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

Abstract

The invention belongs to the technical field of microorganisms, and relates to clostridium for producing phenethyl alcohol and organic acid and application of the clostridium. The strain is preserved in the China General Microbiological Culture Collection Center on July 4, 2023, the preservation number is CGMCC No.1.18018, the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the classification name is Clostridiumsp.mCGM113. The invention further discloses a preparation method of the strain. The clostridium can effectively utilize phenylalanine to quickly synthesize phenethyl alcohol under a strict anaerobic condition, can utilize various sugars in a cellar to synthesize various organic acids, can be used for microorganism enhancement and improvement of a new cellar (cellar mud), and effectively improves the flavor and quality of white spirit.

Description

Technical Field

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

[0002] β-Phenylethyl alcohol, with a molecular formula of C8H10O and a molecular weight of 122.17, is a colorless viscous liquid with a boiling point of 219°C, a relative density of 1.0230, and a refractive index of 1.5310-1.5340. It is found in natural plants such as apples, almonds, bananas, peaches, pears, strawberries, cocoa, and honey. 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-phenylethyl alcohol is also an important flavoring substance in traditional fermented foods such as liquor, rice wine, sake, fruit wine, grape wine, soy sauce, bread, and cheese. For example, it is the main aroma in rice-flavored liquor. Moreover, β-phenylethyl alcohol is not only an important characteristic flavor substance in Luzhou-flavor liquor, Maotai-flavor liquor, Chi-flavor 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, and the substrates are phosphoenolpyruvate in the glycolysis pathway and erythrose 4-phosphate in 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 an aerobic environment. The microbial system for brewing Maotai-flavor liquor is complex, and anaerobic microorganisms in the cellar mud require more stringent separation 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.

[0004] Based on this, the present invention aims to provide a Clostridium that can produce high levels of phenylethanol and a variety of organic acids. Summary of the invention

[0005] The purpose of the present invention is to provide a clostridium that can metabolize and produce abundant organic acids, phenylethanol and other substances under an actual brewing environment. The clostridium is screened and separated from cellar mud, and can use various sugars and lactic acid to quickly convert into various organic acids, and can also effectively use phenylalanine to quickly synthesize phenylethanol under strictly anaerobic conditions. On the one hand, the characteristics of the bacteria can be used in the brewing process, which is beneficial to improve the flavor and quality of liquor; on the other hand, the clostridium mCGM113 can be used to effectively use phenylalanine to quickly synthesize phenylethanol under strictly anaerobic conditions. The characteristics of phenylalanine can be used in the brewing process to convert the phenylalanine accumulated in the cellar into phenylethanol, that is, it is used in the brewing cellar, and it also has important value in the maturation and improvement of cellar mud.

[0006] In one aspect, the present invention provides a Clostridium sp. mCGM113.

[0007] 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.1.18018. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the classification name is Clostridium sp.mCGM113.

[0008] 1) Strain source: The Clostridium obtained in the present invention is screened and isolated from the brewing process of Maotai-flavor liquor, specifically from the wine cellar mud, and has the characteristics of rapid growth, good adaptability, and the ability to produce rich organic acids, phenylethanol and other substances with high yield.

[0009] 2) Colony characteristics: After 72 hours of plate culture, the Clostridium mCGM113 presents smooth, semi-transparent, round colonies with a diameter of 1-2 mm and neat edges, which are easy to pick.

[0010] 3) Bacterial characteristics: The Clostridium mCGM113 is Gram-positive, produces spores, has terminal flagella, is motile, and has a rod-shaped body, 2-4 um in length and 0.4-0.5 um in width.

[0011] 4) Physiological and biochemical characteristics: The physiological and biochemical characteristics of the Clostridium mCGM113 were analyzed by the Biolog AN microplate anaerobic identification system, and the results showed that the Clostridium mCGM113 can utilize D-fructose, D-galactose, D-galacturonic acid, α-D-glucose, D-mannose, isomaltulose, β-hydroxybutyric acid, methyl pyruvate and L-glutamic acid, etc.

[0012] 5) Tolerance: The most suitable growth temperature of the Clostridium mCGM113 is 37-42°C, and the tolerable temperature exceeds 45°C; the Clostridium strain mCGM113 can grow and reproduce in the pH range of 4.5-8, and the optimal growth pH is 7.0; the maximum tolerant salinity of the Clostridium strain mCGM113 is 2%, and the optimal growth salinity is 0.5%.

[0013] On the other hand, the present invention also provides a fermentation agent or pit mud comprising the Clostridium mCGM113.

[0014] In some embodiments, the fermentation agent comprises living cells, dry bacteria, wet bacteria, immobilized cells, liquid fermentation agent, solid fermentation agent, or Clostridium mCGM113 in any other form.

[0015] In some embodiments, the fermentation culture also contains other types of bacteria or fungi and / or yeast and / or Aspergillus.

[0016] On the other hand, the present invention also provides a use of the Clostridium mCGM113 in the fermentation field.

[0017] In some embodiments, the fermentation field includes the preparation of white wine, yellow wine, sake, fruit wine, grape wine, soy sauce, bread, and cheese.

[0018] In some embodiments, the fermentation field includes: fermentation winemaking.

[0019] In some embodiments, the winemaking includes brewing white wine, yellow wine, sake, fruit wine, and grape wine; preferably, the brewing white wine includes brewing strong-flavor white wine, sauce-flavor white wine, and soy-flavor white wine.

[0020] On the other hand, the present invention also provides a use of the Clostridium mCGM113 in producing phenylethanol and / or organic acids.

[0021] In some embodiments, producing phenylethanol comprises: producing phenylethanol via the shikimate pathway or the Ehrlich pathway.

[0022] In some embodiments, producing phenylethanol comprises: producing phenylethanol by fermentation using sugar or phenylalanine or L-phenylalanine.

[0023] In some embodiments, the producing the organic acid comprises utilizing a carbon source to produce the organic acid.

[0024] In some embodiments, the organic acid comprises at least one or more of isopropanol, acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, phenylacetic acid, phenylpropionic acid, palmitic acid, and stearic acid.

[0025] The present invention also provides use of the Clostridium mCGM113 in artificial pit mud cultivation.

[0026] On the other hand, the present invention also provides a method for cultivating artificial pit mud, comprising the step of adding the Clostridium mCGM113.

[0027] In some embodiments, the artificial pit mud cultivation includes maturation and / or improvement of the pit mud.

[0028] In some embodiments, the method comprises the steps of:

[0029] (1) taking cellar mud, preparing it into a cellar mud water suspension, and sterilizing it;

[0030] (2) Adding the bacterial liquid of the Clostridium mCGM113 into the cellar mud water suspension for anaerobic culture to obtain cultivated cellar mud.

[0031] 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.

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

[0033] In some embodiments, in step (2), the bacterial solution of Clostridium mCGM113 is prepared by the following steps:

[0034] 1) Take Clostridium mCGM113 cultured to the logarithmic phase, centrifuge under anaerobic conditions, and discard the upper suspension;

[0035] 2) resuspending with 5-15 times the volume of the Clostridium mCGM113 cells in anaerobic PBS, centrifuging at high speed, and repeating 2-5 times;

[0036] 3) taking the centrifuged bacterial cells and resuspending them again to obtain the bacterial solution.

[0037] In some embodiments, the anaerobic PBS is obtained by adding 0.1% cysteine ​​hydrochloride to the PBS and then sterilizing it.

[0038] In some embodiments, the high-speed centrifugation speed is 6000g-10000g.

[0039] In summary, the present application includes at least one of the following beneficial technical effects:

[0040] (1) The present invention screened a clostridium mCGM113 that can produce phenylethanol and a variety of organic acids from the pit mud of liquor brewing. The bacterium has the characteristic of producing phenylethanol under anaerobic conditions. In a strictly anaerobic pit mud environment, it can utilize relatively limited nutrients to transform carbon sources or amino acids therein to produce abundant organic acids and phenylethanol, and can be used for microbial improvement of new pits (or pit mud) to quickly convert various sugars and lactic acid in new pits into various organic acids. Among them, butyric acid and acetic acid are important substrates for synthesizing caproic acid, so the concentration of caproic acid, ethyl butyrate and ethyl caproate can be indirectly increased to improve the flavor and quality of liquor.

[0041] (2) On the one hand, the characteristics of this bacterium can be utilized in the winemaking process to improve the flavor and quality of liquor. On the other hand, Clostridium mCGM113 can effectively utilize phenylalanine to rapidly synthesize phenylethanol under strictly anaerobic conditions. It can be used in the winemaking process to convert phenylalanine accumulated in the cellar into phenylethanol, that is, it can be used in the winemaking cellar. It also has important value in the maturation and improvement of cellar mud. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIG. 1 is an electron microscope image of Clostridium mCGM113 in an embodiment of the present invention; FIG. 1(A) is a transmission electron microscope image of Clostridium mCGM113, and FIG. 1(B) is a scanning electron microscope image of Clostridium mCGM113;

[0043] Figure 2 The phylogenetic tree result in Example 2 of the present invention;

[0044] FIG3 is a product detection result of strain mCGM113 obtained by fermentation in CNM original medium in Example 4 of the present invention; wherein FIG3(A) is a product chromatogram of strain mCGM113 fermented in original CNM medium, and FIG3(B) is a search result of the corresponding product peak in the spectral library;

[0045] Figure 4 The yield of phenylethanol corresponding to fermentation with phenylalanine at different concentrations (3 g / L, 6 g / L, 12 g / L) in Example 4 of the present invention;

[0046] FIG5 shows the results of fermentation by adding glucose and phenylalanine to the original culture medium CNM, respectively; FIG5(A) is a chromatogram comparison of the fermentation products after additionally adding 10 g / L and 20 g / L glucose to the original CNM culture medium and the original CNM culture medium, and FIG5(B) is a chromatogram comparison of the fermentation products after additionally adding 3 g / L, 6 g / L, and 12 g / L phenylalanine to the original CNM culture medium and the original culture medium;

[0047] Figure 6 is a comparison chart of the fermentation results of the experimental group with mCGM113 bacterial solution added to the cellar mud suspension and the control group without bacterial solution; wherein, Figure 6 (A), Figure 6 (B), Figure 6 (D), Figure 6 (F), and Figure 6 (H) are chromatographic comparison charts of the experimental group with mCGM113 bacterial solution added to the cellar mud suspension and the control group, and Figure 6 (C), Figure 6 (E), Figure 6 (G), and Figure 6 (I) are the search results of the corresponding product peaks in the spectral library of the experimental group with mCGM113 bacterial solution added and the control group without bacterial solution; Figure 6 (B) shows that the experimental group has obvious fermentation product peaks at 25.6-26.2min compared with the control group, and Figure 6 (C ) shows that the corresponding product peak in the spectral library search result is phenylethanol; Figure 6 (D) shows that the experimental group has an obvious fermentation product peak compared with the control group at 9.1-9.6min, and Figure 6 (E) shows that the corresponding product peak in the spectral library search result is isovaleric acid; Figure 6 (F) shows that the experimental group has an obvious fermentation product peak compared with the control group at 19.7-20.0min, and Figure 6 (G) shows that the corresponding product peak in the spectral library search result is butyric acid; Figure 6 (H) shows that the experimental group has an obvious fermentation product peak compared with the control group at 20.7-20.9min, and Figure 6 (I) shows that the corresponding product peak in the spectral library search result is n-hexanoic acid. DETAILED DESCRIPTION

[0048] 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.

[0049] Unless otherwise specified, the equipment, materials, etc. used in the following examples can be obtained from commercial or public sources.

[0050] The Clostridium sp. mCGM113 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.1.18018, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and is classified and named Clostridium sp. mCGM113.

[0051] The Clostridial Nutrient Medium (CNM) used in the embodiment of the present invention has the following formula: beef extract 10 g / L, glucose 5 g / L, casein peptone 5 g / L, yeast extract 3 g / L, soluble starch 1 g / L, sodium chloride 5 g / L, sodium acetate 3 g / L, cysteine ​​0.5 g / L, pH 6.8.

[0052] The method for determining the fermentation product in the fermentation broth by GCMS in the embodiment of the present invention is as follows:

[0053] 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.

[0054] Example 1 Screening of strains

[0055] (1) Bacterial enrichment

[0056] 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.

[0057] 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 carry out enrichment culture at 30°C for 7-8 days. Use GCMS to detect the 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 pit mud enriched solution.

[0058] (2) Initial screening of bacterial strains

[0059] 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.

[0060] (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.

[0061] (4) Strain purity inspection

[0062] 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.

[0063] (5) Rescreening of bacterial strains

[0064] 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.

[0065] Example 2 Identification and preservation of strains

[0066] (1) Morphological characteristics identification

[0067] Colony characteristics: After 72 hours of plate culture on RCM medium, the strain mCGM113 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.

[0068] Bacterial characteristics: Gram-positive, spore-forming, terminal flagella, motile, rod-shaped, 2-8 μm long, 0.4-0.6 μm in diameter. The scanning electron microscope image is shown in Figure 1.

[0069] (2) Identification of physiological and biochemical characteristics

[0070] The biochemical characteristics of the bacteria were analyzed by the Biolog AN microplate anaerobic identification system: the strain mCGM113 can utilize D-fructose, D-galactose, D-galacturonic acid, α-D-glucose, D-mannose, isomaltulose, β-hydroxybutyrate, methyl pyruvate and L-glutamic acid.

[0071] (3) Molecular identification

[0072] The genomic DNA of the screening strain mCGM113 was extracted using the QIAGEN bacterial genome extraction kit, and the 16S rRNA gene of mCGM113 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 mCGM113 was compared with the BLAST sequence, and a phylogenetic analysis was performed. The results Figure 2 And as shown in Table 1.

[0073] according to Figure 2The phylogenetic tree and the comparison results in Table 1 show that the 16S rDNA sequence of the newly screened strain mCGM113 is most closely related to the Clostridium luticellar in the database, with a similarity of 96.66%.

[0074] According to the important criteria for judging new bacterial species, if the similarity of 16S rRNA sequence with known species 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 ). Combined with the results of colony morphology, physiological and biochemical identification: the strain mCGM113 is rod-shaped or rod-shaped, 2-8 microns long and 0.4-0.6 microns in diameter; the main fatty acids are C14:0 and C16:0, and the polar lipids are diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine; the G+C content is 35.70 mol%; that is, the colony morphology, microscopic morphology of the bacteria, the main carbon source that can be utilized, and the G+C content of the strain mCGM113 are similar to those of Clostridium luticellar in the genus Clostridium (Clostridium cluster I) (for relevant information about Clostridium luticellar, see the literature: Wang et al., Clostridium luticellarii sp.nov., isolated from a mud cellar used for producing strong aromatic liquors, 2015, IJSEM).

[0075] Based on the above morphological characteristics, physiological and biochemical results, and 16S rDNA sequence comparison results, it was further demonstrated that the strain mCGM113 screened by the above method was a new species of the genus Clostridium in the family Clostridiaceae, named Clostrdium sp. mCGM113.

[0076] Table 1 Model strains related to mCGM113 and their sequence similarities

[0077]

[0078]

[0079] (4) Patent procedures and deposit of strains

[0080] The obtained pure strain of mCGM113 (Clostridium sp.mCGM113) was deposited in the General Microbiology Center of 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.1.18018.

[0081] Example 3 Growth ability of mCGM113 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, 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 the mCGM113 strain under different fermentation temperatures, different acidities, and salinities.

[0083] Temperature tolerance experiment: Prepare a Henle tube containing 5 mL CNM liquid culture medium, and inoculate the mCGM113 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 incubator at 16°C, 25°C, 30°C, 37°C, 42°C, 45°C, and 50°C for 72 hours. Take 100uL of culture solution every 12 hours to detect the OD600 reading of the bacterial solution on an ELISA reader, and fill in the table with the highest OD value detected in the tube bacterial solution within 72 hours (the results are shown in Table 2).

[0084] Acidity 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, and 8, respectively, and inoculate the mCGM113 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 100 uL of the culture solution every 12 hours and detect the OD600 reading of the bacterial solution on an ELISA reader. Fill in the table with the highest OD value detected in the tube bacterial solution within 72 hours (the results are shown in Table 3).

[0085] Salinity tolerance experiment: Prepare Henle tubes containing 5mL CNM liquid culture medium, adjust the salinity of the culture medium to 0, 0.5%, 1%, 1.5%, 2%, 2.5%, and 3%, respectively, and add the mCGM113 bacterial solution grown to the logarithmic phase into the tube at a ratio of 1:50, and place the anaerobic Henle tube in a 37℃ constant temperature incubator for 72 hours. Take 100uL of the culture solution every 12 hours to detect the OD600 reading of the bacterial solution on the microplate reader, and fill in the table with the highest OD value detected in the tube bacterial solution within 72 hours. (The results are shown in Table 4).

[0086] 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%, 17%, and 19% to the culture medium, respectively, inoculate the mCGM113 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, fill in the table with the highest OD value detected in the tube bacterial solution within 72 hours (the results are shown in Table 5).

[0087] According to the results presented in Tables 2-5, the tolerant temperature of Clostridium strain mCGM113 exceeds 45°C, and the most suitable growth temperature is 37-42°C; Clostridium strain mCGM113 can grow and reproduce in the pH range of 4.5-8, and the most suitable growth pH is 7.0; the maximum tolerant salinity of Clostridium strain mCGM113 is 2%, and the most suitable growth salinity is 0.5%; the maximum ethanol tolerance of Clostridium strain mCGM113 is 15% vol.

[0088] Table 2 Temperature tolerance test

[0089] temperature 16℃ 25℃ 30℃ 37℃ 42℃ 45℃ 50℃ OD value 0.08 0.17 0.31 0.40 0.41 0.35 0.27

[0090] Table 3 Acidity tolerance experiment

[0091] pH 4 4.5 5 5.5 6 6.5 7 7.5 8 OD value 0.08 0.15 0.25 0.29 0.35 0.35 0.42 0.24 0.15

[0092] Table 4 Salinity tolerance experiment

[0093] salinity 0 0.5 1 1.5 2 2.5 3 OD value 0.31 0.42 0.25 0.22 0.13 0.07 0.03

[0094] Table 5 Ethanol tolerance test

[0095]

[0096] Example 4 Study on the ability of mCGM113 to produce phenylethanol by fermentation

[0097] At present, two pathways for microbial metabolism to produce phenylethanol have been discovered, mainly the shikimic acid pathway and the Ehrlich pathway:

[0098] 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 mCGM113 obtained by the above screening has the ability to ferment to produce phenylethanol, as follows:

[0099] After culturing mCGM113 in 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 retrieval (see Figure 3B ), after quantitative analysis, the yield of phenylethanol was about 0.17 mg / L.

[0100] On the basis of the original CNM medium, 10 g / L and 20 g / L glucose were added to the mCGM113 medium and fermented for 72 hours. The phenylethanol yield in the fermentation broth was then tested, reaching 0.29 and 0.99 mg / L, respectively. Figure 5A ), which means that mCGM can synthesize phenylethanol from scratch via the shikimic acid pathway, but the yield is not high.

[0101] 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 22.70, 43.05, and 90.89 mg / L, respectively. Figure 4 and Figure 5B shown).

[0102] The above experiments further indicate that mCGM113 may produce phenylethanol mainly through the Ehrlich pathway.

[0103] Example 5: Research on the fermentation products of mCGM113

[0104] This example explores the fermentation products of mCGM113 as follows:

[0105] (1) After culturing mCGM113 in CNM medium for 72 hours, the fermentation products in the fermentation broth were detected by GCMS; (2) On the basis of the original CNM medium, 10 g / L and 20 g / L glucose were added, respectively, and mCGM113 was inoculated and fermented for 72 hours, and the fermentation products in the fermentation broth were detected by GCMS; (3) On the basis of the original CNM medium, phenylalanine at concentrations of 3, 6, and 12 g / L was added, respectively, and mCGM113 was inoculated and fermented for 72 hours, and the fermentation products in the fermentation broth were detected by GCMS.

[0106] The results are shown in Figure 5, Table 6 and Table 7: Figure 5 (A) and Table 6 show the chromatograms of fermentation products obtained by GCMS detection of the corresponding fermentation broths after mCGM113 was cultured in the original CNM fermentation medium, the original CNM+10g / L glucose fermentation medium and the original CNM+20g / L glucose fermentation medium for 72 hours, as well as the peak areas of the fermentation products at the corresponding retention times; Figure 5 (B) and Table 7 show the chromatograms of fermentation products obtained by GCMS detection of the corresponding fermentation broths after mCGM113 was cultured 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, as well as the peak areas of the fermentation products at the corresponding retention times.

[0107] The above results show that the fermentation products of mCGM113 in CNM medium include not only phenylethanol, but also isopropanol, acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, phenylacetic acid, phenylpropionic acid, palmitic acid and polyhydroxystearic acid. Among them, the concentration of short-chain fatty acids such as acetic acid, butyric acid and isovaleric acid increased significantly with the increase of glucose concentration in the fermentation broth, and the concentration of long-chain fatty acids such as palmitic acid and stearic acid also increased with the increase of glucose concentration; while the concentration of phenylethanol, phenylacetic acid and phenylpropionic acid increased significantly with the increase of phenylalanine concentration in the fermentation broth.

[0108] Table 6 Peak area results of fermentation products of mCGM113 in fermentation medium with different glucose concentrations

[0109]

[0110]

[0111] Table 7 Peak area results of fermentation products of mCGM113 in fermentation medium with different phenylalanine concentrations

[0112]

[0113] Example 6 Application of strain mCGM113 in winemaking

[0114] 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;

[0115] Take 50mL of mCGM113 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.

[0116] The results are shown in Figure 6: Compared with the control group without bacterial solution, the experimental group incubated with bacterial solution for 2 days had more fermentation products of various bacteria in the mud suspension, among which the content of phenylethanol increased by 0.35 mg / L ( Figure 6B , C); at the same time, butyric acid in the suspension ( Fig. 6F , G), isovaleric acid ( Fig.6D , E) and caproic acid ( Figure 6H , I) also increased significantly.

[0117] 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.

[0118] 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.

[0119] Sequence Listing

[0120] SEQ ID NO.1 (mCGM11316S rRNA full-length sequence):

[0121]

Claims

1. A strain of Clostridium sp. mCGM113 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.1.18018.

2. A starter culture comprising Clostridium mCGM113 as claimed in 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 mCGM113.

4. Use of the Clostridium mCGM113 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 the preparation of white wine, yellow wine, sake, fruit wine, grape wine, soy sauce, bread and cheese.

6. The use according to claim 4, characterized in that The fermentation field includes: fermentation wine making; or / The brewing includes brewing white wine, yellow wine, sake, fruit wine, and grape wine; or / The brewed liquor includes brewing Luzhou-flavor liquor, Maotai-flavor liquor and Chi-flavor liquor.

7. Use of the Clostridium mCGM113 according to claim 1 or the fermentation agent according to any one of claims 2 to 3 in fermentation to produce phenylethanol and / or organic acids; or / 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 acids includes utilizing carbon sources to produce organic acids.

8. The use according to claim 7, characterized in that The organic acid includes at least one or more of isopropanol, acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, phenylacetic acid, phenylpropionic acid, palmitic acid and stearic acid.

9. Use of Clostridium mCGM113 as claimed in claim 1 in artificial pit mud cultivation; or / The artificial pit mud cultivation includes maturation and / or improvement of the pit mud.

10. A method for cultivating artificial pit mud, characterized in that: Comprising the step of adding the Clostridium mCGM113 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 mCGM113 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 immerse 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 mCGM113 is prepared by the following steps: 1) Take Clostridium mCGM113 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 mCGM113 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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