Screening and application of common thermoactinomycetes from Maotai-flavor Daqu

Abstract

The invention discloses a thermoactinomycete with the characteristics of producing pyrazine, acetoin and cellulase, which is characterized in that the preservation number of the strain is CCTCC (China Center For Type Culture Collection) M 2025443, and the name of the strain is common thermoactinomycete 4.019. The invention also discloses a preparation method of the thermoactinomycete with the characteristics of producing pyrazine, acetoin and cellulase, and the thermoactinomycete with the characteristics of producing pyrazine, acetoin and cellulase is characterized in that the preservation number of the strain is CCTCC M 2025443. The strain has typical thermophilic growth characteristics; under a liquid culture condition, the strain has relatively good cellulase synthesis capability; on a solid-state Daqu simulated fermentation substrate, the strain can generate flavor components with pyrazine, acetoin and the like as typical representatives, which indicates that the strain is a strain with excellent fermentation characteristics, and has good application prospects in the aspects of promoting saccharification and degradation of wine making raw materials and improving the flavor quality of Maotai-flavor Daqu in the future.

Description

Technical Field

[0001] The present invention relates to a high-temperature actinomycete strain 4.019 with the functions of producing pyrazine, acetoin and cellulase. This strain was isolated from high-temperature sauce-flavor Daqu in Xiuwen production area, central Guizhou. After polyphasic taxonomic identification, it was determined that the strain 4.019 is a species under the genus Thermoactinomyces, and was named Thermoactinomyces vulgaris 4.019. Background Art

[0002] As a typical representative of traditional Chinese solid-state fermentation distilled liquor, the unique flavor of sauce-flavor Baijiu is closely related to the growth and metabolism of complex microbial communities in high-temperature Daqu. The quality of high-temperature Daqu directly determines the stability of the liquor quality. Pyrazine substances are the general name of a series of six-membered heterocyclic compounds containing two symmetric nitrogen atoms. Among the aroma components of sauce-flavor Baijiu, pyrazine components are the most abundant, and their content is more than ten times that detected in other types of Baijiu, making them one of the main flavor contributing components. As an important flavor precursor for the synthesis of pyrazine components in sauce-flavor Baijiu, acetoin is also an important flavor component in the brewing system of sauce-flavor liquor, and makes a potentially important contribution to the formation of the complex sauce flavor. Analyzed from the source, high-temperature sauce-flavor Daqu is the main source of components such as pyrazine and acetoin in the liquor body, which are produced by the metabolism of microorganisms in the Daqu. The microbial groups in the fermentation environment of sauce-flavor Daqu are very complex and the koji-making process is carried out in a high-temperature environment. Therefore, thermophilic bacterial groups represented by high-temperature actinomycetes are highly enriched, thus becoming the main source of acetoin and pyrazine substances in the fermentation system. Cellulase is an important class of hydrolases in high-temperature Daqu and has important potential effects in aspects such as raw material degradation, saccharification and full utilization of koji-making raw materials. Cellulase is also mainly produced by the metabolism of microorganisms in the Daqu. In recent years, with the increasing number of microorganisms discovered in high-temperature sauce-flavor Daqu, the sources of cellulase production also show diverse characteristics, and many microorganisms including high-temperature actinomycetes have been confirmed as new sources of cellulase synthesis in high-temperature sauce-flavor Daqu.

[0003] Traditional high-temperature Daqu is made from 100% wheat, and the microorganisms in it mainly come from natural inoculation of raw materials, the environment and the production process; at an ultra-high koji-making temperature of 60-65°C, a very special thermophilic microbial flora is formed in Daqu, making thermophilic bacteria such as Bacillus sp., Oceanobacillus sp. and Virgibacillus sp. the dominant groups. Further research has also shown that representative strains such as Bacillus subtilis and Bacillus licheniformis can effectively promote biochemical reaction processes such as saccharification, proteolysis and flavor synthesis in the Daqu fermentation environment through their own growth and metabolic activities, thus making an important contribution to the formation of the complex sauce aroma during the brewing process. In addition, high-temperature sauce-flavor Daqu can also be regarded as a "treasure house" of various functional microorganisms. The microorganisms it contains are not only numerous in species but also have very good metabolic potential, with great value for large-scale application. Among them, due to the good culturable characteristics of Bacillus, there are currently more studies and resource development on this group, and its role in enhancing the flavor quality of sauce-flavor Daqu has also been widely recognized in the industry. However, generally speaking, except for Bacillus, nearly 90% of the functional microbial resources in sauce-flavor Daqu have not been effectively studied and explored due to the limitation of culturing methods. In recent years, through microbial metagenomics analysis technology, various uncultured thermophilic bacteria represented by the high-temperature actinomycete group have been widely detected in sauce-flavor Daqu. Analyzing from the abundance, high-temperature actinomycetes even become the dominant bacterial group in the system during the high-temperature Daqu fermentation stage of sauce-flavor Baijiu, which implies from one side that high-temperature actinomycetes are likely to be similar to Bacillus and are a type of microorganism with potential aroma-producing characteristics in the brewing environment of sauce-flavored liquor. Therefore, the effective exploration of the high-temperature actinomycete group resources in sauce-flavor Daqu is extremely valuable.

[0004] The Thermoactinomycetaceae group was first discovered in 1899. From an evolutionary perspective, this group generally lies between bacteria and actinomycetes, showing an independent evolutionary status. In terms of physiological and biochemical characteristics, thermoactinomycetes are a group of Gram-positive, aerobic microorganisms with aerial and substrate mycelia similar to those of actinomycetes, but their metabolic characteristics and living habits are closer to those of bacterial groups. The most prominent feature of this group is its thermophilic property, and the optimal growth temperature range of the discovered strains generally lies within the high-temperature range of 45 - 60 °C. The Thermoactinomycetaceae group is widely distributed in terrestrial and aquatic high-temperature habitats (such as hot springs, submarine volcanoes, high-temperature composts, and marine sediments), and generally has the functions of producing thermophilic enzyme systems (such as high-temperature-resistant amylase and protease) and bioactive substances (such as antibiotics and anti-tumor compounds). In recent years, with the progress of analysis techniques, thermoactinomycetes represented by the genus Kroppenstedtia sp. and Thermoactinomyces sp. have also been found to be dominant microbial groups in natural brewing and fermentation environments (such as high-temperature Daqu substrates), and have been preliminarily proven to be closely related to the degradation and transformation of Daqu raw materials and the generation of characteristic flavors, becoming a class of thermophilic extremophilic microbial resources with good potential application value in the liquor-making environment. However, up to now, in the entire research field of sauce-flavored liquor brewing, the research on the Thermoactinomycetaceae group is still very scarce and mostly fragmented. The research content is limited to simple studies such as the isolation and identification of strains, and there is a severe lack of research on the functions of thermoactinomycetes in aspects such as flavor production, enzyme production, and promotion of the fermentation process during brewing, which is very unfavorable for the development and application of thermoactinomycete resources in Daqu. In the future, systematic exploration of uncultured thermoactinomycete strain resources in the sauce-flavored liquor brewing environment, and clarification of the functional characteristics of thermoactinomycetes in producing flavor components such as pyrazine and enzyme production under solid-state fermentation conditions will not only provide more fermentation flavor-producing strain resources for the future sauce-flavored liquor brewing industry but also effectively contribute to the development needs of the future traditional sauce-flavored liquor industry. Summary of the Invention

[0005] The technical problem to be solved by the present invention is: to solve the problem of the development of thermoactinomycete strain resources with good application potential in the environment of sauce-flavored high-temperature Daqu. Through the isolation and screening of strains, physiological and biochemical analysis, and the evaluation of fermentation and metabolic functions under solid-state simulated fermentation conditions, a common thermoactinomycete strain capable of effectively metabolizing and producing cellulase and pyrazine under brewing conditions is provided for the current sauce-flavored liquor brewing industry.

[0006] The technical solution of the present invention is as follows: A thermophilic actinomycete with the characteristics of producing cellulase, pyrazine, and acetoin is isolated from Jiangxiang high-temperature Daqu. This strain has currently completed the preservation work at the preservation center, and the preservation number is: CCTCC M2025443. The taxonomic name of the strain is: Thermoactinomyces vulgaris 4.019. The preservation unit is the China Center for Type Culture Collection, and the preservation address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, with a postal code of 430072. The preservation date of the strain is March 12, 2025.

[0007] The aerial hyphae and conidia of strain 4.019 are both milky white. The hyphae are relatively developed, and the substrate mycelium is light yellow in color without the production of soluble pigments.

[0008] The colony shape of the said strain on TSA medium is notched circular, slightly raised in the middle of the colony, with obvious radial folds on the surface of the colony and different colors in the inner and outer circles of the colony.

[0009] The growth temperature range of the said strain 4.019 is between 45°C and 60°C. Among them, the optimum growth temperature is 55°C, showing very typical thermophilic growth characteristics.

[0010] The strain Thermoactinomyces vulgaris 4.019 screened in the present invention is a Gram-positive bacterium. When cultured on TSA medium, the macroscopic colony shape is notched circular, slightly raised in the middle of the colony with obvious radial folds on the surface. The texture of the colony is generally dry and dense, with a certain color difference between the inner and outer circles. The colony is not easy to pick up. The aerial hyphae are milky white and the mycelium is relatively developed. The overall diameter of the colony is in the range of 10 mm to 12 mm, without the production of soluble pigments. The microscopic morphological observation of this strain is carried out under a scanning electron microscope, and the main characteristics are as follows: The mycelium of strain 4.019 is composed of medium-length dendritic hyphae. The conidia are irregularly round, about 1 μm in diameter, directly borne on the aerial hyphae, and the density of conidia at the end of the conidiophore is greater and aggregated into a cluster.

[0011] Molecular biological identification is carried out on strain 4.019, and it is determined that it belongs to the species under the order Bacillale, family Thermoactinomycetaceae, and genus Thermoactinomyces. The full length of the nucleotide sequence of the 16S rRNA of strain 4.019 is 1455 bp, as shown in SEQ ID NO.1 for details.

[0012] The enzyme production ability of the strain was evaluated on a liquid enrichment medium. The results showed that Thermoactinomyces vulgaris 4.019 had good cellulase production characteristics under the liquid fermentation conditions, and the measured average enzyme activity could reach 60.08 U / mL. Among them, the inoculation amount of the strain fermentation was 3-4%, the temperature range for enzyme production by liquid fermentation of this strain was 50°C-55°C, and the time for enzyme production by liquid fermentation was 54-72 h.

[0013] Furthermore, the composition and preparation conditions of the above liquid enrichment medium included: glucose 10 g / L, mannitol 10 g / L, peptone 4 g / L, yeast powder 4 g / L, dipotassium hydrogen phosphate 4 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 0.5 g / L, calcium carbonate 0.2 g / L, ferrous sulfate heptahydrate 0.03 g / L, pH 7.0-7.2, sterilized at 121°C for 20 min.

[0014] On the simulated solid-state fermentation substrate of Daqu made of pure wheat (100%), the strain 4.019 was inoculated at an inoculation amount of 5%, and after 3 days of fermentation at 55°C, flavor components such as 2,5-dimethylpyrazine, 2-ethyl-3-methylpyrazine, 2-ethenyl-5-methylpyrazine, and acetoin could be mainly produced, and their average contents reached 31.48 μg / g Daqu, 8.07 μg / g Daqu, 1.53 μg / g Daqu, and 15.51 μg / g Daqu respectively. This result clarified the fermentation characteristics of Thermoactinomyces vulgaris 4.019 in synthesizing flavor components such as pyrazine and acetoin in the simulated Daqu fermentation environment, and also indicated that this strain had good application prospects in Daqu strengthening, Baijiu brewing, food fermentation, etc.

[0015] Furthermore, the composition and preparation conditions of the above simulated solid-state fermentation substrate of Daqu included: selecting 100% wheat special for Daqu making, crushing it with a grinder, sterilizing it at 121°C under high pressure for 30 min, and then adding 38% sterile water under sterile conditions and stirring well to mix evenly.

[0016] The present invention provides the application of Thermoactinomyces vulgaris 4.019 in the preparation of 2,5-dimethylpyrazine, 2-ethyl-3-methylpyrazine, 2-ethenyl-5-methylpyrazine, and acetoin.

[0017] The present invention provides the application of the above strain Thermoactinomyces vulgaris 4.019 in the strengthening of Jiangxiang Daqu.

[0018] The present invention provides the application of the above strain Thermoactinomyces vulgaris 4.019 in the brewing of traditional Maotai-flavor Baijiu.

[0019] Advantages of the present invention: Through the method for isolating, screening and purifying thermophilic actinomycetes from high-temperature Maotai-flavor Daqu described in this invention, the screening and rapid cultivation of thermophilic actinomycetes under laboratory conditions are effectively realized, thus providing important reference for the development of more rare microbial resources in Maotai-flavor Daqu in the future.

[0020] The present invention isolates and screens a strain of thermophilic actinomycetes from high-temperature Maotai-flavor Daqu. The preservation number of this strain is: CCTCC M 2025443. Through taxonomic identification, this strain should be classified into the genus Thermoactinomyces sp. and is named Thermoactinomyces vulgaris 4.019. This strain has very typical thermophilic growth characteristics and still has good growth and metabolic capabilities in a high-temperature environment; under liquid expansion culture conditions, it has good cellulase synthesis characteristics, and the cellulase enzyme activity reaches 60.08 U / mL; on the Daqu simulated solid fermentation substrate, this strain has the ability to produce flavor components such as pyrazines and acetoin. The average contents of flavor components such as 2,5-dimethylpyrazine, 2-ethyl-3-methylpyrazine, 2-ethenyl-5-methylpyrazine, and acetoin are 31.48 μg / g Daqu, 8.07 μg / g Daqu, 1.53 μg / g Daqu, and 15.51 μg / g Daqu respectively.

[0021] It can be seen from this that the thermophilic actinomycetes strain Thermoactinomyces vulgaris 4.019 isolated from Maotai-flavor Daqu has good characteristics of producing cellulase, pyrazines and acetoin under high-temperature (45 - 60 °C) liquid and solid fermentation conditions. It is a strain with typical enzyme-producing and flavor-producing capabilities and has potential value in promoting the saccharification and degradation of brewing raw materials and improving the flavor of the Maotai-flavor Baijiu brewing system; at the same time, it also has good application prospects in the fields of strengthening the quality of high-temperature Daqu, enhancing the flavor of Maotai-flavor Baijiu products and improving the quality of fermented foods in the future. Description of the Drawings

[0022] Figure 1 It is a description diagram of the colony morphology;

[0023] Figure 2 It is a microscopic scanning electron microscope picture of the strain;

[0024] Figure 3 It is a phylogenetic analysis diagram of the strain;

[0025] Figure 4It is a graph showing the enzyme production of strain 4.019 under liquid fermentation conditions;

[0026] Figure 5 It is a graph analyzing the yields of pyrazine and acetoin of strain 4.019 on a pure wheat daqu simulated solid fermentation substrate. Specific implementation manners

[0027] The following part further elaborates on the present invention application in combination with the specific implementation manners of the present invention. Unless otherwise defined, all scientific and technical terms used herein have the same meanings understood by those skilled in the technical field of this research, aiming to better explain the present invention, but not to limit the present invention.

[0028] 1. Experimental scheme:

[0029] 1.1 Experimental materials

[0030] The Jiangxiang high-temperature daqu for screening high-temperature actinomycete strains is sourced from a certain Jiangjiu production enterprise in the central Guizhou production area. The Jiangxiang daqu used in this research is the aged koji powder (stored for 6 months) after being crushed and mixed evenly and waiting for feeding. In each koji pile, it is divided into four equal parts according to the quartering method. Five samples are randomly taken from each part, and a total of 20 samples are taken, mixed and packaged to ensure the representativeness of the samples taken.

[0031] The strain isolation and screening medium adopts the modified TSA medium: tryptone 10 g / L, soy peptone 5 g / L, sodium chloride 5 g / L, daqu extract 10 g / L, mannitol 10 g / L, gellan gum 8 g / L, pH about 7.3. The sterilization conditions of the medium: 121 °C, 15 min.

[0032] The purification culture of the screened strain and the slant preservation of the strain both adopt the TSA medium: tryptone 15 g / L, soy peptone 5 g / L, sodium chloride 5 g / L, agar 15 g / L, pH about 7.3. The sterilization conditions of the medium: sterilize at 121 °C for 15 min.

[0033] The enlarged culture of the strain adopts the liquid enlarged culture medium: glucose 10 g / L, peptone 4 g / L, yeast powder 4 g / L, dipotassium hydrogen phosphate 4 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 0.5 g / L, calcium carbonate 0.2 g / L, ferrous sulfate heptahydrate 0.03 g / L, mannitol 10 g / L, pH 7.0 - 7.2. The sterilization conditions of the medium: sterilize at 121 °C for 20 min.

[0034] The simulated solid-state fermentation experiment of the strain was carried out on a Daqu simulated fermentation substrate made of pure wheat (100%) as the substrate. This medium is a self-designed natural medium, and the specific production process is as follows: Select special wheat for making koji as the raw material, take 50 g of wheat and add 5% boiling water (above 95 °C) for 1 h of grain moistening treatment. Subsequently, use a pulverizer to break the wheat. The degree of pulverization of the wheat should be appropriate with the skin, and the fine powder can pass through a 20-mesh sieve. Then place the pulverized wheat in a beaker and sterilize it sealed at 121 °C for 30 min. Finally, add 38% - 40% sterile water and mix well under sterile conditions to complete the production of the solid-state simulated fermentation substrate.

[0035] 1.2 Pretreatment process of Daqu samples

[0036] The specific treatment method of the Daqu sample is as follows: First, perform dry heat treatment. Take 50 g of the Daqu sample and put it into a sterilized beaker and seal it. Place it in an oven and heat it at 80 °C for 1 h, then take it out. Subsequently, add 5 g of sodium carbonate powder in a sterile environment to achieve the effect of specifically enriching the spores of thermophilic actinomycetes. Then add 15% sterile physiological saline to the sample, mix well, and place it in an incubator at 30 - 35 °C under saturated humidity conditions for 3 - 4 d, and observe whether there are obvious contaminating bacteria growing in the sample. If there are no contaminating bacteria growing in the sample, take 25 g of the koji sample and put it into a sterile conical flask with glass beads, add physiological saline (250 ml in total) according to the ratio of Daqu sample: physiological saline of 1:10, and then place it in a constant temperature shaker at 45 °C and shake it rapidly at a speed of 180 - 200 rpm for 30 min.

[0037] 1.3 Screening, purification and preservation of strains

[0038] Take the concentration of the bacterial suspension of the pretreated Daqu sample as 10 -1 , and then respectively pipette 1 mL of the bacterial suspension and dilute it with a 10-fold dilution unit, so that the dilution multiples of the bacterial suspension are 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 and 10 -7, set aside. After the modified TSA medium is sterilized, add three kinds of bacteria inhibitors, nystatin (30 mg / L), neomycin (10 mg / L) and gentamicin (10 mg / L) filtered through a 0.22 μm organic sterile filter membrane, and Daqu extract (10 g / L) at about 55-60°C, and shake gently to mix. Use the pouring method to pour the plate, take 0.2 mL from the bacterial suspension with different dilution multiples into the blank culture dish, and then pour the prepared modified TSA medium into each, and shake the culture dish gently to make the Daqu sample evenly distributed in the culture medium. Make 5 parallel groups for each concentration gradient. After the culture medium is allowed to stand and solidify, transfer it to a 45°C constant temperature and humidity incubator and invert it for 3, 7, 10, and 14 days to carry out the screening of high-temperature actinomycete strains.

[0039] After a single colony grows on the screening plate, according to the description of the characteristics of thermoactinomycetes in the Bergey's Manual of Bacterial Identification, the colonies with highly suspected morphology are picked, and then purified by 3-zone streaking on the TSA culture medium dish. Each suspected colony is purified at least twice, and duplicate strains are removed by observing morphological characteristics. Finally, a single colony in the third zone on the plate is picked to obtain a purified strain.

[0040] The single strains obtained after purification were inoculated into TSA slant culture medium for preservation, and the strains were preserved at 4°C; at the same time, the long-term preservation of the strains was carried out by glycerol tube preservation, and the specific steps were as follows: the strain cells after 48 hours of expansion in liquid expansion medium were collected in a sterile 30% (W / V) glycerol solution and stored for a long time at -80°C.

[0041] 1.4 Morphological observation and identification of strains

[0042] The isolated strains were inoculated on TSA culture medium and cultured in a constant temperature incubator at 45℃ for 5-7 days. The colony shape, base hyphae, aerial hyphae color and soluble pigment production were observed and recorded, and the colony size was measured.

[0043] The microscopic morphological observation of the strains was carried out under a scanning electron microscope with a magnification of 15,000 times, and the size of mycelium and conidia was measured with the ruler provided by the microscope. The microscopic morphological identification of the selected strains was completed by observing and describing the mycelium morphology, conidia shape, attachment, distribution and other microscopic characteristics, and combining with the Bergey's Bacterial Identification Manual.

[0044] 1.5 Determination of the growth temperature range of strains

[0045] After culturing the screened Thermoactinomyces strains in a liquid culture medium for 48 h (culture conditions: 45 °C, 150 rpm), 0.5 mL of fresh primary seed liquid was taken and spread on TSA medium for 5 days of culture. They were respectively placed at 6 temperature gradients of 30, 37, 45, 50, 55 and 60 °C for 48 h of culture. By directly observing the growth status of the formed colonies, the growth temperature range and the optimum growth temperature of the screened strains were determined.

[0046] 1.6 Physiological and biochemical identification of strains

[0047] According to "Bergey's Manual of Determinative Bacteriology" or the actinomycetes identification manual, physiological and biochemical identification of the screened strains was carried out respectively from indicators such as carbon and nitrogen source utilization, gelatin liquefaction, starch hydrolysis, milk coagulation / peptonization, nitrate reduction, cellulose decomposition, melanin production, hydrogen sulfide production and Gram staining, and the physiological and biochemical results of the strains were recorded in detail.

[0048] 1.7 Molecular biological identification of strains

[0049] The amplification of the strain characteristic identification sequence was completed using the universal 16S rRNA primers for bacteria; among them, the upstream primer was 27F: 5’-AGAGTTTGATCCTGGCTCAG-3’, and the downstream primer was 1492R: 5’-GGTTACCTTGTTACGACTT-3’. The specific amplification conditions for PCR were: pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 2 min, annealing at 57 °C for 1 min, extension at 72 °C for 3 min, for a total of 35 cycles; finally, extension at 72 °C for 8 min. After obtaining the 16S rRNA amplification sequence of the strain, it was sent to Sangon Biotech (Shanghai) Co., Ltd. to complete the sequencing work, and the 16S rRNA gene sequence information of strain 4.019 was obtained. Subsequently, the ContigExpress software was used for quality control and splicing of the gene sequence to obtain the assembled 16S rRNA sequence.

[0050] The sequencing results of the strain were subjected to homologous alignment analysis using the BLAST tool of the NCBI database (http: / / www.ncbi.nlm.nih.gov / blast) and the online bacterial gene alignment service of the EzBiocloud database (http: / / www.eztaxon.org / ). The 16S rDNA gene sequences of typical strains with relatively high sequence alignment similarity and homology were selected as references. Multiple sequence alignment was performed using MEGA 7.0 software, and the neighbor-joining method was used to construct the phylogenetic tree of the screened strains. The topological structure of the tree was evaluated based on 1000 bootstrap analyses of sampling, and the taxonomic status of the screened strains was determined using the sequence of Bacillus thermolactis R-6488 as an outgroup.

[0051] 1.8 Strain preservation

[0052] The strain has now been preserved at the preservation center with the preservation number: CCTCC M2025443. The taxonomic name of the strain is: Thermoactinomyces vulgaris 4.019. The preservation unit is the China Center for Type Culture Collection, and the preservation address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, Postcode: 430072. The strain preservation date is March 12, 2025.

[0053] 1.9 Evaluation of the cellulase-producing ability of the strain

[0054] The preserved fresh strain was inoculated on TSA medium and cultured at 45°C for 24 h for activation. After activation three times, the activated strain was inoculated into a triangular flask containing 50 mL of liquid expansion medium and cultured at 45°C and 160 r / min for 24 h. Then, 5 mL of the primary seed liquid was taken into another triangular flask containing 50 mL of liquid expansion medium and cultured at 45°C and 180 r / min for 24 h to obtain the enzyme-producing liquid of the strain. Subsequently, the fermentation broth was centrifuged at 10000 r / min for 15 min, and the supernatant was taken to obtain the crude enzyme liquid.

[0055] The enzyme activity of pectinase in the crude enzyme liquid of the strain was determined using the classical DNS method (dinitrosalicylic acid method). Among them, the preparation of the enzyme reaction substrate solution was as follows: Weigh 1% sodium carboxymethylcellulose and dissolve it in acetic acid-sodium acetate buffer solution with pH 4.6. Subsequently, take 1 mL of glucose standard solutions with different concentrations (concentration range: 0.2 - 2.0 mg / mL), add 2 mL of DNS reagent, and perform color development in a boiling water bath for 5 min. After cooling, measure the absorbance at 540 nm and plot the glucose concentration-absorbance standard curve.

[0056] The process for measuring the enzyme activity of the crude enzyme solution sample of the strain is as follows: Pipette 0.5 mL of the crude enzyme solution of the strain into a 25-mL stoppered colorimetric tube, place it in a water bath at 50 °C for 10 min, add 1.5 mL of a 1% sodium carboxymethylcellulose substrate solution by mass fraction, vortex, then place it in a water bath at 50 °C for 30 min. Subsequently, add 3 mL of DNS reagent, vortex, and place it in a boiling water bath for 5 min. Immediately add 4 mL of 1 mol / L NaOH to terminate the reaction. Wait for the reaction system to cool to room temperature, make up the volume to the 25-mL graduation line, shake well, measure the OD value at 540 nm, and use the sample that has been pre-treated in a boiling water bath as the blank control. The definition of cellulase enzyme activity under this method is: The amount of enzyme required to hydrolyze 1 μg of reducing sugar (calculated as glucose) from the substrate sodium carboxymethylcellulose per minute under the conditions of 50 °C and pH 4.8 is defined as 1 enzyme activity unit.

[0057] 1.10 Analysis of flavor characteristics produced by the strain under simulated solid-state fermentation conditions of Daqu

[0058] Inoculate the simulated solid-state fermentation medium of Daqu with 5% - 8% of the secondary seed culture solution of the strain, and culture it at 45 °C for 3 d to obtain the solid-state fermentation sample of the strain under the simulated Daqu environment. Set 3 parallels for each sample.

[0059] The flavor compounds in the solid-state fermentation sample of the strain were analyzed by headspace solid-phase microextraction (HS-SPME) (fiber type: DVB / CAR / PDMS, Supelco) and gas chromatography-mass spectrometry (GC-MS) platforms. Put the weighed 5 g of the solid-state fermentation sample into a 20-mL headspace vial, and add 2 μL of 2-octanol with a concentration of 5 mg / L as the internal standard. Insert the aged three-phase extraction head into the headspace of the sample vial, enrich and adsorb at 50 °C for 30 min. After adsorption, insert the extraction head into the gas chromatography injection port and desorb at 250 °C for 3 min, and start the instrument to collect data simultaneously. Use an Agilent 7890 gas chromatography system and a 5977B mass spectrometer for gas chromatography-mass spectrometry analysis. This system uses DB-Wax (30 m × 0.25 mm × 0.15 μm, Agilent). Inject in splitless mode, and the desorption time is 4 min. The carrier gas is helium (purity greater than 99.995%), and the gas flow rate through the chromatographic column is 1 mL / min. The programmed temperature rise process is as follows: Keep the column temperature at 40 °C for 4 min, then rise to 245 °C at a rate of 5 °C / min and keep it for 5 min. The injection temperature, transfer line temperature, ion source temperature, and quadrupole temperature are 250, 250, 230, and 150 °C, respectively. The ionization energy of the electron impact (EI) mass spectrometry is set at 70 eV (m / z: 20 - 400).

[0060] The ChromaTOF workstation was used to collect, control, and process the raw data. The mass spectra of the compounds were compared with the Nist database for compound identification. The peak areas were normalized and used to determine the relative content of flavor compounds based on the content of the internal standard.

[0061] 2. Experimental results:

[0062] 2.1 Morphological description of strain 4.019

[0063] Macroscopically, the colony shape of strain 4.019 was generally notched-round, slightly raised in the middle, with obvious radial folds on the colony surface. The colony texture was relatively dry and dense, with a certain color difference between the inner and outer circles. The colony was generally not easy to pick up. The aerial mycelium was milky white and the mycelium was relatively developed. The colony diameter was in the range of 10 mm to 12 mm, and no soluble pigment was produced (attached Figure 1 ).

[0064] Microscopically, the morphological characteristics of strain 4.019 are as shown in the attachment Figure 2 . The results showed that the mycelium of this strain was composed of medium-length dendritic hyphae. The conidia were irregularly round, about 1 μm in diameter, directly attached to the aerial mycelium with a high density, and the conidia density was even higher and aggregated in clusters at the end of the conidiophores. The conidiophores of the strain were relatively short. Generally speaking, the microscopic characteristics of strain 4.019 were consistent with those of known thermophilic actinomycetes.

[0065] 2.2 Growth temperature range and optimum growth temperature of the strain

[0066] The growth temperature range of strain 4.019 was between 45°C and 60°C, and the optimum growth temperature was 55°C, indicating that this strain had very typical thermophilic growth characteristics. Most strains in the thermophilic actinomycetes group were thermophilic microorganisms, and this result was also consistent with this characteristic.

[0067] 2.3 Analysis of the physiological and biochemical characteristics of the strain

[0068] Judging from the results, strain 4.019 was a Gram-positive bacterium, showing positive results in physiological and biochemical indicators such as gelatin liquefaction, milk coagulation and peptone formation, and starch hydrolysis, while negative results in melanin production and hydrogen sulfide production. In terms of carbon and nitrogen source utilization, strain 4.019 could utilize glucose, trehalose, lactose, and D-mannitol as its own growth carbon sources, and could also effectively utilize ammonium sulfate, threonine, glutamic acid, and tyrosine as growth nitrogen sources.

[0069] 2.4 Molecular biological identification of the strain

[0070] The phylogenetic tree analysis of the 16S rRNA of the strain is shown in the attachment Figure 3As shown, the valid species with the highest similarity to strain 4.019 is Thermoactinomyces vulgaris. The sequence similarity with the type strain Thermoactinomyces vulgaris KCTC 9076 is 98.62%, and they are clustered on a phylogenetic branch with extremely small evolutionary distance differences. Therefore, combining the above-mentioned morphological characteristics and physiological and biochemical identification results, it is determined that strain 4.019 belongs to the species under the genus Thermoactinomyces in the order Bacillale, family Thermoactinomycetaceae, and is thus named Thermoactinomyces vulgaris 4.019.

[0071] 2.5 Analysis of the cellulase production ability of the strain

[0072] The analysis results show (attached Figure 4 ) that the strain Thermoactinomyces vulgaris 4.019 has good characteristics of producing cellulase under this liquid fermentation condition, and the average enzyme activity measured for the strain can reach 60.08 U / mL. Cellulase is closely related to the degradation of macromolecules in raw materials during Baijiu brewing and can effectively promote the saccharification of raw materials. The research results also show again that strain 4.019 has a certain function of promoting the saccharification and degradation of brewing raw materials, and its potential for producing cellulase under liquid conditions is also worthy of further exploration in the future.

[0073] 2.6 Analysis of flavor production by the strain under simulated solid-state fermentation conditions of Daqu

[0074] Judging from the analysis results, strain 4.019 can produce a total of 47 flavor components under simulated solid-state fermentation conditions of Daqu. Mainly included are 13 aldehyde and ketone compounds, 8 aromatic compounds, 5 pyrazine compounds, 5 esters, 5 nitrogen-containing heterocyclic compounds, 3 organic acids, 1 alcohol, and 5 other flavor components. Among them, 2,5-dimethylpyrazine, 2-ethyl-3-methylpyrazine, 2-ethenyl-5-methylpyrazine, acetoin, etc. are relatively typical flavor components, and the average contents reach 31.48 μg / g Daqu, 8.07 μg / g Daqu, 1.53 μg / g Daqu, 15.51 μg / g Daqu respectively (attached Figure 5)。The analysis results are also very close to the flavor-producing characteristics of Bacillus subtilis and Bacillus licheniformis in Jiangxiang Daqu. Generally speaking, both pyrazine and acetoin are important aroma components of high-temperature Jiangxiang Daqu. Pyrazine can effectively endow the Daqu with unique baking and burnt aroma, while acetoin is not only an important precursor for pyrazine synthesis, but also can endow a certain light and elegant creamy aroma by itself.

[0075] This result undoubtedly implies that the common high-temperature actinomycete strain 4.019 (Thermoactinomyces vulgaris 4.019) has good ability to produce pyrazine and acetoin flavor components under solid-state fermentation conditions, and it is a strain with excellent fermentation characteristics in high-temperature Jiangxiang Daqu. This also lays an important research foundation for the large-scale production and application of this strain in the liquor industry in the future.

[0076] The specific embodiments of the present invention have been described above. It should be noted in particular that the specific features, structures, materials, etc. involved in the present invention are not limited to the above specific embodiments, and those skilled in the art can make various deformations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A thermophilic actinomycete with the characteristics of producing pyrazine, acetoin and cellulase, characterized in that: The preservation number of this strain is: CCTCC M 2025443, and the name is: Thermoactinomyces vulgaris 4.

019.

2. The thermoactinomycetes according to claim 1, characterized in that: The aerial hyphae and conidia of the described strain are both milky white, and the colony shape is a certain notched circle.

3. The thermoactinomycetes according to claim 1, characterized in that: The growth temperature range of the described Thermoactinomyces is 45°C to 60°C.

4. Application of the Thermoactinomyces according to claim 1 in enhancing the flavor of Jiangxiang Daqu.

5. Application of the Thermoactinomyces according to claim 1 in the brewing of traditional Jiangxiang-flavor Baijiu.

6. Application of the Thermoactinomyces according to claim 1 in the synthesis of pyrazine or acetoin.

7. Application of the Thermoactinomyces according to claim 1 in the preparation of cellulase.

8. The application according to claim 5 or 6, characterized in that: Add the described Thermoactinomyces to the fermentation process of Jiangxiang Daqu.

9. The application according to claim 5 or 6, characterized in that: Inoculate Thermoactinomyces vulgaris 4.019 into a liquid expansion culture medium to prepare a seed liquid, and the inoculation amount is 3-5% (v / v); further, the liquid fermentation temperature is 48°C, and the liquid fermentation time is 48-54 h.

10. The application according to claim 5 or 6, characterized in that: Inoculate Thermoactinomyces vulgaris 4.019 into a solid pure wheat fermentation medium, and the inoculation amount is 5-8% w / v; further, the solid fermentation temperature is 50-55°C, and the fermentation time is 4-5 days; the solid pure wheat medium is prepared by adding 38% water to 100% wheat dedicated for koji making and sterilizing at 121°C for 30 min.

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