CN117004532A - Preparation method of high-temperature Daqu seed microbial inoculum - Google Patents

Preparation method of high-temperature Daqu seed microbial inoculum Download PDF

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CN117004532A
CN117004532A CN202311024708.6A CN202311024708A CN117004532A CN 117004532 A CN117004532 A CN 117004532A CN 202311024708 A CN202311024708 A CN 202311024708A CN 117004532 A CN117004532 A CN 117004532A
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parts
temperature
bacillus
bacterial
seed
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张楷正
侯耀川
张家彬
刘青松
卢昱洁
李红梅
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Sichuan University of Science and Engineering
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Sichuan University of Science and Engineering
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Abstract

The application provides a preparation method of a high-temperature Daqu seed microbial inoculum, and relates to the technical fields of bioengineering and brewing. The preparation method of the seed bacterial agent is a lyophilization method, and the preparation at least comprises the following steps: preparing a microbial bacterial liquid, wherein the microbial bacterial liquid comprises 21 high-temperature Daqu functional microorganisms, and centrifugally concentrating the seed bacterial liquid into a high-concentration bacterial liquid; crushing and sieving bran, rice husk and corncob, mixing according to a certain proportion to obtain a microbial carrier, and mixing with concentrated bacterial liquid; the added protective agent comprises the following components in parts by weight: 5-8 parts of trehalose, 4-7 parts of mannitol, 6-9 parts of sodium thiosulfate and 8-10 parts of skimmed milk powder. According to the application, the liquid nitrogen is added after freezing at the temperature of minus 20 ℃ to perform adaptive vitrification prefreezing on the thalli, so that the microbial inoculum is more stable, and the survival rate of the thalli is greatly improved. The freeze-dried microbial inoculum can be inoculated with bran as a mother yeast to participate in the production of high-temperature Daqu, so that the dependence on external microbial sources is greatly reduced.

Description

Preparation method of high-temperature Daqu seed microbial inoculum
Technical Field
The application relates to the technical field of bioengineering and wine brewing, in particular to a preparation method of a high-temperature Daqu seed microbial inoculum.
Background
The Maotai-flavor type Chinese liquor is one of four large-flavor types, the high-temperature Daqu is an important saccharification starter and a fragrance generator of the Maotai-flavor type Chinese liquor, and the quality of the high-temperature Daqu has important influence on the liquor yield and the quality of the liquor.
The existing production of traditional high-temperature Daqu, namely open production, mainly has the following problems: (1) the open production mode leads Daqu to be easy to be contaminated with miscellaneous bacteria and to generate mildew (2) the mass of Daqu is uneven (3) the aged yeast stacked strains are passaged more so as to cause degradation, the fermentation capacity is reduced (4) the yeast flies are scattered in disorder, and the working environment is high in humidity and climax.
Freeze-drying is one of the most convenient and successful methods for preserving bacteria, yeasts and fungal spores. The freeze drying has the advantages of being not easy to be polluted in storage, keeping the bacterial activity after long-term storage, and being easy to be packaged, transported and rehydrated.
Inoculating the seed microbial inoculum as a primary microbial inoculum onto bran, and directly taking the bran koji as a secondary microbial inoculum into the preparation of the high-temperature Daqu by using the mother koji. The bran contains a carbon source, a nitrogen source, a small amount of vitamins, minerals and other trace components for microorganism growth, and also contains a large amount of lignin, so that the bran is a basic substance for producing ferulic acid, vanillic acid and coumaric acid. Furthermore, the bran also contains a certain amount of polyphenol compounds such as pyrazines, furans, tannins and the like, and the substances can generate, synthesize or convert into high-boiling aromatic compounds in the Maotai-flavor wine in the stacking and fermenting processes. The bran is not only a carrier of the seed microbial inoculum, but also a good raw material for brewing wine.
Disclosure of Invention
The application aims to provide a preparation method of a high-temperature Daqu seed microbial inoculum, which aims to solve the technical problems existing in a high-temperature Daqu starter making environment in the prior art and improve aroma components of white spirit. The preferred technical solutions of the technical solutions provided by the present application can produce a plurality of technical effects described below.
In order to achieve the above object, the technical scheme of the present application for solving the above technical problems is as follows:
the preparation method of the high-temperature Daqu seed microbial inoculum at least comprises the following steps:
(1) Respectively inoculating the core functional microorganism bacterial suspension in a plurality of high-temperature Daqus into corresponding liquid culture mediums for constant-temperature culture;
(2) Pulverizing testa Tritici, testa oryzae, and corncob, sieving, mixing with water at a certain ratio to obtain microorganism carrier, sterilizing, and cooling to obtain seed bacterial agent carrier;
(3) Taking and sterilizing the protective agent according to the weight parts of the components for later use;
(4) Centrifuging the liquid culture medium, discarding the supernatant, respectively adding sterile physiological saline to prepare concentrated bacterial suspension, respectively adding the protective agent and the carrier, uniformly mixing, and culturing at constant temperature;
(5) Placing the bacterial suspension cultured in the step (4) into a refrigerator at the temperature of minus 20 ℃, and then, putting the bacterial suspension into liquid nitrogen for prefreezing;
(6) Transferring the pre-frozen bacterial suspension in the step (5) to a freeze dryer, and vacuum drying to obtain the seed bacterial agent freeze-dried powder.
Further, in a preferred embodiment of the present application, in the step (1), the liquid medium is prepared by the following method:
taking 2-6 parts by weight of beef extract, 6-18 parts by weight of peptone, 3.5-10 parts by weight of NaCl and 1000 parts by weight of distilled water, mixing, and sterilizing for 15-25min at the pH of 7.2-7.4 and the temperature of 110-130 ℃ to prepare a beef extract peptone liquid culture medium; taking 8-10 parts by weight of yeast extract powder, 15-20 parts by weight of peptone, 18-25 parts by weight of glucose and 1000 parts by weight of distilled water, mixing, and sterilizing for 15-25 minutes at the pH of 7.2-7.4 and the temperature of 110-130 ℃ to prepare a yeast extract powder peptone glucose liquid culture medium; according to the weight portions, 4 to 8 portions of potato soaked powder, 18 to 25 portions of glucose, 0.1 to 0.3 portion of chloramphenicol and 1000 portions of distilled water are mixed, and then sterilized for 15 to 25 minutes under the conditions of pH of 7.2 to 7.4 and 110 to 130 ℃ to prepare the potato glucose liquid culture medium.
Preferably, 5 parts of beef extract, 10 parts of peptone, 10 parts of NaCl and 1000 parts of distilled water are mixed according to parts by weight, and then sterilized for 20 minutes under the conditions of pH7.2 and 110 ℃ to prepare a beef extract peptone liquid culture medium; taking 10 parts by weight of yeast extract powder, 20 parts by weight of peptone, 20 parts by weight of glucose and 1000 parts by weight of distilled water, mixing, and sterilizing at the pH of 7.2 and 110 ℃ for 20min to obtain a yeast extract powder peptone glucose liquid culture medium; the potato dextrose liquid culture medium is prepared by mixing 6 parts by weight of potato soaked powder, 20 parts by weight of dextrose, 0.1 part by weight of chloramphenicol and 1000 parts by weight of distilled water, and sterilizing at the pH of 7.2 and 110 ℃ for 20 minutes.
Preferably, taking 6 parts by weight of beef extract, 15 parts by weight of peptone, 8 parts by weight of NaCl and 1000 parts by weight of distilled water, mixing, and sterilizing for 15min at the pH of 7.2 and the temperature of 110 ℃ to prepare a beef extract peptone liquid culture medium; taking 8 parts by weight of yeast extract powder, 20 parts by weight of peptone, 25 parts by weight of glucose and 1000 parts by weight of distilled water, mixing, and sterilizing for 15min at the pH of 7.2 and the temperature of 110 ℃ to prepare a yeast extract powder peptone glucose liquid culture medium; the potato dextrose liquid culture medium is prepared by mixing 8 parts of potato soaked powder, 25 parts of dextrose, 0.1 part of chloramphenicol and 1000 parts of distilled water according to weight parts, and then sterilizing for 15min at the pH of 7.2 and the temperature of 110 ℃.
Further, in a preferred embodiment of the present application, in the step (1), the culture conditions are as follows: culturing at 36-38deg.C under shaking table of 130-160r/min for 14-18 hr.
Preferably, the above culture conditions are that the culture is carried out at 36 ℃ and 160r/min in a shaking table for 18 hours; alternatively, culturing at 38deg.C under a shaking table of 130r/min for 14 hr; alternatively, the culture was carried out at 37℃for 16 hours in a shaking table of 150 r/min.
Further, the freeze-dried carrier in the step (2) is bran, rice husk and corncob, and the weight parts of the freeze-dried carrier are 45-60 parts of bran, 10 parts of rice husk and 30-45 parts of corncob.
Preferably, the compounding proportion is 60 parts of bran, 10 parts of rice husk and 30 parts of corncob;
preferably, the compounding proportion is 50 parts of bran, 10 parts of rice husk and 40 parts of corncob;
preferably, the compounding ratio is 45 parts of bran, 10 parts of rice husk and 45 parts of corncob.
Further, the protective agent in the step (3) is trehalose, mannitol, sodium thiosulfate and mannitol, and the added amount of the trehalose is 5-7 parts, the added amount of the mannitol is 3-5 parts, the added amount of the sodium thiosulfate is 6-8 parts and the added amount of the skim milk powder is 8-10 parts by weight.
Preferably, the vacuum drying conditions in step (6) are: drying pressure is 10-45 pa, partition temperature is 40-10 ℃, cold trap temperature is 212-76 ℃ and drying time is 45-120min.
According to a preferred embodiment, the core-function microorganism comprises a filamentous fungus, a yeast and a bacterium, and the filamentous fungus comprises aspergillus oryzae CPCC460006, xie Washi aspergillus CBS522.65, rhizopus oryzae CBS112.07, mucor circinelloides FCBS195.68; the yeasts include the sacculus tectorial membrane yeasts NRRLY-2388 and Pichia anomala NRRL Y-5396; the bacteria include bacillus licheniformis JF802183.1, bacillus subtilis JF783990.1, bacillus amyloliquefaciens DQ658169.1, bacillus megaterium ATCC 14581 (T), bacillus cereus ATCC 14579 (T), bacillus albopictus B8W22 (T), bacillus methylotrophicus KCTC 13105 (T), staphylococcus saprophyticus, lactobacillus bread LMG23699 (T), weissella antrum KACC 11862 (T), lactobacillus pentosus JCM 1558 (T), bacillus acetate NBRC 14818 (T), bacillus cereus LMG 1625 (T) and pseudomonas CIP 104663 (T).
According to a preferred embodiment, the filamentous fungus of the core-functional microbial flora: yeast: the initial live bacteria ratio of the bacteria is 4:1:5.
according to a preferred embodiment, the content of each filamentous fungus in the core-functional microbial flora is (10 8 -10 9 )CFU/g；
The content of each yeast in the core functional microbial flora was (10 8 -10 9 )CFU/g；
The content of each bacterium in the core functional microbial flora was (10 8 -10 9 )CFU/g。
The high-temperature Daqu seed microbial inoculum prepared by the preparation process is prepared.
The high-temperature Daqu seed microbial inoculum disclosed by the application is prepared before refrigeration: water content of 8-12%, pH of 6.8, average viable count of 7×10 9 cfu/g. Under the condition of no air leakage and bag expansion, the quality guarantee period of the seed bacterial agent can reach 4 years.
The specific process of the high-temperature Daqu seed microbial inoculum applied in starter propagation is as follows:
the preparation of the secondary microbial inoculum adopts bran as a culture carrier, a 1000mL triangular flask is selected, 250g of bran and 60% of water are added, pH7.2-7.4 is regulated, high-pressure steam sterilization is carried out for 30min at 121 ℃, fungi, yeast and bacteria are respectively added for inoculation after cooling to 50 ℃, and the adding mode is that the powder freeze-dried microbial inoculum is added, and the adding amount is 6%.
Further, after the addition was completed, the mixture was shaken well and placed in a constant temperature incubator at 35℃for cultivation. The triangular flask was taken out of the incubator every 48 hours and gently shaken to maintain the bulk of the bran in the flask. Observing the color of the secondary microbial inoculum, and obtaining a finished microbial inoculum: the inoculated bacteria are yellow brown, the inoculated yeast has obvious wine fragrance, and a large number of hyphae appear in the triangular flask inoculated with the fungi.
Further, the secondary microbial inoculum is dried at 50 ℃ to obtain the moisture content of 4-6%, and then vacuum sealing is carried out.
In the secondary microbial inoculum, the bacterial biomass is (4-7) multiplied by 10 9 cfu/g, yeast biomass (1-3). Times.10 9 cfu/g, fungal biomass of (3-5). Times.10 9 cfu/g。
The secondary microbial inoculum is used as the mother starter to prepare the high-temperature Daqu, and the starter charge is 5-7%.
Based on the technical scheme, the preparation method of the high-temperature seed bacterial agent has at least the following technical effects:
after the preparation of the high-temperature seed microbial inoculum is finished, the high-temperature seed microbial inoculum is vacuum packed and put into a refrigerator with the temperature of 5 ℃ for refrigeration, so that the microbial performance can be effectively maintained, and the influence of strain degradation on fermentation is reduced. The complex microbial flora comprises a core functional microbial strain required in the corresponding high temperature Daqu. Therefore, the secondary microbial inoculum inoculated and manufactured by using the seed microbial inoculum is produced in a clean and closed environment by using the composite flora of the core functional microbial strains required by the corresponding high-temperature Daqu as the mother yeast in the process of making the yeast, and natural microorganisms are not required to be reused, so that the mixing of external mixed bacteria and the interference of the yeast can be effectively avoided, and the food safety of the Daqu is improved.
On the other hand, the use method of the high-temperature seed bacterial agent is convenient for preparing the composite microbial flora and has simple and convenient use method.
The method for using the high-temperature seed microbial inoculum disclosed by the application is fast in dressing in the culture process of the Daqu, and can be subjected to the fermentation process of 'front slow, middle stile and rear slow falling' as the traditional Daqu, but compared with the traditional Daqu, the total bending time of the production method disclosed by the application is shortened, and the production efficiency is improved.
In the application method of the high-temperature seed microbial agent, the microbial source is completely from the representative high-temperature Daqu, and the prepared composite microbial agent can be stored for a long time in a refrigerating or freezing environment, is convenient to transport and carry, and can be not limited by a white spirit producing area in the process of starter propagation.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the technical solutions of the present application will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the application. All other embodiments, based on the examples herein, which are within the scope of the application as defined by the claims, will be within the scope of the application as defined by the claims. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The application provides a preparation method of a high-temperature seed microbial agent, which is based on research results, the inventor compounds 21 kinds of microorganisms to prepare a compound microorganism freeze-dried microbial agent, and a secondary microbial agent prepared from bran is inoculated with a high-temperature Daqu embryo for culture fermentation to carry out clean production of high-temperature Daqu white spirit.
Preferably, the core functional microorganism includes filamentous fungi, yeasts and bacteria.
The filamentous fungi comprise Aspergillus oryzae CPCC460006, xie Washi Aspergillus oryzae CBS522.65, rhizopus oryzae CBS112.07, mucor circinelloides F CBS195.68;
the yeasts include the sacculus tectorial membrane yeasts NRRLY-2388 and Pichia anomala NRRL Y-5396;
the bacteria include bacillus licheniformis JF802183.1, bacillus subtilis JF783990.1, bacillus amyloliquefaciens DQ658169.1, bacillus megaterium ATCC 14581 (T), bacillus cereus ATCC 14579 (T), bacillus albopictus B8W22 (T), bacillus methylotrophicus KCTC 13105 (T), staphylococcus saprophyticus, lactobacillus bread LMG23699 (T), weissella antrum KACC 11862 (T), lactobacillus pentosus JCM 1558 (T), bacillus acetate NBRC 14818 (T), bacillus cereus LMG 1625 (T) and pseudomonas CIP 104663 (T).
Example 1:
the preparation method of the high-temperature seed bacterial agent comprises the following steps:
(1) Inoculating the bacterial suspension of the functional microorganism into corresponding liquid culture mediums for constant temperature culture, wherein the culture conditions are as follows: culturing at 37deg.C under 150r/min shaking table for 16 hr.
The liquid culture medium is prepared by the following method:
taking 5 parts by weight of beef extract, 10 parts by weight of peptone, 10 parts by weight of NaCl and 1000 parts by weight of distilled water, mixing, and sterilizing for 20 minutes at the pH of 7.2 and the temperature of 110 ℃ to prepare a beef extract peptone liquid culture medium; taking 10 parts by weight of yeast extract powder, 20 parts by weight of peptone, 20 parts by weight of glucose and 1000 parts by weight of distilled water, mixing, and sterilizing at the pH of 7.2 and 110 ℃ for 20min to obtain a yeast extract powder peptone glucose liquid culture medium; the potato dextrose liquid culture medium is prepared by mixing 6 parts by weight of potato soaked powder, 20 parts by weight of dextrose, 0.1 part by weight of chloramphenicol and 1000 parts by weight of distilled water, and sterilizing at the pH of 7.2 and 110 ℃ for 20 minutes.
(2) The freeze-dried carrier is bran, rice husk and corncob, the compounding ratio is 60 parts of bran, 10 parts of rice husk and 30 parts of corncob.
(3) The protective agent is prepared from 7 parts by weight of trehalose, 5 parts by weight of mannitol, 8 parts by weight of sodium thiosulfate and 10 parts by weight of skimmed milk powder.
(4) Transferring the pre-frozen bacterial suspension in the step (5) to a freeze dryer, and vacuum drying to obtain the seed bacterial agent freeze-dried powder. The vacuum drying conditions are as follows: drying pressure is 10pa, separator temperature is 40 ℃ below zero, cold trap temperature is 76 ℃ below zero, and drying time is 120min.
The survival rate of the activated seed bacterial agent prepared in the example is 87.58%.
Example 2:
the preparation method of the high-temperature seed bacterial agent comprises the following steps:
(1) Inoculating the bacterial suspension of the functional microorganism into corresponding liquid culture mediums for constant temperature culture, wherein the culture conditions are as follows: culturing at 37deg.C under 150r/min shaking table for 12 hr.
The liquid culture medium is prepared by the following method:
taking 6 parts by weight of beef extract, 18 parts by weight of peptone, 8 parts by weight of NaCl and 1000 parts by weight of distilled water, mixing, and sterilizing for 20 minutes at the pH of 7.2 and the temperature of 110 ℃ to prepare a beef extract peptone liquid culture medium; taking 8 parts by weight of yeast extract powder, 20 parts by weight of peptone, 18 parts by weight of glucose and 1000 parts by weight of distilled water, mixing, and sterilizing at the pH of 7.2 and 110 ℃ for 20min to obtain a yeast extract powder peptone glucose liquid culture medium; the potato dextrose liquid culture medium is prepared by mixing 8 parts of potato soaked powder, 25 parts of dextrose, 0.1 part of chloramphenicol and 1000 parts of distilled water according to weight parts, and then sterilizing for 20min under the conditions of pH7.2 and 110 ℃.
(2) The freeze-dried carrier is bran, rice husk and corncob, the compounding ratio is 50 parts of bran, 10 parts of rice husk and 40 parts of corncob.
(3) The protective agent is prepared from 5 parts by weight of trehalose, 3 parts by weight of mannitol, 6 parts by weight of sodium thiosulfate and 8 parts by weight of skimmed milk powder.
(4) Transferring the pre-frozen bacterial suspension in the step (5) to a freeze dryer, and vacuum drying to obtain the seed bacterial agent freeze-dried powder. The vacuum drying conditions are as follows: drying pressure is 10pa, separator temperature is 40 ℃ below zero, cold trap temperature is 76 ℃ below zero, and drying time is 120min.
The survival rate of the activated seed bacterial agent prepared in the example is 85.58%.
Example 3:
the preparation method of the high-temperature seed bacterial agent comprises the following steps:
(1) Inoculating the bacterial suspension of the functional microorganism into corresponding liquid culture mediums for constant temperature culture, wherein the culture conditions are as follows: culturing at 37deg.C under 150r/min shaking table for 16 hr.
The liquid culture medium is prepared by the following method:
taking 5 parts by weight of beef extract, 18 parts by weight of peptone, 8 parts by weight of NaCl and 1000 parts by weight of distilled water, mixing, and sterilizing for 20 minutes at the pH of 7.2 and the temperature of 110 ℃ to prepare a beef extract peptone liquid culture medium; taking 10 parts by weight of yeast extract powder, 15 parts by weight of peptone, 25 parts by weight of glucose and 1000 parts by weight of distilled water, mixing, and sterilizing at the pH of 7.2 and 110 ℃ for 20min to obtain a yeast extract powder peptone glucose liquid culture medium; the potato dextrose liquid culture medium is prepared by mixing 8 parts of potato soaked powder, 18 parts of dextrose, 0.1 part of chloramphenicol and 1000 parts of distilled water according to weight parts, and then sterilizing for 20min under the conditions of pH7.2 and 110 ℃.
(2) The freeze-dried carrier is bran, rice husk and corncob, the compounding ratio is 45 parts of bran, 10 parts of rice husk and 45 parts of corncob.
(3) The protective agent is prepared from 7 parts by weight of trehalose, 5 parts by weight of mannitol, 8 parts by weight of sodium thiosulfate and 10 parts by weight of skimmed milk powder.
(4) Transferring the pre-frozen bacterial suspension in the step (5) to a freeze dryer, and vacuum drying to obtain the seed bacterial agent freeze-dried powder. The vacuum drying conditions are as follows: drying pressure is 10pa, separator temperature is 40 ℃ below zero, cold trap temperature is 76 ℃ below zero, and drying time is 120min.
The survival rate of the activated seed bacterial agent prepared in the example is 84.35%.
Example 4:
the preparation method of the high-temperature seed bacterial agent comprises the following steps:
(1) Inoculating the bacterial suspension of the functional microorganism into corresponding liquid culture mediums for constant temperature culture, wherein the culture conditions are as follows: culturing at 37deg.C under 150r/min shaking table for 12 hr.
The liquid culture medium is prepared by the following method:
taking 5 parts by weight of beef extract, 10 parts by weight of peptone, 10 parts by weight of NaCl and 1000 parts by weight of distilled water, mixing, and sterilizing for 20 minutes at the pH of 7.2 and the temperature of 110 ℃ to prepare a beef extract peptone liquid culture medium; taking 10 parts by weight of yeast extract powder, 20 parts by weight of peptone, 20 parts by weight of glucose and 1000 parts by weight of distilled water, mixing, and sterilizing at the pH of 7.2 and 110 ℃ for 20min to obtain a yeast extract powder peptone glucose liquid culture medium; the potato dextrose liquid culture medium is prepared by mixing 6 parts by weight of potato soaked powder, 20 parts by weight of dextrose, 0.1 part by weight of chloramphenicol and 1000 parts by weight of distilled water, and sterilizing at the pH of 7.2 and 110 ℃ for 20 minutes.
(2) The freeze-dried carrier is bran, rice husk and corncob, the compounding ratio is 60 parts of bran, 10 parts of rice husk and 30 parts of corncob.
(3) The protective agent is prepared from 7 parts by weight of trehalose, 3 parts by weight of mannitol, 8 parts by weight of sodium thiosulfate and 10 parts by weight of skimmed milk powder.
(4) Transferring the pre-frozen bacterial suspension in the step (5) to a freeze dryer, and vacuum drying to obtain the seed bacterial agent freeze-dried powder. The vacuum drying conditions are as follows: drying pressure is 45pa, the temperature of the partition plate is 40 ℃ below zero, the temperature of the cold trap is 76 ℃ below zero, and the drying time is 100min.
The survival rate of the activated seed bacterial agent prepared in the example is 87.58%.
The application researches the microbial flora composition of the white spirit high-temperature Daqu by using a microbiology technology, analyzes, summarizes and determines 21 core functional microbial strains commonly existing in the high-temperature Daqu, compounds, expands and cultures the core functional microbial strains and selects a proper carrier to prepare a compound microbial flora, and then uses the compound microbial flora as a seed microbial agent to culture and ferment the Daqu, thereby realizing clean production of the high-temperature Daqu and having positive significance for sustainable development of the white spirit industry.
The foregoing is merely illustrative of the present application, and the present application is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.

Claims (11)

1. The preparation method of the high-temperature Daqu seed microbial inoculum is characterized by comprising the following steps of:
(1) The 21 kinds of microorganisms are divided into bacteria, filamentous fungi and yeast; bacteria include the following microorganisms: bacillus licheniformis JF802183.1, bacillus subtilis JF783990.1, bacillus amyloliquefaciens DQ658169.1, bacillus megaterium ATCC 14581 (T), bacillus cereus ATCC 14579 (T), bacillus albolabris B8W22 (T), bacillus methylotrophicus KCTC 13105 (T), staphylococcus saprophyticus, lactobacillus bread LMG23699 (T), weissella antrum KACC 11862 (T), lactobacillus pentosus JCM 1558 (T), bacillus acetate NBRC 14818 (T), bacillus cereus LMG 1625 (T), pseudomonas CIP 104663 (T); filamentous fungi include the following microorganisms: aspergillus oryzae CPCC460006, xie Washi Aspergillus oryzae CBS522.65, rhizopus oryzae CBS112.07, mucor circinelloides FCBS195.68; the yeasts include the following microorganisms: the saccharum tectorum NRRLY2388 and Pichia anomala NRRLY5396;
(2) Respectively inoculating bacterial suspensions of 15 microorganisms in bacterial microorganisms into beef extract peptone liquid culture medium for constant temperature culture; inoculating bacterial suspensions of 4 kinds of microorganisms in filamentous fungus microorganisms into potato glucose liquid culture medium for constant temperature culture; respectively inoculating bacterial suspensions of 2 microorganisms in the microzyme microorganisms into a yeast powder-immersed glucose liquid culture medium for constant-temperature culture;
(3) Grinding testa Tritici, testa oryzae and corncob, mixing, sterilizing, and taking as carrier;
(4) Taking and sterilizing the protective agent according to the weight parts of the components for later use;
(5) Centrifuging the liquid culture medium, discarding the supernatant, respectively adding sterile physiological saline to prepare concentrated bacterial suspension, respectively adding the protective agent and the carrier, uniformly mixing, and culturing at constant temperature;
(6) Placing the bacterial suspension cultured in the step (5) into a refrigerator at the temperature of minus 20 ℃, and then, putting the bacterial suspension into liquid nitrogen for prefreezing;
(7) Transferring the pre-frozen bacterial suspension in the step (6) to a freeze dryer, and vacuum drying to obtain the seed bacterial agent freeze-dried powder.
2. The method for preparing the high-temperature Daqu seed bacterial agent according to claim 1, which is characterized in that: in the step (1), 21 microorganism bacteria are well preserved and have no degradation and bacteria infection; the 21 kinds of microorganisms are classified into bacteria, filamentous fungi and yeast; bacteria include the following microorganisms: bacillus licheniformis JF802183.1, bacillus subtilis JF783990.1, bacillus amyloliquefaciens DQ658169.1, bacillus megaterium ATCC 14581 (T), bacillus cereus ATCC 14579 (T), bacillus albolabris B8W22 (T), bacillus methylotrophicus KCTC 13105 (T), staphylococcus saprophyticus, lactobacillus bread LMG23699 (T), weissella antrum KACC 11862 (T), lactobacillus pentosus JCM 1558 (T), bacillus acetate NBRC 14818 (T), bacillus cereus LMG 1625 (T), pseudomonas CIP 104663 (T); filamentous fungi include the following microorganisms: aspergillus oryzae CPCC460006, xie Washi Aspergillus oryzae CBS522.65, rhizopus oryzae CBS112.07, mucor circinelloides FCBS195.68; the yeasts include the following microorganisms: the bursa-covered yeasts nrrliy 2388 and pichia anomala nrrliy 5396.
3. The method for preparing a high-temperature Daqu seed bacterial agent according to claim 2, wherein in the step (2), the bacterial culture conditions are as follows: inoculating the bacterial suspension of 15 bacteria into beef extract peptone liquid culture medium, and shake culturing at 36-38 deg.c and 130-160rpm for 14-18 hr.
4. The method for preparing a high temperature Daqu seed inoculant according to claim 2, wherein in the step (2), the cultivation conditions of the filamentous fungus are as follows: inoculating the bacterial suspension of 4 kinds of filamentous fungi into potato dextrose liquid culture medium, and carrying out shaking culture at the temperature of 36-38 ℃ and the speed of 130-160rpm for 14-18h.
5. The process for preparing the high-temperature Daqu secondary microbial inoculum according to claim 2, wherein in the step (2), the yeast culture conditions are as follows: inoculating the bacterial suspension of 2 kinds of saccharomycetes into a saccharomycete powder soaking glucose liquid culture medium, and carrying out shaking culture at the temperature of 36-38 ℃ and the rpm of 130-160 for 14-18h.
6. A method as claimed in claim 1, characterized in that: the protective agent is trehalose, mannitol, sodium thiosulfate and skimmed milk powder; the concentration of the concentrated bacterial suspension in the step (5) is 4-7 multiplied by 10 9 cfu/mL。
7. A method as claimed in claim 1, characterized in that: the ratio of the carrier to the bacterial suspension in the step (3) is bacterial liquid: the carrier (V: W) is 1.5-2:1.
8. A method as claimed in claim 1, characterized in that: in the step (6), the pre-cooling temperature of the refrigerator is minus 20 ℃ and the pre-cooling time is 40min; the pre-freezing time in liquid nitrogen is 8-12 h.
9. The high temperature Daqu seed bacterial agent prepared by the preparation method of any one of claims 1-8.
10. The use of the high temperature Daqu seed inoculant of claim 9 in a high temperature Daqu secondary inoculant.
11. The use according to claim 10, wherein the seed bacterial agent is added in an amount of 0.5 to 0.7% when the secondary bacterial agent is produced.
CN202311024708.6A 2023-08-15 2023-08-15 Preparation method of high-temperature Daqu seed microbial inoculum Pending CN117004532A (en)

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