Lactobacillus aging cellar, compound microbial agent thereof, pit maintenance liquid and application of compound microbial agent and pit maintenance liquid

Abstract

The invention discloses lactobacillus aging cellar, a compound microbial agent thereof, a cellar maintenance liquid and application, and belongs to the technical field of microorganisms. According to the invention, a strain of lactobacillus ageing pit IM332801 is screened from white wine pit mud, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.19561. The strain provided by the invention can be used for producing white spirit flavor substances, can also provide rich flavor substances in white spirit brewing and balance the ratio of ethyl acetate to ethyl hexanoate at the same time, achieves the effects of reducing ethyl acetate and increasing hexanoate, maintains the typical characteristics of Luzhou-flavor white spirit, and can also be used for preparing a compound microbial agent and white spirit cellar maintenance liquid. The method plays an important role in improvement of flavor substances of the white spirit, industrial production and quality maintenance of the white spirit, pit maintenance of the white spirit, improvement of a brewing process and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to an old cellar lactobacillus, a composite microbial agent and a cellar curing liquid comprising the lactobacillus, and applications thereof. Background Art

[0002] Luzhou-flavor liquor is also known as Luzhou-flavor liquor. It has a strong cellar aroma, a soft taste, and a long aftertaste, and is widely loved by consumers. The fermentation process of Luzhou-flavor liquor is a complex fermentation process involving multiple strains and enzyme systems. Sorghum (raw material) and Daqu (saccharifying agent) are the main materials for brewing Luzhou-flavor liquor, which is brewed through multiple complex processes such as solid-state fermentation, solid-state distillation, aging, and blending. Some microorganisms that can be used for liquor fermentation exchange substances and metabolize through the fermented grains to generate various metabolites and flavor substances, ultimately forming the unique composition and flavor characteristics of Luzhou-flavor liquor.

[0003] Among the components of liquor, only 2% of trace components affect the style and flavor of liquor, and promote the transformation of liquor quality. Esters, as important aromatic substances in liquor, can provide liquor with special aroma. Among them, ethyl acetate, ethyl caproate, ethyl lactate, and ethyl butyrate are the four major esters of liquor. The relationship between their content and proportion plays an important role in balancing the flavor quality of liquor. Especially in the brewing process of strong-flavor liquor, ethyl caproate is the main aroma of strong-flavor liquor. The increase of its content is of great significance to the transformation of liquor quality, and the same is true for balancing ester substances in liquor. For example, an appropriate amount of ethyl acetate can give liquor a special cellar aroma and fruity aroma, while too much content will bring astringency and roughness, affecting the taste of liquor. In addition, too much ethyl acetate will promote the transformation of strong-flavor liquor to light-flavor liquor, which will overshadow the main flavor, unbalance the liquor quality, and lose the typical characteristics of strong-flavor liquor. In this regard, balancing the ratio between ethyl acetate and ethyl caproate can control the taste and flavor of Luzhou-flavor liquor to remain at an appropriate threshold, balance the wine quality and coordinate the taste in the brewing process of Luzhou-flavor liquor, so as to maintain the high quality and typical characteristics of Luzhou-flavor liquor.

[0004] Traditional microbial cultivation technology refers to the separation, purification and cultivation of microorganisms to obtain microorganisms, and then study the physiological characteristics of microorganisms. It is based on the traditional cultivation technology research of microorganisms such as Lactobacillus and yeast that deepens the understanding of ecological brewing of liquor, and gradually makes it clear that Lactobacillus is an important strain in the liquor brewing process. Lactobacillus is widely present in nature, often present in fermentation raw materials, and has high fermentation activity. The current liquor fermentation process is mainly based on the control of raw material components and brewing process in the cellar, and rarely relies on the intervention of exogenous strains to control the quality of liquor. As an important bacterium existing in the cellar and even in the fermentation raw materials, Lactobacillus metabolizes flavor substances such as ethyl lactate, ethyl caproate, and ethyl acetate, which can give the liquor a layered taste. The increase or decrease of related metabolites can orient the quality of liquor and maintain the balance of wine quality.

[0005] Therefore, obtaining a new lactobacillus strain that can be suitable for the brewing process of Luzhou-flavor liquor is of great significance to the quality and industrial production of liquor. Summary of the invention

[0006] The invention aims to provide a strain of Lactobacillus laojiaoensis IM332801, which can provide flavor substances and balance the contents of ethyl acetate and ethyl caproate during the brewing process of Luzhou-flavor liquor, thereby achieving the effect of "reducing ethyl and increasing hexanoate".

[0007] To achieve the above-mentioned purpose, in the first aspect, the present invention provides a kind of Lactobacillus laojiao, whose strain name is Lactobacillus laojiao (Lactobacillus laojiao) IM332801, which has been deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on April 10, 2020, and its deposit number is CGMCC No.19561.

[0008] The 16S rDNA sequence of the above-mentioned Lactobacillus laojiao is shown in SEQ ID NO:1.

[0009] SEQ ID NO:1 The 16S rDNA sequence of Lactobacillus laojiaoensis IM332801 is as follows:

[0010] GCCTATAATGCAGTCGACGCGTCCCTCGTTTAAACAATTGGTGCTTGCACCGAGCGTT

[0011] TTAAACATTGGGACGAGTGGCGAACTGGTGAGTAACACGTGGGTAACCTACCCTAAAGC

[0012] AGGGGATAACACTTGGAAACAGGTGCTAATACCGTATAACAACTGAAACCGCATGGTTT

[0013] CGGTTTAAAAGGTGGTTTTGCTACCACTTTAGGATGGACCCGCGGCGCATTAGCTAGTTG

[0014] GTGAGATAATAGCCCACCAAGGCAATGATGCGTAGCCGACCTGAGAGGGTAATCGGCCA

[0015] CATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCA

[0016] CAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGT

[0017] AAAACTCTGTTGTTGGAGAAGAACAGGTGTGATAGTAACTGATCATGCTTTGACGGTAT

[0018] CCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCA

[0019] AGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTG

[0020] AAAGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACTGGGAGACTTGAGTGCAGAAG

[0021] AGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGT

[0022] GGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGA

[0023] ACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGAGGG

[0024] TTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCG

[0025] CAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTT

[0026] TAATTCGATGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTCTGCCAACCTCAGAG

[0027] ATGAGGCGTTCCCTTCGGGGACAGAATGACAGGTGGTGCATGGCTGTCGTCAGCTCGTG

[0028] TCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCCAGCAT

[0029] TTAGTTGGGCACTCTAGCAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACG

[0030] TCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACG

[0031] AGTCGCGAAGTCGCGAGGCTAAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGATTGCAG

[0032] GCTGCAACTCGCCTGCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGCATGCCACGG

[0033] TGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACC

[0034] CAAAGTCGGTGAGGTAACCTTTTGGGGCCAGCCGCCTAAGGTGGGACAGATGATTAGG

[0035] GGAAGTCGAGCAAGA。

[0036] In a second aspect, the present invention also provides a method for isolating and culturing the above-mentioned Lactobacillus laojiao, which comprises the following steps:

[0037] Cellar mud treatment: suspend the liquor cellar mud in sterile water, shake (in a shaking incubator), and fully mix to obtain a cellar mud suspension for Lactobacillus screening;

[0038] Lactobacillus screening: dilute the cellar mud suspension with sterile water to 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 and 10 -7 Take each gradient bacterial liquid and evenly spread it on the mixed system of GMRS solid culture medium and nystatin, culture it statically at 30-37°C in a facultative anaerobic environment, and screen out IM332801.

[0039] Among them, in the above-mentioned method for isolating and culturing old cellar lactobacillus, the liquor cellar mud is Luzhou-flavor liquor cellar mud.

[0040] Among them, in the above-mentioned isolation and culture method of Laojiao Lactobacillus, the liquor cellar mud is suspended in sterile water at a mass volume ratio of 1.5-3g:50-100mL.

[0041] Wherein, in the above-mentioned method for isolating and culturing Lactobacillus laojiao, the shaking time is 12 to 24 hours.

[0042] Among them, in the above-mentioned isolation and culture method of Lactobacillus laojiao, the mixed system of GMRS solid culture medium and nystatin is prepared by the following method: after the GMRS solid culture medium is sterilized, it is poured into a plate, and nystatin with a concentration of 20 to 30 mg / mL is coated on the GMRS solid culture medium plate at a volume ratio of 10 to 30:0.05.

[0043] In order to quickly determine whether the growing colony is a possibility of lactobacillus, in the above-mentioned isolation and culture method of Lactobacillus laojiaoensis, the mixed system of GMRS solid culture medium and nystatin also contains 0.1-1.0% bromocresol green; the volume ratio of 0.1-1.0% bromocresol green to GMRS solid culture medium is 1:10-30.

[0044] Wherein, in the above-mentioned method for isolating and culturing Lactobacillus laojiao, the volume ratio of each gradient bacterial liquid to the mixed system of the GMRS solid culture medium and nystatin is 0.1:10-30.

[0045] Wherein, in the above-mentioned method for isolating and culturing Lactobacillus laojiao, the time of static culture is 2 to 3 days.

[0046] In a third aspect, the present invention also provides the use of the above-mentioned Lactobacillus laojiaoensis in the production of liquor flavor substances, liquor brewing or cellar maintenance.

[0047] Among them, in the application of the above-mentioned Laojiao Lactobacillus, the white wine flavor substances include alcohol substances and / or ester substances, the alcohol substances include ethanol, isopentanol, hexanol and / or phenylethanol, and the ester substances include ethyl acetate, ethyl caproate, ethyl lactate and / or ethyl palmitate.

[0048] Wherein, in the application of the above-mentioned Lactobacillus laojiaoensis, the liquor brewing is the Luzhou-flavor liquor brewing.

[0049] Among them, in the application of the above-mentioned Lactobacillus laojiaoensis, the cellar maintenance is the cellar maintenance of Luzhou-flavor liquor.

[0050] In a fourth aspect, the present invention also provides a process for producing liquor flavor substances by fermenting the above-mentioned Lactobacillus laojiaoensis, which comprises the following steps: inoculating the glycerol tube strain IM332801 into an MRS liquid culture medium, culturing at 30-37° C. for 24-48 hours, and then streaking on an MRS solid culture medium plate; picking a monoclonal colony on the MRS solid culture medium and inoculating it into an MRS liquid culture medium, culturing at 30-37° C. for 24-48 hours, and obtaining a seed solution; and then inoculating it into an MRS liquid culture medium at an inoculum amount of 1-2%, culturing at 30-37° C. for 24-48 hours, and obtaining a fermentation liquid.

[0051] In a fifth aspect, the present invention also provides a method for in-situ culture and fermentation of the above-mentioned Lactobacillus laojiao in liquor brewing, which comprises the following steps: adding water to moisten the sorghum, spreading it to dry, and then mixing it evenly with Daqu, according to the inoculation amount of 1.5-2.0×10 8 CFU was inoculated with IM332801 and cultured at a controlled temperature for 25 to 30 days. The temperature was set at 25 to 30°C for the first 15 to 20 days and then adjusted to 20 to 26°C for the next 5 to 15 days.

[0052] In the present invention, when Lactobacillus laojiao is used for in-situ culture and fermentation in liquor brewing, conventional raw materials, koji medicines and dosages in the field of liquor brewing can be used. For example, the amount of the koji added can be 10-12% of the mass of sorghum; the koji can be medium-high temperature koji.

[0053] In a sixth aspect, the present invention further provides a composite microbial agent comprising IM332801, Clostridium kluyveri, Clostridium tachyzoites, Pichia kudriavzevii and Aspergillus niger.

[0054] In a seventh aspect, the present invention also provides a method for preparing the composite microbial agent, which comprises the following steps: culturing IM332801, Clostridium kluyveri, Clostridium tachyzogenes, Pichia kudriavzevii and Aspergillus niger respectively to obtain pure bacterial fermentation liquid, wherein the concentration of IM332801 fermentation liquid is 10 8 ~10 10 CFU / mL, the concentration of Clostridium kluyveri fermentation broth was 10 8 ~10 10 CFU / mL, the concentration of Clostridium fastidiosa fermentation broth was 10 8 ~10 10 CFU / mL, the concentration of Pichia kudriavzevii fermentation broth was 10 6 ~10 7 CFU / mL, the concentration of Aspergillus niger fermentation broth was 10 6 ~10 7 CFU / mL, and mix the fermentation broths in a volume ratio of 1-3:1-2:1-2:1:1 to obtain.

[0055] Among them, in the preparation method of the above-mentioned composite microbial agent, IM332801 is cultured in MRS liquid culture medium.

[0056] Wherein, in the preparation method of the composite microbial agent, ethanol sodium acetate medium is used to culture Clostridium kluyveri.

[0057] Wherein, in the preparation method of the above-mentioned composite microbial agent, ethanol sodium acetate culture medium is used to culture fast-growing Clostridium.

[0058] Among them, in the preparation method of the above-mentioned composite microbial agent, potato liquid culture medium is used to culture Pichia kudriavzevii.

[0059] Wherein, in the preparation method of the composite microbial agent, Aspergillus niger is cultured in potato liquid culture medium.

[0060] Wherein, in the preparation method of the composite microbial agent, during the culture, the inoculation amount of IM332801 is 5-10%.

[0061] Wherein, in the preparation method of the composite microbial agent, during the culture, the inoculation amount of Clostridium kluyveri is 5-10%.

[0062] Wherein, in the preparation method of the composite microbial agent, during the culture, the inoculation amount of Clostridium fast-growing is 5-10%.

[0063] Wherein, in the preparation method of the composite microbial agent, during the culture, the inoculation amount of Pichia kudriavzevii is 5-10%.

[0064] Wherein, in the preparation method of the composite microbial agent, during the culture, the inoculation amount of Aspergillus niger is 5-10%.

[0065] Among them, in the preparation method of the above-mentioned composite microbial agent, the culture conditions of IM332801 are: 37°C, shaking on a shaker at 120 rpm for 24 to 36 hours.

[0066] Among them, in the preparation method of the above-mentioned composite microbial agent, the culture conditions of Clostridium kluyveri are: 34°C, static culture for 5 to 7 days.

[0067] Among them, in the preparation method of the above-mentioned composite microbial agent, the culture conditions of the fast-growing Clostridium are: 34°C, static culture for 5 to 7 days.

[0068] Among them, in the preparation method of the above-mentioned composite microbial agent, the culture conditions of Pichia kudriavzevii are: 30°C, shaking on a 120rpm shaker for 36 to 48 hours.

[0069] Among them, in the preparation method of the above-mentioned composite microbial agent, the culture conditions of Aspergillus niger are: 30°C, shaking table 120rpm for 48 to 72 hours.

[0070] In an eighth aspect, the present invention further provides the use of the above-mentioned composite microbial agent or the composite microbial agent prepared by the above-mentioned preparation method in the production of liquor flavor substances or liquor brewing.

[0071] In the ninth aspect, the present invention also provides a process for brewing liquor using the above-mentioned composite microbial agent or the composite microbial agent prepared by the above-mentioned preparation method, wherein the brewing production is carried out according to the brewing process of Luzhou-flavor liquor, and when Daqu powder is added to the brewing raw materials, the composite microbial agent is added thereto, and the volume mass ratio of the composite microbial agent to Daqu powder is 50-250mL:1kg.

[0072] In the tenth aspect, the present invention also provides a liquor cellar curing liquid, which is prepared by the following method: adjusting the pH of yellow water to 7.2±0.5, taking 70-80 parts of water and 5-20 parts of yellow water after adjusting the pH by weight, mixing, and sterilizing to obtain a base liquid; inoculating IM332801 in the base liquid at an inoculation rate of 5-15v / m%, and culturing at 30-37°C to obtain the curing liquid.

[0073] Among them, in the above-mentioned liquor cellar curing liquid, sodium hydroxide is used to adjust the pH of yellow water.

[0074] Among them, in the above-mentioned liquor cellar curing liquid, the sterilization is: sterilization at 115℃ for 15 minutes.

[0075] Wherein, in the above-mentioned liquor cellar curing liquid, the culturing time is 5 to 7 days.

[0076] Among them, in the above-mentioned liquor cellar curing liquid, the number of viable bacteria of IM332801 in the liquor cellar curing liquid is ≥10 7 CFU / mL.

[0077] In an eleventh aspect, the present invention also provides the use of the above-mentioned liquor cellar curing liquid in cellar curing.

[0078] Among them, in the application of the above-mentioned liquor cellar curing liquid, the cellar curing is the Luzhou-flavor liquor cellar curing.

[0079] Among them, in the application of the above-mentioned liquor cellar curing liquid, when curing the cellar, the spraying amount of the cellar curing liquid is 0.02-0.20 kg / m 2 .

[0080] Beneficial effects of the present invention:

[0081] The IM332801 provided by the present invention is a lactobacillus strain that can provide standard flavor substances in liquor brewing. It can balance the ratio of ethyl acetate and ethyl caproate while providing rich flavor substances, thereby achieving the effect of "reducing ethyl and increasing hexanoate", maintaining the typical characteristics of Luzhou-flavor liquor, and having important significance for the quality and industrial production of liquor.

[0082] The role of IM332801 in liquor brewing is mainly reflected in: (1) providing ethyl hexanoate, the main aroma of Luzhou-flavor liquor, reducing the excessive output of ethyl acetate, avoiding the imbalance of the flavor of the wine, and maintaining the typical characteristics of Luzhou-flavor liquor; (2) increasing the content of some flavor substances in liquor, giving Luzhou-flavor liquor a layered taste and flavor; (3) IM332801 or a composite microbial agent including IM332801 can be cultured in situ during liquor brewing, and the changes in alcohol content and related metabolites in the mash can be monitored at any time to achieve real-time monitoring of the flavor of the liquor; (4) In addition to improving the level of liquor flavor substances, it can also play an important role in the industrial production and quality maintenance of liquor, the cellar maintenance and brewing process improvement of Luzhou-flavor liquor, and the lactobacillus-assisted liquor brewing industry.

[0083] The preservation number of the Lactobacillus laojiao of the present invention is CGMCC No. 19561. The preservation time is April 10, 2020, and the preservation center is: General Microbiological Center of China Microbiological Culture Collection Administration (CGMCC), the address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, postal code 100101. The classification name is Lactobacillus laojiaoIM332801. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 This is an electron microscope scanning image of Lactobacillus laojiao IM332801.

[0085] Figure 2 This is a bar graph showing the content of volatile metabolites in the mash after 0 days of in situ cultivation.

[0086] Figure 3 This is a bar graph showing the content of volatile metabolites in the mash after 2-day in situ culture.

[0087] Figure 4 This is a bar graph showing the content of volatile metabolites in the mash after in situ culture for 4 days.

[0088] Figure 5 This is a bar graph showing the content of volatile metabolites in the mash cultured in situ for 8 days.

[0089] Figure 6 This is a bar graph showing the content of volatile metabolites in the mash cultured in situ for 12 days.

[0090] Figure 7 This is a bar graph showing the content of volatile metabolites in the mash cultured in situ for 20 days.

[0091] Figure 8 This is a bar graph showing the content of volatile metabolites in the mash cultured in situ for 28 days.

[0092] Fig. 9 This is a bar graph showing the content of volatile metabolites in yellow water after 28 days of in situ culture. DETAILED DESCRIPTION

[0093] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0094] GMRS liquid medium: peptone 10g, beef powder 8g, yeast powder 4g, glucose 25g, dipotassium hydrogen phosphate 2g, diammonium hydrogen citrate 2g, sodium acetate 5g, magnesium sulfate 0.2g, manganese sulfate 0.04g, Tween 80 1g, water 1000mL, pH adjusted to 7.0±0.2. High pressure steam sterilization at 115℃ for 20 min.

[0095] GMRS solid medium: peptone 10g, beef powder 8g, yeast powder 4g, glucose 25g, dipotassium hydrogen phosphate 2g, diammonium hydrogen citrate 2g, sodium acetate 5g, magnesium sulfate 0.2g, manganese sulfate 0.04g, Tween 80 1g, agar 15g, water 1000mL, pH adjusted to 7.0±0.2. High pressure steam sterilization at 115℃ for 20 min.

[0096] 25 mg / mL Nystatin: Dissolve 0.25 g of Nystatin in dimethyl sulfoxide, dilute to 10 mL, and freeze at -20°C.

[0097] 0.5% bromocresol green: Dissolve 0.5 g bromocresol green in anhydrous ethanol and store at 4°C until use.

[0098] MRS liquid medium: peptone 10g, beef powder 8g, yeast powder 4g, glucose 20g, dipotassium hydrogen phosphate 2g, diammonium hydrogen citrate 2g, sodium acetate 5g, magnesium sulfate 0.2g, manganese sulfate 0.04g, Tween 80 1g, water 1000mL. Autoclave at 115℃ for 20 minutes.

[0099] MRS solid medium: peptone 10g, beef powder 8g, yeast powder 4g, glucose 20g, dipotassium hydrogen phosphate 2g, diammonium hydrogen citrate 2g, sodium acetate 5g, magnesium sulfate 0.2g, manganese sulfate 0.04g, Tween 80 1g, agar 15g, water 1000mL. Autoclave at 115℃ for 20 minutes.

[0100] Potato liquid culture medium: 6 g potato extract powder, 20 g glucose, 1000 mL water. Sterilize with high pressure steam at 115°C for 20 minutes.

[0101] Ethanol sodium acetate medium: 5g sodium acetate, 0.5g ammonium sulfate, 0.4g potassium hydrogen phosphate, 0.2g magnesium sulfate, 10g yeast extract, 20mL anhydrous ethanol (after sterilization, add before inoculation), 10g dry heat sterilized CaCO3 (same as ethanol), 1000mL water. Autoclave at 115℃ for 20 minutes.

[0102] Example 1: Isolation and Identification of IM332801 in Pit Mud

[0103] Sampling of cellar mud: Using the stratified sampling method, the cellar mud in the Luzhou Laojiao Luzhou-flavor liquor cellars was first divided into different layers or categories, such as cellar sealing mud, shallow cellar wall mud, deep cellar wall mud, and cellar bottom mud. Then, cellar mud samples were randomly collected from each layer.

[0104] Treatment of cellar mud: The cellar mud stored in sterile sealed bags at 4°C was packaged in an ultra-clean workbench, and an appropriate amount was taken out with a sterile medicine spoon. 1.5 g of cellar mud was weighed and suspended in 50 mL of sterile water in a 250 mL conical flask. The cellar mud was shaken at 25°C in a shaking incubator for 24 h, and after thorough mixing, it was used for Lactobacillus screening.

[0105] Lactobacillus screening: Take 1 mL of the cellar mud suspension and dilute it to 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6and 10 -7 , take 0.1mL of each gradient and spread it evenly on GMRS + 0.5% bromocresol green + 25mg / mL nystatin solid screening medium (that is, after the GMRS solid medium is sterilized, add 1mL of 0.5% bromocresol green, mix thoroughly and pour the plate, spread 50μL of 25mg / mL nystatin on the plate containing 20mL GMRS solid medium, let it stand for 10min, and then spread 0.1mL of sample dilution). In a facultative anaerobic environment, culture at 37℃ for 2-3 days. (Bromocresol green is the most commonly used acid-base indicator. Its color change can indicate whether the strain has the acid-producing characteristics. Without detecting the lactic acid content, it can preliminarily determine whether the growing colony is lactic acid bacteria.)

[0106] Lactobacillus identification: PCR technology was used to amplify the 16S rDNA fragment of the Lactobacillus strain, and gel agar electrophoresis was used to detect the amplification and product size. The sample was sent to a biological company for sequencing, and its specific species was determined through NCBI comparison.

[0107] PCR reaction program: 98°C, 5 min; 98°C, 10 s, 30 cycles; 50-60 (Tm±3)°C, 15 s, 30 cycles; 72°C, 1-2 kb / min, 30 cycles; 72°C 10 min;

[0108] Primers used in PCR reaction:

[0109] 338F (DNA sequence as shown in SEQ ID NO: 2: ACTCCTACGGGAGGCAGCAG);

[0110] 806R (DNA sequence is shown in SEQ ID NO: 3: GGACTACHVGGGTWTCTAAT).

[0111] PCR reaction system (50 μL): contains 2×Taq PCR Master Mix 25 μL, primer 338F (10 μM) 2.5 μL, primer 806R (10 μM) 2.5 μL, and sterile deionized water 20 μL.

[0112] The PCR products were separated and tested by 1.0% agarose gel electrophoresis at 120V, 120A for 20min.

[0113] The 16S rDNA fragment obtained by sequencing was compared with NCBI's BLAST, and the strain was determined to be a new species of the genus Lentilactobacillus, named Lactobacillus laojiao IM332801. It is also the smallest genome lactobacillus (1.24Mb) that has been fully sequenced in the database so far. Its electron microscope scanning image is shown in the figure below. Figure 1 shown.

[0114] Example 2: Determination of the ability of IM332801 to metabolize volatile flavor substances by headspace-solid phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS)

[0115] Take the glycerol tube strain IM332801 stored in the ultra-low temperature refrigerator, inoculate it into MRS liquid culture medium, culture it at 37℃ for 24h, and then streak it on the MRS solid culture medium plate. Pick the monoclonal colony on the MRS solid culture medium and inoculate it into the MRS liquid culture medium at 37℃ for 24-48h to obtain the seed liquid. Then inoculate it into a 10mL extraction bottle (containing 7mL MRS liquid culture medium) at an inoculum of 1-2%, culture it at 37℃ for 24-48h, and obtain the fermentation liquid. Heat the fermentation liquid in a constant temperature water bath at 80℃, insert the extraction needle into the bottle, and extend the extraction fiber needle and place it above the liquid surface. The extraction needle is an Rtx-Wax capillary column (60m×0.25mm×0.25μm, RESTEK, Bellefonte, PA), and adsorb for 1h. After the extraction, desorption is performed at 230℃ at the GC injection port.

[0116] The method for determining volatile metabolites was as follows: 35°C for 5 min, then heated to 230°C at 5°C (F.oxysporum) or 2°C / min, and held for 15 min. Helium was used as carrier gas, and the temperature of the injector, transfer line, and ion source was set at 230°C. The electron ionization source was set at 70 eV.

[0117] The mass spectrum distribution and retention index of the volatile substances were compared with the data in the NIST11 library to obtain the types of flavor substances metabolized by IM332801 during the growth process.

[0118] The detection results showed that the metabolites produced by the strain IM332801 during the fermentation process included alcohols and esters. The alcohols included ethanol, isopentanol, hexanol and phenylethanol, and the esters included ethyl acetate, ethyl caproate, ethyl lactate and ethyl palmitate.

[0119] The results of the determination of volatile flavor substances metabolized by IM332801 are shown in Table 1.

[0120] Table 1 Volatile substances contained in IM332801 fermentation broth (μg / ml)

[0121]

[0122]

[0123] As shown in Table 1, the metabolites measured in the culture solution of IM332801 are highly correlated with the flavor substances of Luzhou-flavor liquor. It can be seen that Lactobacillus IM332801 screened from the cellar mud has the ability to produce volatile metabolites related to liquor, including but not limited to one alcohol substance and ester substance. A single strain may improve the quality and flavor of liquor.

[0124] Example 3: In situ culture and fermentation simulated liquor habitat analysis of the role of IM332801 in liquor brewing

[0125] In situ culture technology was used to simulate the habitat of liquor, and CK (blank control group) and IM332801 (experimental strain group) were set up to verify the role of Lactobacillus laojiao IM332801 in liquor brewing.

[0126] The raw materials for in situ cultivation (sorghum and medium-high temperature Daqu powder) are provided by the production workshop of Luzhou Laojiao.

[0127] In situ culture method: Pour the sorghum on the ground covered with a clean plastic film, add hot water to stir evenly, adjust the moisture content to 58% (w / w), and spread it to room temperature. Divide the sorghum into 3 equal parts, each of which is used as the initial fermented grains for subsequent fermentation studies. Mix the sorghum and Daqu evenly after spreading. The storage time of Daqu is 3 months, and the qu blocks are crushed into uniform qu powder before use. The amount of Daqu added is 11.85% of the mass of the raw materials before steaming. Put the mixed samples into an appropriate number of clean glass containers (180mm in diameter) respectively. Each container is filled with 2kg of fermented grains, and 300g of clean glass beads are placed at the bottom in advance to reserve sedimentation space for yellow water (a byproduct of liquor fermentation). For the control group, after filling, the upper edge of the fermented grains is flush with the container mouth, and the container mouth is covered with a double-layer polyethylene film, the sealing film is fixed with a rubber band, and the lid is closed. In the experimental group, after filling, the IM332801 enriched bacterial solution cultured overnight at 30°C was taken, the culture medium was washed off with pure water, and the bacterial solution concentration was adjusted to 2.0×10 8 CFU, inoculated into the mash, stirred evenly, and the subsequent treatment was the same as the control group. The containers of the control group and the experimental group were placed in an incubator for 28 days with a temperature control. The temperature was set at 28°C for the first 20 days and 24°C for the last 8 days.

[0128] Sampling: Sampling was performed on the 0th, 2nd, 4th, 8th, 12th, 20th and 28th days of fermentation. When sampling, the fermented grains were stirred evenly in the container, and appropriate amount of samples were collected and placed in a 50mL sterile tube, immediately frozen in liquid nitrogen, and then stored at -80℃. On the 28th day, the yellow water material accumulated in the lower layer of the fermentation container was collected and the volatile substances were determined together with the fermented grains.

[0129] The sample processing method is as follows: Use high-temperature sterilized distilled water to shake the fermented grains sample, sample 5g of fermented grains, 20ml of distilled water, and shake for 2h. Use the headspace-solid phase microextraction method to determine the volatile metabolites in fermented grains and yellow water. The treated sample needs to pass through a 0.22μm pore size filter membrane, heat in a constant temperature water bath at 80℃ for 1h, and be tested on the machine after extraction for 1h. The detection method is shown in Example 2.

[0130] Test results such as Figure 2 to Figure 9 As shown, the left Y-axis represents the ethanol content, and the right Y-axis represents the content of other metabolites (isoamyl alcohol, hexanol, phenylethanol, ethyl acetate, ethyl caproate, ethyl lactate and ethyl palmitate) except ethanol.

[0131] Depend on Figure 2 to Figure 9 It can be seen that the metabolites in the mash and yellow water fermented by in situ culture of IM332801 are highly similar to the metabolites produced by the single bacteria in Example 2, and the content is significantly increased compared with the single strain of IM332801.

[0132] Among the metabolized alcohol substances, the content of ethanol in the in-situ fermented mash and yellow water was the highest, and the content of hexanol was the lowest. Except for hexanol, the alcohol substances in the CK group were higher than those in the IM332801 experimental group. Among the metabolized ester substances, with the increase of in-situ fermentation time, the content of ethyl hexanoate in the mash increased, and the content of ethyl acetate in the mash decreased. On the 28th day of fermentation, the concentration of ethyl hexanoate in the IM332801 experimental group (0.10170±0.02300μg / ml) was higher than that in the CK group (0.06353±0.00130μg / ml). The content of ethyl hexanoate in the yellow water of the IM332801 experimental group on the 28th day was 0.04872±0.00682μg / ml, and no ethyl hexanoate content was found in the yellow water of the CK experimental group. The ethyl acetate content in the fermented grains and yellow water of the IM332801 experimental group after 28 days of fermentation was significantly lower than that of the CK experimental group. The ethyl acetate content in the fermented grains of the IM332801 experimental group was 2.99020±0.30150μg / ml, which was lower than 8.68361±0.42809μg / ml in the CK group. The ethyl acetate content in the yellow water of the IM332801 experimental group was 9.32568±0.68020μg / ml, which was lower than 13.54428±0.49357μg / ml in the CK. In addition, the content of ethyl lactate and ethyl palmitate in the IM332801 experimental group also showed a similar trend, which was higher than the content of ester substances in the CK. This shows that IM332801 focuses more on the generation of flavor substances in liquor fermentation, and can play a certain role in improving the quality and aroma of liquor.

[0133] The IM332801 of the present invention was inoculated into the in-situ cultured liquor brewing microenvironment as required, and the experiment proved that the volatile flavor substances in the fermented grains were significantly improved compared with the single strain in Example 2. Among them, ethyl caproate, as the main aroma of Luzhou-flavor liquor, had a significantly higher content than the CK group. From the early to the late fermentation stage, ethyl caproate was maintained at a higher concentration compared with the CK experimental group, indicating that IM332801 can provide the typical aroma of Luzhou-flavor liquor and has a certain effect on improving the quality of liquor.

[0134] Other studies have shown that excessive ethyl acetate can easily cause the body of the wine to be unbalanced, which will cause it to turn into a light-flavor liquor and affect the quality of the liquor. The content of ethyl acetate can affect the quality of liquor to a certain extent. In this regard, the ratio of ethyl acetate to ethyl caproate is particularly important for the quality of strong-flavor liquor. The intervention of IM332801 made the ethyl acetate in the liquor habitat lower than that of the CK group, and the concentration of ethyl caproate was higher than that of the CK group, which indirectly showed that IM332801 is a lactobacillus that can provide standard flavor substances in liquor brewing. While providing rich flavor substances, it balances the ratio of ethyl acetate and ethyl caproate, achieving the effect of "lowering ethyl and increasing ethyl", and maintaining the typical characteristics of strong-flavor liquor. IM332801 may play an important role in the industrial production and quality maintenance of liquor, the maintenance of the cellar and brewing process of strong-flavor liquor, and the lactobacillus-assisted liquor brewing industry, and has a good application prospect.

[0135] Example 4: Preparation of composite microbial inoculants and their application in liquor brewing

[0136] The composite microbial strains include: Lactobacillus laojiao IM332801, Clostridium kluyveri, Clostridium fast-growing, Pichia kudriavzevii, and Aspergillus niger.

[0137] Preparation of composite microbial agent: The above strains were cultured according to the culture conditions in Table 2 to obtain strain seed solution. The final concentration of the fermentation liquid of Lactobacillus laojiao IM332801, Clostridium kluyveri and Clostridium swiftii reached 10 8 ~10 10 CFU / mL, the final concentration of Pichia kudriavzevii and Aspergillus niger fermentation broth reached 10 6 ~10 7 CFU / mL, and the bacterial suspensions were mixed in equal volume ratios to prepare a composite microbial agent that can increase the content of ethyl caproate in Luzhou-flavor liquor.

[0138] Table 2 Strain seed solution culture conditions

[0139]

[0140]

[0141] Application of compound microbial agents in liquor brewing:

[0142] According to the brewing process of Luzhou-flavor liquor, when Daqu powder is added to the brewing raw materials, the experimental group adds the composite microbial agent according to 10% (v / m) of the mass of Daqu, and the control group is not added with the composite microbial agent. After the fermentation, the original liquor of the experimental group and the control group is taken, and the ethyl hexanoate and ethyl acetate therein are analyzed and detected. The detection method is shown in Example 2, and the detection results are shown in Table 3. The ethyl hexanoate content in the experimental group is 1.91g / L, which is 11% higher than that of the control group, and the ethyl hexanoate ratio is increased from 1:0.98 to 1:0.80.

[0143] Table 3 Comparison of data between the experimental group and the control group

[0144] Ethyl hexanoate(g / L) Ethyl acetate (g / L) Hexane-Ethyl Experimental Group 1.91 1.52 1：0.80 Control group 1.72 1.68 1：0.98

[0145] Example 5: Preparation and application of Luzhou-flavor liquor cellar curing liquid

[0146] The preparation method of cellar curing solution is as follows:

[0147] Add sodium hydroxide to the yellow water to adjust the pH value to 7.2 ± 0.5. Prepare the base solution by weight, take 80 parts of pure water and 5 parts of yellow water, mix and sterilize at 115℃ for 15min. Inoculate the base solution with 5% (v / m) inoculation of Lactobacillus laojiao IM332801, culture at 37℃ for 5 days, and the number of viable bacteria of Lactobacillus laojiao IM332801 is ≥10 7 CFU / mL.

[0148] Application of cellar curing liquid:

[0149] After leaving the cellar, clean the cellar wall and cellar bottom to remove residual lees; drill holes around the cellar wall and cellar bottom, with each row of small holes arranged in a staggered manner, with an average hole diameter of 8mm, a hole spacing of 50-80mm, and a hole depth of 40mm. The holes on the cellar wall are tilted upward at 45°, with sparse holes at the bottom and dense holes at the top. Spray the cellar curing liquid slowly and evenly from top to bottom, with a spraying amount of 0.05kg / m 2 After the curing liquid is completely absorbed by the cellar mud, the pool wall and cellar bottom are smoothed. Then the brewing production is carried out according to the production process of Luzhou-flavor liquor.

[0150] Taking the cellar without curing liquid as the control group, the raw wine data of the experimental group and the control group in different rounds are shown in Table 4. There are changes between different rounds in the experimental group. The wine yield is improved in each round, and the rate of superior wine is significantly improved, from the original 37.33% to 46.22%. The average ethyl caproate content finally reached 3.58g / L, while there was basically no change between different rounds in the control group; the ratio of ethyl acetate and ethyl caproate in the experimental group increased.

[0151] Table 4 Analysis of raw liquor data for experimental group and control group

[0152]

[0153] In summary, the strain of the present invention can achieve positive regulation of volatile metabolites in the in-situ culture environment, not only can it provide standard flavor substances in liquor brewing, but also can balance the ratio of ethyl acetate and ethyl caproate, achieve the effect of "reducing ethyl and increasing hexanoate", and maintain the typical characteristics of Luzhou-flavor liquor. Avoid imbalance in the flavor of the wine. The content of some liquor flavor substances is increased, giving Luzhou-flavor liquor a layered taste and flavor. However, the strain of the present invention is not limited to the level of improvement of liquor flavor substances, or can play an important role in the industrial production and quality maintenance of liquor, the cellar maintenance and brewing process improvement of Luzhou-flavor liquor, and the lactobacillus-assisted liquor brewing industry, and has good application prospects.

Claims

1. Lactobacillus spp., characterized in that: The strain of Lactobacillus laojiao is named as Lactobacillus laojiao IM332801, and is deposited in the General Microbiological Center of China National Committee for Microbiological Culture Collection, with a deposit number of CGMCC No.19561.

2. The separation and culture method of the described old cellar lactobacillus of claim 1, characterized in that: The following steps are involved: Cellar mud treatment: suspend the liquor cellar mud in sterile water, shake it, and mix it thoroughly to obtain a cellar mud suspension for Lactobacillus screening; Lactobacillus screening: dilute the cellar mud suspension with sterile water to 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 and 10 -7 Take each gradient bacterial liquid and evenly spread it on the mixed system of GMRS solid culture medium and nystatin, culture it statically at 30-37°C in a facultative anaerobic environment, and screen out IM332801.

3. the separation and culture method of Lactobacillus spp. according to claim 2, is characterized in that: At least one of the following is met: The liquor cellar mud is Luzhou-flavor liquor cellar mud; Suspend the liquor cellar mud in sterile water at a mass volume ratio of 1.5-3 g: 50-100 mL; The shaking time is 12 to 24 hours; The mixed system of GMRS solid culture medium and nystatin is prepared by the following method: after the GMRS solid culture medium is sterilized, it is poured into a plate, and the volume ratio is 10 to 30: 0.05, on GMRS solid medium plates, spread nystatin at a concentration of 20-30 mg / mL; The mixed system of the GMRS solid culture medium and nystatin also contains bromocresol green with a concentration of 0.1 to 1.0%; the volume ratio of bromocresol green with a concentration of 0.1 to 1.0% to the GMRS solid culture medium is 1:10 to 30; The volume ratio of each gradient bacterial solution to the mixed system of the GMRS solid culture medium and nystatin is 0.1:10-30; The static culture time is 2 to 3 days.

4. Application of the Lactobacillus laojiaoensis described in claim 1 in producing liquor flavor substances, liquor brewing or cellar maintenance.

5. The use according to claim 4, characterized in that: At least one of the following is met: The liquor flavor substances include alcohol substances and / or ester substances, the alcohol substances include ethanol, isopentanol, hexanol and / or phenylethanol, and the ester substances include ethyl acetate, ethyl caproate, ethyl lactate and / or ethyl palmitate; The liquor brewing is Luzhou-flavor liquor brewing; The cellar maintenance described is the cellar maintenance of Luzhou-flavor liquor.

6. The process for producing liquor flavor substances by fermentation with the lactobacillus laojiao described in claim 1 is characterized in that: The method comprises the following steps: inoculating the glycerol tube strain IM332801 into an MRS liquid culture medium, culturing at 30-37°C for 24-48 hours, and then streaking on an MRS solid culture medium plate; picking a monoclonal colony on the MRS solid culture medium and inoculating it into an MRS liquid culture medium, culturing at 30-37°C for 24-48 hours, and obtaining a seed solution; and then inoculating it into an MRS liquid culture medium at an inoculation amount of 1-2%, culturing at 30-37°C for 24-48 hours, and obtaining a fermentation solution.

7. A method for in-situ culture and fermentation of the Lactobacillus laojiaoensis according to claim 1 in liquor brewing, characterized in that: The following steps are involved: After sorghum is moistened with water and spread out to dry, mix it evenly with Daqu and inoculate it at a rate of 1.5-2.0×10 8 CFU was inoculated with IM332801 and cultured at a controlled temperature for 25 to 30 days. The temperature was set at 25 to 30°C for the first 15 to 20 days and then adjusted to 20 to 26°C for the next 5 to 15 days.

8. A composite microbial agent, characterized in that: The composite microbial agent comprises IM332801, Clostridium kluyveri, Clostridium tachyphyllum, Pichia kudriavzevii and Aspergillus niger.

9. The method for preparing the composite microbial agent according to claim 8, characterized in that: The following steps are involved: IM332801, Clostridium kluyveri, Clostridium tachyzoites, Pichia pastoris and Aspergillus niger were cultured separately to obtain pure bacterial fermentation broth, wherein the concentration of IM332801 fermentation broth was 10 8 ~10 10 CFU / mL, the concentration of Clostridium kluyveri fermentation broth was 10 8 ~10 10 CFU / mL, the concentration of Clostridium fastidiosa fermentation broth was 10 8 ~10 10 CFU / mL, the concentration of Pichia kudriavzevii fermentation broth was 10 6 ~10 7 CFU / mL, the concentration of Aspergillus niger fermentation broth was 10 6 ~10 7 CFU / mL, and mix the fermentation broths in a volume ratio of 1-3:1-2:1-2:1:1 to obtain.

10. The method for preparing the composite microbial agent according to claim 9, characterized in that: At least one of the following is met: IM332801 was cultured in MRS liquid medium; Clostridium kluyveri was cultured in ethanol sodium acetate medium; Ethanol sodium acetate medium was used to culture Clostridium tachyphyllum; Pichia kudriavzevii was cultured in potato liquid medium; Aspergillus niger was cultured in potato liquid medium; During cultivation, the inoculum amount of IM332801 was 5-10%; When culturing, the inoculum amount of Clostridium kluyveri is 5-10%; When culturing, the inoculum amount of Clostridium tachyphyllum is 5-10%; During the culture, the inoculation amount of Pichia kudriavzevii is 5-10%; During cultivation, the inoculum amount of Aspergillus niger is 5-10%; The culture conditions of IM332801 were: 37°C, shaking at 120 rpm for 24–36 h; The culture conditions of Clostridium kluyveri are: 34°C, static culture for 5 to 7 days; The culture conditions of fast-growing Clostridium are: 34°C, static culture for 5 to 7 days; The culture conditions of Pichia kudriavzevii were as follows: 30°C, shaking at 120 rpm for 36 to 48 h; The culture conditions of Aspergillus niger are: 30°C, shaking in a 120 rpm incubator for 48 to 72 hours.

11. Use of the composite microbial agent according to claim 8 or the composite microbial agent prepared by the preparation method according to claims 9 to 10 in producing liquor flavor substances or liquor brewing.

12. A process for brewing liquor using the composite microbial agent according to claim 8 or the composite microbial agent prepared by the preparation method according to claims 9 to 10, characterized in that: The brewing process of Luzhou-flavor liquor is used for brewing. When Daqu powder is added to brewing raw materials, the composite microbial agent is added thereto. The volume mass ratio of the composite microbial agent to Daqu powder is 50-250 mL: 1 kg.

13. Liquor cellar curing liquid, characterized by: The preparation method is as follows: the pH value of yellow water is adjusted to 7.2±0.5, 70 to 80 parts of water and 5 to 20 parts of yellow water after the pH value is adjusted are taken by weight, the mixture is sterilized, and a base solution is obtained; IM332801 is inoculated into the base solution at an inoculation rate of 5 to 15 v / m%, and the base solution is obtained after being cultured at 30 to 37° C.

14. The liquor cellar curing liquid according to claim 13, characterized in that: At least one of the following is met: Sodium hydroxide was used to adjust the pH of yellow water; The sterilization is as follows: sterilization at 115°C for 15 minutes; The culture time is 5 to 7 days; In the liquor cellar curing solution, the number of IM332801 live bacteria is ≥ 10 7 CFU / mL.

15. Use of the liquor cellar curing liquid according to claim 13 or 14 in cellar curing.

16. The use according to claim 15, characterized in that: At least one of the following is met: The cellar maintenance is the cellar maintenance of Luzhou-flavor liquor; When maintaining the cellar, the spraying amount of the cellar maintenance liquid is 0.02-0.20 kg / m 2 .

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