CN106701518B

CN106701518B - Daqu strengthening method for reducing Daqu dosage and improving vinegar quality

Info

Publication number: CN106701518B
Application number: CN201611063373.9A
Authority: CN
Inventors: 霍乃蕊; 王如福; 李宝霞; 华锦; 张也; 侯红萍; 武朝霞; 朱芷葳; 唐中伟
Original assignee: Shanxi Agricultural University
Current assignee: Shanxi Agricultural University
Priority date: 2016-11-28
Filing date: 2016-11-28
Publication date: 2020-08-21
Anticipated expiration: 2036-11-28
Also published as: CN106701518A

Abstract

The invention belongs to the technical field of biological materials and application thereof, and provides a yeast strengthening method for reducing the using amount of yeast and improving the quality of vinegar in order to solve the problems of low saccharification capacity, low wine-making capacity and low vinegar yield of pure yeast vinegar. Isolation of fungal strains including high cellulase and amylase production: culturing single strain, adding yeast for enhancing Daqu by Penicillium chrysogenum and Saccharomycopsis fibuligera, and performing acetic fermentation. No need of adding exogenous catalyst, reduced yeast consumption, and improved raw material utilization rate. The method has simple operation, maintains the unique flavor and quality of the traditional Shanxi mature vinegar, improves the yield, improves the vinegar quality, reduces the cost and increases the benefit. The application and popularization of the method bring the change of the traditional Shanxi mature vinegar process, assist the development of the Shanxi mature vinegar industry, generate corresponding economic and social benefits and have wide application and popularization prospects.

Description

Daqu strengthening method for reducing Daqu dosage and improving vinegar quality

Technical Field

The invention belongs to the technical field of biological materials and application thereof, and particularly relates to a yeast strengthening method for reducing the using amount of yeast and improving the quality of vinegar.

Background

Shanxi is the biggest province of producing and consuming vinegar in China, the yield of vinegar reaches 70 ten thousand tons, and accounts for 1/8 of the total amount of vinegar in China. Shanxi mature vinegar is the first of four famous Chinese vinegars, and the traditional brewing process is characterized in that the yeast is used for replacing grains, and the using amount of the yeast is at least more than 40%. The Daqu is a saccharifying agent and a fermenting agent. The special Daqu for Shanxi mature vinegar is prepared by using barley and peas as raw materials and performing microbial natural fermentation, the production cycle is about 21 days, the quality of the Daqu determines the yield and the quality of vinegar, the Daqu is a core material for brewing the vinegar and is the soul of the vinegar, and the Daqu and a brewing process consisting of 80 processes jointly endow the Shanxi mature vinegar with a plurality of excellent qualities which cannot be compared with other vinegar serving as the first vinegar in the world. The pure Daqu vinegar has good flavor, but low saccharification capacity, alcoholization capacity and vinegar yield.

The national standard of Shanxi mature vinegar has higher and higher requirements on acidity, the national standard of Shanxi mature vinegar issued and executed in 10/1/2014 is increased from the original lowest 4.5g/L to 6g/L, and the use amount of Daqu is required to be increased to more than 62.5%, so that the edible vinegar with the pH of 3.6-3.9 and the final total acidity of more than 6 ℃ can be produced, thereby ensuring that the Shanxi mature vinegar can be stored for a long time without adding any preservative, and eliminating the quality guarantee period. The starter preparation of Shanxi mature vinegar takes about 21 days, the vinegar needs more than 20 days from the raw material to the leaching, and the starter is aged for at least 1 year, so the production period is longer, and the cost is increased by replacing the grain with the starter. How to use modern biotechnology to develop, optimize and standardize traditional technology of Shanxi mature vinegar has been proposed as a agenda.

In order to modify the traditional production process behind Shanxi mature vinegar, Shanxi four-eyed well brewing industry Co., Ltd introduces enzyme engineering technology into the process of first fermenting and then solidifying the Shanxi mature vinegar, and amylase is added in the raw material liquefaction stage to convert long-chain starch in the raw material into short-chain starch; adding saccharifying enzyme and red yeast rice in the saccharifying stage to further convert the raw materials into glucose, and adding a certain amount of red yeast rice to maintain the flavor of the traditional Shanxi mature vinegar and promote the saccharifying process; adding 40-50% of Daqu and immobilized yeast liquid in a alcoholization stage. The invention patent of the method for producing Shanxi mature vinegar by adopting multiple strains (patent number: ZL 200610027012.9) and the method and equipment for producing Shanxi mature vinegar by adopting the process of pre-mash and post-solidification (patent number: ZL 200610048373.1) are applied to the technologies.

Said invention introduces the enzyme engineering technology into the Shanxi mature vinegar fermentation process, and can reduce the use amount of Daqu, raise raw material utilization rate, reduce cost and raise benefit, but its operation process is complex, and the amylase, saccharifying enzyme, red yeast and Daqu are respectively added in different stages, and its operation is complex, and in particular, the addition of Daqu is late, and can be used as saccharifying agent in the Shanxi mature vinegar brewing process, and can limit the action of several microorganisms and their enzymes. In the above patent, before the addition of yeast, the raw materials are converted into fermentable sugar positions in large quantity, after yeast is inoculated, various microorganisms are rapidly activated, especially yeast is rapidly fermented, the product temperature is rapidly increased due to violent fermentation, the alcohol content is rapidly increased, the metabolism of a large number of microorganisms is limited, and the release and accumulation of various aroma components and functional components are influenced. The Shanxi mature vinegar has mellow taste, and is soft, sour, fragrant, sweet and fresh, and the quality is mainly caused by the action of abundant microorganism systems in the Daqu except the exquisite materials, so the Shanxi mature vinegar completely depends on the natural fermentation of microorganisms in the whole brewing process and does not have the intervention of any chemical catalyst.

Disclosure of Invention

The invention provides a yeast strengthening method for reducing the using amount of yeast and improving the quality of vinegar, aiming at solving the problems of low saccharification capacity, low alcoholization capacity and low vinegar yield of pure yeast vinegar.

The invention is realized by the following technical scheme: a yeast strengthening method for reducing the dosage of yeast and improving the quality of vinegar comprises the following steps:

(1) strain separation: the fungus strain for separating and screening high-yield cellulase and amylase from the special yeast for Shanxi mature vinegar comprises the following specific separation methods: collecting 10g of fermented grains fermented by alcohol for 24h fermented by conventional Shanxi mature vinegar process in a triangular flask containing 90ml of sterile water, and shaking with sterile glass beads for 20min to obtain bacterial suspension; the pipette sucks 1ml of the bacterial suspension into 9ml of sterile waterIn the test tube, oscillating for 1-2 min to mix them uniformly, and preparing into 10% concentration in turn^-1～10^-7A graded bacterial suspension; sucking and coating 100 mul of each diluted concentration bacterial suspension on a Martin culture medium plate, paralleling each dilution by 2, standing for 10min, and culturing for 3d at 30 ℃; selecting a single colony with obvious colony characteristics, selecting mould, purifying for 2-3 times by using a Martin flat plate until a pure strain is obtained, preserving the pure strain by using a PDA culture medium inclined plane for later use, wherein the formula of the Martin culture medium is as follows: 0.33ml of Bengal (1 mg/ml), 1.2g of agar powder, 7g of glucose, 0.5g of peptone and KH₂PO₄·3H₂O 0.1g、MgSO₄·7H₂0.05g of O and 100mL of water, the pH value is natural, 2mL of 2 percent deoxysodium cholate solution and 0.33mL of streptomycin solution (1 ten thousand units/mL) which are sterilized in advance are added before use, and the mixture is sterilized for 25min at 112 ℃ for standby; after two batches of separation and purification experiments, a high-yield cellulase strain is obtained, and is finally identified as follows through microscopic examination, colony morphology observation and sequence analysis and identification of a rRNA gene D1/D2 region: penicillium chrysogenum (Penicillium chrysogenum) M4, Saccharomycopsis fibuligera (C.), (Saccharomycopsis fibuligera)M8；

(2) Preparation of Penicillium chrysogenum (Penicillium chrysogenum) M4 and Saccharomycopsis fibuligera (C.), (Saccharomycopsis fibuligera) M8 bran koji: mixing bran, rice hull and water at a mass ratio of 20:1:16, stirring uniformly, steaming for 20min, taking out, scattering while hot, and cooling to 30 ℃; respectively inoculating with the same volume concentration of 10⁷Uniformly stirring spore suspension or bacterial suspension of M4 and M8 strains per ml, culturing at 30 ℃ for 12-15 h to grow white hyphae, then buckling a bottle for culturing for 2-5 h, and taking out for later use when the surface is full of white hyphae or produces a small amount of spores;

(3) acetic acid fermentation: adopting the M4 and M8 bran koji prepared in the step (2) to strengthen Daqu, carrying out acetic fermentation, wherein during fermentation and batching, the total addition amount of the Daqu and the bran koji is 40% of the main material used for fermentation, wherein the Daqu accounts for 25.1% of the main material, the addition amount of the M4 bran koji accounts for 8.09% of the main material, the addition amount of the M8 bran koji accounts for 6.81% of the main material, the addition amount of the Angel yeast accounts for 0.06% of the main material, and the water addition amount is 3 times of the main material; steaming the main material according to the traditional mature vinegar process, fermenting for 7 days at 25 ℃, continuously performing acetic acid solid state fermentation on the thin mash, and filling the auxiliary materials of rice hulls and bran, wherein the ratio of the rice hulls to the bran to the main material is 15:20: 12; then continuing to ferment for 16 days at 25 ℃, after the solid state fermentation of the acetic acid is finished, pouring the vinegar by using water with the volume 2 times of the total volume of the vinegar grains, pouring out the first vinegar by using 70 percent of the total water volume, using the rest 30 percent of the water for next vinegar pouring, and then combining the vinegar liquid.

The main materials used for fermentation are corn, a corn and sorghum mixture or sorghum with a mass ratio of 3: 7. Adding 5% of salt into the vinegar-pouring water.

Said Penicillium chrysogenum (A)Penicillium chrysogenum) M4 is fast in growth on a wort agar medium, the culture is carried out for 7 days under the dark condition of 25 ℃, the diameter of a bacterial colony is 32-40mm, the texture is villiform, a large number of spore-forming structures are formed, the spore surface is dark grey green, the back of the bacterial colony is light brown, no water-soluble pigment is contained, the specialization of a conidiophor is not obvious, the width is 2.5-4.0 mu M, the wall is smooth, 3-4 rounds of broomcorn branches are generated and loose, the phialide is 7.0-11.7 × 2.2.2-3.0 mu M, the conidiophore is nearly spherical, wide oval, light green, the surface is smooth, 2.8-4.0 mu M, no sexual spore is found, the bacterial colony is white in the initial stage and gradually changes to dark green on a PDA medium, the conidiophore and the hypha are all provided with transverse partitions, the broomcorn body is single round or multiple branches, symmetrical or asymmetrical, and the penicillium chrysogenum (a) is free from podium chrysPenicillium chrysogenum) M4 deposit number is: CGMCC NO.12375, the preservation unit is China general microbiological culture Collection center, the address is No. 3 of Xilu No.1 of Beijing, Chaoyang, and the preservation date is 2016, 4 and 28 days.

The Saccharomycopsis fibuligera (A), (B) and (C)Saccharomycopsis fibuligera) M8 is cultured in wort liquid culture medium at 25 deg.C for three days to obtain cells with spherical, oval and sausage shape (4.0-7.0) × (3.6-7.0) mum), and cultured on wort agar slant at 25 deg.C for one month to obtain tough, white, fluffy, non-reflective, and edge whisker colony, cultured on corn flour agar Dalmau plate to produce fungus filament, and cultured on PDA culture medium to obtain colony with central bulge, radial and straw hat shape, white surface and colony diameter of 19.3-19.8mm, and cultured in solid wortBasically takes the shape of ester mud or involucra and takes the shape of white; culturing in liquid wort culture medium for 1d to produce white pellicle; has fungal hypha, some have branches and transverse septa; the node spores are single or chain, are square, long cylindrical or oval, and have two blunt ends; the Saccharomycopsis fibuligera (A), (B) and (C)Saccharomycopsis fibuligera) M8 deposit number is: CGMCC No.12408, the preservation unit is China general microbiological culture Collection center, the address is No. 3 of West Lu No.1 of North Chen of the Korean-Yang district in Beijing, and the preservation date is 2016, 4 and 28 days.

The M4 and M8 bacterial strains are separated from the traditional special Daqu for Shanxi mature vinegar, and are identified as follows by the institute of microbiology in Chinese academy of sciences: penicillium chrysogenum (Penicillium chrysogenum) M4, Saccharomycopsis fibuligera (C.), (Saccharomycopsis fibuligera) M8。

Before the new national standard of Shanxi mature vinegar is issued and implemented (10.1.2014), the use amount of Daqu in general vinegar plants is about 50 percent. The invention carries out single factor test on the using amount of the Daqu when the corn is used as the main material, takes the alcoholic strength as an investigation index, the higher the adding amount of the Daqu is, the better the Daqu is, 40 percent is the best, the using amount of the Daqu is only 20.08 percent through bran koji reinforcement, the using amount of the Daqu is greatly reduced, the experimental result shows that the using amount of sorghum can be reduced, the utilization rate of raw materials is improved, and the production cost is saved. The total acid content, the non-volatile acid content, the soluble solid content, the amino nitrogen content and the total ester content in the vinegar are all improved compared with the single fermentation of the Daqu, and the vinegar quality is improved. When sorghum is used as a main material, the actual production process of a vinegar factory is simulated under laboratory conditions without fumigating and aging processes, the acidity can reach over 6g/L, and the production cost is saved.

The method does not need to add an exogenous catalyst, improves the traditional vinegar brewing process from the microbiological perspective, separates and screens fungus strains with high yield of cellulase and amylase from the special Daqu for Shanxi mature vinegar, prepares bran koji, strengthens the Daqu, achieves the same effect as that of exogenously adding amylase and saccharifying enzyme, reduces the using amount of the Daqu, improves the utilization rate of raw materials, and improves the contents of total acid, non-volatile acid, reducing sugar, soluble solid, amino nitrogen and total ester in new vinegar. The method is simple and convenient to operate, only needs to prepare the M4 and M8 bran koji, and adds the bran koji, the Daqu and the Angel yeast powder into the main material without adding the bran koji and the Angel yeast powder in sections. Not only maintains the unique flavor and quality of the traditional Shanxi mature vinegar, but also improves the yield, reduces the cost and increases the benefit. The application and popularization of the method bring the change of the traditional Shanxi mature vinegar process, assist the development of the Shanxi mature vinegar industry, generate corresponding economic and social benefits and have wide application and popularization prospects.

Drawings

FIG. 1 shows the effect of different feed water ratios on alcoholic fermentation; FIG. 2 shows the influence of the addition of Daqu on the alcohol content; FIG. 3 shows the effect of the addition ratio of Daqu and bran koji on the alcohol content; FIG. 4 shows the enhanced co-fermentation of Daqu alcohol by each strain; FIG. 5 is a graph of the response of the addition ratio of M8 bran koji, M4 bran koji and Daqu to the alcohol content; FIG. 6 is an iso-plot showing the effect of the addition ratio of M8 bran koji, M4 bran koji and Daqu on the alcohol content; FIG. 7 shows the colony characteristics and individual morphology of M4 on PDA medium; FIG. 8 is a characteristic of colonies of M8 on PDA medium and wort medium; FIG. 9 shows the individual morphology of M8 on wort medium.

Detailed Description

Example 1: a yeast strengthening method for reducing the dosage of yeast and improving the quality of vinegar comprises the following steps:

(1) strain separation: the fungus strain for separating and screening high-yield cellulase and amylase from the special yeast for Shanxi mature vinegar comprises the following specific separation methods: collecting 10g of fermented grains fermented by alcohol for 24h fermented by conventional Shanxi mature vinegar process in a triangular flask containing 90ml of sterile water, and shaking with sterile glass beads for 20min to obtain bacterial suspension; sucking 1ml of the bacterial suspension by a pipette gun, oscillating for 1-2 min in a test tube filled with 9ml of sterile water, fully mixing the bacterial suspension and the sterile water uniformly, and sequentially preparing the bacterial suspension with the concentration of 10^-1～10^-7A graded bacterial suspension; sucking and coating 100 mul of each diluted concentration bacterial suspension on a Martin culture medium plate, paralleling each dilution by 2, standing for 10min, and culturing for 3d at 30 ℃; selecting single colony with obvious colony characteristic, picking mold, purifying with Martin plate for 2-3 times until pure strain is obtained, preserving the pure strain with PDA culture medium slant for useThe medium Martin culture medium formula is as follows: 0.33ml of Bengal (1 mg/ml), 1.2g of agar powder, 7g of glucose, 0.5g of peptone and KH₂PO₄·3H₂O 0.1g、MgSO₄·7H₂0.05g of O and 100mL of water, the pH value is natural, 2mL of 2 percent deoxysodium cholate solution and 0.33mL of streptomycin solution (1 ten thousand units/mL) which are sterilized in advance are added before use, and the mixture is sterilized for 25min at 112 ℃ for standby; through two batches of separation and purification experiments, high-yield cellulase strains are obtained, wherein M4, M5, M6 and M8 are high-yield cellulase (M4) and glucoamylase (M5, M6 and M8) strains which are separated and screened; m4 has the strongest cellulase producing capability in all the cellulase producing strains, and the cellulase enzyme activities at 30 ℃ and 25 ℃ are 57.90 +/-2.65U/mL and 82.29 +/-0.27U/mL respectively;

the carbohydrase activity of M6, M5, M8, M15 and M2 is higher than that of a control strain M0 (AS 3.4309) at 30 ℃, the carbohydrase activity is 3582.49 +/-30.67U/mL (M6), 1326.53 +/-18.56U/mL (M5) and 1108.35 +/-14.21U/mL (M8) and 1069.08 +/-20.41U/mL (M15) beta 842.17 +/-20.44U/mL (M2) respectively, and the M0 is 754.90 +/-21.82U/mL. The saccharifying enzyme activity of M4 is 658.90 + -22.40U/mL, which is slightly lower than M0. M8 also has the ability to produce ethanol.

The main material used for fermentation is corn; adding 5% of salt into the vinegar-pouring water.

Said Penicillium chrysogenum (A)Penicillium chrysogenum) M4 is fast in growth on a malt extract agar medium, the culture is carried out for 7 days under the dark condition of 25 ℃, the diameter of a colony is 32-40mm, the texture is villiform, a large number of spore-forming structures are formed, the spore surface is dark grey green, the back of the colony is light brown, no water-soluble pigment exists, the specialization of a conidiophor is not obvious, the width is 2.5-4.0 mu M, the wall is smooth, the broomcorn branches are 3-4 rounds and loose, the phialide is 7.0-11.7 × 2.2.2-3.0 mu M, the conidiophor is nearly spherical, wide elliptical, light green, the surface is smooth, 2.8-4.0 mu M, sexual spores are not seen, the colonies are fallen into felty on a PDA medium, the colonies are raised, the hyphae is compact, the initial stage of the colonies are white, the colonies are gradually changed into dark green, transverse partitions are arranged on the conidiophores, the broomcorn bodies are single round or multiple branches.

The Saccharomycopsis fibuligera (A), (B) and (C)Saccharomycopsis fibuligera) M8 is cultured in wort liquid culture medium at 25 deg.C for three days, the cells are spherical, oval and sausage-shaped with the size of (4.0-7.0) × (3.6-7.0) mum, the malt agar is cultured on a slope at 25 deg.C for one month, the colony is tough, white, villous on the surface, non-reflective and tough at the edge, the corn flour agar Dalmau is cultured on a plate to produce fungus filament, the colony on PDA culture medium is radial and straw-hat-shaped, the color is white, the surface is in the shape of a skin membrane, the diameter of the colony is 19.3-19.8mm, the colony on solid malt liquid culture medium is in the shape of ester mud or skin membrane and is white, the fungus filament is cultured in liquid malt liquid culture medium for 1d to produce white fungus filament, some fungus filaments have branches and transverse septa, and spores are single or chain-grown and have square, long cylinder or ellipse shapes, and two ends are round.

Example 2: a Daqu strengthening method for reducing the using amount of Daqu and improving the quality of vinegar is characterized in that during acetic fermentation, a mixture of corn and sorghum with the mass ratio of 3:7 is selected as a main material, and the rest methods are the same as the preparation method in example 1.

Example 3: a method for enhancing Daqu yeast to reduce the amount of Daqu yeast and improve the quality of vinegar comprises selecting sorghum as the main material during acetic acid fermentation, and the rest of the methods are the same as the preparation method described in example 1.

After the alcoholic fermentation is finished, when the corn is used as the main material, the alcohol content of the wine mash reaches 11.2 percent, and the mass ratio is 3: the alcohol content of the corn and the sorghum as the main materials is 11.8 percent, the alcohol content of the sorghum as the main material is 12.2 percent, and the good alcohol content is provided for acetification and stirring, while the alcohol content of a common vinegar plant is 9-11 percent. The alcohol content is improved, which shows that the utilization rate of the raw materials is improved, and the content of the starch in the vinegar residue is high after the fermentation is determined, so that the utilization rate of the raw materials is improved in the alcoholization process, and the problem of insufficient starch in the subsequent acetic fermentation process can not be caused. The utilization rates of the raw materials in the alcoholic fermentation stage are respectively 55.28% (corn), 57.76% (corn + sorghum) and 59.98% (sorghum), and the total utilization rates of the raw materials are respectively 83.42% (corn flour), corn flour + sorghum flour (84.63%) and sorghum flour (85.39%).

Experimental example 1: making bran koji of each test strain, and determining the optimum feed-water ratio

M0 was used AS a control strain in this study, and was a quick-starter strain AS in the production of Shanxi mature vinegar. 3.34309, it was found that M0 does not produce cellulase.

Preparation of bran koji for each test strain: the mass ratio of the bran to the rice hull to the water is 20:1:16, the mixture is uniformly stirred and steamed for 20min, the mixture is taken out and then scattered while being hot, and the mixture is cooled to 30 ℃. Inoculating spore suspension or bacterial suspension (10) of each selected M0, M2, M4, M5, M6, M8 and M15 strains with equal volume⁷Uniformly stirring, culturing at 30 ℃ for 12-15 h to grow white hyphae, then buckling a bottle for culturing for 2-5 h, and taking out for later use when the surface is full of white hyphae or produces a small amount of spores.

The concentration of the LIAO liquid has great influence on alcohol fermentation, and three material-water ratio gradients are set according to the production process of a vinegar factory at 1:3, 1: 4 and 1: 5. The alcohol fermentation process of a vinegar factory comprises the following steps: weighing 80g of corn flour in a 50ml beaker, adding 48ml of water, uniformly mixing, moistening the corn flour for 12h at room temperature, steaming for 1h, cooling by blowing in an ultra-clean workbench, performing aseptic operation, adding the steamed corn flour, 0.05g of yeast, 14g of Daqu and 15.5g of bran koji into a 500ml triangular flask filled with 240ml, 320ml and 400ml of sterile distilled water, uniformly stirring, sealing by using eight layers of gauze, and culturing in a constant-temperature biochemical incubator at 25 ℃. Stirring once every day for the first 3 days, sealing with double-layer plastic cloth after stirring for 3 days, culturing at 25 deg.C for 4 days, and measuring alcohol content on 8 days.

The alcohol fermentation alcohol content of each strain inoculated under different feed-water ratio fermentation conditions is shown in figure 1, and the results show that: when the ratio of raw material to water is 1:3, the alcoholic strength of the fermented Liao liquid is highest, and the alcoholic strength of the fermented Liao liquid inoculated with M8, M6 and M4 is higher than that of M0, and the alcoholic strength is remarkably different. When the water addition amount is higher, the substrate concentration is lower, which is not beneficial to the growth and metabolism of the strain. The same feed water ratio had different effects on each strain. In order to improve the utilization rate and yield of raw materials, a proper ratio of feed to water must be selected to provide the most favorable environment for the growth, metabolism and enzyme production of microorganisms. According to Liu faith research results, the optimal raw material-water ratio is 1:3, which is consistent with research results in the paper, because the fermentation liquid is too thick and alcohol fermentation is not performed, when the raw material-water ratio is lower than 1: 3.

Experimental example 2: determination of Daqu addition amount

Setting the addition amounts of Daqu as 20%, 30%, 40% and 50%, the feed-water ratio as 1:3, 80g of corn flour and 0.05g of yeast, adding no bran koji, fermenting at 25 deg.C for 7d, measuring alcohol content on day 8, and performing the steps as in experimental example 1.

The yeast is a main saccharification leavening agent in the alcohol fermentation stage, the addition of the yeast can not only improve the wine yield but also improve the flavor of vinegar, but also has high preparation cost and long production period, the fermentation time is prolonged due to the small addition of the yeast in the alcohol fermentation process, the waste is caused due to the excessive addition of the yeast, and the production cost is increased, so that the determination of the addition amount of the yeast through experiments is particularly important, the raw materials can be utilized to the maximum extent for fermentation, the using amount of the yeast can be reduced, and the cost is reduced. The results are shown in FIG. 2, and from the results, the alcohol content increased and then decreased with the increase of the addition amount of the Daqu, and reached the maximum of 10.1% (v/v) at 40% of the addition amount of the Daqu. When the addition amount of the Daqu is small, the alcoholic fermentation is incomplete or the fermentation period is prolonged, and when the addition amount is too high, the growth and metabolism of microorganisms are not facilitated, so that the optimal addition amount of the Daqu is 40%.

Experimental example 3: determination of addition ratio of Daqu and bran koji

One of the purposes of this experiment is to replace part of Daqu with bran koji, reduce the amount of Daqu, increase the utilization rate of raw materials and improve the quality of vinegar. In the current production process of a vinegar factory, the adding amount ratio of the Daqu to the bran koji is about 1:1, in the invention, under the condition of the same total inoculation amount, M0 bran koji is used for replacing partial Daqu, the adding amount ratio of the Daqu to the bran koji is set to four gradients of 2:1, 1:2 and 1:3, the material-water ratio is 1:3, and the rest is the same as in experiment example 1.

In order to reduce the cost and the using amount of the Daqu, the experiment tries to reduce the using amount of the Daqu by increasing the adding amount of the bran koji. The study was conducted using M0 bran koji as a representative, and the total amount of addition of Daqu and bran koji was 40% (32 g) of the raw material, and the results of the test are shown in FIG. 3. As can be seen from the figure, when the addition amount of Daqu and bran koji is 2:1, the alcohol fermentation alcohol content is close to that of only 40% Daqu, the difference is not significant and the method is economical, so the addition ratio of Daqu to bran koji in the following test is 2: 1.

Experimental example 4: alcohol fermentation under optimal ratio of Daqu and bran koji

Each strain was made into bran koji as described in example 1. The alcohol fermentation is carried out according to the following adding amounts of 80g of corn flour, 240ml of water, 0.05g of yeast, 21.3g of Daqu, 10.7g of bran koji, and the ratio of the Daqu to the bran koji of 2:1 (the total adding amount is 40 percent of the raw materials), the fermentation temperature is 25 ℃, the fermentation time is 7d, the alcohol content is measured on the 8 th day, and the operation steps are as in the experimental example 1.

Fermenting with bran koji and Daqu. The alcohol contents were measured as shown in FIG. 4. It can be seen that M8, M6, and M4 are the highest in alcohol content. Except M8, the alcohol fermentation alcohol content of other 5 strains is equal to the activity of the carbohydrase. The M8 alcohol content is the highest, and the analysis reason is that the strain can utilize glucose to generate alcohol, and the saccharifying enzyme has higher activity, so that the glucose conversion rate is improved. The analytical result of the saccharifying enzyme producing capability shows that the saccharifying enzyme producing capability of the M6 strain is highest; m4 also has a higher glucoamylase-producing ability than M0. M8 and M4 are selected as mixed fermentation strains because M4 has the strongest cellulase producing capability and M6 does not produce cellulase.

TABLE 1 quality index of alcoholic fermented wine with higher alcohol content strain

As can be seen from Table 1, except for total acid, other index values in the liquor fermented by Daqu alone are all lower than the index of Daqu + M8 co-fermentation; the indexes of Daqu + M0 are all higher than those of Daqu + M4, Daqu + M6 and Daqu + M8 except total ester and amino nitrogen; daqu + M6 has the highest amino nitrogen content. Therefore, the next step is to prepare bran koji from M8 and M4, add the bran koji and Daqu into alcohol fermentation according to a certain proportion, and test the quality of the vinegar produced while improving the alcohol content.

The raw material utilization rates of Daqu, Daqu + M0, Daqu + M4, Daqu + M6 and Daqu + M8 in the alcohol fermentation stage are respectively 21.24%, 46.52%, 46.94%, 50.07% and 53.05%, and are respectively increased by 25.28% (M0), 25.70% (M4), 28.83% (M6) and 31.81% (M8). When the addition ratio is 2:1, the use amount of the Daqu can be saved by at least 37.25%.

Experimental example 5: determination of optimal addition ratio of Daqu to M4 and M8

Respectively preparing bran koji from M4 and M8, inoculating the bran koji and the Daqu in a certain ratio in alcohol fermentation, determining the addition ratio of the bran koji, the Daqu and the Daqu by a Minitab16 mixing material regression design, and determining the alcoholic strength of the three by the operation steps of experiment example 1, 80g of corn flour, 240ml of water, 0.05g of yeast, 32g of total amount of the Daqu, fermentation temperature of 25 ℃, fermentation time of 7d and the 8 th day.

The optimal addition ratio of Daqu and bran koji is 2:1, so the extreme apex design method is adopted, the upper limit of Daqu is 1, the lower limit is 0.5, and the upper limit of M8 bran koji and M4 bran koji is 0.4, and the lower limit is 0.1. The total addition amount of Daqu and bran koji is 40% of the raw materials. Mixing material recovery by utilizing Minitab16Determination of M8 bran koji (X) by design₁) M4 bran koji (X)₂) And Daqu (X)₃) The amounts added and the test results are shown in Table 2.

TABLE 2 results of alcohol fermentation test by regression design of M8 bran koji, M4 bran koji, Daqu Minitab16 blend

As can be seen from Table 2, the alcohol content was high when the amount of Daqu added was 0.5 to 0.6. The addition amount of M8 koji had a greater effect on the alcohol content than M4 koji.

The mixed material design can observe the influence of the addition of each additive on the alcohol content according to the ternary contour line of each additive. FIG. 5 is a response curve of the alcohol content of M8 bran koji, M4 bran koji and Daqu in different addition ratios, and it can be seen that the alcohol content of the three additives inoculated in alcohol fermentation is higher than that of the M8 bran koji, M4 bran koji and Daqu inoculated separately. FIG. 6 is the equivalent curves of the alcohol content of M8 bran koji, M4 bran koji and Daqu at different addition ratios, and the interaction between M8 bran koji, M4 bran koji and Daqu is more obvious. The impact of M8 and M4 koji on alcohol fermentation alcohol content was greater at lower addition rates. This may be because the species of the microorganism in the yeast is more, when the addition is excessive, the microorganism fermentation is not facilitated due to the limitation of the substrate concentration, or the microbial population structure in the yeast is changed by a large amount of foreign species, the growth and reproduction of some microorganisms are inhibited, and the fermentation is not facilitated; however, the addition of a small amount of M8 koji or M4 koji can promote fermentation. The alcohol degree of the alcohol fermentation is increased and then reduced along with the increase of the addition amount of the yeast; the addition of a small amount of M8 koji or M4 koji contributes to the improvement of the alcohol content.

TABLE 3 analysis of variance results of regression equation

Table 3 Variance analysis of regression modle equation of alcoholcontent

Variance from regression equationAs a result of the analysis, it is possible to see,P﹤0.01the model selected by the experiment is highly significant, so that the model can be used for determining M8 mouldy bran (X)₁) M4 bran koji (X)₂) And Daqu (X)₃) The addition ratio of (2). The coefficients of the secondary terms are positive numbers from the regression equation model, which shows that the interaction between M8 mouldy bran and M4 mouldy bran, M8 mouldy bran and Daqu, and the interaction between M4 mouldy bran and Daqu are obvious, and the absolute value of the regression coefficient of the secondary terms is X₁X₃﹥X₂X₃﹥X₁X₂. It is demonstrated that the addition ratio of M8 bran koji and Daqu has the greatest effect on the alcohol content, the addition ratio of M4 bran koji and Daqu has the second least effect on the alcohol content, and the addition ratio of M8 bran koji and M4 bran koji has the least effect on the alcohol content during alcohol fermentation.

Performing regression fitting analysis on the table 2 test data by using Minitab16 software to obtain a primary interactive regression equation model as follows: alcohol content-63.31X₁－58.7X₂－7.73X₃＋72.95X₁X₂＋157.19X₁X₃＋153.56X₂X₃。

Solving the equation by using linear programming, wherein the constraint conditions are as follows: x₁＋X₂＋X₃＝1.0；0≤X₁≤0.1；0≤X₂≤0.1；0≤X₃Less than or equal to 0.5; solving to obtain X₁＝0.170249，X₂＝0.202217，X₃＝0.627534，

That is, 32g of the raw material 80g, 20.08g of Daqu, 5.45g of M8 bran koji and 6.47g of M4 bran koji were added to 32g of the koji at a ratio of 40%, and the ternary quadratic equation of the alcohol content was maximized.

Namely, when the ingredients are fermented, the total adding amount of the Daqu and the bran koji accounts for 40 percent of the main material, wherein the Daqu accounts for 25.1 percent of the main material, the adding amount of the M8 bran koji accounts for 6.81 percent of the main material, the adding amount of the M4 bran koji accounts for 8.09 percent of the main material, the adding amount of the Angel yeast accounts for 0.06 percent of the main material, and the adding amount of water accounts for 3 times of the main material, so that the maximum alcohol content can be obtained.

Experimental example 6: fermentation test under optimal addition ratio of Daqu to M4 and M8

The raw materials in the alcoholic fermentation are corn flour, but the raw materials in the traditional Shanxi mature vinegar production are sorghum, so that the corn flour, the sorghum flour and the sorghum flour are respectively used as raw materials for fermentation tests. The proportion of the corn flour and the sorghum flour is obtained from a vinegar factory. 80g of corn flour, 240ml of water, 0.05g of yeast, 20.08g of Daqu, 6.47g of M4 bran koji and 5.45g of M8 bran koji, the fermentation temperature is 25 ℃, the fermentation time is 7 days, and the alcoholic strength is measured on the 8 th day. Fermenting the rest of the solution with acetic acid, mixing main material, testa oryzae and testa Tritici at a ratio of 12: 15:20 in 8L barrel, inoculating fermented grains of 1/6 raw materials, fermenting for 24 hr, covering, and culturing at 25 deg.C. And (3) turning the fermented grains when the temperature is raised to about 40 ℃, and fermenting for 16d 1-2 times every day. Adding 5% of salt and 2 times of water, soaking for 24 hr, and pouring vinegar (twice, adding 7 for the first time, soaking for 12 hr, pouring out the first vinegar, adding 3 for the second time, soaking for 12 hr, and then pouring out). Mixing the twice-drenched vinegar, and measuring the quality index of the vinegar. Because of the laboratory conditions, fumigation is not performed. The results of acetic fermentation and quality index thereof at the optimum addition ratio of Daqu to M4 and M8 are shown in Table 4.

TABLE 4 quality index of mash wine

Under the optimal addition proportion, different raw materials are used for fermentation, and the alcohol degree of the alcohol fermentation is more than 11% (v/v) and is consistent with the value predicted by the equation of a three-way equation. Under the same conditions, the alcohol contents of the fermented product are respectively 7.7% (v/v), 7.9% (v/v) and 8.2% (v/v) by fermenting with the Daqu alone. It can be seen that when partial daqu is replaced by M8 and M4 bran koji, the alcoholic strength is not reduced, but is increased, and the index values except for the total ester are increased with the increase of the sorghum consumption.

TABLE 5 comparison of quality indexes of vinegar prepared by different main materials and enhanced Daqu fermentation

Note: the unit of other indexes is g/100mL except that the unit of total ester is g/L; a represents the single fermentation of the Daqu, B represents the co-fermentation of M8+ M4+ Daqu; the corn flour and the sorghum flour are mixed according to the ratio of 3: 7.

As can be seen from Table 5, the contents of total acid, non-volatile acid, reducing sugar, soluble solid, amino nitrogen and total ester in the vinegar prepared by fermenting the different main materials with the enhanced Daqu are higher than those in the vinegar prepared by fermenting the Daqu alone; along with the increase of the sorghum dosage, the index values except the total ester are correspondingly increased, and the total ester content in the fermented vinegar taking the corn flour as the raw material is the highest. In sensory evaluation, the new drenched vinegar smells more fragrant with the increase of the sorghum dosage, and the index values are correspondingly increased with the increase of the sorghum dosage, which explains the process essence of using the sorghum as the main material in the traditional brewing technology of Shanxi mature vinegar. When corn flour is used as a raw material, the index values of the enhanced Daqu fermented vinegar are very close to the quality of the corn flour and sorghum flour Daqu fermented vinegar, and further, the M8+ M4+ Daqu fermentation can reduce the Daqu dosage in the brewing process of the aged vinegar and can also reduce the sorghum dosage. The vinegar sample obtained in the experiment is the vinegar sample which is obtained by soaking fermented white vinegar for 24 hours, filtering the fermented white vinegar by using gauze, standing the filtrate for 24 hours and taking supernatant fluid. The soluble solids content may be higher than it is practical. Due to test conditions limitations, the absence of fumigation resulted in less total esters and some aroma.

And the raw material utilization rate is detected and displayed: in every 80g of raw materials, the addition amount of the yeast is only 20.08g, the dosage of 37.25% ((32-20.08)/32 multiplied by 100%) is reduced, and the production cost is reduced. And under the same fermentation conditions, when corn is used as a raw material for fermentation, the alcohol content of the Daqu + M8 bran koji + M4 bran koji after fermentation is 11.2% (v/v) > Daqu + M8 bran koji is 10.9% (v/v) > Daqu + M4 bran koji is 10.3% (v/v) > Daqu + M0 bran koji is 8.4% (v/v). In the alcohol fermentation stage, the utilization rate of each raw material is 55.28 percent (corn flour), 57.76 percent (corn flour + sorghum flour) and 59.98 percent (sorghum flour), and the raw material rate of the sorghum flour is 4.70 percent higher than that of the corn flour. The total utilization rate of the raw materials for fermentation is 83.42% of corn flour, 84.63% of corn flour and sorghum flour and 85.39% of sorghum flour.

The above experimental results show that: the addition amount of the yeast is 40 percent, and when the ratio of the materials to the water is 1:3, the alcohol degree of the alcohol fermented mash is the highest. In order to reduce the consumption of Daqu, the Daqu is replaced by the bran koji, the ratio of the Daqu to the bran koji (AS.3.4309) is 2:1, and the alcohol content is the highest when the total amount of the added Daqu is 40%. M2, M4, M5, M6, M8 and M15 are prepared into bran koji, the bran koji is matched with Daqu according to the proportion of 1:2 (the total addition amount is 40 percent), and the material-water ratio is 1:3 for fermentation, and the results show that the alcohol degree in the enhanced Daqu alcohol fermented mash of the M4, the M6 and the M8 is obviously higher than that of other strains. In order to improve the utilization rate of raw materials, M4 and M8 are selected for double strain strengthening research, considering that M4 and M8 can produce cellulase but M6 cannot. The results of Minitab16 mixed material regression design experiments show that the alcohol degree of the alcohol fermented mash can exceed 11% (V/V) when the addition amount of the Daqu is 25.1% of the main material, the addition amount of the M8 mouldy bran is 6.81% of the main material and the addition amount of the M4 mouldy bran is 8.09% of the main material. The using amount of the yeast is reduced from 40% to 25.1%. The test results of the verification test by respectively taking corn, corn and sorghum (3: 7) and sorghum as main materials and taking Daqu single fermentation as a reference show that the contents of total acid, non-volatile acid, reducing sugar, soluble solid, amino nitrogen and total ester in the vinegar liquid are higher than the reference. With the increase of the sorghum dosage, all index values except the total ester content have a rising trend, and the total utilization rate of the raw materials is obviously improved, so that the Daqu is enhanced by the M4 and the M8 together, the Daqu and sorghum dosage can be reduced in the vinegar brewing process, the utilization rate of the raw materials is improved, the vinegar quality is improved, and the cost is further reduced.

The method for measuring the alcohol content in the experimental example comprises the following steps: weighing 100ml of fermentation liquor into a distillation flask filled with 5-6 grains of zeolite, wetting a measuring cylinder with 150ml of water for 2-3 times, adding the washing liquor into the distillation flask together, heating to boil, turning to soft fire, and collecting 100ml of distillate by using a drying measuring cylinder. And (3) putting the alcohol degree meter and the thermometer into the measuring cylinder at the same time, keeping for about 5min, recording the alcohol degree after the alcohol degree meter and the thermometer are stable, and checking the table to correct the alcohol degree to be 20 ℃.

The method for measuring the quality index in the experimental example comprises the following steps: the measurement of each index in the vinegar liquid is carried out according to the method specified by the national standard, wherein the measurement of total acid is carried out according to GB/T5009.41-2003; determination of the fixed acids according to GB 18187-2000; the reducing sugars were determined according to GB 19777-2005; the amino nitrogen is determined according to GB/T5009.39-1996, and the total ester content is determined according to GB 19777-2005.

The method for measuring the utilization rate of the raw materials in the experimental example comprises the following steps: weighing 5g of total material (10 g of distiller's grains and vinegar residue) in a 250ml triangular flask, adding 80ml of distilled water and 20ml of 20% hydrochloric acid, shaking up, connecting with a condenser tube (about 1 m), boiling in a water bath for 30min, immediately cooling, filtering with de-esterified cotton, and adjusting the volume of the filtrate to 500 ml. And (3) taking 20.0ml of filtrate to measure the content of reducing sugar, wherein the measuring method is measured according to the method of GB 19777-2005. Drying the vinasse and the vinegar residue at 80 ℃. The total material is prepared according to the proportion of fermentation raw materials, auxiliary materials and filling materials.

Utilization ratio of raw materials (%) (alcoholic fermentation) = (total material reducing sugar content-vinasse reducing sugar content)/total material reducing sugar content x 100%;

the total raw material utilization ratio (%) = (total material reducing sugar content-vinegar residue reducing sugar content)/total material reducing sugar content x 100%.

Claims

1. A yeast strengthening method for reducing the dosage of yeast and improving the quality of vinegar is characterized in that: the method comprises the following steps:

(1) strain separation: the fungus strain for separating and screening high-yield cellulase and amylase from the special yeast for Shanxi mature vinegar comprises the following specific separation methods: collecting 10g of fermented grains fermented by alcohol for 24h fermented by conventional Shanxi mature vinegar process in a triangular flask containing 90ml of sterile water, and shaking with sterile glass beads for 20min to obtain bacterial suspension; sucking 1ml of the bacterial suspension by a pipette gun, oscillating for 1-2 min in a test tube filled with 9ml of sterile water, fully mixing the bacterial suspension and the sterile water uniformly, and sequentially preparing the bacterial suspension with the concentration of 10^-1～10^-7A graded bacterial suspension; sucking and coating 100 mul of each diluted concentration bacterial suspension on a Martin culture medium plate, paralleling each dilution by 2, standing for 10min, and culturing for 3d at 30 ℃; selecting a single colony with obvious colony characteristics, selecting mould, purifying for 2-3 times by using a Martin flat plate until a pure strain is obtained, preserving the pure strain by using a PDA culture medium inclined plane for later use, wherein the formula of the Martin culture medium is as follows: bengal 1mg/ml 0.33ml, agar powder 1.2g, glucose 7g, peptone 0.5g, KH₂PO₄·3H₂O 0.1g、MgSO₄·7H₂O0.05g, water 100ml, natural pH, and pre-sterilized 2% sodium deoxycholate solution 2ml and chain 0.33ml before useSterilizing the solution of the mycin at the temperature of 112 ℃ for 25min for later use, wherein the concentration of the solution of the mycin is 1 ten thousand units/mL; after two batches of separation and purification experiments, a high-yield cellulase strain is obtained, and is finally identified as follows through microscopic examination, colony morphology observation and sequence analysis and identification of a rRNA gene D1/D2 region: penicillium chrysogenum (Penicillium chrysogenum) M4 deposit number is: CGMCC number 12375, wherein the preservation unit is China general microbiological culture Collection center, the address is No. 3 of Xilu-Tai-Ji-3 of Chaoyang district in Beijing, and the preservation date is 2016, 4 and 28 days; saccharomycopsis fibuligera (C.)), (Saccharomycopsis fibuligera) M8 deposit number is: CGMCC NO.12408, the preservation unit is China general microbiological culture Collection center, the address is No. 3 of Xilu No. Beijing north Chen of the Korean-yang district in Beijing, and the preservation date is 2016, 4 and 28 days;

(2) preparation of Penicillium chrysogenum (Penicillium chrysogenum) M4 and Saccharomycopsis fibuligera (C.), (Saccharomycopsis fibuligera) M8 bran koji: mixing bran, rice hull and water at a mass ratio of 20:1:16, stirring uniformly, steaming for 20min, taking out, scattering while hot, and cooling to 30 ℃; respectively inoculating with the same volume concentration of 10⁷Penicillium chrysogenum (I) per mlPenicillium chrysogenum) M4 and Saccharomycopsis fibuligera (C.), (Saccharomycopsis fibuligera) Uniformly stirring spore suspension or bacterial suspension of the M8 strain, culturing at 30 ℃ for 12-15 h to grow white hypha, then buckling a bottle for culturing for 2-5 h, and taking out for later use when the surface is full of white hypha or produces a small amount of spores;

(3) acetic acid fermentation: penicillium chrysogenum (II) prepared by the step (2)Penicillium chrysogenum) M4 and Saccharomycopsis fibuligera (C.), (Saccharomycopsis fibuligera) The M8 bran koji strengthens Daqu, acetic fermentation is carried out, when ingredients are fermented and blended, the total adding amount of the Daqu and the bran koji is 40 percent of the main material used for fermentation, wherein the Daqu accounts for 25.1 percent of the main material, the adding amount of the Penicillium chrysogenum M4 bran koji accounts for 8.09 percent of the main material, the adding amount of the buckling bag compound yeast M8 bran koji accounts for 6.81 percent of the main material, the adding amount of Angel yeast accounts for 0.06 percent of the main material, and the adding amount of water accounts for 3 times of the main material; steaming main material according to conventional mature vinegar process, fermenting at 25 deg.C for 7 days, fermenting diluted mash with acetic acid, and filling with rice husk as adjuvantAnd bran, wherein the ratio of the rice hull to the bran to the main material is 15:20: 12; then continuing to ferment for 16 days at 25 ℃, after the solid state fermentation of the acetic acid is finished, pouring the vinegar by using water with the volume 2 times of the total volume of the vinegar grains, pouring out the first vinegar by using 70 percent of the total water volume, using the rest 30 percent of the water for next vinegar pouring, and then combining the vinegar liquid.

2. The yeast strengthening method for reducing the use amount of yeast and improving the quality of vinegar according to claim 1, wherein: the main materials used for fermentation are corn, a corn and sorghum mixture or sorghum with a mass ratio of 3: 7.

3. The yeast strengthening method for reducing the use amount of yeast and improving the quality of vinegar according to claim 1, wherein: adding 5% of salt into the vinegar-pouring water.

4. The yeast strengthening method for reducing the use amount of yeast and improving the quality of vinegar according to claim 1, wherein: said Penicillium chrysogenum (A)Penicillium chrysogenum) M4 is fast in growth on a malt extract agar medium, the culture is carried out for 7 days under the dark condition of 25 ℃, the diameter of a colony is 32-40mm, the texture is villiform, a large number of spore-forming structures are formed, the surface of the spore is dark grey and green, the back of the colony is light brown, no water-soluble pigment exists, the specialization of a conidiophor is not obvious, the width is 2.5-4.0 mu M, the wall is smooth, the broomcorn branches are 3-4 rounds and loose, the phialide is 7.0-11.7 × 2.2.2-3.0 mu M, the conidiophore is nearly spherical, wide elliptical, light green, the surface is smooth, the diameter is 2.8-4.0 mu M, sexual spores are not seen, the mycelia are not seen on a PDA medium, the mycelia fall into a felty shape, the bulges, the mycelia are compact, the initial stage of the colony is white, the gradual change is indigo green, the mycelia and the conidiophores are all provided with transverse partitions.

5. The yeast strengthening method for reducing the use amount of yeast and improving the quality of vinegar according to claim 1, wherein: the Saccharomycopsis fibuligera (A), (B) and (C)Saccharomycopsis fibuligera) M8 was cultured in malt liquid medium at 25 ℃ for three days in the form of cells of spherical, ovoid, and sausage with a size of (4)0-7.0) × (3.6-7.0) mum, culturing on malt extract agar slant at 25 deg.C for one month to obtain tough and white colony with villous surface and no reflection, culturing on corn powder agar Dalman flat plate to obtain fungal filament, culturing on PDA culture medium to obtain radial and straw hat shaped colony with white color, skin shape and diameter of 19.3-19.8mm, culturing on solid malt extract culture medium to obtain ester mud or skin shape with white color, culturing in liquid malt extract culture medium for 1d to obtain white fungal filament with branches and transverse partitions, and sporozoites with single or chain growth, square, long cylinder or ellipse, and round two ends.