CN112940975B - Bacillus subtilis compost subspecies and application thereof in vinegar brewing - Google Patents

Bacillus subtilis compost subspecies and application thereof in vinegar brewing Download PDF

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CN112940975B
CN112940975B CN202110224178.4A CN202110224178A CN112940975B CN 112940975 B CN112940975 B CN 112940975B CN 202110224178 A CN202110224178 A CN 202110224178A CN 112940975 B CN112940975 B CN 112940975B
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vinegar
bacillus subtilis
fermentation
compost
subspecies
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CN112940975A (en
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康雪梅
黄静
樊君
李范宇
伍学明
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Qianhe Condiment And Food Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12J—VINEGAR; PREPARATION OR PURIFICATION THEREOF
    • C12J1/00—Vinegar; Preparation or purification thereof
    • C12J1/04—Vinegar; Preparation or purification thereof from alcohol
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
    • C12N9/2457—Pullulanase (3.2.1.41)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01041—Pullulanase (3.2.1.41)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W30/00—Technologies for solid waste management
    • Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse

Abstract

The invention discloses a Bacillus subtilis compost subspecies and application thereof in vinegar brewing, wherein the Bacillus subtilis compost subspecies (Stercoris) is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms with the preservation name QH-20010, and the preservation number is as follows: CGMCC NO.21615. The bacillus subtilis compost subspecies QH-20010 can simultaneously produce strains of acid-resistant granular amylase, protease and pullulanase, and when the strains are used for brewing vinegar, the strains can improve the utilization rate of starch and the content of non-volatile acid, and are beneficial to reducing the cost of brewing vinegar and enhancing the flavor and taste.

Description

Bacillus subtilis compost subspecies and application thereof in vinegar brewing
Technical Field
The invention relates to the technical field of microorganisms, in particular to a bacillus subtilis compost subspecies and application thereof in vinegar brewing.
Background
Most of the traditional Chinese vinegar brewing adopts the solid-state method open environment fermentation production, and simultaneously adopts raw materials for fermentation. The required raw materials comprise starch raw materials such as rice, sticky rice, sorghum, wheat and the like, and the chaff is taken as an auxiliary material, and leavening agents such as wine medicine, lump koji, bran koji, yeast and the like are added for fermentation production. Mainly comprises the procedures of raw material treatment, starch saccharification, alcohol fermentation and acetic acid fermentation. In the current brewing process, bran, chaff and the like are directly fermented by raw materials without being cooked and gelatinized, a large amount of protein and starch are not fully utilized, so that the fermentation efficiency is reduced, and finally, vinegar pouring generates a large amount of precipitates, so that the waste of raw materials is caused, the final appearance of the product is influenced, and the product value is reduced.
At present, modes of crushing raw materials, adding a complex enzyme preparation, pouring vinegar grains at high temperature, adding cane sugar, lactic acid and the like are mostly adopted in industrial production to improve the utilization rate of the raw materials in vinegar brewing, improve and enhance the flavor and taste and the like, although certain improvement effect is achieved, the production cost is improved, the concept of zero addition increasingly pursued by people is contradicted, and the production and the application of high-quality vinegar are not facilitated.
Disclosure of Invention
The invention aims to provide a bacillus subtilis compost subspecies and application thereof, wherein the bacillus subtilis compost subspecies can simultaneously produce strains of acid-resistant granular amylase, protease and pullulanase, and when the strains are used for brewing vinegar, the strains can improve the starch utilization rate and the non-volatile acid content, and are beneficial to reducing the vinegar brewing cost and enhancing the flavor and taste.
The invention is realized by the following technical scheme:
a Bacillus subtilis subsp. Stercoris is preserved in China general microbiological culture Collection center with the preservation name QH-20010 and the preservation number is as follows: CGMCC NO.21615.
The preservation information of the preserved Bacillus subtilis compost subspecies QH-20010 related to the invention is as follows: the preservation unit: china general microbiological culture Collection center; the preservation address is as follows: the microbial research institute of western road 1, 3, national academy of sciences, north-south, morning-yang, beijing, zip code: 100101; the preservation date is as follows: 1 month and 13 days 2021; the preservation number is: CGMCC NO.21615 with the preservation name of QH-20010; and (3) classification and naming: stercoris.
The bacillus subtilis compost subspecies QH-20010 related to preservation in the invention is separated from vinegar grains by the research and development center of Qian standing grain food, and has the capability of producing acid-resistant granular amylase, protease and pullulanase through detection.
The bacillus is a bacterium with strong tolerance, and part of the bacillus has tolerance to acetic acid and high temperature. The bacillus subtilis is a food-grade safe strain, has rich enzyme systems, can participate in various catalytic reactions, and has higher commercial value. However, the research on applying the starch in vinegar brewing to improve the starch utilization rate and enhance the taste and flavor of vinegar is only reported.
The bacillus subtilis compost subspecies QH-20010 can simultaneously produce granular amylase, pullulanase and protease activities, and the produced granular amylase and pullulanase have better pH tolerance and can keep higher enzyme activity under the condition of low pH; the protease is neutral protease or acidic protease.
When the bacillus subtilis compost subspecies QH-20010 is used for brewing vinegar, the utilization rate of starch and the content of non-volatile acid can be improved, the cost of brewing vinegar is reduced, and the flavor and taste are enhanced.
A Bacillus subtilis compost subspecies is used for preparing a biological enzyme catalyst.
A Bacillus subtilis compost subspecies is used for preparing a microbial fermentation inoculant.
An application of Bacillus subtilis compost subspecies or a microbial fermentation inoculant of a biological enzyme catalyst in the field of food.
An application of Bacillus subtilis compost subspecies or a microbial fermentation inoculant as a biological enzyme catalyst in vinegar brewing.
Further, the vinegar is cellar vinegar, bran vinegar, aromatic vinegar or rice vinegar.
When the bacillus subtilis compost subspecies QH-20010 is used for brewing vinegar, the starch utilization rate and the non-volatile acid content are both improved compared with the contrast:
in cellar vinegar brewing, the starch utilization rate is improved by 12.17%, in bran vinegar brewing, the starch utilization rate is improved by 16.09%, and in Zhenjiang aromatic vinegar brewing, the starch utilization rate is improved by 19.47%.
In cellar vinegar brewing, the content of non-volatile acid is increased by 16.32%, in bran vinegar brewing, the content of non-volatile acid is increased by 18.60%, and in Zhenjiang aromatic vinegar brewing, the content of non-volatile acid is increased by 9.88%.
Further, fermentation broth obtained by fermenting and culturing bacillus subtilis compost subspecies is used as enzyme, and the enzyme is added into vinegar mash to be used as a biological enzyme catalyst for catalysis, so that the hydrolysis of raw materials and the generation of products ethanol and acetic acid are promoted, namely the bacillus subtilis compost subspecies is used for preparing the biological enzyme catalyst.
Specifically, the method comprises the following steps:
fermenting and culturing bacillus subtilis compost subspecies to obtain fermentation liquor as enzyme, adding the fermentation liquor into the vinegar culture, catalyzing starch and protein in grain raw materials in the vinegar culture to hydrolyze to generate fermentable sugar and amino acid, providing carbon, nitrogen sources and substrates required by growth and metabolism of saccharomycetes, acetic acid bacteria and the like, and promoting the generation of ethanol and acetic acid.
Further, fermenting and culturing bacillus subtilis compost subspecies to obtain fermentation liquor serving as seed liquor, inoculating the seed liquor into the vinegar culture, continuing to grow and reproduce the bacillus subtilis compost subspecies in the vinegar culture environment to generate enzyme, promoting the hydrolysis of grain raw materials and the generation of products ethanol and acetic acid, and using the bacillus subtilis compost subspecies to prepare the microbial fermentation inoculum.
Specifically, the method comprises the following steps:
fermenting and culturing bacillus subtilis compost subspecies to obtain fermentation liquor as seed liquor, inoculating the seed liquor into the vinegar culture, continuously growing, reproducing and metabolizing the bacillus subtilis compost subspecies in the vinegar culture environment, further producing enzymes such as amylase, protease and pullulanase, secreting the enzymes into the extracellular vinegar culture environment, and promoting hydrolysis of starch, protein and the like in grain raw materials to generate fermentable sugar and amino acid.
Further, the preparation process of the fermentation liquor is as follows:
s1, slant culture: inoculating bacillus subtilis compost subspecies to a slant culture medium to obtain slant thalli;
specifically, the method comprises the following steps:
inoculating bacillus subtilis compost subspecies to a slant culture medium, and culturing for 48h at 35 ℃ and 200rpm to obtain slant thalli; the final concentration of the slant culture medium is as follows: 10-25g/L glucose, 3-15g/L peptone, 2-10g/L yeast powder and Na 2 HPO 4 0.2-2.0g/L，K 2 HPO 4 0.2-1.8g/L，MgSO 4 0.03-0.15g/L, 20.0g/L agar, deionized water as solvent, and 5.0-6.5 pH value;
s2, seed culture: the method comprises the following steps of primary seed culture and secondary seed culture:
first-stage seed culture: inoculating the slant thallus to a seed culture medium to obtain a primary seed solution;
specifically, the method comprises the following steps:
selecting one strain of the thallus on the inclined plane, inoculating the strain to a seed culture medium, and culturing at 35 ℃ for 24 hours to obtain a primary seed solution; the final concentration composition of the seed culture medium is as follows: 10-25g/L glucose, 3-15g/L peptone, 2-10g/L yeast powder and Na 2 HPO 4 0.2-2.0g/L，K 2 HPO 4 0.2-1.8g/L，MgSO 4 0.03-0.15g/L, 20.0g/L agar, deionized water as solvent, and 5.0-6.5 pH value;
secondary seed culture: inoculating the first-stage seed liquid into a second-stage seed culture medium by an inoculation amount with the volume concentration of 1-10% to obtain a second-stage seed liquid;
specifically, the method comprises the following steps:
inoculating the primary seed solution into a secondary seed culture medium in an inoculation amount with the volume concentration of 1-10%, and culturing at 35 ℃ for 24-48h to obtain a secondary seed solution; the final concentration of the secondary seed culture medium comprises: 10-25g/L of raw corn starch, 3-15g/L of peptone, 2-10g/L of yeast powder and Na 2 HPO 4 0.2-2.0g/L，K 2 HPO 4 0.2-1.8g/L，MgSO 4 0.03-0.15g/L of agar, 20.0g/L of agar, deionized water as a solvent and 5.0-6.5 of pH value;
s3, fermentation culture: selecting a liquid fermentation tank, adding water, stirring, simultaneously adding rice flour and high-temperature alpha-amylase to obtain mash, cooling the mash to 45-55 ℃, adding saccharifying enzyme under the condition of slow stirring, preserving heat, sterilizing, adding peptone and yeast powder, cooling after sterilizing, inoculating secondary seed liquid, ventilating, stirring and fermenting to obtain fermentation liquor;
specifically, the method comprises the following steps:
selecting a liquid fermentation tank, adding water, stirring, simultaneously adding rice flour and high-temperature alpha-amylase, wherein the dosage of the high-temperature alpha-amylase is 0.01-0.2% of the mass of the rice flour, stirring and heating to 90-95 ℃, uniformly stirring for about 30min to obtain mash, cooling the mash to 45-55 ℃, adding saccharifying enzyme under the condition of slow stirring, keeping the temperature for about 20min, wherein the dosage of the saccharifying enzyme is 0.02-0.4% of the mass of the rice flour, sterilizing, adding 5-15g/L of peptone and 2-10g/L of yeast powder, sterilizing, cooling to 33-37 ℃, inoculating the secondary seed liquid according to the inoculation amount of 2-10%, ventilating, stirring, and fermenting for 20-48h under the condition of 33-40 ℃ and maintaining the pressure.
Vinegar brewed based on bacillus subtilis compost subspecies.
Specifically, the method comprises the following steps:
when pit vinegar is prepared, inoculating fermentation liquor according to the inoculation amount of 2-10% to prepare vinegar pit mud, simultaneously inoculating the fermentation liquor into vinegar grains according to the inoculation amount of 2-10% in the initial fermentation stage, completing acetic fermentation through a grain overturning process, and further moving the vinegar pit mud to a vinegar pit mud pool to complete pit vinegar fermentation to prepare corresponding pit vinegar. When preparing the bran vinegar or the aromatic vinegar, the fermentation liquor is inoculated into the vinegar culture according to the inoculation amount of 2-10%, and the corresponding bran vinegar or the aromatic vinegar is prepared by fermenting and producing according to the process of the bran vinegar or the aromatic vinegar after the fermentation liquor is turned and mixed uniformly. When preparing the rice vinegar, inoculating the prepared rice flour mash according to the inoculation amount of 2-10%, and performing fermentation production according to a rice vinegar process to prepare the corresponding rice vinegar.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the invention provides a strain-Bacillus subtilis subsp.
2. The strain QH-20010 provides a new enzyme source and a beneficial microorganism fermentation strain for the vinegar brewing industry, provides a new improvement idea for solving the problem of low raw material utilization rate faced by the vinegar industry integrally, promotes the overall progress of the industry, ensures the full utilization of grain resources and avoids unnecessary waste.
3. The invention provides a method for hydrolyzing starch and protein into reducing sugar and amino acid in vinegar brewing by using fermentation broth obtained by fermenting and culturing bacillus subtilis compost subspecies as a biological enzyme catalyst, and also provides a method for using the bacillus subtilis compost subspecies as a microbial fermentation microbial inoculum applied to the field of vinegar brewing.
4. The invention improves the utilization rate of starch by 12.17 percent in cellar vinegar brewing, improves the utilization rate of starch by 16.09 percent in bran vinegar brewing, and improves the utilization rate of starch by 19.47 percent in Zhenjiang aromatic vinegar brewing.
5. The invention improves the non-volatile acid content by 16.32 percent in cellar vinegar brewing, improves the non-volatile acid content by 18.60 percent in bran vinegar brewing, and improves the non-volatile acid content by 9.88 percent in Zhenjiang aromatic vinegar brewing.
6. The strain QH-20010 is applied to rice vinegar brewing, and can enable the content of a flavor substance acetoin to reach 30mg/L from nothing to 21.5mg/L and the content of 2, 3-butanediol to reach 21.5mg/L.
7. The strain QH-20010 is applied to vinegar brewing, so that the leached vinegar is black and bright in color, clear in body form and superior to a control group in fragrance and taste.
Drawings
The accompanying drawings, which are included to provide a further understanding of the embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention. In the drawings:
FIG. 1 is a schematic diagram showing the colony morphology of the strain QH-20010 of the present invention;
FIG. 2 is a breeding flow chart of the strain QH-20010 of the invention;
FIG. 3 is a graph showing the results of pH effects on the activity of soluble amylase and raw amylase;
FIG. 4 is a graph showing the effect of pH on pullulanase activity.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.
Example 1:
screening of Bacillus subtilis subsp (Stercoris) QH-20010
11. Preliminary screening
As shown in figure 2, the method selects vinegar grains fermented for 2, 4, 6, 8, 10, 12, 14, 6, 18, 20, 22 and 24 days from vinegar grains fermenting ponds in natural fermentation states of Qian He Wei food shares GmbH and Zhen Jiang Jinshan Temple food GmbH respectively, and the sampling mode is that the samples of the vinegar grains are vertically sampled from the surface to the bottom of the vinegar grains around the fermenting ponds, and then the samples of the vinegar grains in different fermentation periods are uniformly mixed to obtain strain screening samples. The screening method comprises the following specific steps: weighing 100g of a vinegar mash sample, placing the vinegar mash sample in 1000mL of 0.85% physiological saline, shaking, standing, taking supernatant into an enrichment medium, and carrying out shaking culture at 30 ℃ and 150r/min for 2-3 days. 10mL of the enriched solution is added into 100mL of fresh enrichment medium, and separation and purification are carried out after the process is repeated for 3 times.
And selecting a screening culture medium to carry out primary screening on the strains, carrying out gradient dilution on the enriched bacterial liquid, coating the diluted bacterial liquid on a solid screening culture medium flat plate, culturing for 48 hours at 35 ℃, and selecting bacterial colonies generated by the obviously hydrolyzed transparent ring to carry out further secondary screening. The screening media used were: 20g/L of soluble starch, 5g/L of yeast powder, 10g/L of peptone and Na 2 HPO 4 0.5g/L，K 2 HPO 4 0.5g/L, agar 20g/L and distilled water as solvent.
12. Double sieve
The secondary screening is carried out by using granular amylase producing bacteria and pullulanase producing bacteria.
Re-screening granular amylase producing bacteria: selecting a single colony with an obvious transparent hydrolysis ring during primary screening, dibbling the single colony on a granular starch re-screening culture medium, culturing for 48 hours at 35 ℃, observing the generation condition of the hydrolysis transparent ring around the single colony, selecting a colony with the diameter of the hydrolysis transparent ring larger than that of the single colony, and further performing separation and purification on the granular starch re-screening culture medium to obtain a single colony of a pure-breed granular amylase producing strain. The granular starch re-screening culture medium comprises: 20g/L of raw corn starch, 5g/L of yeast powder, 10g/L of peptone and Na 2 HPO 4 0.5g/L，K 2 HPO 4 0.5g/L, 20g/L agar and distilled water as solvent, wherein the raw corn starch is weighed into a weighing bottle separately, dried and heat-sterilized in an oven at 107 ℃ for 2h, added into the sterilized culture medium before use, mixed evenly and poured onto a flat plate.
Re-screening pullulanase producing bacteria: and (2) selecting a single colony with an obvious hydrolysis transparent ring during primary screening, dibbling the single colony on a pullulanase re-screening culture medium, culturing for 48 hours at 35 ℃, adding 5mL of absolute ethyl alcohol to the surface of the culture medium, placing for 2 hours in a refrigerator at 4 ℃, taking out and observing the generation condition of the transparent ring around the colony, selecting the colony with the diameter of the transparent ring larger than that of the single colony, and further carrying out separation and purification to obtain the pure pullulanase producing strain. The pullulanase re-screening culture medium comprises: 20g/L of pullulanose, 5g/L of yeast powder, 10g/L of peptone and Na 2 HPO 4 0.5g/L，K 2 HPO 4 0.5g/L, 20g/L agar and distilled water as solvent.
13. Determination of enzyme-producing Activity of Strain
Inoculating the strain screened in the step 2 into a slant culture medium, culturing at 30 ℃ for 48h, and storing in a refrigerator at 4 ℃. The slant culture medium is as follows: 20g/L glucose, 10g/L peptone, 5g/L yeast powder and Na 2 HPO 4 0.5g/L，K 2 HPO 4 0.5g/L，MgSO 4 0.1g/L, 20.0g/L agar, deionized water as solvent, and pH of 6.0. The strain preserved on the slant was inoculated into a seed medium and cultured at 30 ℃ for 24 hours. Inoculating the seed liquid into a fermentation culture medium according to the inoculation amount with the volume concentration of 1%, carrying out shaking culture at 30 ℃,150rpm for 60h, centrifuging for 5min under 12000g, separating fermentation liquid from wet thalli, taking the fermentation liquid as enzyme liquid to carry out corresponding enzyme activity determination, and carrying out further research on the obtained strain with higher enzyme activity.
And (3) measuring the enzyme activity of amylase: soluble starch was used as a substrate, and acetic acid-sodium acetate buffer (50mM, pH 5.8) was used as a solvent to dissolve the substrate, thereby preparing a substrate solution of 20 g/L. Taking 5mL of substrate solution in a test tube, preserving heat for 5min at 40 ℃, adding 30 mu L of enzyme solution which is preserved heat for 5min at the temperature, reacting for 30min at 200rpm, stopping the reaction by 200 mu L of 200mM NaOH, and reacting for 5min at 200rpm to obtain reaction solution. The control reaction was: the substrate solution was incubated at 40 ℃ for 5min, reacted at 200rpm for 30min, quenched with 200. Mu.L of 200mM NaOH, added with 30. Mu.L of the enzyme solution incubated at 40 ℃ for 5min, and reacted at 200rpm for 5min. The amount of glucose produced in the reaction solution and the control reaction solution was measured by DNS method, and the amylase activity was calculated.
Determination of granular amylase enzyme activity: a substrate solution of 20g/L was prepared by dissolving a substrate in raw corn starch as a substrate and acetic acid-sodium acetate buffer (50mM, pH 5.8) as a solvent. Taking 5mL of substrate solution, preserving heat for 5min at 40 ℃ in a test tube, adding 30 mu L of enzyme solution which is preserved heat for 5min at the temperature, reacting for 30min at 200rpm, stopping the reaction by 200 mu L of 200mM NaOH, and reacting for 5min at 200rpm to obtain reaction solution. The control reaction was: the substrate solution was incubated at 40 ℃ for 5min, reacted at 200rpm for 30min, quenched with 200. Mu.L of 200mM NaOH, added with 30. Mu.L of the enzyme solution incubated at 40 ℃ for 5min, and reacted at 200rpm for 5min. And measuring the generation amount of glucose in the reaction solution and the reference reaction solution by using a DNS method, and calculating the enzyme activity of the granular amylase.
The enzyme activity of the pullulanase is determined as follows: a substrate solution of 20g/L was prepared by dissolving a substrate in pullulan as a substrate and acetic acid-sodium acetate buffer (50mM, pH 5.8) as a solvent. Taking 5mL of substrate solution and a test tube, preserving heat for 5min at 40 ℃, adding 30 mu L of enzyme solution which is preserved for 5min at the same temperature, reacting for 30min at 200rpm, stopping the reaction by 200 mu L of 200mM NaOH, and reacting for 5min at 200rpm to obtain reaction liquid. The control reaction was: the substrate solution was incubated at 40 ℃ for 5min, reacted at 200rpm for 30min, quenched with 200. Mu.L of 200mM NaOH, added with 30. Mu.L of the enzyme solution incubated at 40 ℃ for 5min, and reacted at 200rpm for 5min. And measuring the generation amount of glucose in the reaction solution and the reference reaction solution by using a DNS method, and calculating the enzyme activity of the pullulanase.
The DNS method comprises the following steps: reacting glucose standard solutions with different concentration gradients with a DNS (Domain name System) reagent, namely adding 2mL of enzymatic reaction liquid into a 2mL of DNS reagent, after 5min of boiling water bath, adding distilled water to a constant volume of 25mL, uniformly mixing, taking a DNS reaction of a control group as a blank, measuring absorbance of the enzymatic reaction DNS reaction liquid at lambda =520nm, and calculating enzyme activity according to the absorbance.
The amylase activity is defined as: at 40 ℃, the amount of enzyme required to release 1 μ g of reducing sugar (calculated on glucose) per 1min using soluble starch as substrate and acetic acid-sodium acetate (50mM, pH 5.8) as buffer was taken as one unit of enzyme activity and is recorded as 1U.
The granular amylase enzyme activity is defined as: at 40 ℃, the enzyme amount required for releasing 1 μ g of reducing sugar (calculated on glucose) per 1min with corn raw starch as substrate and acetic acid-sodium acetate (50mM, pH 5.8) as buffer is 1U.
The pullulanase enzyme activity is defined as: at 40 ℃, pullulan is taken as a substrate, acetic acid-sodium acetate (50mM, pH 5.8) is taken as a buffer solution, and the enzyme amount required for releasing 1 mu g of reducing sugar (calculated by glucose) every 1min is taken as one enzyme activity unit and is marked as 1U.
And (3) protease activity determination: casein (20 g/L) and NaH are used as substrates 2 PO 4 -Na 2 HPO 4 (50mM, pH 7.2) is slowThe neutral protease activity was measured under the condition of washing, and the acid protease activity was measured under the condition of a sodium lactate-50mM, pH 3.8 buffer. Preheating 2mL of enzyme solution at 40 ℃ for 5min, adding 2mL of substrate solution, reacting for 10min under heat preservation, adding 4mL of trichloroacetic acid (0.4M) solution, and continuing to preserve heat for 20min to obtain reaction solution. The contrast reaction is that after the enzyme solution is preheated, 4mL of trichloroacetic acid (0.4M) solution is kept warm for 10min, then 2mL of substrate solution is added, and the temperature is kept for 20min to obtain the contrast reaction solution. The enzyme activity was determined and calculated by the forskolin-phenol method.
The neutral protease enzyme activity is defined as: at 40 deg.C, using casein as substrate, naH 2 PO 4 -Na 2 HPO 4 (50mM, pH 7.2) the amount of enzyme required to release 1. Mu.g of tyrosine per 1min in the buffer system was one unit of enzyme activity and was 1U.
The acid protease enzyme activity is defined as: the enzyme amount required for releasing 1. Mu.g of tyrosine per 1min in a lactic acid-sodium lactate (50mM, pH 3.8) buffer system with casein as substrate at 40 ℃ is one enzyme activity unit and is 1U.
TABLE 1 determination of Bacillus subtilis subsp
Figure GDA0003956506680000071
By measuring the change conditions of enzyme activities under different pH conditions, the amylase, granular amylase and pullulanase produced by the strain can be tolerant to a lower pH environment, the highest enzyme activity is possessed under the condition of pH 5.0, and the soluble amylase, granular amylase, pullulanase and the like can keep more than 50% of activity as the pH is reduced to 3.8 (figures 3 and 4), so that the tolerance of the enzyme produced by the subspecies of bacillus subtilis to the low pH environment is further verified, and the feasibility of applying the enzyme to vinegar brewing is further verified.
Example 2:
identification of strain QH-20010
21. And (3) morphological identification:
the strain QH-20010 obtained by screening in example 1 is inoculated on a solid culture medium, and after 24 hours of culture at 37 ℃, the strain is irregular in shape, soft in texture, flat in colony and irregular in edge, and is clung to a light yellow colony on the surface of the culture medium, and the diameter of the light yellow colony is 3-5mm, as shown in figure 1. And (3) gram staining observation: pink short rod shape, no spores. Solid medium composition: 10g/L of sodium chloride, 10g/L of peptone, 5g/L of yeast powder, 20g/L of agar and deionized water as a solvent.
22. Physiological and biochemical identification:
strain QH-20010 was phenotyped 94 tests using the Biolog (GEN iii) automated microorganism identification system, including 71 carbon source utilization assays and 23 chemosensitivity assays: inoculating strain QH-20010 to BUG plate culture medium (BIOLOG UNIVERSAL GROWTH AGAR), culturing at 33 deg.C for 2 days, washing off thallus on the plate with sterile cotton swab, mixing with inoculum (IF-A), making into bacterial suspension, and adjusting to 91% T/IF-A with turbidimeter. The bacterial suspensions were added to each well of the BiologGEN iii microwell assay plate using an 8-well electric applicator, 100 μ L per well. The plate was placed in a 33 ℃ incubator and read on a Biolog reader after 12h, 24h, 36h, 48h incubation, respectively.
Metabolic fingerprints of the strain QH-20010 are analyzed by a Biolog reader, 65 carbon sources can be strongly utilized by the strain QH-20010, and other 6 carbon sources cannot be utilized or are weak in utilization capacity; the strain QH-20010 is sensitive to 21 chemical substances. The 48h identification results given by the Biolog system are shown in tables 2 and 3.
TABLE 2 ability of Strain QH-20010 to utilize 71 carbon sources on BiologGEN III plates
Figure GDA0003956506680000081
Figure GDA0003956506680000091
Notes:+,positive；-,negative；B,borderline
TABLE 3 chemosensitivity of Strain QH-20010 to 23 chemicals on BiologGEN III plates
Figure GDA0003956506680000092
Notes:+,positive；-,negative；B,borderline
23. Molecular biology identification:
the 16S rDNA gene of the strain was amplified and sequenced using the primers P1:5 'AGAGTTTGATCCTGGCTCCAG-3' and P2:5 'AAGGAGGTGATCCAGCCGCA 3' using the total DNA of the strain QH-20010 as a template to obtain the 16S rDNA sequence of the strain, and the 16S rDNA gene sequences of the related strains in GenBank were searched on the NCBI website using BLAST and subjected to homology alignment. The strain QH-20010 has the highest homology with Stercoris (homology of 99% in basic on 16S ribosomal RNAgene) of Bacillus subtilis strain subspecies, and the strain is basically identified to belong to a control bacterium based on the 16S rDNA homology of more than 95% according to the microbial genetics identification principle. Therefore, the strain QH-20010 is Bacillus subtilis subsp.Stercoris (Bacillus subtilis subsp.Stercoris) QH-20010 which is prepared by preserving in China general microbiological culture Collection center (CGMCC No): 21615, preservation date 2021, month 1, day 13, preservation address: the microbial research institute of western road 1, 3, national academy of sciences, north-south, morning-yang, beijing, zip code: 100101.
example 3:
preparation of fermentation broth and seed liquid
31. Slant culture:
inoculating bacillus subtilis compost subspecies to a slant culture medium, and culturing at 35 ℃ for 48h to obtain slant thalli; the final concentration of the slant culture medium is as follows: 20g/L glucose, 10g/L peptone, 5g/L yeast powder and Na 2 HPO 4 0.5g/L，K 2 HPO 4 0.5g/L，MgSO 4 0.1g/L, 20.0g/L agar, deionized water as solvent, and pH of 6.0.
32. Seed culture
The method comprises first-stage seed culture and second-stage seed culture.
First-order seed culture: selecting one strain of the thallus on the inclined plane, inoculating the strain to a seed culture medium, and culturing at 35 ℃ for 24 hours to obtain a primary seed solution; the final concentration composition of the primary seed culture medium is as follows: 10g/L of glucose, 10g/L of peptone, 5g/L of yeast powder and Na 2 HPO 4 0.5g/L，K 2 HPO 4 0.5g/L，MgSO 4 0.1g/L, 20.0g/L of agar, deionized water as a solvent and 6.0 of pH value; the control group was a medium of the same procedure but without inoculation of QH-20010.
Secondary seed culture: inoculating the primary seed solution into a secondary seed culture medium by an inoculation amount with the volume concentration of 1-10%, and culturing at 35 ℃ for 24-48h to obtain a secondary seed solution, wherein the preferred inoculation amount is 5%; the final concentration of the secondary seed culture medium comprises: preferably, the final concentration of the secondary seed culture medium is as follows: 20g/L of raw corn starch, 10g/L of peptone, 5g/L of yeast powder and Na 2 HPO 4 0.5g/L，K 2 HPO 4 0.5g/L，MgSO 4 0.1g/L, 20.0g/L agar, deionized water as solvent, and pH of 6.0. The control group was the same composition and was not inoculated with QH-20010 medium.
33. Fermentation culture
Selecting a liquid fermentation tank, adding water and stirring, simultaneously adding rice flour and high-temperature alpha-amylase, wherein the dosage of the high-temperature alpha-amylase is 0.05 percent of the mass of the rice flour, stirring and heating to 90-95 ℃, uniformly stirring for about 30min to obtain mash, cooling the mash to 45-55 ℃, adding saccharifying enzyme under the condition of slow stirring and keeping the temperature for about 20min, wherein the dosage of the saccharifying enzyme is 0.1 percent of the mass of the rice flour, adding 1g/L of peptone and 0.5g/L of yeast powder, cooling to 33-37 ℃ after sterilization, inoculating the secondary seed liquid according to 5 percent of inoculation quantity, ventilating and stirring, and carrying out pressure-maintaining fermentation at 33-40 ℃ for 20-52h. After the fermentation is finished, the obtained fermentation liquor is enzyme liquor, and the obtained bacterial liquor (fermentation liquor) is also used as seed liquor for inoculating fermented grains of vinegar for fermentation. The control group was a sugar mash of the same treatment but not inoculated with QH-20010.
Example 4:
influence of different pH values on growth of Bacillus subtilis compost subspecies QH-20010
LB culture medium (peptone 10g/L, yeast powder 5g/L, naCl 10 g/L) is prepared, lactic acid is used for adjusting different pH values, and the strain can grow under the condition of pH when the culture medium turns turbid after being cultured for a certain time at 35 ℃ and 200 rpm. The results are shown in Table 4. Wherein, the bacterial strain grows for about 8-12h after inoculation under the conditions of pH 5.5 and pH 4.8, the bacterial strain grows for 16-24h under the conditions of pH 4.4 and pH 4.0, and the bacterial strain grows for 24h under the condition of pH 3.8.
TABLE 4 growth of enzyme-producing strains under different pH conditions
Figure GDA0003956506680000101
Example 5:
application of bacillus subtilis compost subspecies QH-20010 in cellar vinegar brewing
51. Preparation of vinegar pit mud
Taking out pit mud at the bottom and on the side of the existing pit mud pool, taking out pit mud positions to be spread over the whole pit mud pool, taking out about 100kg in total, adding 10kg of secondary vinegar, 20kg of fermented fresh vinegar grains fermented in the pit mud pool, 25kg of bran leachate and 20kg of strain fermentation liquor prepared in the embodiment 3, uniformly mixing, and carrying out stacking culture at 30-35 ℃ for 5 days to obtain the vinegar pit mud. The mature vinegar cellar mud is spread and coated on the bottom and the side surface of the vinegar cellar mud pool, and the coating thickness is 10cm. In the control group 1, the vinegar pit mud is used as the vinegar pit mud existing in the pit mud pool.
52. Preparation of wine mash
Weighing 300kg of rice, grinding into slurry, milling into powder, adding 900kg of water while stirring, adding 1kg of alpha-amylase, heating to 90-95 ℃, uniformly stirring for about 30min to obtain mash, cooling the mash to 45-55 ℃, adding lactic acid under slow stirring to adjust the mash pH of 4.7, adding 2kg of saccharifying enzyme, keeping the temperature for about 20min, cooling to 33-37 ℃, inoculating 5kg of active dry yeast, standing and culturing at normal temperature for 12-16h to obtain the yeast activated wine mash.
53. Expanding culture of strain
The fermentation broth was prepared according to the method for preparing the fermentation broth and the seed solution of example 3.
54. Inoculating fermented grains of vinegar for fermentation
Control group 1: 540kg of rice husks are paved from bottom to top inside a fermentation pool, 4600kg of bran, 200kg of Daqu and 250kg of bran koji are added, 8300kg of the fermented mash prepared in the step 52 is added (wherein the temperature of the fermented mash is about 33 ℃), meanwhile, the control sugar mash prepared in the example 3 is added, the dosage of the control sugar mash is 5 percent of the total amount of the mash, after the fermented mash is soaked into the new fermented grains, 100kg of fresh vinegar fermented grains fermented for 9-11 days are inoculated on the surfaces of the new fermented grains and are uniformly paved on the surfaces of the new fermented grains for manual fermented grain overturning. Turning over the fermented grains every day 3 days before the fermentation period, and then turning over the fermented grains every other day. And naturally fermenting after the fermentation is finished.
Experimental group 1: 540kg of chaff is laid inside the fermentation tank from bottom to top, 4600kg of bran, 200kg of Daqu and 250kg of bran koji are added, the fermentation liquor prepared in the example 3 is inoculated into the wine mash prepared in the step 52 and is uniformly mixed to form the wine mash containing bacillus, and the sugar mash containing the bacillus is inoculated into the fermentation tank (wherein the temperature of the mixed wine mash is about 33 ℃), and the inoculation amount is 5 percent of the total amount of the mash. After the fermented grains are immersed into the new grains, 100kg of fresh vinegar grains fermented for 9-11 days are inoculated on the surfaces of the new grains, evenly spread on the surfaces of the new grains and manually turned. Turning over the fermented grains every day 3 days before the fermentation period, and then turning over the fermented grains every other day. And naturally fermenting after the fermentation is completed. Taking vinegar grains and marinade in the fermentation process to measure related physicochemical indexes.
55. Secondary fermentation of vinegar cellar mud pool
And (5) transferring the fermented vinegar unstrained spirits obtained in the step (4) into a vinegar cellar mud pool, compacting, further sealing by using mature vinegar cellar mud, and continuously maintaining sealed fermentation for 25 days to obtain mature cellar vinegar unstrained spirits. The experimental group 1 vinegar culture was transferred to a newly prepared pit mud pool containing subspecies of bacillus subtilis, and sealed with the correspondingly prepared vinegar pit mud, while the control group 1 vinegar culture was transferred to the original pit mud pool and sealed with the original vinegar pit mud.
56. Drench vinegar
Pouring vinegar by a sleeve pouring method to obtain the vinegar. And (3) shoveling all the fermented vinegar grains and the marinade in a vinegar spraying pool, spraying vinegar in the previous round, soaking for 2h, taking the vinegar to obtain head vinegar, and putting the head vinegar into a storage tank. And then the vinegar is poured and drenched by the vinegar in the previous round and the vinegar in the second round, the vinegar is soaked for 2 hours to obtain the vinegar in the first round, and the vinegar is put into a transfer tank for the vinegar pouring and drenching in the next round. And then, soaking the fermented grains of the vinegar for 2 hours by using tap water to obtain the secondary vinegar, and putting the secondary vinegar into a transfer tank for the next round of pouring and pouring the primary vinegar.
57. Settling, sterilizing and filling
And (3) performing high-temperature instantaneous sterilization on the obtained vinegar head through a pipeline, settling in a storage tank, extracting upper-layer vinegar liquid after settling, performing filter pressing on the upper-layer vinegar liquid through a plate frame, and entering a refining and filling process to finally obtain finished vinegar.
58. Total acid content monitoring
And (4) taking vinegar grains and the marinade at the bottom of the pond every other day from the first day of fermentation to the end of fermentation, and detecting. The total acid is measured as acetic acid by acid-base titration. After the fermentation was completed, the total fermented grain acid content of the control group 1 was 5.32g/100g (wet fermented grains), and the total fermented grain acid content of the experimental group 1 was 5.82g/100g (wet fermented grains). The total acid content of the pit mud bottom filtrate of the control group 1 is 6.82g/100mL, and the total acid content of the pit mud bottom filtrate of the experimental group 1 is 7.76g/100mL.
59. Detection of non-volatile acid content
After volatile acid is removed by adopting a distillation mode, the content of the non-volatile acid is measured by adopting an acid-base titration mode. After fermentation in the cellar mud pool is finished, the content of the non-volatile acid in the cellar mud pool bottom filtrate of the control group 1 is 3.74g/100mL, the content of the non-volatile acid in the cellar mud pool bottom filtrate of the experimental group 1 is 4.95g/100mL, the proportion of the non-volatile acid is 54.83 percent and 63.78 percent respectively, and the proportion of the non-volatile acid in the experimental group 1 is improved by 16.32 percent compared with the proportion in the control group 1.
510. Starch content detection
The content of starch in the vinegar mash is determined by an enzymolysis method according to GB 5009.9-2016. The starch content of the vinegar grains of the control group 1 and the experimental group 1 is 4.78g/100g and 3.95g/100g respectively on the day of fermentation. The initial starch content is 11.6g/100g, the starch utilization rate is 58.79 percent and 65.95 percent respectively, and the starch utilization rate is improved by 12.17 percent.
512. Detection of precipitate content
The sediment content in the finished vinegar is measured by a centrifugal method, the sediment contents of the control group 1 and the experiment group 1 are 332mg/100mL and 258mg/100mL respectively, and the sediment content of the experiment group 1 is reduced by 28.68 percent compared with the sediment content of the control group 1.
513. Sensory index analysis of finished vinegar
The finished vinegar of the experimental group 1 is darker and brighter in color, clearer in body state, softer and fuller in sour taste and longer in aftertaste, and the result shows that the quality of the finished vinegar can be obviously improved by adding bacillus subtilis in the acetic acid fermentation stage and the cellar vinegar fermentation stage of the cellar vinegar.
Example 6:
application of bacillus subtilis compost subspecies QH-20010 in bran vinegar brewing
61. Preparation of wine mash
The same procedure was followed as in the preparation of the mash of example 5.
62. Expanding culture of strain
The fermentation broth was prepared according to the method for preparing the fermentation broth and the seed solution of example 3.
63. Inoculating fermented grains of vinegar and fermenting
The operation steps of the inoculation and fermentation of the vinegar substrate in the control group 2 and the experimental group 2 are the same as those in the control group 1 and the experimental group 1 in the example 5.
During the fermentation process, the vinegar culture and the marinade are taken to measure relevant physicochemical indexes.
64. Salt seal
And (3) uniformly spreading NaCl on the surface of the vinegar grains at the 24 th day of the fermentation period, turning the vinegar grains, and spreading NaCl on the surface of the vinegar grains after the turning is finished. The total NaCl dosage is 1 percent of the total amount of the feed water of the fermentation tank. And (5) pouring vinegar after 3 days of salt sealing.
65. Drench vinegar
The vinegar pouring step was the same as in example 5.
66. Settling, sterilizing and filling
And (3) performing high-temperature instantaneous sterilization on the obtained vinegar head through a pipeline, settling in a storage tank, extracting upper-layer vinegar liquid after settling, performing filter pressing on the upper-layer vinegar liquid through a plate frame, and entering a refining and filling process to finally obtain finished vinegar.
67. Total acid content monitoring
And (4) taking vinegar grains and the marinade at the bottom of the pond every other day from the first day of fermentation to the end of fermentation, and detecting. The total acid is measured as acetic acid by acid-base titration. After the fermentation is finished, the total fermented grain acid content of the control group 2 is 5.06g/100g (wet fermented grains), the total fermented grain acid content of the experimental group 2 is 5.45g/100g (wet fermented grains), the total marinade acid content of the control group 2 is 6.52g/100mL, and the total marinade acid content of the experimental group 2 is 7.03g/100mL.
68. Detection of non-volatile acid content
After volatile acid is removed by adopting a distillation mode, the content of the non-volatile acid is measured by adopting an acid-base titration mode. After the fermentation is finished, the non-volatile acid content of the marinade of the control group 2 is 3.66g/100mL, the non-volatile acid content of the marinade of the experimental group 2 is 4.68g/100mL, the non-volatile acid proportion is respectively 56.13% and 66.57%, and the non-volatile acid proportion of the experimental group 2 is improved by 18.60% compared with that of the control group 2.
69. Starch content detection
The content of starch in the vinegar mash is determined by an enzymolysis method according to GB 5009.9-2016. The starch content of the vinegar mash of the control group 2 and the vinegar mash of the experimental group 2 are 5.76g/100g and 4.82g/100g respectively on the day of the end of fermentation. The initial starch content is 11.6g/100g, the starch utilization rates are respectively 50.34% and 58.44%, and the starch utilization rate is improved by 16.09%.
610. Detection of precipitate content
The sediment content in the finished vinegar is measured by adopting a centrifugal method, the sediment contents of the control group 2 and the experiment group 2 are 398mg/100mL and 287mg/100mL respectively, and the sediment content of the experiment group 2 is reduced by 27.89 percent compared with the sediment content of the control group 2.
611. Sensory index analysis of finished vinegar
The finished vinegar of the experimental group 2 is reddish and bright in color, clearer in body state and softer and fuller in sour taste, which indicates that the quality of the finished vinegar can be obviously improved by adding the bacillus subtilis in the fermentation stage.
Example 7:
application of bacillus subtilis compost subspecies QH-20010 in Zhenjiang aromatic vinegar brewing
71. Preparation of wine mash
Weighing 935kg of glutinous rice, soaking in water for 1h, grinding, adding water to 3272.5kg while stirring, adding 1kg of alpha-amylase, heating to 90-95 ℃, uniformly stirring for about 30min to obtain mash, cooling the mash to 45-55 ℃, adding lactic acid under slow stirring to adjust the pH of the mash to 4.7, adding 2kg of saccharifying enzyme, keeping the temperature for about 30min, supplementing water until the total mass of the mash is 4800kg, cooling to 33-37 ℃, inoculating 1kg of active dry yeast, 2kg of ammonium sulfate and 0.3kg of acid protease, and standing, culturing and fermenting for 3-4 days to obtain the wine mash.
72. Expanding culture of strain
The fermentation broth was prepared according to the method for preparing the fermentation broth and the seed solution of example 3.
73. Inoculating fermented grains of vinegar and fermenting
Control group 3: 96kg of rice husks are paved on the bottom layer in the fermentation pool, 1600kg of bran is added, 4800kg of the wine mash prepared in the step 71 is inoculated, the sugar mash prepared in the control group in the embodiment 3 is inoculated, the inoculation amount is 5% of the total amount of the feed liquid, after the wine mash is soaked in the new fermented grains, the mixture is turned and stirred uniformly, 100kg of fresh vinegar fermented grains fermented to the 9 th to 11 th days are inoculated on the surface of the fresh vinegar fermented grains, the fresh vinegar fermented grains are uniformly paved on the surface of the new fermented grains, and 72kg of rice husks are uniformly paved on the surface of the new fermented grains.
Experimental group 3: 96kg of rice husks are paved on the bottom layer in the fermentation pool, 1600kg of bran is added, 4800kg of the fermented mash prepared in the step 71 is inoculated, the bacterial liquid (fermentation liquid) prepared in the embodiment 3 is inoculated, the inoculation amount is 5% of the total amount of the feed liquid, after the fermented mash is soaked in the new fermented grains, the fermented grains are turned and stirred uniformly, 100kg of fresh vinegar fermented grains fermented to 9-11 days are inoculated on the surface of the fermented grains, the fresh vinegar fermented grains are uniformly paved on the surface of the new fermented grains, and 72kg of rice husks are uniformly paved on the surface of the new fermented grains.
According to a layered seed expansion mode, under the condition that fermentation culture is turned every day, 72kg of chaff is inoculated every day in the first 7 days, and then solid state fermentation of regular fermentation culture turning every day is carried out until the fermentation is finished.
74. Sealing fermented grains
Compacting the fermented vinegar, covering with a plastic film, sealing with 100kg NaCl for about 3 days.
75. Fermented grain pouring vinegar
Uncovering the plastic film, mixing with NaCl, turning and stirring evenly, and taking the mixture into a vinegar spraying pool for vinegar spraying. Soaking the drenched vinegar with primary vinegar, adding parched rice color 4% of the raw materials, and collecting primary vinegar after soaking. The pouring of vinegar was performed in the same manner as in example 5.
76. Frying vinegar and filling
Sterilizing raw vinegar at high temperature for instant, settling, taking out upper layer vinegar liquid, adding 2% white sugar, decocting at 100 deg.C for about 30min, and packaging when the decoction is finished.
77. Total acid content detection
And (4) taking the vinegar substrate and the marinade at the bottom of the pool every other day from the first day of fermentation to the end of fermentation. The total acid is measured as acetic acid by acid-base titration. After the fermentation is finished, the total fermented grain acid content of the control group 3 is 5.38g/100g (wet fermented grains), the total fermented grain acid content of the experimental group 3 is 5.65g/100g (wet fermented grains), the total marinade acid content of the control group 3 is 7.43g/100mL, and the total marinade acid content of the experimental group 3 is 7.95g/100mL.
78. Detection of non-volatile acid content
After volatile acid is removed by adopting a distillation mode, the content of the non-volatile acid is measured by adopting an acid-base titration mode. After the fermentation is finished, the content of the non-volatile acid in the marinade of the control group 3 is 1.82g/100mL, the content of the non-volatile acid in the marinade of the experimental group 3 is 2.14g/100mL, the proportion of the non-volatile acid is 24.50 percent and 26.92 percent respectively, and compared with the control group 3, the content of the non-volatile acid in the experimental group 3 is improved by 17.58 percent and the proportion of the non-volatile acid in the experimental group 3 is improved by 9.88 percent.
79. Starch content detection
The starch content of the vinegar grains is determined by an enzymolysis method according to GB 5009.9-2016. The starch content of the vinegar grains of the control group 3 and the experimental group 3 is 6.86g/100g and 5.32g/100g respectively on the day of fermentation. The content of the starch in the initial raw material is 14.01g/100g, the utilization rate of the starch is 51.03 percent and 62.03 percent respectively, and the utilization rate of the starch is improved by 19.47 percent.
710. Detection of precipitate content
The sediment content in the finished vinegar is measured by adopting a centrifugal method, the sediment contents of a control group 3 and an experimental group 3 are 492mg/100mL and 385mg/100mL respectively, and the sediment content of the experimental group 3 is reduced by 21.75 percent relative to the sediment content of the control group 3.
711. Sensory index analysis of finished vinegar
The finished vinegar of the experimental group 3 has reddish and bright color, clearer body state and softer sour taste, which shows that the product quality can be obviously improved by adding the bacillus subtilis in the fermentation stage.
Example 8:
application of bacillus subtilis compost subspecies QH-20010 in rice vinegar brewing
81. Expanding culture of strain
The fermentation broth was prepared according to the method for preparing the fermentation broth and the seed solution of example 3.
82. Preparation of wine mash
Weighing 935kg of glutinous rice, soaking in water for 1h, grinding, adding water to 3272.5kg while stirring, adding 1kg of alpha-amylase, heating to 90-95 ℃, uniformly stirring for about 30min to obtain mash, cooling the mash to 45-55 ℃, adding lactic acid under slow stirring to adjust the pH of the mash to 4.7, adding 2kg of saccharifying enzyme, keeping the temperature for about 30min, supplementing water until the total mass of the mash is 4800kg, cooling to 33-37 ℃, inoculating 1kg of active dry yeast, 2kg of ammonium sulfate and 0.3kg of acid protease, inoculating 5 percent of inoculum size into the zymogen liquid prepared in the step 1, ventilating, stirring and culturing (200 rpm) in the first day, standing and culturing for three days to obtain the wine mash of the experimental group 4. Control group 4 was inoculated with active dry yeast alone without the zymogen solution.
83. Acetic acid fermentation
Inoculating activated acetic acid bacteria CGMCC 1.508 with inoculum size of 5%, culturing at 30 deg.C under aeration and stirring at 200rpm and aeration rate of 0.34vvm, and fermenting with acetic acid. The accuracy of the mash to be fermented is lower than 3vol%, the fermentation liquor prepared in the step 81 is inoculated into the experimental group 4 according to the inoculation amount of 5%, and the acetic fermentation is continued. Control group 4 was not inoculated with the fermentation broth.
84. Total acid determination
And (3) measuring the total acid content by adopting an acid-base titration method. After the fermentation is finished, the total acid content in the mash of the experimental group 4 is 11.2g/100mL, and the total acid content in the comparison group 4 is 10.87g/100mL.
85. Sensory evaluation
The sensory evaluation of the experimental group 4 and the control group 4 was conducted, and it was found that the sour taste of the experimental group 4 was softer and had a more prominent sweet taste than the control group 4. Through GC-MS analysis of the flavor substance compositions, the experimental group 4 contains 30mg/L of acetoin and 21.5mg/L of 2, 3-butanediol, but the experimental group 4 does not detect the acetoin and the 2, 3-butanediol. The simultaneous addition of the bacillus subtilis in the alcohol fermentation and acetic acid fermentation stages can obviously improve the product quality.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (9)

1. Bacillus subtilis compost subspecies (B., (B.))Bacillus subtilis subsp. Stercoris) The bacillus subtilis compost subspecies is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation name QH-20010 and the preservation number is as follows: CGMCC NO.21615.
2. A bio-enzyme catalyst comprising a fermentation broth prepared from the bacillus subtilis compost subspecies of claim 1.
3. A microbial fermentation inoculum comprising the bacillus subtilis compost subspecies of claim 1.
4. The use of a Bacillus subtilis subsp compost species as claimed in claim 1 or a biological enzyme catalyst as claimed in claim 2 or a microbial fermentation inoculum as claimed in claim 3 in the food field.
5. Use of a bacillus subtilis compost subspecies as claimed in claim 1 or a bio-enzyme catalyst as claimed in claim 2 or a microbial fermentation inoculum as claimed in claim 3 in vinegar brewing.
6. Use according to claim 5, characterized in that the vinegar is cellar vinegar, bran vinegar, aromatic vinegar or rice vinegar.
7. The application of the compound enzyme as claimed in claim 5, wherein Bacillus subtilis compost subspecies QH-20010 is subjected to fermentation culture to obtain fermentation broth as enzyme, and the enzyme is added into vinegar mash as biological enzyme catalyst to perform catalysis, so as to promote hydrolysis of raw materials and generation of ethanol and acetic acid.
8. The application of the bacillus subtilis preparation of claim 5, wherein the bacillus subtilis compost subspecies QH-20010 is subjected to fermentation culture to obtain a fermentation liquid as a seed liquid, and after the seed liquid is inoculated into the vinegar culture, the bacillus subtilis compost subspecies QH-20010 continues to grow and reproduce in the vinegar culture environment to produce enzyme, so that the hydrolysis of the cereal raw material and the production of ethanol and acetic acid are promoted.
9. The use according to claim 5, wherein the fermentation broth is prepared as follows:
s1, slant culture: inoculating bacillus subtilis compost subspecies QH-20010 to a slant culture medium to obtain slant thalli;
s2, seed culture: the method comprises the following steps of primary seed culture and secondary seed culture:
first-order seed culture: inoculating the slant thallus to a seed culture medium to obtain a primary seed solution;
secondary seed culture: inoculating the primary seed liquid into a secondary seed culture medium by an inoculation amount with the volume concentration of 1-10% to obtain a secondary seed liquid;
s3, fermentation culture: selecting a liquid fermentation tank, adding water, stirring, simultaneously adding rice flour and high-temperature alpha-amylase to obtain mash, cooling the mash to 45-55 ℃, adding saccharifying enzyme under the condition of slow stirring, preserving heat, sterilizing, adding peptone and yeast powder, sterilizing, cooling, inoculating secondary seed liquid, ventilating, stirring and fermenting to obtain fermentation liquor.
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