Clostridium for producing butyric acid and application thereof

Abstract

The invention belongs to the technical field of microorganisms and brewing, and relates to clostridium for producing butyric acid and application thereof. The clostridium is Clostridium sp.mCGM5 and is preserved in the China General Microbiological Culture Collection Center on July 4, 2023, and the preservation number of the clostridium is CGMCC No. 1.18016. The clostridium has the advantages that the clostridium is clostridium sp.mCGM5, and the clostridium is clostridium sp.mCGM5; the novel bacterium Clostridium sp.mCGM5 provided by the invention can produce butyric acid, and butyric acid is an important precursor for synthesizing ester substances such as hexanoic acid and ethyl butyrate and can be used in a wine brewing process, so that the flavor and quality of white wine are improved; and secondly, the strain can effectively utilize substances such as sugar and lactic acid in the cellar to synthesize various organic acids, so that the strain can be used for microbial enhancement and improvement of a new cellar (cellar mud).

Description

Technical Field

[0001] The invention belongs to the field of microorganisms and brewing technology, and particularly relates to a butyrate-producing clostridium and application thereof. Background Art

[0002] The cellar mud used for winemaking is inhabited by a large number of functional microorganisms for winemaking. After long-term continuous production, the cellar mud microorganisms have gradually been domesticated and stabilized. Firmicutes and Bacteroidetes are the dominant microorganisms in high-quality cellar bottoms. These bacterial communities and the interactions between them have established a unique microbial system for the cellar mud, and also endowed the cellar mud microorganisms with functionality and importance in liquor. Therefore, the cellar mud not only has an important influence on the brewing of Luzhou-flavor liquor, but also plays an important role in the formation of the unique flavor of Maotai-flavor liquor.

[0003] The brewing process of Maotai-flavor liquor is a process of fermentation by multiple strains, and the microbial system is complex. Anaerobic microorganisms in cellar mud require relatively strict separation and culture conditions. Propionic acid bacteria, butyric acid bacteria, and caproic acid bacteria are important brewing microbial groups in cellar mud. Butyric acid bacteria provide precursors for important aroma substances in liquor brewing, and are therefore considered the most important functional bacteria among brewing microorganisms. Butyric acid and other organic acids produced by butyric acid bacteria are important precursors for the synthesis of caproic acid and ethyl butyrate. At present, the butyric acid bacteria isolated from the bottom of Maotai-flavor liquor cellars are mainly Clostridium tyobutyricum and Clostridium pabulibutyricum. The above two types of clostridia will produce the odor substance 4-methylphenol under certain conditions. Therefore, separating butyric acid bacteria that do not produce odor substances but produce an appropriate amount of butyric acid from the bottom of Maotai-flavor liquor cellars is important for improving the flavor of Maotai-flavor liquor.

[0004] Based on this, the present invention aims to provide a Clostridium that does not produce odorous substances and produces high butyric acid. Summary of the invention

[0005] The purpose of the present invention is to provide a new bacterium (Clostridium sp.) of the genus Clostridium that can metabolize and efficiently produce organic acids such as butyric acid under actual brewing conditions. The bacterium has been deposited in the General Microbiological Center of the China Association for the Collection of Microbiological Cultures on July 4, 2023, with a deposit number of CGMCC No.1.18016, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and is classified and named Clostridium sp.mCGM5.

[0006] In one aspect, the present invention provides a strain of Clostridium mCGM5. The strain has been deposited in the General Microbiological Center of the China Association for the Preservation of Microbiological Cultures on July 4, 2023, with a deposit number of CGMCC No. 1.18016, and a deposit location of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and is classified and named Clostridium sp. mCGM5.

[0007] 1) Strain source: The Clostridium mCGM5 was screened and isolated from the brewing process of Maotai-flavor liquor, specifically, isolated from the cellar mud of Maotai-flavor liquor brewing cellar.

[0008] 2) Colony characteristics: After 72 hours of plate culture, the colonies of the Clostridium mCGM5 are colorless and translucent, round, smooth, 2-3 mm in diameter, with neat edges, easy to pick, and Gram-positive.

[0009] 3) Bacterial characteristics: Under a projection electron microscope, the cells of the Clostridium mCGM5 strain were rod-shaped, spore-forming, flagella-free, about 1-2 um long, and mostly 0.4-0.5 um wide.

[0010] 4) Physiological and biochemical characteristics: The Clostridium mCGM5 can utilize D-galactose, gentiobiose, α-D-glucose, glycerol, lactulose, methyl glucoside, turanose, L-alanyl-histidine, etc.

[0011] 5) Tolerance: The temperature tolerance range of Clostridium mCGM5 is 16-65℃, and the optimal growth temperature is 37℃-42℃. When the temperature is 37℃ and 42℃, the OD of Clostridium mCGM5 is 600 can reach 0.79; Clostridium mCGM5 has a pH tolerance range of 4-9, and the optimal growth pH is 7. When the pH is 7, the OD of Clostridium mCGM5 600 can reach 0.88; the tolerance concentration range of salinity is 0-2.5%, the optimal growth salinity is 0.5%, when the salinity is 0.5%, the OD of Clostridium mCGM5 600 can reach 0.79; the tolerance concentration range of ethanol is 0-19%, and the optimal growth ethanol concentration is 3%. When the ethanol concentration is 3%, the OD of Clostridium mCGM5 600 It can reach 0.84.

[0012] In another aspect, the present invention provides a fermentation culture comprising the Clostridium mCGM5.

[0013] In some embodiments, the fermentation agent comprises 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 mCGM5.

[0014] On the other hand, the present invention provides a use of the Clostridium mCGM5 or the fermentation agent in the field of fermentation.

[0015] In some embodiments, the fermentation field includes fermentation winemaking.

[0016] On the other hand, the present invention also provides a use of the Clostridium mCGM5 or the fermentation agent in fermenting and producing organic acids.

[0017] In some embodiments, producing the organic acid comprises: utilizing a carbon source to produce the organic acid.

[0018] In some embodiments, the carbon source comprises sugars and / or acids; preferably, the sugars comprise glucose; and the acids comprise lactic acid.

[0019] In some embodiments, the organic acid comprises at least one or more of isoacetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and phenylacetic acid.

[0020] On the other hand, the present invention also provides a use of the Clostridium mCGM5 in artificial pit mud cultivation.

[0021] In some embodiments, the artificial pit mud cultivation includes the maturation and / or improvement of new pits and / or pit mud.

[0022] On the other hand, the present invention also provides a method for cultivating artificial pit mud, comprising the step of adding the Clostridium mCGM5.

[0023] In some embodiments, the method comprises the steps of:

[0024] (1) taking cellar mud, preparing it into a cellar mud water suspension, and sterilizing it;

[0025] (2) Adding the bacterial liquid of Clostridium mCGM5 into the cellar mud water suspension for anaerobic culture to obtain cultivated cellar mud.

[0026] 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.

[0027] In some embodiments, the anaerobic distilled water is obtained by adding 0.1% cysteine ​​hydrochloride to distilled water and then sterilizing it.

[0028] In some embodiments, in step (2), the bacterial solution of Clostridium mCGM5 is prepared by the following steps:

[0029] 1) Take Clostridium mCGM5 cultured to the logarithmic phase, centrifuge under anaerobic conditions, and discard the upper suspension;

[0030] 2) resuspending with 5-15 times the volume of the Clostridium mCGM5 bacteria in anaerobic PBS, centrifuging at high speed, and repeating 2-5 times;

[0031] 3) taking the centrifuged bacterial cells and resuspending them again to obtain the bacterial solution.

[0032] In some embodiments, the anaerobic PBS is obtained by adding 0.1% cysteine ​​hydrochloride to the PBS and then sterilizing it.

[0033] In some embodiments, the high-speed centrifugation speed is 6000g-10000g; preferably, the high-speed centrifugation speed is 7000g-9000g.

[0034] In summary, the present application includes at least one of the following beneficial technical effects:

[0035] (1) The new bacterium Clostridium sp.mCGM5 provided by the present invention can effectively utilize sugars and lactic acid in the pit to synthesize various organic acids. Based on this, the bacterium Clostridium sp.mCGM5 can be used for microbial improvement of new pits (or pit mud), and various sugars and lactic acid in the new pits can be quickly converted into various organic acids by the bacterium.

[0036] (2) The new bacterium Clostridium sp.mCGM5 provided by the present invention can effectively utilize the carbon source in the cellar to synthesize a variety of organic acids, including isoacetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, phenylacetic acid, etc. Among them, butyric acid and acetic acid are important substrates for synthesizing caproic acid. Therefore, the use of the bacterium Clostridium sp.mCGM5 in the winemaking process can indirectly increase the concentration of caproic acid, ethyl butyrate, ethyl caproate, etc., thereby improving the flavor and quality of liquor. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIG. 1 is an electron microscope image of Clostridium mCGM5 in an embodiment of the present invention; wherein FIG. 1(A) is a transmission electron microscope image of Clostridium mCGM5, and FIG. 1(B) is a scanning electron microscope image of Clostridium mCGM5;

[0038] Figure 2 The phylogenetic tree result in Example 2 of the present invention;

[0039] Figure 3The product chromatograms after fermentation of the mCGM5 bacterial solution inoculated with CNM medium containing different concentrations of glucose (5 g / L, 20 g / L) in Example 4 of the present invention and the search results of the corresponding product peaks in the spectrum library; wherein, in the figure, "①" represents acetic acid, "②" represents propionic acid, "③" represents 2-methyl-propionic acid, "④" represents butyric acid, "⑤" represents 3-methyl-butyric acid, and "⑥" represents hexanoic acid;

[0040] FIG4 shows the fermentation results of the experimental group with mCGM5 bacterial solution added to the cellar mud suspension and the control group without bacterial solution; Figure 4(A) , 4(B) The chromatograms of the control group and experimental group of mCGM5 bacterial liquid at different elution times (0-26min, 26-47min) and the search results of the corresponding product peaks in the spectral library are shown respectively. In the fermentation product peak results, the relatively obvious fermentation product peaks added in the experimental group and the corresponding spectral library search results are highlighted; in Figure 4 (A), "①" represents butanol, "②" represents isopentanol, "③" represents octanol, "④" represents isobutyric acid, "⑤" represents n-butyric acid, and "⑥" represents isovaleric acid; in Figure 4 (B), "①" represents phenylethanol, "②" represents 2,6-di-tert-butyl-p-methylphenol, and "③" represents tridecanol. DETAILED DESCRIPTION

[0041] 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.

[0042] Unless otherwise specified, the equipment, materials, etc. used in the following examples can be obtained from commercial or public sources.

[0043] The Clostridium sp. mCGM5 in the embodiment of the present invention is a new species of the genus Clostridium of the family Clostridiaceae isolated from the cellar mud of the wine-making cellar of Maotai-flavor liquor. The strain has been deposited in the General Microbiological Center of the China Association for the Collection of Microbiological Cultures on July 4, 2023, with a deposit number of CGMCC No. 1.18016, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and is classified and named Clostridium sp. mCGM5.

[0044] The formula of the Clostridial Nutrient Medium (CNM) 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.

[0045] The method for determining the fermentation products in the fermentation broth by GCMS in the embodiment of the present invention is as follows: Fermentation products such as short-chain fatty acids and phenylethanol are determined by gas chromatography-mass spectrometry. 1 mL of culture is extracted with 1 mL of ethyl acetate, and the supernatant is centrifuged for GC-MS analysis, which is performed on the GCMS using an automatic sampler and a DB capillary column (30 m, 0.25 mmi.d., 0.25 μm film thickness). The temperature of the oven is set to rise from 80°C to 140°C at a rate of 20°C / min, and maintained for 1 minute; then raised to 290°C at a rate of 3.5°C / min and maintained for 15 minutes. 1 μL of sample is injected at 280°C. The carrier gas helium flows at a rate of 1.2 ml / min. The electron impact is recorded as 70 eV.

[0046] Example 1 Screening and enrichment of strains

[0047] (1) Bacterial enrichment

[0048] 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.

[0049] Take 1g of pit 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℃ for 7-8 days. Use GCMS to detect the substances in the enriched culture solution. If the product peak of short-chain organic acids such as butyric acid is detected, it is determined to be the target pit mud enriched solution.

[0050] (2) Initial screening of bacterial strains

[0051] 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.

[0052] (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.

[0053] (4) Strain purity inspection

[0054] 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.

[0055] (5) Rescreening of bacterial strains

[0056] The strain was inoculated into CNM liquid culture medium and cultured for 2-3 days, and GCMS was used to detect whether the strain produced organic acids such as butyrate.

[0057] Example 2 Identification and preservation of strains

[0058] (1) Morphological characteristics identification

[0059] Colony characteristics: After 72 hours of plate culture on CNM medium, the strain mCGM5 obtained by the above method appears as translucent round colonies, which are smooth, 1-2 mm in diameter, with neat edges and easy to pick.

[0060] Bacterial characteristics: Gram-positive, spore-forming, no flagella, rod-shaped, 1-2um long, 0.4-0.5um wide. Transmission electron microscopy and scanning electron microscopy images are shown in Figure 1 (A) and Figure 1 (B).

[0061] (2) Identification of physiological and biochemical characteristics

[0062] The physiological and biochemical characteristics of the bacteria were analyzed by Biolog AN microplate anaerobic identification system: strain mCGM5 can utilize D-galactose, gentiobiose, α-D-glucose, glycerol, lactulose, methyl glucoside, turanose, etc.

[0063] 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) and identified 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, and then 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) butanol solution. Phospholipids were detected with Zinzadze reagent (molybdenum blue spray reagent, 1.3%; Sigma), and glycolipids were detected with 0.5% α-naphthol reagent. The results showed that the main cellular fatty acid of mCGM5 was C14:0 (43.9%). The polar lipids are diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine.

[0064] (3) Molecular identification

[0065] The mCGM5 genomic DNA was extracted using the QIAGEN bacterial genome extraction kit, and the 16S rRNA gene of mCGM5 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 NCBI official website. The obtained 16S rDNA sequence of mCGM5 was compared with the BLAST sequence, and phylogenetic analysis was performed. The results are shown in Figure 2 And as shown in Table 1.

[0066] The genomic DNA library was sequenced using the Illumina Hiseq X-ten platform. All paired reads of good quality were assembled using SPAdes software (v3.9.0). The average nucleotide identity (ANI) value was calculated using OAT software http: / / www.ezbiocloud.net / sw / oat. The genomic distance, digital DNA-DNA hybridization (dDDH), was calculated by using the genome-to-genome distance calculator (GGDC; http: / / ggdc.dsmz.de / ), and the results are shown in Tables 2 and 3.

[0067] According to the phylogenetic tree and the comparison results in Table 1: the 16S rDNA sequence of the newly screened strain mCGM5 is most closely related to Clostridium luticellar in the database, with a similarity of 97.35%. According to the important criteria for judging new bacterial species, if the 16S rRNA gene sequence similarity with the closest model strain is less than 98.65%, it 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 ).

[0068] Combined with the results of strain morphology, physiological and biochemical identification: strain mCGM5 is a rod-shaped bacterium with a size of about 2-6μm×0.8-1.2μm, and metabolizes D-galactose, gentiobiose, α-D-glucose, D, L-α-glycerol, lactulose, β-methyl-D-glucoside, and turanose. The main cellular fatty acid is C14:0 (43.9%). The polar lipids are diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. The whole genome size is 3661612bp, and the G+C content is 32.85mol%.

[0069] Based on the above physiological and biochemical characteristics, combined with Table 1, the 16S rDNA gene sequence of the newly screened strain mCGM5 is 97.34% similar to the closest model strain in the database, Clostridium luticellar, which is lower than 98.65%. In addition, the ANI% of this strain and the currently preserved standard strains are both lower than 95%, and the dDDH% is lower than 70% (Tables 2, 3).

[0070] Based on the morphological characteristics, physiological and biochemical identification characteristics, and 16S rDNA sequence comparison of the above-mentioned model strain Clostridium luticellar, which is the most closely related, the genome comparison results further determined that the strain mCGM5 screened by the above method is a new species of the genus Clostridium in the family Clostridiaceae, named Clostrdium sp. mCGM5.

[0071] In addition, among the clostridium strains screened here, two new strains of the same genus Clostridium were also screened: Clostridium sp. mCGM14 and Clostridium sp. mCGM113, with the preservation numbers CGMCC No.1.18017 and CGMCC No.1.18018 (patents have been applied for respectively). After calculation and comparison: the 16S rRNA gene sequence similarity between mCGM5 and strain mCGM113 (CGMCC No.1.18018) is 96.28%, ANIb% is 78.92%, and dDDH% is 22.2%; the 16S rDNA gene sequence similarity between mCGM5 and strain mCGM14 (CGMCC No.1.18017) is 97.35%, ANIb% is 82.18%, and dDDH% is 25.4%. That is, the 16S rRNA gene sequence similarity of the new strain mCGM5 for which protection is sought in the present invention with that of mCGM14 and mCGM113 is less than 98.65%, the ANI% is less than 95%, and the dDDH% is less than 70%. It is completely different from the other two new strains and is a new strain of the genus Clostridium.

[0072] Table 1. Type strains related to mCGM5 and their sequence similarities

[0073]

[0074]

[0075] Table 2 Calculation of average nucleotide identity (ANIb) values

[0076] 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

[0077] Table 3 Digital DNA-DNA hybridization calculations

[0078] 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) n.d n.d n.d 25.4 mCGM5 (CGMCC1.18016) n.d n.d n.d n.d

[0079] (4) Strain preservation procedures

[0080] The obtained pure strain of mCGM5 (Clostridium sp.mCGM5) was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration Committee for patent strain preservation. The specific address is: No. 3, Yard No. 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.1.18016.

[0081] Example 3 Growth ability of mCGM5 strain under different culture conditions

[0082] 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 mCGM5 under different fermentation temperatures, different acidities, different salinities, and different ethanol conditions.

[0083] Temperature tolerance experiment: Prepare Henle tubes containing 5 mL CNM liquid culture medium, and inoculate the mCGM5 bacterial solution cultured to the logarithmic phase into the anaerobic tube at a ratio of 1:50. Place the anaerobic tubes in a constant temperature incubator at 16°C, 25°C, 30°C, 37°C, 42°C, 45°C, 50°C, 55°C, 60°C, and 65°C for 72 hours. Take 100uL of the culture solution every 12 hours and detect the OD value of the bacterial solution on an ELISA reader. 600 The results are shown in Table 4.

[0084] 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, 9, respectively, and inoculate the mCGM5 bacterial solution cultured to the logarithmic phase into the tube at a ratio of 1:50. Place the anaerobic Henle tube in a 37°C incubator for 72 hours, and take 100uL of the culture solution every 12 hours to detect the OD value of the bacterial solution on an ELISA reader. 600 The results are shown in Table 5.

[0085] 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%, and 2.5%, respectively, and inoculate the mCGM5 bacterial solution cultured to the logarithmic phase into the tube at a ratio of 1:50. Place the anaerobic Henle tube in a 37°C incubator for 72 hours, and take 100uL of the culture medium every 12 hours to detect the OD of the bacterial solution on an ELISA reader. 600 The results are shown in Table 6.

[0086] Ethanol tolerance experiment: Prepare Henle tubes containing 5 mL of CNM liquid culture medium, add ethanol at final concentrations of 0, 1%, 3%, 5%, 7%, 9%, 11%, 13%, 15%, 17%, and 19% to the culture medium, inoculate the mCGM5 bacterial solution cultured 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. The results are shown in Table 7.

[0087] Table 4. Temperature tolerance test

[0088] Temperature 16℃ 25℃ 30℃ 37℃ 42℃ 45℃ 50℃ 55℃ 60℃ 65℃ <![CDATA[OD 600 ]]> 0.19 0.54 0.66 0.79 0.79 0.73 0.43 0.21 0.06 0.04

[0089] Table 5. Acidity tolerance test

[0090] pH 4 4.5 5 5.5 6 6.5 7 7.5 8 9 <![CDATA[OD 600 ]]> 0.16 0.24 0.63 0.66 0.72 0.82 0.88 0.84 0.11 0.07

[0091] Table 6. Salinity tolerance experiment

[0092] Salinity 0 0.5 1 1.5 2 2.5 <![CDATA[OD 600 ]]> 0.68 0.79 0.60 0.13 0.06 0.04

[0093] Table 7. Ethanol tolerance test

[0094] Ethanol 0％ 1％ 3％ 5％ 7％ 9％ 11％ 13％ 15％ 17％ 19％ <![CDATA[OD 600 ]]> 0.79 0.82 0.84 0.75 0.52 0.43 0.31 0.25 0.16 0.10 0.06

[0095] According to the results presented in Tables 4-7 above, the temperature tolerance range of Clostridium mCGM5 is 16-65°C, and the optimal growth temperature is 37°C-42°C. When the temperature is 37°C and 42°C, the OD of Clostridium mCGM5 600 can reach 0.79; Clostridium mCGM5 has a pH tolerance range of 4-9, and the optimal growth pH is 7. When the pH is 7, the OD of Clostridium mCGM5 600 can reach 0.88; the tolerance concentration range of salinity is 0-2.5%, the optimal growth salinity is 0.5%, when the salinity is 0.5%, the OD of Clostridium mCGM5 600 can reach 0.79; the tolerance concentration range of ethanol is 0-19%, and the optimal growth ethanol concentration is 3%. When the ethanol concentration is 3%, the OD of Clostridium mCGM5 600 It can reach 0.84.

[0096] Example 4: Research on the fermentation products of mCGM5

[0097] In order to further explore the fermentation capacity of the newly screened strain mCGM5, the following experiments were carried out:

[0098] (1) After culturing mCGM5 in CNM medium for 72 hours, the fermentation products in the fermentation broth were detected by GCMS; (2) 5 g / L and 20 g / L glucose were added to the original CNM medium, and mCGM5 was inoculated and fermented for 72 hours, and the fermentation products in the fermentation broth were detected by GCMS.

[0099] The results are as follows Figure 3As shown in Table 8: After mCGM5 was cultured and fermented in CNM medium for 72 hours, organic acids such as acetic acid, propionic acid, isobutyric acid, butyric acid, and isovaleric acid were detected in the fermentation broth by GCMS, and the concentration increased with the increase of glucose concentration added to the fermentation broth. According to the quantitative results in Table 8, it can be seen that the butyric acid content of Clostridium mCGM5 in the original CNM medium can reach 85.08 mg / L, and in the CNM medium with an additional addition of 20 g / L of glucose, the amount of butyric acid produced increased by 2-3 times, up to 192.60 mg / L. In addition, no odor substances such as 4-methylphenol, which are easily fermented by Clostridium bacteria, were detected in the fermentation broth of the bacteria.

[0100] Table 8. Qualitative and quantitative results of fermentation products of mCGM5 in fermentation medium with different glucose concentrations

[0101]

[0102] Example 5 Application of Clostridium mCGM5

[0103] 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;

[0104] Take 50mL of mCGM5 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.

[0105] The results are shown in Figure 4. 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. Butanol, isopentanol and octanol were also detected in the suspension. In addition, isobutyric acid, butyric acid and isovaleric acid ( Figure 4A ) also increased significantly, and a small amount of phenylethanol ( Figure 4B ).

[0106] This application example proves that in a strictly anaerobic cellar mud environment, the bacteria can utilize relatively limited nutrients to convert carbon sources or amino acids into a variety of medium- and short-chain alcohols and 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.

[0107] 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 by 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.

[0108]

Claims

1. A strain of Clostridium sp. mCGM5 was deposited in the General Microbiology Center of China Association for Microbiological Culture Collection on July 4, 2023, with the deposit number CGMCC No.1.18016.

2. A starter culture comprising the Clostridium mCGM5 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 mCGM5.

4. Use of the Clostridium mCGM5 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 wine making.

6. Use of the Clostridium mCGM5 according to claim 1 or the starter according to any one of claims 2 to 3 in fermenting to produce organic acids.

7. The use according to claim 6, characterized in that 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 isoacetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and phenylacetic acid.

9. Use of Clostridium mCGM5 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 mCGM5 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 mCGM5 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 mCGM5 is prepared by the following steps: 1) Take Clostridium mCGM5 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 mCGM15 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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