Clostridium for producing phenethyl alcohol and organic acid and application of clostridium

Abstract

The invention belongs to the technical field of microorganisms and wine brewing, and relates to clostridium for producing phenethyl alcohol and organic acid and application of the clostridium. The clostridium sp. MCGM14 is preserved in the China General Microbiological Culture Collection Center on July 4, 2023, and the preservation number of the clostridium sp. MCGM14 is CGMCC No. 1.18017. The clostridium sp. MCGM14 can be used for preparing the clostridium sp. The strain can effectively utilize phenylalanine to rapidly synthesize phenethyl alcohol under a strict anaerobic condition, and utilize various sugars and lactic acid in a pit to synthesize various organic acids, so that the strain can be used in a wine brewing process, the flavor and quality of white wine are improved, and microbial enhancement and improvement of a new pit (pit mud) are realized.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and brewing, and relates to a clostridium capable of producing phenylethanol and organic acid and a use thereof. Background Art

[0002] β-Phenylethyl alcohol, also known as phenylethyl alcohol, has a molecular formula of C8H 10 O, molecular weight is 122.17. It is a colorless viscous liquid, boiling point 219℃, relative density 1.0230, refractive index 1.5310~1.5340. It is found in natural plants such as apples, almonds, bananas, peaches, pears, strawberries, cocoa, honey, etc. It has a soft, pleasant and lasting rose aroma, and is widely used as a spice in food flavors, cosmetics, and washing products. At the same time, β-phenylethanol is also an important flavoring substance in traditional fermented foods such as liquor, rice wine, sake, fruit wine, grape wine, soy sauce, bread, cheese, etc. For example, it is the main aroma in rice-flavored liquor, and it is not only an important characteristic flavor substance in strong-flavored liquor, sauce-flavored liquor, soy-flavored liquor and rice wine, but also plays a synergistic and synergistic role in other flavor substances in these fermented foods.

[0003] At present, two pathways for microbial metabolism to produce phenylethanol have been discovered, mainly the shikimate pathway and the Ehrlich pathway: the shikimate pathway is a metabolic pathway for Saccharomyces cerevisiae to synthesize β-phenylethanol from scratch, with the substrates being phosphoenolpyruvate in the glycolysis pathway and 4-phosphate erythrose in the pentose phosphate pathway; the Ehrlich pathway is a pathway in which yeast uses L-phenylalanine as a precursor to synthesize β-phenylethanol through aminotransferases and decarboxylases. Previously, research on microorganisms that ferment to produce phenylethanol mainly focused on the production of phenylethanol by various yeasts in an aerobic environment.

[0004] The brewing microbial system of Maotai-flavor liquor is complex, and anaerobic microorganisms in the cellar mud require relatively strict isolation and culture conditions. There are few reports on the isolation, screening and application of phenylethanol-producing bacteria in the anaerobic environment of the cellar bottom. Organic acids also play a vital role in the brewing process.

[0005] Based on this, the present invention aims to provide a Clostridium that can produce high levels of phenylethanol and multiple organic acids. Summary of the invention

[0006] The present invention aims to provide a clostridium that can metabolize and produce abundant organic acids, phenylethanol and other substances under actual brewing environment. The clostridium is screened and separated from wine cellar mud. The bacterium can effectively utilize phenylalanine to rapidly synthesize phenylethanol under strictly anaerobic conditions, and utilize various sugars and lactic acid in the cellar to synthesize various organic acids. It can then be used in the brewing process to improve the flavor and quality of liquor, as well as the microbial enhancement and improvement of new cellars (cellar mud), and has important utilization value.

[0007] On the one hand, the present invention provides a strain of Clostridium sp. mCGM14, which was deposited in the General Microbiology Center of China Association for the Collection of Microbiological Cultures on July 4, 2023, with the deposit number CGMCC No.1.18017.

[0008] This strain has been deposited in the General Microbiology Center of China Association for Microbiological Culture Collection on July 4, 2023, with the deposit number CGMCC No.1.18017. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The classification name is Clostridium sp.mCGM14.

[0009] 1) Strain source: The Clostridium obtained in the present invention was screened and isolated from the brewing process of Maotai-flavor liquor, specifically from the wine cellar mud, and obtained by enrichment and gradient dilution separation. It has the characteristics of being able to produce a variety of organic acids, as well as phenylethanol, isopropanol and other substances in high yield.

[0010] 2) Colony characteristics: After 72 hours of plate culture, the colonies of the Clostridium mCGM14 are colorless or ivory, translucent, round, smooth, 1-2 mm in diameter, with neat edges, easy to pick, and Gram-positive.

[0011] 3) Bacterial characteristics: Under transmission electron microscopy, the cells of the Clostridium mCGM14 strain are rod-shaped, produce spores, have flagella at the ends, are about 2-5 um long, and are mostly 0.4-0.6 um wide.

[0012] 4) Physiological and biochemical characteristics: The physiological and biochemical characteristics of the bacteria were analyzed by the BiologAN microplate anaerobic identification system: the Clostridium mCGM14 can utilize D-fructose, D-galactose, α-D-glucose, glycerol, D-mannose, methyl pyruvate and L-glutamic acid, etc.

[0013] 5) Tolerance: The Clostridium strain mCGM14 has a temperature tolerance range of 16-55°C, an optimum growth temperature of 37°C, and an OD value of 0.35 at 37°C; a pH tolerance range of 4-9, an optimum growth pH of 5.5, and an OD value of 0.45 at a pH of 5.5; a salinity tolerance range of 0-3.0%, an optimum growth salinity of 0.5%, and an OD value of 0.31 at a salinity of 0.5%; and an ethanol tolerance range of 0-17%, with an optimum growth ethanol concentration of 0-1%.

[0014] On the other hand, the present invention also provides a starter culture of the Clostridium mCGM14.

[0015] In some embodiments, the living cells and / or dry bacteria and / or wet bacteria and / or immobilized cells and / or liquid bacterial agent and / or solid bacterial agent of the Clostridium mCGM14 are contained.

[0016] On the other hand, the present invention also provides a use of the Clostridium mCGM14 or the fermentation agent in the field of fermentation.

[0017] In some embodiments, the fermentation field includes: fermentation for winemaking and / or fermentation for food preparation and / or fermentation for cosmetics preparation and / or fermentation for cleaning products preparation and / or fermentation for flavors preparation, and also includes the preparation of various other products requiring phenylethanol.

[0018] In some embodiments, the fermentation field includes the preparation of white wine, yellow wine, sake, fruit wine, grape wine, soy sauce, bread, and cheese.

[0019] In some embodiments, the winemaking includes brewing white wine, yellow wine, sake, fruit wine, and grape wine.

[0020] In some embodiments, the brewed liquor includes brewing Luzhou-flavor liquor, Maotai-flavor liquor, and Chi-flavor liquor, and also includes liquors of various other flavors.

[0021] On the other hand, the present invention also provides a use of the Clostridium mCGM14 or the fermentation agent in fermentation to produce alcohols and / or organic acids.

[0022] In some embodiments, the alcohol production includes: phenylethanol production and / or isopropanol production.

[0023] In some embodiments, producing phenylethanol comprises: producing phenylethanol via the shikimate pathway or the Ehrlich pathway.

[0024] In some embodiments, producing phenylethanol comprises: producing phenylethanol by fermentation using sugar or phenylalanine or L-phenylalanine.

[0025] In some embodiments, producing the organic acid comprises: utilizing a carbon source to produce the organic acid.

[0026] In some embodiments, the organic acid includes at least one or more of acetic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, caproic acid, phenylacetic acid, palmitic acid, and polyhydroxystearic acid.

[0027] On the other hand, the present invention also provides a use of the Clostridium mCGM14 in artificial pit mud cultivation.

[0028] In some embodiments, the artificial pit mud cultivation includes the maturation and / or improvement of new pits and / or pit mud.

[0029] On the other hand, the present invention also provides a method for cultivating artificial pit mud, comprising the step of adding the Clostridium mCGM14.

[0030] In some embodiments, the method comprises the steps of:

[0031] (1) taking cellar mud, preparing it into a cellar mud water suspension, and sterilizing it;

[0032] (2) Adding the bacterial liquid of the Clostridium mCGM14 into the cellar mud water suspension for anaerobic culture to obtain cultivated cellar mud.

[0033] In some embodiments, in step (1), the method for preparing the cellar mud water suspension comprises the following steps: placing the cellar mud in an anaerobic bottle, adding anaerobic distilled water to submerge the cellar mud, and shaking violently to obtain the cellar mud water suspension.

[0034] In some embodiments, the anaerobic distilled water is obtained by adding 0.1% cysteine ​​hydrochloride to distilled water and then sterilizing it.

[0035] In some embodiments, in step (2), the bacterial solution of Clostridium mCGM14 is prepared by the following steps:

[0036] 1) Take Clostridium mCGM14 cultured to the logarithmic phase, centrifuge under anaerobic conditions, and discard the upper suspension;

[0037] 2) resuspending with 5-15 times the volume of the Clostridium mCGM14 cells in anaerobic PBS, centrifuging at high speed, and repeating 2-5 times;

[0038] 3) taking the centrifuged bacterial cells and resuspending them again to obtain the bacterial solution.

[0039] In some embodiments, the anaerobic PBS is obtained by adding 0.1% cysteine ​​hydrochloride to the PBS and then sterilizing it.

[0040] In some embodiments, the high-speed centrifugation speed is 6000g-10000g; preferably, the high-speed centrifugation speed is 7000g-9000g.

[0041] In summary, the present application includes at least one of the following beneficial technical effects:

[0042] (1) The present invention provides a new strain of Clostridium, which can effectively utilize phenylalanine to rapidly and efficiently synthesize phenylethanol under strictly anaerobic conditions. Based on this, the new strain can be used in the winemaking process to convert phenylalanine accumulated in the cellar into phenylethanol.

[0043] (2) The new bacteria provided by the present invention can be used to efficiently ferment and produce phenylethanol. For example, in Example 4, the phenylethanol yield of the Clostridium mCGM14 in a CNM medium supplemented with 12 g / L phenylalanine can be as high as 123.84 mg / L. Based on this, it can be used to prepare food flavors, cosmetics, washing products and other fields that require spices; at the same time, phenylethanol is also an important flavoring substance in traditional fermented foods such as white wine, yellow wine, sake, fruit wine, grape wine, soy sauce, bread, cheese, etc., so it can be used to ferment and prepare various foods.

[0044] (3) The new bacterium mCGM14 provided by the present invention can utilize various sugars and lactic acid in the cellar to synthesize various organic acids. Based on this, the new bacterium can be used for microbial improvement of new cellars (or cellar mud) to quickly convert various sugars and lactic acid in the new cellar into various organic acids.

[0045] (4) The new bacteria mCGM14 provided by the present invention can utilize various sugars and lactic acid in the cellar to synthesize various organic acids, including acetic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, caproic acid, phenylacetic acid, palmitic acid, and polyhydroxystearic acid, etc. Among them, butyric acid and acetic acid are important substrates for synthesizing caproic acid, and caproic acid is a precursor substance for microbial metabolism to produce ethyl caproate. Therefore, it can be used in the winemaking process to indirectly increase the concentration of caproic acid, ethyl butyrate, and ethyl caproate, and further improve the flavor and quality of liquor. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIG. 1 is an electron microscope image of Clostridium mCGM14 in an embodiment of the present invention; FIG. 1(A) is a transmission electron microscope image of Clostridium mCGM14, and FIG. 1(B) is a scanning electron microscope image of Clostridium mCGM14;

[0047] Figure 2 The phylogenetic tree result in Example 2 of the present invention;

[0048] FIG3 shows the fermentation product results of strain mCGM14 obtained by fermentation using CNM original medium in Example 4 of the present invention; wherein FIG3(A) is a chromatogram of the product fermented using the original CNM medium, and FIG3(B) is a search result of the corresponding product peak in the spectral library;

[0049] Figure 4 The average phenylethanol yield corresponding to the fermentation of the original CNM medium with different concentrations of phenylalanine (3 g / L, 6 g / L, 12 g / L) in Example 4 of the present invention;

[0050] Figure 5 is a chromatogram of the product peaks of the strain mCGM14 obtained by adding different concentrations of glucose and different concentrations of phenylalanine to the CNM original medium for fermentation in Example 4 of the present invention, and the search results of the corresponding product peaks in the spectrum library; wherein Figure 5 (A) is a chromatogram of the product peaks of the strain mCGM14 obtained by adding 0g / L, 5g / L, and 15g / L of glucose to the CNM original medium for fermentation, and Figure 5 (B) is a chromatogram of the product peaks of the strain mCGM14 obtained by adding 0g / L, 3g / L, 6g / L, and 12g / L of phenylalanine to the CNM original medium for fermentation;

[0051] Figure 6 shows the fermentation results of the experimental group in which mCGM14 bacterial solution was added to the cellar mud suspension and the control group in which no bacterial solution was added; wherein, Figure 6(A) is the chromatogram of the experimental group in which mCGM14 bacterial solution was added to the cellar mud suspension and the control group, Figure 6(B) marks the relatively obvious fermentation product peaks and spectral library search results in the experimental group in the time interval of 0-22min, and Figure 6(C) shows the relatively obvious fermentation product peaks and spectral library search results in the experimental group in the time interval of 19-34min; in Figure 6(B), "①" represents isopentanol, "②" represents n-hexanol, "③" represents acetic acid, "④" represents isobutyric acid, "⑤" represents n-butyric acid, and "⑥" represents isovaleric acid; in Figure 6(C), "①" represents n-hexanoic acid, "②" represents phenylethanol, and "③" represents 2,6-di-tert-butyl-p-methylphenol. DETAILED DESCRIPTION

[0052] The technical solution of the present invention is further described below by specific embodiments, which do not limit the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0053] Unless otherwise specified, the equipment, materials, etc. used in the following examples can be obtained from commercial or public sources.

[0054] The Clostridium sp. mCGM14 used in the embodiment of the present invention is a new species of the genus Clostridium of the family Clostridiaceae obtained by screening and separation from wine cellar mud. The strain has been deposited in the General Microbiology Center of the China Association for the Collection of Microbiological Cultures on July 4, 2023, with a deposit number of CGMCC No.1.18017, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and is classified and named Clostridium sp. mCGM14.

[0055] The CNM medium formula used in the embodiment of the present invention is: beef extract 10g / L, glucose 5g / L, casein peptone 5g / L, yeast extract 3g / L, soluble starch 1g / L, sodium chloride 5g / L, sodium acetate 3g / L, cysteine ​​0.5g / L, pH 6.8.

[0056] The method for determining the fermentation product in the fermentation broth by GCMS in the embodiment of the present invention is as follows:

[0057] Fermentation products such as short-chain fatty acids and phenylethanol were determined by gas chromatography-mass spectrometry. 1 mL of culture was extracted with 1 mL of ethyl acetate, and the supernatant was centrifuged for GC-MS analysis, which was performed on a GCMS with an autosampler and a DB capillary column (30 m, 0.25 mm id, 0.25 μm film thickness). The temperature of the oven was set to rise from 80°C to 140°C at a rate of 20°C / min, maintained for 1 minute, and then raised to 290°C at a rate of 3.5°C / min and maintained for 15 minutes. 1 μL of sample was injected at 280°C. The carrier gas helium flowed at a rate of 1.2 mL / min. The electron impact was recorded as 70 eV.

[0058] Example 1 Screening and enrichment of strains

[0059] (1) Bacterial enrichment

[0060] 500 mL of CNM culture medium was placed in a 1000 mL anaerobic serum bottle and sealed. The gas above the liquid surface was replaced with nitrogen using a vacuum ventilator. The bottle was sterilized at 115°C for 20 minutes and placed in an anaerobic glove box.

[0061] Take 1 g of cellar mud, treat it with 70% alcohol for 6 hours, rinse and resuspend it with distilled water three times, centrifuge it and place it in the above anaerobic serum bottle containing culture medium for enrichment culture, and enrich and culture it at 30°C for 7-8 days. Use GCMS to detect the volatile substances in the enriched culture solution. If the product peaks of short-chain organic acids and phenylethanol are detected, it is determined to be the target cellar mud enriched solution.

[0062] (2) Initial screening of bacterial strains

[0063] After the CNM medium is prepared, 15 g agar is added per liter, oxygen is removed from the medium, and the medium is sterilized by high pressure to prepare the medium plate in an anaerobic glove box. -3 , 10 -4 , 10 -5 , 10 -6 The cells were diluted in series, spread on CNM medium plates, and cultured anaerobically in a 37°C incubator for 2-3 days.

[0064] (3) Select a plate with 100-200 colonies on it and a suitable gradient plate on which the colonies do not overlap each other. Select colonies with white or light yellow surface, translucent or opaque, smooth edges or outward diffusion, and a villous network. Pick them and streak them onto a freshly prepared CNM plate for streak purification.

[0065] (4) Strain purity inspection

[0066] The pure strain was determined based on the colony morphology and distribution on the streaking plate. The pure strain was further tested by colony PCR and first generation sequencing. The obtained pure colonies were streaked or punctured on anaerobic slants for storage.

[0067] (5) Rescreening of bacterial strains

[0068] The strain was inoculated into CNM liquid culture medium containing 3 g / L phenylalanine and cultured for 2-3 days. GCMS was used to detect whether the strain produced organic acid or phenylethanol.

[0069] Example 2 Identification and preservation of strains

[0070] (1) Morphological characteristics identification

[0071] Colony characteristics: After 72 hours of plate culture on CNM medium, the strain mCGM14 obtained by the above method was translucent or ivory, round, smooth, 1-2 mm in diameter, with neat edges and easy to pick.

[0072] Bacterial characteristics: Gram-positive, spore-producing, flagella-possessing, motile, rod-shaped, 2-8um long, 0.4-0.7um wide. The transmission electron micrograph is shown in Figure 1(A), and the scanning electron micrograph is shown in Figure 1(B).

[0073] (2) Identification of physiological and biochemical characteristics

[0074] The physiological and biochemical characteristics of the bacteria were analyzed by Biolog AN microplate anaerobic identification system: strain mCGM14 can utilize D-fructose, D-galactose, α-D-glucose, glycerol, D-mannose, methyl pyruvate and L-glutamate.

[0075] Fatty acid and polar lipid determination: Cellular fatty acids were extracted and methylated according to the standard protocol of MIDI (Sherlock Microbiological Identification System, version 6.0). Identification was performed by GC (HP 6890 Series GC System; Agilent). Polar lipids were separated by two-dimensional thin layer chromatography (TLC plates coated with silica gel, 1010 cm; Merck). Chromatography used chloroform / methanol / water (volume ratio of 65:25:4) as the first dimension, followed by chloroform / ethanol / acetic acid / water (volume ratio of 80:12:15:4) for the second dimension. Total lipids were detected with 10% ethanol molybdophosphoric acid (Sigma). Amino lipids were detected with 0.4% ninhydrin (Sigma) in butanol. Phospholipids were detected with Zinzadze reagent (molybdenum blue spray reagent, 1.3%; Sigma), and glycolipids were detected with 0.5% α-naphthol reagent.

[0076] The results showed that the main cellular fatty acids of mCGM14 were C14:0 (40.8%) and C16:0 (15.5%), and the polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine.

[0077] (3) Molecular identification

[0078] The genomic DNA of the screening strain mCGM14 was extracted using the QIAGEN bacterial genome extraction kit, and the 16S rRNA gene of mCGM14 was amplified using the 16S rRNA universal primers 27F and 1492R. The amplified fragment of about 1500 bp was sent to a sequencing company for sequencing. The sequence obtained by sequencing (as shown in SEQ ID NO.1) was compared with the BLAST sequence on the official website of NCBI. The obtained 16S rDNA sequence of mCGM14 was compared with the BLAST sequence, and a phylogenetic analysis was performed. The results are as follows: Figure 2 And as shown in Table 1.

[0079] Genomic DNA libraries were sequenced using the Illumina Hiseq X-ten platform. All paired reads of good quality were assembled using SPAdes software (v3.9.0). Average nucleotide identity (ANI) values ​​were calculated using OAT software http: / / www.ezbiocloud.net / sw / oat. Genomic distances, digital DNA-DNA hybridization (dDDH), were calculated using the genome-to-genome distance calculator (GGDC; http: / / ggdc.dsmz.de / ).

[0080] According to the phylogenetic tree and the comparison results in Table 1: the 16S rRNA gene sequence of the newly screened strain mCGM14 is most closely related to the Clostridium luticellar in the database, with a similarity of 97.34%. According to the important criteria for judging new bacterial species, a 16S rRNA gene sequence with a similarity of less than 98.65% with a known species can be judged as a new species (reference for new species judgment criteria: Kim, et al. Chapter 4 - 16S rRNA Gene - Based Identification of Bacteria and Archaea using the EzTaxon Server,2014,IJSEM ), that is, the similarity between the 16S rRNA gene sequence of strain mCGM14 and the 16S rRNA gene sequence of known species is less than 98.65%.

[0081] Furthermore, the genome comparison results of mCGM14 and Clostridium sphaeroides showed that the ANIb% of mCGM14 and Clostridium sphaeroides was 83.93% (as shown in Table 2), and the dDDH% was 28.6% (as shown in Table 3). That is, the ANI% of the bacterium mCGM14 and the currently preserved standard strains was less than 95%, and the dDDH% was less than 70%.

[0082] Combined with the colony morphology of the strain, as well as the results of physiological and biochemical identification, the main cellular fatty acids of strain mCGM14 are C14:0 and C16:0. The polar lipids are diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine, and the secondary lipids include three unknown phospholipids and two unknown glycolipids. The genome size is 4,326,664bp, and the G+C content is 33.78mol%. The microscopic morphology of the bacteria, the main carbon source that can be utilized, and the G+C content are similar to those of Clostridium luticellar in Clostridium cluster I (Wang et al., Clostridium luticellari isp.nov., isolated from a mud cellar used for producing strong aromatic liquors, 2015, IJSEM).

[0083] Based on the comparison results of gene sequence similarity, ANI%, dDDH%, morphological characteristics, physiological and biochemical identification characteristics of the above-mentioned strain mCGM14 and the most closely related model strain Clostridium luticellar, it was further determined that the strain mCGM14 screened by the above method was a new species of the genus Clostridium in the family Clostridiaceae, named Clostrdium sp. mCGM14.

[0084] In addition, among the clostridium strains screened here, two new strains of the same genus Clostridium were also screened: Clostridium sp. mCGM113 and Clostridium sp. mCGM5, with the preservation numbers CGMCC No.1.18018 and CGMCC No.1.18016, respectively, and patents have been applied for. After calculation and comparison: the 16S rRNA gene sequence similarity between mCGM14 and strain mCGM113 (CGMCC No.1.18018) is 96.28%, ANIb% is 81.83%, and dDDH% is 25.4%; the 16S rDNA gene sequence similarity between mCGM14 and strain mCGM5 is 97.35%, ANIb% is 82.18%, and dDDH% is 25.4%. That is, the 16S rRNA gene sequence similarity between the new strain mCGM14 for which protection is sought in the present invention and mCGM113 and mCGM5 is less than 98.65%, the ANI% is less than 95%, and the dDDH% is less than 70%. The new bacterium mCGM14 for which protection is sought in the present invention is also completely different from the other two new strains and is a new bacterium of the genus Clostridium.

[0085] Table 1 Model strains related to mCGM14 and their sequence similarities

[0086] strain Accession Similarity Clostridium luticellarii NR_145907 97.34％ Clostridium ljungdahlii DSM 13528 NR_117113 95.27％ Clostridium autoethanogenum DSM 10061 NR_121758 95.26％ Clostridium kluyveri NBRC 12016 NR_074447 94.57％ Clostridium autoethanogenum DSM 10061 NR_119283 95.14％ Clostridium algifaecis NR_134004 94.59％ Clostridium kluyveri DSM 555 NR_074165 94.39％ Clostridium pabulibutyricum NR_159224 94.43％ Clostridium tyrobutyricum NR_044718 93.61％ Clostridium rectalis NR_179639 91.76％ Clostridium fermenticellae NR_171400 91.55％ Clostridium tepidum NR_157639 91.34％

[0087] Table 2 Calculation of average nucleotide identity (ANIb%) values

[0088] ANIb(%) mCGM113 C.luticellarii mCGM14 mCGM5 mCGM113(CGMCC1.18018) n.d 79.71 81.83 78.92 Clostridium luticellarii n.d n.d 83.93 81.83 mCGM14(CGMCC1.18017) n.d n.d n.d 82.18 mCGM5(CGMCC1.18016) n.d n.d n.d n.d

[0089] ANIb(%) mCGM113 C.luticellarii mCGM14 mCGM5 mCGM113(CGMCC1.18018) n.d 79.71 81.83 78.92 Clostridium luticellarii n.d n.d 83.93 81.83 mCGM14(CGMCC1.18017) n.d n.d n.d 82.18 mCGM5(CGMCC1.18016) n.d n.d n.d n.d

[0090] Table 3 Digital DNA-DNA hybridization calculations

[0091] dDDH(%) mCGM113 C.luticellarii mCGM14 mCGM5 mCGM113(CGMCC1.18018) n.d 23.4 25.4 22.2 Clostridium luticellarii n.d n.d 28.6 25.1 mCGM14(CGMCC1.18017) nd nd nd 25.4 mCGM5 (CGMCC1.18016) nd nd nd nd

[0092] (4) Patent deposit of strains

[0093] The obtained strain mCGM14 (Clostridium sp.mCGM14) was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration for patent culture preservation. The specific address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101. After strain review and activity testing, the collection center issued a patent strain deposit certificate on July 4, 2023, numbered CGMCC No.1.18017.

[0094] Example 3 Growth ability of mCGM14 strain under different culture conditions

[0095] The brewing environment has a very important influence on the growth and metabolism of microorganisms. The fermentation temperature, substrate acidity, fermentation salinity, fermentation ethanol concentration, etc. in the brewing process of liquor are constantly changing, causing the environment in which the microorganisms are located to change all the time. This embodiment further explores the growth of strain mCGM14 under different fermentation temperatures, different acidities, different salinities, and different ethanol conditions.

[0096] Temperature tolerance experiment: Prepare a Henle tube containing 5 mL CNM liquid culture medium, and add the mCGM14 bacterial solution grown to the logarithmic phase into the anaerobic tube at a ratio of 1:50. Place the anaerobic tube in a constant temperature box at 16℃, 25℃, 30℃, 37℃, 42℃, 45℃, 50℃, and 55℃ for 72 hours. Take 100uL of the culture solution every 12 hours and detect the OD of the bacterial solution on the microplate reader. 600 The results are shown in Table 4.

[0097] Acid tolerance experiment: Prepare Henle tubes containing 5 mL CNM liquid culture medium, adjust the pH value of the culture medium to 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, and 9, respectively, add mCGM14 bacterial solution grown to the logarithmic phase into the tubes at a ratio of 1:50, place the anaerobic Henle tubes in a 37°C incubator and culture for 72 hours, take 100uL of the culture medium every 12 hours and detect the OD of the bacterial solution on an ELISA reader 600 The results are shown in Table 5.

[0098] Salinity tolerance experiment: Prepare Henle tubes containing 5 mL CNM liquid culture medium, adjust the salinity of the culture medium to 0, 0.5%, 1%, 1.5%, 2%, 2.5%, and 3%, respectively, add the mCGM14 bacterial solution grown to the logarithmic phase into the tube at a ratio of 1:50, place the anaerobic Henle tube in a 37°C incubator and culture for 72 hours, take 100uL of the culture medium every 12 hours and detect the OD of the bacterial solution on an ELISA reader 600 The results are shown in Table 6.

[0099] Ethanol tolerance experiment: Prepare Henle tubes containing 5 mL CNM liquid culture medium, add ethanol with final concentrations of 0, 1%, 3%, 5%, 7%, 9%, 11%, 13%, 15%, and 17% to the culture medium, respectively, inoculate the mCGM14 bacterial solution grown to the logarithmic phase into the tubes at a ratio of 1:50, and place the anaerobic Henle tubes in a 37°C incubator for 72 hours. The results are shown in Table 7.

[0100] Table 4 Temperature tolerance test

[0101] temperature 16℃ 25℃ 30℃ 37℃ 42℃ 45℃ 50℃ 55℃ OD value 0.09 0.14 0.28 0.35 0.31 0.28 0.18 0.10

[0102] Table 5 Acidity tolerance test

[0103] pH 4 4.5 5 5.5 6 6.5 7 7.5 8 9 OD value 0.11 0.24 0.42 0.45 0.30 0.31 0.31 0.25 0.15 0.04

[0104] Table 6 Salinity tolerance experiment

[0105]

[0106]

[0107] Table 7 Ethanol tolerance test

[0108] Ethanol 0％ 1％ 3％ 5％ 7％ 9％ 11％ 13％ 15％ 17％ OD value 0.31 0.30 0.27 0.27 0.25 0.20 0.17 0.14 0.10 0.04

[0109] According to the results presented in Tables 4-7 above, the temperature tolerance range of Clostridium strain mCGM14 is 16-55°C, the optimal growth temperature is 37°C, and when the temperature is 37°C, the OD value of strain mCGM14 can reach 0.35; the pH tolerance range is 4-9, the optimal growth pH is 5.5, and when the pH is 5.5, the OD value of strain mCGM14 can reach 0.45; the salinity tolerance concentration range is 0-3.0%, the optimal growth salinity is 0.5%, and when the salinity is 0.5%, the OD value of strain mCGM14 can reach 0.31; the ethanol tolerance concentration range is 0-17%, and the optimal growth ethanol concentration is 0-1%.

[0110] Example 4 Study on the ability of mCGM14 to produce phenylethanol by fermentation

[0111] At present, two pathways for microbial metabolism to produce phenylethanol have been discovered, mainly the shikimic acid pathway and the Ehrlich pathway:

[0112] The shikimate pathway is a metabolic pathway for the de novo synthesis of β-phenylethanol by Saccharomyces cerevisiae, and the substrates are phosphoenolpyruvate of the glycolysis pathway and erythrose 4-phosphate of the pentose phosphate pathway. The Ehrlich pathway is a process in which yeast uses L-phenylalanine as a precursor to synthesize β-phenylethanol through aminotransferases and decarboxylases. Previously, research on microorganisms that ferment to produce phenylethanol mainly focused on the production of phenylethanol by various yeasts in aerobic environments, and there were few reports on the isolation, screening and application of phenylethanol-producing bacteria in anaerobic environments at the bottom of the cellar. The present invention unexpectedly discovered that the new bacterium mCGM14 obtained through the above screening has the ability to ferment to produce phenylethanol, as follows:

[0113] After culturing mCGM14 in the original CNM medium for 72 hours, the fermentation broth was tested by GCMS and a product peak was found at 20.75-21.00 min ( Figure 3A ), which was found to be phenylethanol by spectral library search (see Figure 3B ), after quantitative analysis, the yield of phenylethanol was about 3.02 mg / L.

[0114] On the basis of the original CNM medium, 5g / L and 15g / L glucose were added to the mCGM14 medium and fermented for 72 hours, and then the phenylethanol yield in the fermentation broth was detected, which could reach 3.34mg / L and 5.18mg / L respectively. Figure 5A ). This means that mCGM14 can synthesize phenylethanol from scratch via the shikimic acid pathway, but the yield is not high.

[0115] When phenylalanine was added to the original CNM medium at concentrations of 3, 6, and 12 g / L for 72 hours, the average phenylethanol yields reached 41.85 mg / L, 65.41 mg / L, and 102.21 mg / L, respectively. Figure 5B ). This means that mCGM14 mainly produces phenylethanol via the Ehrlich pathway.

[0116] The above experiments further show that mCGM14 can produce phenylethanol by fermentation in the original CNM medium. The addition of different concentrations of glucose to the original CNM medium can increase the yield of phenylethanol produced by mCGM14 to a certain extent. When different concentrations of phenylalanine are added to the original CNM medium, the yield of phenylethanol produced by mCGM14 is greatly improved, with an increase of about 14-34 times.

[0117] Example 5: Research on the fermentation products of mCGM14

[0118] This example explores the fermentation products of mCGM14 as follows:

[0119] (1) After culturing mCGM14 in CNM medium for 72 hours, the fermentation products in the fermentation broth were detected by GCMS; (2) Glucose of different concentrations of 5 g / L and 15 g / L was added to the original CNM medium, and mCGM14 was inoculated and fermented for 72 hours. The fermentation products in the fermentation broth were detected by GCMS; (3) Phenylalanine of different concentrations of 3 g / L, 6 g / L, and 12 g / L was added to the original CNM medium, and mCGM14 was inoculated and fermented for 72 hours. The fermentation products in the fermentation broth were detected by GCMS.

[0120] The results are shown in Figure 5, Table 8 and Table 9. Figure 5 (A) and Table 8 show the library search results and peak areas of the fermentation products with corresponding retention times obtained by GCMS detection of the corresponding fermentation broths after culturing mCGM14 in the original CNM fermentation medium, the original CNM+5g / L glucose fermentation medium and the original CNM+15g / L glucose fermentation medium for 72 hours; Figure 5 (B) and Table 9 show the library search results and peak areas of the fermentation products with corresponding retention times obtained by GCMS detection of the corresponding fermentation broths after culturing mCGM14 in the original CNM fermentation medium, the original CNM+3g / L phenylalanine fermentation medium, the original CNM+6g / L phenylalanine fermentation medium and the original CNM+12g / L phenylalanine fermentation medium for 72 hours.

[0121] The above results showed that the fermentation products of mCGM14 included not only phenylethanol, but also isopropanol, acetic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, caproic acid, phenylacetic acid, palmitic acid and polyhydroxystearic acid. The concentrations of isobutyric acid, butyric acid and isovaleric acid increased significantly with the increase of glucose concentration in the fermentation broth, while the concentrations of phenylethanol and phenylacetic acid increased significantly with the increase of phenylalanine concentration in the fermentation broth.

[0122] Table 8 Qualitative and quantitative results of fermentation products of mCGM14 in fermentation medium with different glucose concentrations

[0123]

[0124]

[0125] Table 9 Qualitative and quantitative results of fermentation products of mCGM14 in fermentation medium with different phenylalanine concentrations

[0126]

[0127]

[0128] Example 6

[0129] Weigh 200g of each of the mixed pit mud, place it in a 1L anaerobic bottle, add 300mL of pre-prepared anaerobic distilled water (sterilized after adding 0.1% cysteine ​​hydrochloride) to immerse the pit mud, shake it vigorously to prepare a pit mud water suspension, use a suction and ventilation device to replace the air in the bottle with nitrogen repeatedly, and sterilize it at 115°C for 25 minutes;

[0130] Take 50mL of mCGM14 cultured to the logarithmic phase, centrifuge under anaerobism, discard the upper suspension, resuspend with anaerobic PBS (sterilized after adding 0.1% cysteine ​​hydrochloride) 10 times the volume of the bacterial cells, and centrifuge at 8000g for 3 times. Take 0.2g of the wet weight of the bacteria from the centrifuge, resuspend the bacteria with PBS again, inject the bacterial solution into the anaerobic bottle with a sterile syringe, shake and evenly shake, and use the anaerobic bottle of the cellar mud suspension with bacterial solution as the experimental group, and the cellar mud suspension without bacterial solution as the control group. After culturing at 37℃ for 2 days, shake and evenly shake, take the suspension and measure the volatile products therein by GCMS.

[0131] The results are as follows Fig. 6A As shown in the figure, compared with the control group without bacterial solution, the fermentation products of various bacteria increased in the experimental group incubated with bacterial solution for 2 days, among which the content of phenylethanol increased by about 3 mg / L ( Figure 6C ), and at the same time, the contents of butyric acid, isovaleric acid and caproic acid in the suspension also increased significantly ( Figure 6B ).

[0132] This example proves that the bacteria has the characteristic of producing phenylethanol under anaerobic environment. In a strictly anaerobic cellar mud environment, the bacteria can utilize relatively limited nutrients to convert carbon sources or amino acids to produce phenylethanol and medium- and short-chain fatty acids. This characteristic is used in the winemaking process to improve the flavor and quality of the liquor. It is also of great value in the maturation and improvement of cellar mud when used in cellars.

[0133] It is to be understood that the present invention is described by some embodiments, and it is known to those skilled in the art that various changes or equivalent substitutions may be made to these features and embodiments without departing from the spirit and scope of the present invention. In addition, under the teachings of the present invention, these features and embodiments may be modified to adapt to specific circumstances and materials without departing from the spirit and scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of this application are within the scope of protection of the present invention.

[0134]

Claims

1. A strain of Clostridium sp. mCGM14 was deposited in the General Microbiology Center of China Association for Microbiological Culture Collection on July 4, 2023, with the deposit number CGMCC No.1.18017.

2. A starter culture comprising the Clostridium mCGM14 according to claim 1.

3. The leavening agent according to claim 2, characterized in that Contains living cells and / or dry bacteria and / or wet bacteria and / or immobilized cells and / or liquid bacterial agent and / or solid bacterial agent of the Clostridium mCGM14.

4. Use of the Clostridium mCGM14 according to claim 1 or the starter according to any one of claims 2-3 in the field of fermentation.

5. The use according to claim 4, characterized in that The fermentation field includes fermentation for wine making and / or fermentation for food preparation and / or fermentation for cosmetics preparation and / or fermentation for cleaning products preparation and / or fermentation for essence preparation.

6. Use of the Clostridium mCGM14 according to claim 1 or the fermentation agent according to any one of claims 2 to 3 in fermentation to produce alcohols and / or organic acids.

7. The use according to claim 6, characterized in that The alcohol production includes: production of phenylethanol and / or production of isopropanol; The production of phenylethanol comprises: producing phenylethanol via shikimic acid pathway or Ehrlich pathway; or / The production of phenylethanol comprises: using sugar or phenylalanine or L-phenylalanine to ferment and produce phenylethanol; or / The producing of organic acid comprises: producing organic acid by utilizing carbon source.

8. The use according to claim 6, characterized in that The organic acid includes at least one or more of acetic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, caproic acid, phenylacetic acid, palmitic acid and polyhydroxystearic acid.

9. Use of Clostridium mCGM14 as claimed in claim 1 in artificial pit mud cultivation; or / The artificial pit mud cultivation includes the maturation and / or improvement of new pits and / or pit mud.

10. A method for cultivating artificial pit mud, characterized in that: Comprising the step of adding the Clostridium mCGM14 as described in claim 1; or / The method comprises the following steps: (1) taking cellar mud, preparing it into a cellar mud water suspension, and sterilizing it; (2) adding the bacterial liquid of the Clostridium mCGM14 to the cellar mud water suspension for anaerobic culture to obtain cultivated cellar mud; or / In the step (1), the method for preparing the cellar mud water suspension comprises the following steps: placing the cellar mud in an anaerobic bottle, adding anaerobic distilled water to submerge the cellar mud, and violently shaking to obtain the cellar mud water suspension; or / The anaerobic distilled water is obtained by adding 0.1% cysteine ​​hydrochloride to distilled water and then sterilizing it; or / In the step (2), the bacterial solution of Clostridium mCGM14 is prepared by the following steps: 1) Take Clostridium mCGM14 cultured to the logarithmic phase, centrifuge under anaerobic conditions, and discard the upper suspension; 2) resuspending with 5-15 times the volume of the Clostridium mCGM14 cells in anaerobic PBS, centrifuging at high speed, and repeating 2-5 times; 3) taking the centrifuged bacterial cells and resuspending them again to obtain the bacterial solution; or / The anaerobic PBS is obtained by adding 0.1% cysteine ​​hydrochloride to the PBS and then sterilizing it; or / The high-speed centrifugation speed is 6000g-10000g.

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