CN114395513B - Microbial agent for brewing fen-flavor liquor and application thereof - Google Patents

Microbial agent for brewing fen-flavor liquor and application thereof Download PDF

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CN114395513B
CN114395513B CN202210175388.3A CN202210175388A CN114395513B CN 114395513 B CN114395513 B CN 114395513B CN 202210175388 A CN202210175388 A CN 202210175388A CN 114395513 B CN114395513 B CN 114395513B
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吴群
徐岩
曲冠颐
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Jiangnan University
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Abstract

The invention discloses a microbial agent for brewing fen-flavor liquor and application thereof, and belongs to the technical fields of bioengineering and liquor brewing. The invention provides a compound microbial agent, which combines microorganisms for degrading substrate macromolecules, flavor-producing microorganisms and flavor-regulating microorganisms, and can realize the production and regulation of the core flavor substances of the fen-flavor liquor with the least microorganisms by combining the modes. When the microorganisms are compounded, the flavor producing capability of the flavor microorganisms is greatly improved by the flavor regulating microorganisms, and the production of the fen-flavor liquor is realized by the least microorganism species. The microbial inoculum designed by the invention can realize independent production and application without being attached to distiller's yeast, and can improve the stability of fermentation compared with the traditional fermentation inoculation distiller's yeast, and has a promotion effect on the large-scale production of the fen-flavor liquor.

Description

Microbial agent for brewing fen-flavor liquor and application thereof
Technical Field
The invention relates to a microbial agent for brewing fen-flavor liquor and application thereof, belonging to the technical field of bioengineering and liquor brewing.
Background
The white spirit industry is an important component part of the brewing industry, is also one of the pillar industries for national economy development, and is of great importance in development. The fragrant type white spirit comprises Daqu fragrant type white spirit, xiaoqu fragrant type white and dry fragrance type white spirit, and the fragrant pure, mellow, sweet and soft, natural and coordinated, and clean and refreshing aftertaste are widely favored by consumers, so the fragrant type white spirit has great significance in developing fragrant type white spirit production technology.
Volatile flavour substances have a decisive effect on the flavour profile of the white spirit. In the previous report, the main flavor substance of the fen-flavor white spirit is ethyl acetate and ethyl lactate, and the content of other esters is higher; in addition, a small amount of higher alcohol endows the wine body with mellow and full characteristics; the acid substances play a role in balancing and coordinating the aroma substances; the aldehyde ketone phenol substances have great contribution to the floral fragrance. Microorganisms such as yeast and bacteria are related to the production of alcohols, acids and esters in the process of brewing white spirit; meanwhile, mould is generally adopted to degrade macromolecular substrates in the white spirit brewing process so as to supply the growth and metabolism of other microorganisms.
The species of yeast, bacteria, mold and the like are important microorganisms for white spirit fermentation, and although some main fermentation microorganisms such as rhizopus with saccharification, saccharomyces cerevisiae for producing wine, abnormal Wick ham yeast for producing urea with high yield flavor and low yield and the like are discovered, a plurality of unknown microorganisms exist in white spirit production, and the microorganisms have important flavor functions in the fermentation process and also have important regulation effects on the white spirit fermentation. The nature of these microorganisms imparts a beneficial flavor and unique style characteristics to the wine, which must be considered in the construction of the wine fermenting agent.
As is well known, white spirit belongs to a natural fermentation process, and the sources of white spirit fermentation microorganisms are mainly distiller's yeast, and the distiller's yeast is divided into Daqu, xiaoqu, bran koji and the like. Distiller's yeast is mainly enriched for microorganisms by natural processes, and its composition is also largely dependent on environmental microorganisms. Because the adopted raw materials and the manufacturing method are different, the natural conditions of the production area are different, so that the number and the types of microorganisms in the distiller's yeast are different, and the same number and the same types of microorganisms in the distiller's yeast prepared in each batch cannot be ensured, and the large-scale production of the distiller's yeast is difficult to ensure to be safe, high-quality, efficient and stable, therefore, how to find a microbial agent capable of replacing the distiller's yeast becomes a research hot spot and a research difficulty.
Disclosure of Invention
In order to solve the technical problems that the quantity and the types of microorganisms in distiller's yeast are greatly dependent on the environment, so that the fermentation of white spirit is difficult to ensure the safety, high quality, high efficiency, stability and the like of large-scale production in the prior art, the invention provides a compound microbial agent, which contains rhizopus chinensis (Rhizopus chinensis), saccharomyces cerevisiae (Saccharomyces cerevisiae), pichia kudriavzevii (Pichia kudriavzevii), wilkham's yeast (Wickerhamomyces anomalus), lactobacillus acidproof (Lactobacillus acetotolerans), weissella multocida (Weissella parameteroides) and Pediococcus acidilactici (Pediococcus acidilactici). Rhizopus chinensis has saccharification effect, saccharomyces cerevisiae can produce wine, and the microorganisms have excellent flavor substance metabolism activity; meanwhile, the Weissella multocida and the Pediococcus acidilactici have the functions of regulating and controlling the growth of other microorganisms and the metabolism of flavor substances, and are beneficial functional microorganisms.
In one embodiment of the present invention, in the complex microbial inoculant, the rhizopus chinensis: saccharomyces cerevisiae: pichia kudriavzevii: abnormal Weikeham yeast: acid-resistant lactobacillus: weissella multocida: the ratio of the viable count of the pediococcus acidilactici in the composite microbial agent is (1-10): (10-100): (1-100): (1-10): (1-100): (1-10): (1-10).
In one embodiment of the present invention, the total viable count in the composite microbial agent is at least 10 10 CFU/g。
In one embodiment of the invention, the rhizopus chinensis (Rhizopus chinensis) is rhizopus chinensis CGMCC No. 14998.
In one embodiment of the invention, the Saccharomyces cerevisiae (Saccharomyces cerevisiae) is Saccharomyces cerevisiae CGMCC No.8130, saccharomyces cerevisiae CCTCC M2014463 or Saccharomyces cerevisiae CGMCC No.4747.
In one embodiment of the invention, the pichia kudriavzevii (Pichia kudriavzevii) is pichia kudriavzevii CGMCC No.12418 or pichia kudriavzevii CGMCC No.4741.
In one embodiment of the invention, the Wilkinsonii yeast (Wickerhamomyces anomalus) is Wilkinsonii yeast CGMCC No.12416.
In one embodiment of the invention, the acid-resistant lactobacillus (Lactobacillus acetotolerans) is acid-resistant lactobacillus CGMCC No.21135 or acid-resistant lactobacillus CGMCC No.14086.
In one embodiment of the invention, the enteroid Weissella (Weissella parameteroides) is enteroid Weissella CGMCC No.22238; the intestinal membrane-like Weissella CGMCC No.22238 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with the date of 2021,4 and 25, and the preservation address is: no.1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
In one embodiment of the invention, the pediococcus acidilactici (Pediococcus acidilactici) is pediococcus acidilactici CGMCC No.22237; the Pediococcus acidilactici CGMCC No.22237 is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) with the preservation address of 2021,4 and 25: no.1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
In one embodiment of the invention, the composite microbial agent is combined with microorganisms for degrading substrate macromolecules, flavor-producing microorganisms and flavor-regulating microorganisms, and the production and regulation of the core flavor substances of the fen-flavor liquor are realized by combining the mode with the least microorganisms.
In one embodiment of the invention, the degradation of the macromolecular substrate, the provision of other microorganisms for growth, metabolism and flavour production are: rhizopus chinensis; the flavor microorganisms are: saccharomyces cerevisiae, pichia kudriavzevii, wilkinsonii, and Lactobacillus acidophilus; the flavor-controlling microorganisms are: weissella multocida and Pediococcus acidilactici.
In one embodiment of the present invention, the total number of viable yeast in the solid microbial inoculum obtained by the culture method is 10 8 ～10 10 CFU/g, total viable count of lactobacillus reaches 10 8 ～10 9 CFU/g, total spore number of mould reaches 10 8 ～10 10 spores/g。
In one embodiment of the invention, the composite microbial agent is a solid microbial agent, and the solid microbial agent is prepared by the following method:
(1) Respectively preparing rhizopus chinensis microbial solid microbial agent, saccharomyces cerevisiae microbial solid microbial agent, pichia kudriavzevii microbial solid microbial agent, wick-Hanm-anomala microbial solid microbial agent, lactobacillus acidophilus microbial solid microbial agent, intestinal membrane-like Weissella microbial solid microbial agent and Pediococcus acidilactici microbial solid microbial agent;
(2) The prepared rhizopus chinensis microorganism solid microbial agent, saccharomyces cerevisiae microorganism solid microbial agent, pichia kudriavzevii microorganism solid microbial agent, wilker's yeast microorganism solid microbial agent, lactobacillus acidophilus microorganism solid microbial agent, intestinal membrane-like Weissella microorganism solid microbial agent and Pediococcus acidilactici microorganism solid microbial agent are mixed according to the viable count ratio of (1-10): (10-100): (1-100): (1-10): (1-100): (1-10): mixing the components (1-10) in proportion to obtain the composite microbial preparation.
In one embodiment of the invention, the preparation method of each group of microorganism solid-state bacteria comprises the following steps:
(1) The preparation method of the rhizopus chinensis microorganism solid microbial agent comprises the following steps:
inoculating rhizopus chinensis mycelia into a spore culture medium for culture to prepare spores, eluting the spores by using a buffer solution to obtain spore liquid, and inoculating the spore liquid into a solid culture medium for culture to prepare rhizopus chinensis microorganism solid microbial inoculum;
(2) The preparation method of the Saccharomyces cerevisiae microbial solid microbial agent, the Pichia kudriavzevii microbial solid microbial agent and the Wilkham yeast abnormal solid microbial agent comprises the following steps:
respectively inoculating thalli into the liquid culture medium 1 for culture, preparing a culture solution, transferring the culture solution into the liquid culture medium 2 for culture, and preparing a seed solution;
inoculating the prepared seed solution into a solid culture medium for culturing to prepare a microbial agent;
(3) The preparation method of the acid-resistant lactobacillus microorganism solid microbial agent, the intestinal membrane-like Weissella microorganism solid microbial agent and the Pediococcus acidilactici microorganism solid microbial agent comprises the following steps:
respectively inoculating thalli into the liquid culture medium 3 for culture, preparing a culture solution, transferring the culture solution into the liquid culture medium 2 for culture, and preparing a seed solution;
inoculating the prepared seed solution into a solid culture medium for culturing to prepare a microbial agent;
the spore culture medium (g/L): potato powder 200, glucose 20, agar 15-20, natural pH and sterilization treatment;
the liquid culture medium 1 (g/L): yeast powder 10, peptone 20, glucose 20, natural pH and sterilization treatment;
the liquid culture medium 2 is as follows: crushing a sorghum raw material sample, mixing the crushed sorghum raw material sample with water, adding 30-50U/g of high-temperature alpha-amylase, steaming, cooling, adding 10-50U/g of saccharifying enzyme, standing for 2-10 h, filtering, centrifuging the obtained filtrate, adjusting the sugar degree to 6-10 degrees Bx, adjusting the pH to 4-6, and sterilizing;
the liquid culture medium 3 (g/L): beef extract 5, peptone 10, yeast powder 4, mgSO 4 ·7H 2 O0.2, naCl 5, tween 80 1, pH 7.0, and sterilizing;
the solid culture medium is as follows: mixing bran with water, and steaming for 30min;
in one embodiment of the present invention, the method for combining the microbial agents of the above-mentioned types comprises the steps of: bacteria: the total number of the mould is (1-100): (1-100): (1-10), wherein the wine producing yeast: the total number of viable yeast for producing ester is 10: (1-5). And (3) through calculation of the biomass proportion, stirring and mixing the different solid microbial agents according to the mass ratio.
The invention also provides application of the composite microbial agent in white spirit production.
In one embodiment of the invention, the application is that the composite microbial agent is added into brewing raw materials for fermentation to prepare the fen-flavor liquor.
In one embodiment of the invention, the addition amount of the composite microbial agent is 10-20% (w/w) of the mass of the brewing raw material.
In one embodiment of the present invention, the fen-flavor liquor is: daqu fen-flavor liquor, xiaoqu fen-flavor liquor and white spirit.
In one embodiment of the invention, various microbial agents are used as fermentation initiators to be inoculated into cooked grains, and the cooked grains are put into a pool for fermentation to produce core flavor substances of the fen-flavor liquor, so that the fen-flavor liquor has characteristic aroma, and meanwhile, the proportions of substances such as alcohol, acid, ester and the like are generated to accord with the characteristics of the fen-flavor liquor.
Advantageous effects
When the microorganisms are compounded, the flavor producing capability of the flavor microorganisms is greatly improved by the flavor regulating microorganisms, the production of the fen-flavor liquor is realized by using the least microorganism species, and the produced liquor has the typical flavor of the fen-flavor liquor and has good flavor effect. The microbial inoculum designed by the invention can realize independent production and application without being attached to distiller's yeast, and compared with the traditional fermentation inoculation distiller's yeast, the microbial inoculum can improve the stability of fermentation, the total content of fermented and produced wine flavor substances and promote the overall sensory effect. Reduces the dependence of white spirit fermentation on environmental microorganisms, improves the controllability and the repeatability, and has promotion effect on the large-scale production of high-quality fen-flavor white spirit.
Preservation of biological materials
The strain of Weissella multocida is classified and named as Weissella multocida Weissella paramesenteroides, the preservation date is 2021,4 and 25 days, the preservation number is CGMCC No.22238, the preservation unit is China general microbiological culture Collection center, and the preservation address is North Chen West Lu No.1, 3 of the Beijing Korea, and the national academy of sciences of China.
Pediococcus acidilactici is classified and named as Pediococcus acidilactici Pediococcus acidilactici, the preservation date is 2021,4 and 25 days, the preservation number is CGMCC No.22237, the preservation unit is China general microbiological culture Collection center, and the preservation address is North Chen West Lu No.1, 3 of the Korean area of Beijing city, and the institute of microbiology of China academy of sciences.
Drawings
Fig. 1: ethanol yield of fermented grains of different microbial inoculum.
Fig. 2: different microbial inoculum fermented grains have special flavor.
Fig. 3: different microbial agents ferment the flavor structure.
Detailed Description
The Rhizopus chinensis CGMCC No.14998 related in the following examples is described in the Chinese patent application text with the publication number of CN 108441428A; saccharomyces cerevisiae CGMCC No.8130 related in the following examples is described in the text of Chinese patent application with publication number CN 103571763A; pichia kudriavzevii CGMCC No.12418, which is referred to in the following examples, is described in the Chinese patent publication No. CN 105861345A; the abnormal Wikiham yeast CGMCC No.12416 related in the following embodiment is recorded in the Chinese invention patent text with the publication number of CN 105861346A; the acid-resistant lactobacillus CGMCC No.21135 related in the following examples is described in the Chinese patent application text with the publication number of CN 112538450A; the intestinal membrane-like Weissella CGMCC No.22238 related in the following examples is preserved in the China general microbiological culture Collection center (China Committee); pediococcus acidilactici CGMCC No.22237, which is referred to in the following examples, has been preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms).
The detection method involved in the following examples is as follows:
the method for detecting the content of the flavor substances comprises the following steps: adding ultra-pure water into fermented grains, fully and uniformly mixing, and centrifuging to obtain fermented grain leaching liquor. Performing headspace solid-phase microextraction and gas chromatography-mass spectrometry to determine the content of volatile substances in a sample; see paper for specific conditions: peng Wang, qun Wu, xuejian Jiang, zhiqian Wang, jingli Tang, yan xu. Bacillus licheniformis affects the microbial community and metabolic profile in the spontaneous fermentation of Daqu starter for Chinese liquor king. International Journal of Food Microbiology, 2017.
The method for detecting the alpha-amylase activity comprises the following steps: the detection method is referred to the standard GB/T5521-2008.
The detection method of the saccharifying enzyme activity comprises the following steps: the detection method refers to standard GB/T36356-2018.
The detection method of the acid protease activity comprises the following steps: the detection method refers to the standard DB 22/T1819-2013.
Example 1: screening and identification of enterococcus faecalis Weissella and Pediococcus acidilactici
1. Strain screening
Weighing 0.5-5 g of fermented grain sample, dissolving in 20-150 mL of sterile physiological saline, fully vibrating and uniformly mixing for 30 150min, and diluting for 10 5 ～10 7 Coating on MRS solid culture medium, culturing at 37 deg.C for 36-48 hr, and selecting single colony; the single colonies selected were transferred to an acid-producing screening medium, and single colonies were selected in which the medium became significantly yellow around the colonies. And transferring the selected single colony into a basic culture medium 1, culturing for 24-36 h, transferring a basic culture solution into a liquid grain culture medium 2 for fermentation, detecting flavor substances, and selecting strains with excellent flavor characteristics.
The MRS solid state culture medium formula (g/L): beef extract 5, peptone 10, yeast powder 4, mgSO 4 ·7H 2 O0.2, naCl 5, tween 80, pH 7.0, sterilizing for 20min, and pouring the culture medium into a culture dish.
The acid production screening culture medium formula (g/L): yeast powder 2, peptone 2, glucose 20, KH 2 PO 4 0.25，KCl 0.4， CaCl 2 0.1，MgSO 4 0.1，FeCl 3 0.002，MnSO 4 0.002, bromocresol green 0.05, agar 20, pH 5.0, and sterilized for 20min.
2. Identification of strains
Molecular biology identification is carried out on the strains obtained by screening, the 16S rDNA fragments (the 16S rDNA sequences of which are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2) of the microorganisms obtained by screening are amplified by utilizing bacteria classification identification primers, and gel electrophoresis is carried out. The amplified products were sequenced and aligned, and the strains selected above were Pediococcus acidilactici (Pediococcus acidilactici) and Weissella enterica (Weissella paramesenteroides), respectively.
Example 2: preparation of composite microorganism solid microbial inoculum
The method comprises the following specific steps:
1. preparing a culture medium:
spore media (g/L): potato powder 200, glucose 20, agar 15-20, natural pH and sterilization treatment;
liquid Medium 1 (g/L): yeast powder 10, peptone 20, glucose 20, natural pH and sterilization treatment;
the liquid culture medium 2 is: crushing a sorghum raw material sample, mixing the crushed sorghum raw material sample with water, adding 30-50U/g of high-temperature alpha-amylase, steaming and boiling for 1-3 hours, cooling, adding 10-50U/g of saccharifying enzyme, standing for 2-10 hours, filtering, centrifuging the obtained filtrate, adjusting the sugar degree to be 6-12 degrees Bx, adjusting the pH to be 4-6, and sterilizing;
liquid Medium 3 (g/L): beef extract 5, peptone 10, yeast powder 4, mgSO 4 ·7H 2 O0.2, naCl 5, tween 80 1, pH 7.0, and sterilizing;
the solid culture medium is as follows: the bran as raw material is mixed with water and then steamed for 30min at 80-100 ℃.
2. Preparation of solid microbial inoculum of each microorganism
(1) Preparation of rhizopus chinensis CGMCC No.14998 solid microbial inoculum:
inoculating rhizopus ringworm fungus body in spore culture medium, culturing for 48-72 h at 28-32 ℃, eluting spores by buffer solution to obtain spore solution, inoculating the spore solution in fungus solid culture medium, culturing for 24-36 h at 28-32 ℃ to obtain rhizopus ringworm CGMCC No.14998 solid microbial inoculum;
(2) Preparation of Saccharomyces cerevisiae CGMCC No.8130 solid microbial agent, pichia kudriavzevii CGMCC No.12418 solid microbial agent and Wilkameter anomala CGMCC No.12416 solid microbial agent:
inoculating the first-stage fungus body in the liquid basic culture medium 1, activating and culturing for 24 hours at 28-32 ℃, preparing a culture solution, transferring the culture solution into the liquid culture medium 2, and culturing for 16-24 hours at 28-32 ℃ to prepare a seed solution;
inoculating the prepared seed liquid into a solid culture medium, and culturing for 24-36 hours at 28-32 ℃ to respectively obtain a saccharomyces cerevisiae CGMCC No.8130 solid microbial inoculum, a pichia kudriavzevii CGMCC No.12418 solid microbial inoculum and a Wiackham yeast CGMCC No.12416 solid microbial inoculum;
(3) The preparation method of the lactobacillus-resistant CGMCC No.21135 solid microbial inoculum, the intestinal membrane-like Weissella CGMCC No.22238 solid microbial inoculum and the pediococcus acidilactici CGMCC No.22237 solid microbial inoculum comprises the following steps:
inoculating the one-ring bacteria in the liquid culture medium 3, culturing for 24-72 h at 30-37 ℃ to prepare a culture solution, transferring the culture solution into the liquid culture medium 2 for culturing, and culturing for 24-72 h at 30-37 ℃ to prepare a seed solution;
inoculating the seed solution into a solid culture medium, and culturing for 36-48 hours at the temperature of 30-37 ℃ to respectively prepare an acid-resistant lactobacillus CGMCC No.21135 solid microbial inoculum, an intestinal membrane-like Weissella CGMCC No.22238 solid microbial inoculum and a Pediococcus acidilactici CGMCC No.22237 solid microbial inoculum.
3. Performance detection of individual solid state bacterial agents
Experiments were conducted on the solid microbial agents in 50 batches, and in order to ensure that the solid microbial agents produced in each batch are stable and have good effects, the performances of the microbial agents prepared in different batches were detected.
(1) Performance of rhizopus chinensis CGMCC No.14998 solid microbial inoculum:
the spore numbers in the microbial inoculum are respectively detected as follows: 10 8 ～10 10 spores/g。
The degradation capacity of the substrate of the rhizopus chinensis CGMCC No.14998 solid microbial inoculum is detected respectively, the activity of alpha-amylase is measured to be 120-150U/g, the activity of saccharifying enzyme is measured to be 180-210U/g, and the activity of acid proteinase is measured to be 140-170U/g.
(2) Saccharomyces cerevisiae CGMCC No.8130 solid microbial agent, pichia kudriavzevii CGMCC No.12418 solid microbial agent and Wilkham's yeast CGMCC No.12416 solid microbial agent performance
The viable count of the microbial inoculum is respectively detected as follows: 10 8 ～10 10 CFU/g；
The fermentation capacity of the obtained solid microbial inoculum is detected, and the specific steps are as follows: inoculating the solid microbial inoculum into steamed sorghum according to the inoculum size of 1% by mass, and culturing for 72-96 hours at 28-30 ℃ to obtain the following detection results:
after the saccharomyces cerevisiae CGMCC No.8130 solid microbial inoculum is fermented, 33.2-36.8 g/kg of ethanol is produced and 217-240 mg/kg of ethyl acetate is produced;
after the pichia kudriavzevii CGMCC No.12418 solid microbial inoculum is fermented, volatile flavor substances of 2.16-2.84 mg/L are produced, wherein the volatile flavor substances comprise 0.81-1.05 mg/kg of total alcohols (excluding ethanol), 0.46-0.62 mg/kg of total acids (excluding acetic acid and lactic acid), 0.32-0.45 mg/kg of total esters, 0.01-0.02 mg/kg of total aldehydes and ketones and 0.56-0.70 mg/kg of total phenols (semi-quantitative).
After the solid microbial inoculum of the Wilkham yeast CGMCC No.12416 is fermented, 750-900 mg/kg of ethyl acetate is produced;
(3) Lactobacillus-resistant CGMCC No.21135 solid microbial agent, weissella enteroides CGMCC No.22238 solid microbial agent and Pediococcus acidilactici CGMCC No.22237 solid microbial agent
The number of viable bacteria in the microbial inoculum is respectively detected to be 10 8 ～10 9 In the CFU/g range.
The fermentation performance of the obtained solid microbial inoculum is detected, and the specific steps are as follows: inoculating the solid microbial inoculum into steamed sorghum according to the inoculum size of 1% by mass, culturing for 72-96 hours at 30-37 ℃, and detecting the following results:
the lactobacillus-resistant CGMCC No.21135 solid microbial inoculum is fermented to produce 2.67-3.23 mg/kg of total volatile flavor substances, wherein (not counting ethanol) 0.41-0.53 mg/kg of total alcohols substances (not counting lactic acid) 0.70-0.84 mg/kg of total acids substances, 0.50-0.55 mg/kg of total esters substances, 0.03-0.06 mg/kg of total aldehyde ketones substances and 0.63-0.85 mg/kg of total phenols substances (semi-quantitative).
The Weissella multocida CGMCC No.22238 solid microbial inoculum is fermented to produce 2.62-3.04 mg/kg of total volatile flavor substances, wherein (not counting ethanol) 0.46-0.55 mg/kg of total alcohols substances, (not counting lactic acid) 0.56-0.64 mg/kg of total acids substances, 0.28-0.33 mg/kg of total esters substances, 0.03-0.06 mg/kg of total aldehyde ketones substances and 0.48-0.52 mg/kg of total phenols substances (semi-quantitative);
pediococcus acidilactici CGMCC No.22237 solid microbial inoculum is fermented to produce 3-3.6 g/kg of lactic acid and 2.15-2.64 mg/kg of total volatile flavor substances, wherein (without counting ethanol) 0.12-0.35 mg/kg of total alcohols substances, (without counting lactic acid) 0.56-0.64 mg/kg of total acids substances, 0.28-0.33 mg/kg of total esters substances, 0.01-0.02 mg/kg of total aldehyde ketones substances and 0.34-0.41 mg/kg of total phenols substances (semi-quantitative).
The results show that: carrying out experiments on the solid microbial agents in batches of 50, wherein the solid microbial agents of all microorganisms prepared by the method can reach the minimum value of the viable count; the fermentation performance of each microbial agent accords with the range.
Example 3: preparation of composite microorganism solid microbial inoculum
The solid microbial agents of rhizopus chinensis CGMCC No.14998, saccharomyces cerevisiae CGMCC No.8130, pichia kudriavzevii CGMCC No.12418, wilkham yeast CGMCC No.12416, lactobacillus acidophilus CGMCC No.21135, weissella multocida CGMCC No.22238 and Pediococcus acidilactici CGMCC No.22237 prepared in the embodiment 2 are respectively mixed according to the ratio of the viable count of (1-10): (10-100): (1-100): (1-10): (1-100): (1-10): (1-10) mixing to prepare a composite solid microbial inoculum; the method comprises the following specific steps:
the composite solid microbial inoculum 1-4 is prepared respectively, and comprises the following specific components:
(1) Composite solid microbial inoculum 1:
only the solid microbial agent 1 with the microorganism degrading the substrate macromolecules and the flavor microbial agent is added, and the flavor regulating microorganism is not added:
rhizopus chinensis CGMCC No.14998 microbial inoculum, saccharomyces cerevisiae CGMCC No.8130 microbial inoculum, pichia kudriavzevii CGMCC No.12418 microbial inoculum, wilkinsonii yeast CGMCC No.12416 microbial inoculum and lactobacillus acidophilus CGMCC No.21135 microbial inoculum, wherein the viable count is 1:10:10:1:50, wherein the number of viable bacteria in the composite solid microbial inoculum 1 is 1.2 multiplied by 10, and the composite solid microbial inoculum 1 is obtained by detection 10 CFU/g；
(2) Composite solid microbial inoculum 2:
rhizopus chinensis CGMCC No.14998 microbial inoculum, saccharomyces cerevisiae CGMCC No.8130 microbial inoculum, pichia kudriavzevii CGMCC No.12418 microbial inoculum, wilkinsonii yeast CGMCC No.12416 microbial inoculum, lactobacillus acidophilus CGMCC No.21135 microbial inoculum, weissella enterica CGMCC No.22238 microbial inoculum and Pediococcus acidilactici CGMCC No.22237 microbial inoculum, wherein the viable count is 1:10:10:1:50:1:1 to prepare a composite solid microbial inoculum 2, wherein the number of viable bacteria in the composite solid microbial inoculum 2 is 1.6X10 after detection 10 CFU/g；
(3) Composite solid microbial inoculum 3:
rhizopus chinensis CGMCC No.14998 microbial inoculum, saccharomyces cerevisiae CGMCC No.8130 microbial inoculum, pichia kudriavzevii CGMCC No.12418 microbial inoculum, wilkinsonii yeast CGMCC No.12416 microbial inoculum, lactobacillus acidophilus CGMCC No.21135 microbial inoculum, weissella enterica CGMCC No.22238 microbial inoculum and Pediococcus acidilactici CGMCC No.22237 microbial inoculum, wherein the viable count is 1:20:10:5:50:5:5, and preparing solid microbial inoculum 3, wherein the number of viable bacteria in the composite solid microbial inoculum 3 is 1.8x10 after detection 10 CFU/g；
(4) Composite solid microbial inoculum 4:
rhizopus chinensis CGMCC No.14998 microbial inoculum, saccharomyces cerevisiae CGMCC No.8130 microbial inoculum and Curie AzweiPichia pastoris CGMCC No.12418 microbial inoculum, wickham's yeast CGMCC No.12416 microbial inoculum, lactobacillus acidophilus CGMCC No.21135 microbial inoculum, weissella enterokinesis CGMCC No.22238 microbial inoculum and Pediococcus acidilactici CGMCC No.22237 microbial inoculum, wherein the viable count is 5:50:30:5:30:5:5, and preparing the solid microbial inoculum 4, wherein the number of viable bacteria in the composite solid microbial inoculum 4 is 2 multiplied by 10 after detection 10 CFU/g。
(5) Composite solid microbial inoculum 5:
rhizopus chinensis CGMCC No.14998 microbial inoculum, saccharomyces cerevisiae CGMCC No.8130 microbial inoculum, pichia kudriavzevii CGMCC No.12418 microbial inoculum, wilkinsonii yeast CGMCC No.12416 microbial inoculum, lactobacillus acidophilus CGMCC No.21135 microbial inoculum, weissella enterica CGMCC No.22238 microbial inoculum and Pediococcus acidilactici CGMCC No.22237 microbial inoculum, wherein the viable count is 5:50:50:5:50:5:5, wherein the number of viable bacteria in the composite solid microbial inoculum 5 is 2.5X10 after detection 10 CFU/g。
(6) Composite solid microbial inoculum 6:
rhizopus chinensis CGMCC No.14998 microbial inoculum, saccharomyces cerevisiae CGMCC No.8130 microbial inoculum, pichia kudriavzevii CGMCC No.12418 microbial inoculum, wilkinsonii yeast CGMCC No.12416 microbial inoculum, lactobacillus acidophilus CGMCC No.21135 microbial inoculum, weissella enterica CGMCC No.22238 microbial inoculum and Pediococcus acidilactici CGMCC No.22237 microbial inoculum, wherein the viable count is 1:10:1:1:1:1:1 to prepare a solid microbial inoculum 6, wherein the number of viable bacteria in the composite solid microbial inoculum 6 is 1.2 multiplied by 10 after detection 10 CFU/g。
(7) Composite solid microbial inoculum 7:
rhizopus chinensis CGMCC No.14998 microbial inoculum, saccharomyces cerevisiae CGMCC No.8130 microbial inoculum, pichia kudriavzevii CGMCC No.12418 microbial inoculum, wilkinsonii yeast CGMCC No.12416 microbial inoculum, lactobacillus acidophilus CGMCC No.21135 microbial inoculum, weissella enterica CGMCC No.22238 microbial inoculum and Pediococcus acidilactici CGMCC No.22237 microbial inoculum, wherein the viable count is 3:25:25:3:25:3:3 to prepare a solid microbial inoculum 7, wherein the number of viable bacteria in the composite solid microbial inoculum 7 is detected1.8X10 10 CFU/g。
(8) Composite solid microbial inoculum 8:
rhizopus chinensis CGMCC No.14998 microbial inoculum, saccharomyces cerevisiae CGMCC No.8130 microbial inoculum, pichia kudriavzevii CGMCC No.12418 microbial inoculum, wilkinsonii yeast CGMCC No.12416 microbial inoculum, lactobacillus acidophilus CGMCC No.21135 microbial inoculum, weissella enterica CGMCC No.22238 microbial inoculum and Pediococcus acidilactici CGMCC No.22237 microbial inoculum, wherein the viable count is 5:30:25:5:40:5:5, and preparing the solid microbial inoculum 7, wherein the number of viable bacteria in the composite solid microbial inoculum 7 is 2 multiplied by 10 after detection 10 CFU/g。
Example 4: preparation of fen-flavor liquor by adopting solid composite microbial inoculum
The method comprises the following specific steps:
(1) Daqu fen-flavor liquor:
the composite solid microbial agents 2 to 8 prepared in the example 3 are respectively mixed into grains which are steamed and spread to cool by the grits, and are evenly mixed for fermentation. Wherein the solid bacteria dosage is 10-20% (by mass) of the feeding amount, and the solid bacteria is put into a jar for fermentation. Steaming and burning to obtain Daqu fen-flavor liquor 2-8 (specific Daqu production process is described in paper: tian Deyu, ziru, cristalline crystal, guan Junfeng, zhao Guoqun, liu Jinlong. Research progress of fen-flavor liquor brewing microorganism and flavor substance [ J ]. Chinese brewing, 2021,40 (04): 20-25.).
(2) Xiaoqu fen-flavor liquor:
the composite solid microbial inoculum 2 to 8 prepared in the example 3 are respectively mixed into the grains which are steamed and spread to cool, and are piled and saccharified for 1d, and then are evenly mixed and distilled into a tank car to ferment for 14d. Wherein the solid bacterial dosage is 1 percent (by mass) of the feeding amount. Steamed and burned to obtain the Xiaoqu fen-flavor liquor 2-8 (specific Xiaoqu production process is described in papers: qu Guanyi, tang Jie, jiang Jian, yang Jiang, liu Yuancai, wu Qun, chen Shenxi and Xu Yan. Microbial flora synthesis of higher alcohol metabolism characteristics [ J ] in the process of fermenting Xiaoqu fen-flavor liquor, food and fermentation industry, 2021,47 (11): 32-37.).
(3) White spirit with white spirit and dry fragrance
The grains after being steamed and spread for cooling are mixed, and the grains are evenly mixed for re-steaming and spread for cooling. The composite solid microbial agents 2 to 8 prepared in example 3 were mixed with a solid microbial agent amount of 25% by mass of the amount to be fed, and put into a jar for fermentation for 40d. Steaming and mixing, and preparing the white spirit with the dry flavor of 2-8 (the production process of the white spirit with the dry flavor of the white spirit is described in the paper: li Zexia, liu Ningning, zhang Fuyan. Formula optimization of white spirit with dry fragrance of Duoliang Laobai [ J ]. Brewing, 2021,48 (03): 23-25 ].
Comparative example 1:
the specific implementation steps are the same as those of example 4, except that the production distiller's yeast is adopted to replace the compound microbial agent, and the Daqu fen-flavor liquor, xiaoqu fen-flavor liquor and Laobai fen-flavor liquor are prepared according to the corresponding processes respectively.
The distiller's yeast is taken from a winery manufacturing plant. The yeast for producing the Daqu is prepared by crushing barley and pea, soaking in water, treading, compacting into cuboid, stacking in a yeast room, enriching microorganisms in the environment, periodically performing yeast turning operation, distinguishing the quality of distiller's yeast after stacking for at least 3 months, respectively crushing red heart yeast, stubble cleaning yeast and back fire yeast, and mixing according to a certain proportion to obtain the yeast for producing. The yeast for producing small yeast is prepared by soaking rice flour in water, adding mother yeast, pressing into cake, fermenting in yeast room for 10-15 days, and pulverizing to obtain yeast for producing small yeast. The yeast for producing white spirit is produced with wheat and through crushing, adding water, treading, pressing into blocks, piling in a yeast house for 45-60 days, crushing and mixing.
Comparative example 2:
the specific implementation steps are the same as those of example 4, except that the solid microbial inoculum 1 added with only flavor microorganisms is adopted, and the Daqu fen-flavor liquor, xiaoqu fen-flavor liquor and Laobai-fudge-flavor liquor are prepared according to the corresponding processes respectively.
Example 5: content of flavor substances in different fen-flavor distilled spirit
The volatile flavor substances of the fen-flavor liquor prepared in the example 4 and the comparative examples 1 and 2 are detected by using gas chromatography-mass spectrometry, the spectrograms are analyzed, the detected substance types of the composite solid microbial inoculum 2-8 in the production of different fen-flavor liquor are shown in the table 1, and meanwhile, the names and the content ranges of typical aroma substances common to the fen-flavor liquor are listed in the attached table 2.
TABLE 1 flavor species detected by solid state microbial agent protocol
TABLE 2 Table of important volatile flavors
The results show that the composite solid microbial inoculum 2-8 is used for producing Daqu fen-flavor liquor, xiaoqu fen-flavor liquor and bran fen-flavor liquor, the average ethanol yield of the composite solid microbial inoculum 2-8 in different types of fen-flavor liquor and the average ethanol yields of the comparative examples 1 and 2 are shown in figure 1, and the solid microbial inoculum can achieve the ethanol yield in the production of the representative distiller's yeast of fen-flavor liquor.
The total amounts of the flavor substances produced on average by the composite solid-state bacterial agents 2 to 8 and comparative examples 1 and 2 are shown in FIG. 2. The flavor microorganisms and the flavor regulating microorganisms are combined to promote the types and the yields of the flavor substances, the total amount of the volatile flavor substances produced by the multi-flavor regulating microorganism combination is higher, and the flavor producing effect is better. The flavor regulating microorganism is added on the basis of the flavor microorganism, so that the flavor producing effect is more similar to that of distiller's yeast fermentation, and the flavor regulating microorganism has good effect. After the flavor regulating microorganism is added, the flavor generation variety and the total amount of the microbial inoculum are improved, and most of the added flavor substances cannot be generated by single culture of the flavor regulating microorganism, so that the flavor regulating microorganism has the capability of promoting the flavor substances generation of the flavor microorganisms, the importance of the flavor regulating microorganism is shown, and further, the rationality of the solid microbial inoculum composed of the flavor microorganisms and the flavor regulating microorganism is demonstrated, so that the microbial inoculum can better realize the production of the fen-flavor white spirit.
The formula of the composite solid microbial agents 2-8 is used for producing the white spirit with different faint scent types, the average content structures of all the flavor substances of the composite solid microbial agents 2-8 and the comparison examples 1 and 2 are shown in figure 3, and the composition structure of all the substances is basically similar to the group of distiller's yeast fermentation by the combination of flavor microorganisms and flavor control microorganisms, so that the effect of better realizing the flavor of the faint scent type white spirit by utilizing the combined solid microbial agents for fermentation is shown. Similarly, a similar good effect can be achieved by combining the microorganism ratios in the range described in the formulation.
The solid microbial inoculum prepared according to the formula is applied to brewing production, has good effects on ethanol yield, flavor structure and flavor yield, and performs sensory evaluation on white spirit produced by the microbial inoculum, and the results are shown in the following table 3.
TABLE 3 average sensory evaluation of wine produced in solid inoculant brewing applications
Note that: sensory evaluation criteria: stronger 4-5 minutes; typically 2-3 minutes; weaker 1 point; no 0 point
The composite solid microbial inoculum 2-8 prepared by the invention covers different proportions of flavor microorganisms and flavor regulating microorganisms, and illustrates the important effect of the flavor regulating microorganisms on the flavor of the fen-flavor liquor. Meanwhile, the combination mode is applied to the production of the fen-flavor liquor with different processes, and the effect of the liquor production can be achieved.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of Jiangnan
<120> microbial agent for brewing fen-flavor liquor and application thereof
<130> BAA210289A
<160> 2
<170> PatentIn version 3.3
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Claims (9)

1. A composite microbial agent is characterized by comprising rhizopus chinensisRhizopus chinensis) Saccharomyces cerevisiaeSaccharomyces cerevisiae) Pichia kudriavzeviiPichia kudriavzevii) Abnormal Wick ham yeastWickerhamomyces anomalus) Acid-resistant lactobacillusLactobacillus acetotolerans) Weissella multocida @ intestinal membraneWeissella parameteroides) Pediococcus acidilacticiPediococcus acidilactici) Rhizopus chinensis: saccharomyces cerevisiae: pichia kudriavzevii: abnormal Weikeham yeast: acid-resistant lactobacillus: weissella multocida: the ratio of the viable count of the pediococcus acidilactici is (1-10): (10-100): (1-100): (1-10): (1-100): (1-10): (1-10),
the rhizopus chinensis is rhizopus chinensis CGMCC No.14998; the Saccharomyces cerevisiae is Saccharomyces cerevisiae CGMCC No.8130; the Pichia kudriavzevii is Pichia kudriavzevii CGMCC No.12418; the Wikiwim abnormal yeast is Wikiwim abnormal yeast CGMCC No.12416; the acid-resistant lactobacillus is acid-resistant lactobacillus CGMCC No.21135; the intestinal membrane-like Weissella is intestinal membrane-like Weissella CGMCC No.22238; the pediococcus acidilactici is pediococcus acidilactici CGMCC No.22237;
pediococcus acidilactici CGMCC No.22237 is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) with the year 4 and the month 25 of 2021; the intestinal membrane-like Weissella CGMCC No.22238 is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) with the year 2021, month 4 and day 25.
2. The composite microbial agent according to claim 1, wherein the total viable count in the composite microbial agent is at least 10 10 CFU/g。
3. The composite microbial agent according to claim 2, wherein the viable cell concentration of each microorganism in the composite microbial agent is 10 8 ~10 10 CFU/g。
4. The composite microbial agent of claim 3, wherein the composite microbial agent is a solid microbial agent, and the solid microbial agent is prepared by the following steps:
(1) Respectively preparing rhizopus chinensis microbial solid microbial agent, saccharomyces cerevisiae microbial solid microbial agent, pichia kudriavzevii microbial solid microbial agent, wick-Hanm-anomala microbial solid microbial agent, lactobacillus acidophilus microbial solid microbial agent, intestinal membrane-like Weissella microbial solid microbial agent and Pediococcus acidilactici microbial solid microbial agent;
(2) The prepared rhizopus chinensis microbial solid microbial agent, saccharomyces cerevisiae microbial solid microbial agent, pichia kudriavzevii microbial solid microbial agent, wilker's yeast microbial solid microbial agent, lactobacillus acidophilus microbial solid microbial agent, intestinal membrane-like Weissella microbial solid microbial agent and Pediococcus acidilactici microbial solid microbial agent are prepared according to the viable count ratio of (1-10): (10-100): (1-100): (1-10): (1-100): (1-10): and (3) mixing the components in a ratio of (1-10) to prepare the composite microbial preparation.
5. The compound microbial agent according to claim 4, wherein the step (2) is that the prepared rhizopus chinensis microbial solid agent, saccharomyces cerevisiae microbial solid agent, pichia kudriavzevii microbial solid agent, wechat-resistant lactobacillus microbial agent, weissella multocida microbial solid agent and Pediococcus acidilactici microbial solid agent are mixed according to a viable count ratio of 1:10:1:1:1:1:1, and mixing the components according to the proportion to obtain the composite microbial preparation.
6. The composite microbial agent of claim 5, wherein the preparation method of each group of microbial solid state agents is as follows:
(1) The preparation method of the rhizopus chinensis microorganism solid microbial agent comprises the following steps:
inoculating rhizopus chinensis mycelia into a spore culture medium for culture to prepare spores, eluting the spores by using a buffer solution to obtain spore liquid, and inoculating the spore liquid into a solid culture medium for culture to prepare rhizopus chinensis microorganism solid microbial inoculum;
(2) The preparation method of the Saccharomyces cerevisiae microbial solid microbial agent, the Pichia kudriavzevii microbial solid microbial agent and the Wilkham yeast abnormal solid microbial agent comprises the following steps:
respectively inoculating thalli into the liquid culture medium 1 for culture to prepare a primary culture solution, transferring the primary culture solution into the liquid culture medium 2 for culture to prepare a secondary seed solution;
inoculating the prepared secondary seed solution into a solid culture medium for culturing to prepare a microbial agent;
(3) The preparation method of the acid-resistant lactobacillus microorganism solid microbial agent, the intestinal membrane-like Weissella microorganism solid microbial agent and the Pediococcus acidilactici microorganism solid microbial agent comprises the following steps:
respectively inoculating thalli into the liquid culture medium 3 for culture to prepare a primary culture solution, transferring the primary culture solution into the liquid culture medium 2 for culture to prepare a secondary seed solution;
inoculating the prepared secondary seed solution into a solid culture medium for culturing to prepare a microbial agent;
the spore culture medium (g/L): potato powder 200, glucose 20, agar 15-20, natural pH and sterilization treatment;
the liquid culture medium 1 (g/L): yeast powder 10, peptone 20, glucose 20, natural pH and sterilization treatment;
the liquid culture medium 2 is as follows: crushing a sorghum raw material sample, mixing the crushed sorghum raw material sample with water, adding 30-50U/g of high-temperature alpha-amylase raw material, steaming for 1-3 hours, cooling, adding 10-50U/g of saccharifying enzyme raw material, standing for 2-10 hours, filtering, centrifuging to obtain filtrate, and adjusting the sugar degree to 6-12 o Bx, adjusting the pH to 4-6;
the liquid culture medium 3 (g/L): beef extract 5, peptone 10, yeast powder 4, mgSO 4 ·7H 2 O0.2, naCl 5, tween 80 1, pH 7.0, and sterilizing;
the solid culture medium is as follows: mixing bran with water, and steaming for 30min.
7. The application of the composite microbial agent in white spirit production according to any one of claims 1-6.
8. The use according to claim 7, wherein the white spirit is prepared by adding the composite microbial agent to a brewing material or substrate for fermentation.
9. The method of claim 8, wherein the composite microbial agent is added in an amount of 10-20% of the mass of the brewing raw material.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102766576A (en) * 2012-07-19 2012-11-07 江南大学 Brewing function oriented microbe combination method and application of combination bacterium in liquor-making industry
CN103571763A (en) * 2013-11-20 2014-02-12 江南大学 Saccharomyces cerevisiae capable of auto-synthesizing terpenoid substances and applications thereof
CN105861345A (en) * 2016-06-14 2016-08-17 江南大学 Pichia kudriavzevii yeast low in urea yield and capable of producing flavor and application thereof to food fermentation
CN105861346A (en) * 2016-06-14 2016-08-17 江南大学 Wickerhamomyces anomalus strain low in urea yield and capable of producing flavor and application thereof to food production
CN108441428A (en) * 2018-03-22 2018-08-24 江南大学 One plant degradation alcohol soluble protein rhizopus chinensis and its application
CN112538450A (en) * 2020-12-29 2021-03-23 江南大学 Application of high-yield flavor acid-resistant lactobacillus in food production

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102766576A (en) * 2012-07-19 2012-11-07 江南大学 Brewing function oriented microbe combination method and application of combination bacterium in liquor-making industry
CN103571763A (en) * 2013-11-20 2014-02-12 江南大学 Saccharomyces cerevisiae capable of auto-synthesizing terpenoid substances and applications thereof
CN105861345A (en) * 2016-06-14 2016-08-17 江南大学 Pichia kudriavzevii yeast low in urea yield and capable of producing flavor and application thereof to food fermentation
CN105861346A (en) * 2016-06-14 2016-08-17 江南大学 Wickerhamomyces anomalus strain low in urea yield and capable of producing flavor and application thereof to food production
CN108441428A (en) * 2018-03-22 2018-08-24 江南大学 One plant degradation alcohol soluble protein rhizopus chinensis and its application
CN112538450A (en) * 2020-12-29 2021-03-23 江南大学 Application of high-yield flavor acid-resistant lactobacillus in food production

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