regular section, grey white and slightly brown color, and has a yeast fragrance and a sauce fragrance, and the activity of the liquefied amylase is increased by 25.91% (the activity of the unarchified contrast Daqu liquefied amylase is 2.74g/g.h, and the activity of the intensified high-temperature Daqu liquefied amylase is 3.45 g/g.h).
Example six:
preparing the reinforced high-temperature Daqu:
0.1 kg of the solid fermentation powder prepared in the embodiment 2 is weighed, added into 1000 kg of the crushed starter propagation raw materials (the starter propagation raw materials are the conventional high-temperature starter propagation raw materials), uniformly mixed, and prepared into the enhanced high-temperature starter propagation according to the conventional high-temperature starter propagation preparation process.
The viable count of the enhanced high-temperature rhizomucor daquus prepared by the embodiment is 1.0 multiplied by 104CFU/g, brown appearance, regular section, grey white and slightly brown color, and has a yeast fragrance and a sauce fragrance, and the activity of the liquefied amylase is increased by 20.44% (the activity of the unarchified contrast Daqu liquefied amylase is 2.74g/g.h, and the activity of the intensified high-temperature Daqu liquefied amylase is 3.30 g/g.h).
Example seven:
preparing reinforced medium-temperature Daqu:
weighing 10 kg of the solid fermentation powder prepared in the embodiment 2, adding 1000 kg of the crushed starter propagation raw material (the starter propagation raw material is the conventional medium-temperature starter propagation raw material), uniformly mixing, and preparing the enhanced medium-temperature starter propagation according to the conventional medium-temperature starter propagation preparation process.
The viable count of the intensified intermediate-temperature rhizomucor majorana prepared by the embodiment is 1.0 multiplied by 107CFU/g, thin skin, regular section, dense hypha, grey white color, pure yeast aroma and no peculiar smell, and the activity of the liquefied amylase is increased by 62.21% (the activity of the reference Daqu liquefied amylase which is not enhanced is 5.53g/g.h, and the activity of the enhanced medium-temperature Daqu liquefied amylase is 8.97 g/g.h).
Example eight:
when the enhanced medium-temperature Daqu prepared in the third embodiment is applied to brewing of fen-flavor liquor, compared with the common medium-temperature Daqu, the liquor yield is increased by 5%, and the top-grade liquor yield is increased by 4%. When brewing wine, the adding amount of the common medium-temperature yeast and the reinforced medium-temperature yeast is the same, and the specific results are shown in the following table 1:
TABLE 1 wine yield and top grade wine yield results for common medium temperature Daqu and enhanced medium temperature Daqu
| Common medium temperature Daqu | Intensified medium-temperature Daqu | |
| Percentage of alcohol production (%) | 42.7 | 47.7 |
| Percentage of super liquor (%) | 61.4 | 65.4 |
Example nine:
when the enhanced medium-high temperature Daqu prepared in the fourth embodiment is applied to brewing of Luzhou-flavor liquor, compared with the common medium-high temperature Daqu, the liquor yield is increased by 4%, and the top-grade liquor yield is increased by 6%. When brewing wine, the adding amount of the common medium-high temperature yeast and the reinforced medium-high temperature yeast is the same, and the specific results are shown in the following table 2:
TABLE 2 results of the liquor yield and the top grade liquor yield of common middle and high temperature Daqu and enhanced middle and high temperature Daqu
Example ten:
when the enhanced high-temperature Daqu prepared in the fifth embodiment is applied to brewing of Maotai-flavor liquor, compared with the common high-temperature Daqu, the liquor yield is increased by 4%, and the top-grade liquor yield is increased by 3%. When brewing wine, the adding amount of the common high-temperature Daqu and the reinforced high-temperature Daqu is the same, and the specific results are shown in the following table 3:
TABLE 3 wine yield and top grade wine yield results of common high temperature Daqu and enhanced high temperature Daqu
| Common high-temperature Daqu | Intensified high-temp. Daqu | |
| Percentage of alcohol production (%) | 24.4 | 28.4 |
| Percentage of super liquor (%) | 90.7 | 93.7 |
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention.
Claims (10)
1. A Rhizomucor sp TSM10.4 for producing liquefied amylase is preserved in China general microbiological culture collection center with the preservation number of CGMCC No. 17200.
2. A solid fermentation powder prepared from the liquid amylase-producing Rhizomucor fungi of claim 1.
3. The method for preparing solid fermentative bacterial powder according to claim 2, comprising the steps of:
(1) preparing spore suspension by using rhizomucor rhizopus producing liquefied amylase as defined in claim 1 as strain;
(2) inoculating the spore suspension prepared in the step (1) by using the sterilized solid fermentation culture medium, wherein the temperature of the solid fermentation culture medium is 30-50 ℃ during inoculation; culturing at 30-50 deg.C after inoculation;
(3) and (3) drying the culture obtained in the step (2) at the temperature of 30-50 ℃, and crushing to obtain the solid zymophyte powder.
4. The method for producing a solid fermentative bacterial powder according to claim 3, comprising the steps of: in the step (1), the strains need to be selected from strains which are fresh and activated, grow vigorously and have rich spores.
5. The method for producing a solid fermentative bacterial powder according to claim 3 or 4, comprising the steps of: in the step (2), after the spore suspension prepared in the step (1) is inoculated, standing culture is carried out for 2.5 days to 3.5 days;
in the step (3), after the culture is dried, the water content is controlled to be 10-12%.
6. An enhanced koji for brewing comprising the solid fermentation fungus powder as claimed in claim 2.
7. The enhanced brewing yeast for making wine of claim 6, wherein: the viable count of the rhizomucor in the enhanced brewing yeast is 1 multiplied by 104CFU/g-1×107CFU/g。
8. The enhanced brewing yeast for making wine according to claim 6 or 7, characterized in that: the solid fermentative bacteria powder of claim 2 is added in an amount of
9. The enhanced brew of claim 8The liquor yeast is characterized in that: the solid fermentative bacteria powder of claim 2 is added in an amount of
10. A method for increasing the wine yield is characterized in that: brewing with the enhanced brewing koji according to any of claims 6 to 9.
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| CN110628648A (en) | 2019-12-31 |
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