Klebsiella and application thereof

Abstract

The invention discloses Klebsiella and application thereof, and belongs to the technical field of microorganisms. According to the invention, firstly, a strain LLMJ-DNXJ-01 with high yield of tannase is screened from wine-making yeast, the identification result shows that the strain belongs to klebsiella, then the tannase activity of the strain is investigated, and the result shows that the enzyme activity reaches 8.8 + / -0.02 U / mL. In addition, the invention also investigates the application of the strain in degrading sorghum tannin, the sorghum tannin degradation rate reaches 34.95%, and isoamyl alcohol, isopentenol, caryophyllene, phenethyl alcohol, 4-vinyl guaiacol and other volatile aroma substances with Daqu characteristics can be generated through fermentation. Therefore, by integrating the tannase production capability of the strain LLMJ-DNXJ-01 and the sorghum tannin degradation and fermentation aroma production capability, the strain has relatively good development potential in the aspects of manufacturing of reinforced yeast for making hard liquor and quality improvement and aroma enhancement of white spirit.

Description

Technical Field

[0001] The present invention relates to a Klebsiella bacterium and its application, belonging to the field of microbial technology. Background Art

[0002] Chinese liquor is a traditional fermented product in China. Its unique brewing method and special flavor presented are unique among liquor products in the world. During the production of Chinese liquor, various aromatic compounds are produced, which can endow Chinese liquor with special flavors. The reason is that during the fermentation process of Chinese liquor, tannase produced by microorganisms in the koji can degrade tannins in the raw materials to generate flavor substances such as syringic acid, syringaldehyde, and 4-ethylguaiacol. "The koji is the backbone of liquor". During the fermentation process of daqu (a type of starter for Chinese liquor), various microbial flora metabolize substances such as starch and protein in the raw materials to generate various products, forming the unique koji aroma of daqu, which is ultimately brought into the liquor body to form the flavor of Chinese liquor or the precursor substances of Chinese liquor flavor. Research shows that 2,3-butanediol, isobutanol, isoamyl alcohol, benzyl alcohol, phenethyl alcohol, isopentenol, mushroom alcohol, ethyl acetate, ethyl hexanoate, ethyl 2-methylbutyrate, ethyl isovalerate, isoamyl acetate, isovaleraldehyde, 2-nonanone, 3-hydroxy-2-butanone, 3-octanone, 4-ethylguaiacol, 2,6-di-tert-butyl-4-methyl-4-vinylguaiacol, furfuryl alcohol, furfural, 2,3,5,6-tetramethylpyrazine, 2,3,5-trimethylpyrazine, 2,5-dimethylpyrazine, and β-caryophyllene are the main characteristic aroma substances of strong aroma daqu. Since ancient times, it has been widely spread in China that sorghum makes liquor fragrant. The reason is that sorghum contains a large amount of tannins. Although excessive tannins will affect the coordination of yeast and bacteria, appropriate tannins are the substrates for the conversion into heterocyclic aromatic substances such as syringic acid, syringaldehyde, and 4-ethylguaiacol, making the liquor body fragrant and pleasant.

[0003] Tannase was first named by French scientists, and the enzyme capable of degrading tannins is called tannase. Reports related to tannase appeared very early, but progressive research began in 1960. Tannase has a wide range of uses and is particularly valued in the development of new products in the tea beverage industry and the improvement of food flavors. Tannase can break the ester bonds and condensed phenol carboxyl bonds in tannic acid molecules to generate products such as gallic acid, glucose, and aromatic compounds. In the pharmaceutical industry, people use the gallic acid product obtained from the reaction of gallic acid and propanol to synthesize the drug propyl gallate. In addition, tannase can also degrade a type of tannin (catechin) in tea leaves to prepare monomeric catechins, improving the taste and quality of tea. Tannase can also be used as a food additive to develop tea beverages. When tannic acid is present, microorganisms can be induced to produce tannase. Many strains capable of producing tannase have been studied, including the Aspergillus genus, especially Aspergillus niger and Aspergillus oryzae. In addition, there are reports of bacteria, yeasts and other fungi producing tannase at home and abroad. In 1786, Scheele first discovered the existence of tannase, and then people began to pay attention to and study tannase. The research and use of tannase first started in tea beverages. With the in-depth research, tannase has gradually been widely used in industries such as baijiu, food and beverage, beer, wine, and feed.

[0004] During the baijiu brewing process, tannin is a double-edged sword. Excessive tannin will affect the coordination of yeast and bacteria, while appropriate tannin is the substrate for the conversion into heterocyclic aromatic substances such as syringic acid, syringaldehyde, and 4-ethylguaiacol, making the liquor body fragrant and pleasant. Therefore, screening strains that use tannin as the sole carbon source from the koji has important application value for converting the excessive tannin in the brewing raw materials into flavor substances.

[0005] Liu Guangqian et al.'s research on the dynamic evolution of the microbial community in Daqu showed that Klebsiella is one of the main bacterial genera during the Daqu fermentation process, mainly the dominant bacterial community in the early stage of Daqu fermentation. As the fermentation progresses, they almost disappear between 14 and 30 days of fermentation. However, it is unknown whether Klebsiella can affect the volatile aroma substances during the baijiu brewing process, and its mechanism of action on tannin in the brewing raw materials is also unclear.

[0006] Therefore, screening strains that can degrade the tannin in the brewing raw materials and produce typical volatile aroma substances of Daqu has great application value for manufacturing fortified Daqu and improving the quality and aroma of baijiu. Summary of the Invention

[0007] Aiming at the problems existing in the prior art, the present invention provides a Klebsiella producing tannase and its application, aiming to solve the technical problem of the lack of strains capable of degrading the tannin in the brewing raw materials and producing typical volatile aroma substances of Daqu in the prior art.

[0008] The first technical solution provided by the present invention is a strain of Klebsiella sp. LLMJ-DNXJ-01, which was deposited with the China General Microbiological Culture Collection Center on November 25, 2024, and the deposit number is CGMCC No. 32792.

[0009] The Klebsiella sp. LLMJ-DNXJ-01, isolated from Daqu for liquor-making, has the following characteristics:

[0010] When cultured on LB agar medium for 72 h, the colonies are white, opaque, moist, shiny, and have a serrated edge.

[0011] The second technical solution provided by the present invention is a microbial preparation containing the Klebsiella sp. LLMJ-DNXJ-01 described in the first technical solution.

[0012] In some embodiments, the cell concentration of the Klebsiella sp. LLMJ-DNXJ-01 in the microbial preparation is at least 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0013] Furthermore, the cell concentration of the Klebsiella sp. LLMJ-DNXJ-01 in the microbial preparation is at least 1×10 8 CFU / mL or 1×10 8 CFU / g.

[0014] The third technical solution provided by the present invention is a method for producing tannase, which is to ferment and produce tannase using the Klebsiella sp. LLMJ-DNXJ-01 described in the first technical solution or the microbial preparation described in the second technical solution.

[0015] In some embodiments, the method is to inoculate the Klebsiella sp. LLMJ-DNXJ-01 described in the first technical solution or the microbial preparation described in the second technical solution into an enzyme-producing system, culture at 28 - 35 °C for 100 - 150 h, centrifuge, and take the supernatant.

[0016] Furthermore, the inoculation amount of the Klebsiella sp. LLMJ-DNXJ-01 is 5% - 10% (v / v).

[0017] Furthermore, the enzyme-producing system includes 10 g / L tannic acid, 10 g / L sucrose, 1.5 g / L NH4Cl, 0.5 g / L KCl, 1 g / L K2HPO4, 0.5 g / L MgSO4, and 0.5 g / L NaCl.

[0018] The fourth technical solution provided by the present invention is the application of Klebsiella sp. LLMJ-DNXJ-01 described in the first technical solution or the microbial preparation described in the second technical solution in degrading tannins.

[0019] In some embodiments, the tannins are derived from sorghum.

[0020] The fifth technical solution provided by the present invention is a method for producing characteristic volatile aroma substances of Daqu. The method uses sorghum as a raw material and forms a fermentation system with Klebsiella sp. LLMJ-DNXJ-01 described in the first technical solution or the microbial preparation described in the second technical solution to ferment and produce characteristic volatile aroma substances of Daqu.

[0021] In some embodiments, the characteristic volatile aroma substances of Daqu include isoamyl alcohol, isopentenol, caryophyllene, phenethyl alcohol, 4-vinylguaiacol.

[0022] In some embodiments, in the fermentation system, the inoculation amount of Klebsiella sp. LLMJ-DNXJ-01 is 5%-10% (v / v).

[0023] The sixth technical solution provided by the present invention is the application of Klebsiella sp. LLMJ-DNXJ-01 described in the first technical solution or the microbial preparation described in the second technical solution in the field of brewing.

[0024] The technical effects of the present invention are as follows:

[0025] The present invention first screened a strain LLMJ-DNXJ-01 with high tannase productivity from brewing Daqu. The identification results showed that the strain belongs to Klebsiella. Then, the present invention also examined the tannase activity of the strain, and the results showed that the enzyme activity reached 8.8±0.02 U / mL. In addition, this application also examined the application of the strain in degrading sorghum tannins, and the degradation rate of sorghum tannins reached 34.95%. Fermentation can produce characteristic volatile aroma substances of Daqu such as isoamyl alcohol, isopentenol, caryophyllene, phenethyl alcohol, 4-vinylguaiacol. Therefore, considering the ability of strain LLMJ-DNXJ-01 to produce tannase, degrade sorghum tannins, and ferment to produce aroma, it has good development potential in the manufacture of fortified Daqu and improving the quality and aroma of Baijiu.

[0026] Biological material preservation

[0027] A strain of Klebsiella sp. LLMJ-DNXJ-01, taxonomically named Klebsiella sp., was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 25, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 32792. Description of the Drawings

[0028] Figure 1 is the standard curve of absorbance of gallic acid;

[0029] Figure 2 is the standard curve of absorbance of tannin;

[0030] Figure 3 is the comparison peak map of volatile flavor substances. Detailed Embodiments

[0031] The following are the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0032] Raw materials used in the embodiments:

[0033] 1. Screening medium: Soybean protein isolate 15 g / L, agar 5 g / L, bromophenol blue 0.04 g / L, tannic acid 2 g / L (tannic acid should be sterilized separately from other substances).

[0034] 2. Enzyme-producing medium: Tannic acid 10 g / L, sucrose 10 g / L, NH4Cl 1.5 g / L, KCl 0.5 g / L, K2HPO4 1 g / L, MgSO4 0.5 g / L, NaCl 0.5 g / L, natural pH, sterilized at 121 °C for 20 min.

[0035] 3. LB medium: Tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH 7.2, sterilized at 121 °C for 20 min.

[0036] Example 1: Isolation and Identification of Strains

[0037] 1. Isolation and purification of strains

[0038] Take the strong-flavor Daqu, crush it into fine powder. Weigh 1 g of the koji powder, dissolve it in 9 mL of normal saline, shake well until fully mixed. Take 1 mL of the suspension and add it to a test tube containing 9 mL of sterile water, pipette 5 times to mix evenly, which is the sample suspension of 0.01 g / mL. Dilute it successively to 0.001 g / mL. Select the dilution degree of 0.1 - 0.001 g / mL, and take an inoculation amount of 100 μL / plate and spread it on the screening medium. Incubate at 30 °C for 48 h - 72 h. Tannic acid and bromophenol blue indicator are added to the screening plate medium. If the strain produces tannase, it will hydrolyze tannic acid in the medium to produce gallic acid, turning the bromophenol blue indicator around the colony from blue-violet to yellow, thus forming an obvious color-changing circle around the colony. Select the strain with high tannase production according to the color depth and diameter size of the produced color-changing circle, and pick the typical colony with a color-changing circle from the medium with an inoculation loop.

[0039] 2. Strain screening

[0040] According to the morphology of single colonies and the size of the color-changing circles, select 20 strains, streak inoculate them on a new screening medium. After the single colonies grow, use a borer to further transfer them to a new screening medium plate, and select the strain LLMJ-DNXJ-01 with the most obvious color-changing circle. Inoculate the purified 3 strains into 100 mL of LB medium respectively, and culture them in a shaker flask at 30 °C for 72 h. The fermented bacterial liquid is used for DNA extraction. Then, save the bacterial liquid in 40% glycerol at a ratio of 1:1 and store it at -80 °C at low temperature.

[0041] 3. Strain identification

[0042] Send the LLMJ-DNXJ-01 strain to Sangon Biotech (Shanghai) Co., Ltd. for the identification of 16S gene sequence, and the gene sequence is shown as SEQ ID NO:1.

[0043] SEQ ID NO:1:

[0044]

[0045] According to the results of molecular biology identification, the strain LLMJ-DNXJ-01 was identified as Klebsiella sp. LLMJ-DNXJ-01. And the biological preservation of this strain was carried out, and the preservation information is as follows:

[0046] Species name: Klebsiella;

[0047] Latin name: Klebsiella sp.;

[0048] Preservation institution: General Microbiology Center of China Committee for Culture Collection of Microorganisms;

[0049] Abbreviation of preservation institution: CGMCC;

[0050] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0051] Preservation date: November 25, 2024;

[0052] Preservation number: CGMCC No. 32792.

[0053] Example 2: Determination of tannase activity produced by strain LLMJ-DNXJ-01

[0054] 1. Method for determining tannase activity

[0055] (1) Definition of enzyme activity: Under the conditions of 40 °C and pH 5.0, the amount of enzyme required to decompose the substrate propyl gallate to produce 1 μmol of gallic acid per minute is defined as one enzyme activity unit (U / mL);

[0056] (2) Solution preparation:

[0057] 0.1 mol / L citric acid-sodium citrate buffer (pH 5.0): Mix 0.1 mol / L citric acid solution and 0.1 mol / L sodium citrate solution, and adjust the pH to 5.0. Among them, for 0.1 mol / L citric acid solution, accurately weigh 19.21 g of citric acid and make up to 1000 mL volumetric flask with distilled water; for 0.1 mol / L sodium citrate solution, accurately weigh 25.81 g of anhydrous sodium citrate, dissolve it with distilled water and make up to 1000 mL volumetric flask.

[0058] Propyl gallate solution (0.01 mol / L): Accurately weigh 0.212 g of propyl gallate and make up to 100 mL volumetric flask with citric acid buffer solution.

[0059] Methanol rhodanine solution: Accurately weigh 0.334 g of rhodanine and make up to 50 mL volumetric flask with methanol.

[0060] Gallic acid standard solution (5 mg / L): Accurately weigh 0.5 mg of gallic acid and make up to 100 mL in a volumetric flask with citrate-sodium citrate buffer solution.

[0061] KOH standard solution (0.5 mol / L): Accurately weigh 2.805 g of KOH and make up to 100 mL in a volumetric flask with distilled water.

[0062] (3) Determination of enzyme activity:

[0063] Preheat the propyl gallate solution in a 40 °C water bath in advance; take 0.25 mL of crude enzyme solution and add it to 0.25 mL of preheated propyl gallate solution, shake well, react in a 40 °C water bath for 5 min, add 0.3 mL of methanol rhodanine, react in a 40 °C water bath for 5 min, add 0.35 mL of KOH, react in a 40 °C water bath for 5 min, add 3.85 mL of distilled water, and react in a 40 °C water bath for 10 min. Zero with distilled water as the reference, measure the absorbance of the sample at a wavelength of 520 nm with a spectrophotometer, and calculate the enzyme activity according to the formula. For the blank group, add the terminating solution KOH before adding the enzyme solution.

[0064] Result calculation method:

[0065] The formula for enzyme activity determination is: Enzyme activity = △OD 520 ×n×1 / k×1000 / WM×1 / 5×1 / 0.25;

[0066] In the formula, n is the dilution factor during enzyme solution testing (the dilution factor from crude enzyme solution to the enzyme solution used for testing), k is the slope of the fitted standard curve, 5 is the enzyme-catalyzed reaction time, and 1 / 0.5 is converted to 1 mL of enzyme solution.

[0067] Among them, for the standard curve drawing: Prepare standard solutions with different concentration gradients (0 mg / L, 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L) using a 5 mg / L gallic acid solution, and measure the absorbance at 520 nm according to the enzyme activity determination method. Draw a linear standard curve with gallic acid concentration as the abscissa and OD value as the ordinate (see Figure 1 ).

[0068] 2. Enzyme production determination of Klebsiella sp. LLMJ-DNXJ-01

[0069] The process for obtaining the crude enzyme solution in this example is as follows: Inoculate Klebsiella sp. LLMJ-DNXJ-01 obtained in Example 1 into the enzyme-producing medium, culture at 28 - 35 °C for 100 - 150 h, take the culture solution and centrifuge at high speed at 4 °C, and take the supernatant, which is the crude enzyme solution; Dilute the crude enzyme solution to the test enzyme solution at a certain multiple, and measure the enzyme activity according to the above method. Among them, the inoculation amount of Klebsiella sp. LLMJ-DNXJ-01 is 5% (v / v) of the medium.

[0070] The measurement results show that the tannase activity of Klebsiella sp. LLMJ-DNXJ-01 is 8.8 ± 0.02 U / mL.

[0071] Example 3: Application of strain LLMJ-DNXJ-01 in degrading sorghum tannins

[0072] Prepare a tannin standard solution with a concentration of 0.100 mg / mL, and successively pipette 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 mL and add them to 50 mL volumetric flasks containing 25 mL of distilled water. Then, successively add 2.5 mL of Folin color reagent and 10 mL of Na2CO3 solution, shake well and make up the volume. Let it stand at room temperature for 30 min. Take the reaction system without adding the standard solution as the control group, and measure the absorbance value at a wavelength of 760 nm to establish a standard curve (see Figure 2 ).

[0073] Weigh 100 g of sorghum into a 500 mL Erlenmeyer flask, add 60 mL of distilled water preheated to 60 °C, stir evenly, sterilize at 121 °C for 20 min, cool to room temperature, inoculate 5% of the experimental bacterial solution, stir evenly, culture at 35 °C for 5 d. The blank control is not inoculated, and the tannin content is detected after the culture ends.

[0074] Pipette 0.5 mL of the extract and add it to a volumetric flask (50 mL) containing 25 mL of distilled water. Add 2.5 mL of F-D reagent and 10 mL of Na2CO3 solution, shake well and make up the volume. Let it stand at room temperature for 30 min, and measure the OD value of the extract at 760 nm. Calculate the tannin concentration according to the linear regression equation of the tannin standard curve.

[0075] Crush the sorghum sample and pass it through a 40-mesh sieve, dry it to a constant weight, accurately weigh 1 g, place it in an Erlenmeyer flask and add 20 mL of 50% ethanol as the solvent, extract at a constant temperature of 45 °C for 1 h, continuously repeat the extraction until adding 1% ferric chloride to the extract does not turn green. After the extraction ends, perform suction filtration, and finally evaporate the solvent with a rotary evaporator, measure the tannin amount each time, and accumulate and calculate the total tannin content. After measurement, the tannin degradation rate is 34.95%.

[0076] Example 4: Application of Strain LLMJ-DNZJ-01 in Producing Aroma during Sorghum Fermentation

[0077] Weigh 100 g of sorghum into a 500 mL Erlenmeyer flask respectively, add 60 mL of distilled water preheated to 60 °C, stir evenly, sterilize at 121 °C for 20 min, cool to room temperature, inoculate with 5% experimental bacterial solution, stir evenly, and culture at 35 °C for 5 d. The blank control is not inoculated, and the volatile flavor substances are detected after the culture ends.

[0078] Stir the fermented sorghum evenly, weigh 2 g of sorghum into a 20 mL headspace extraction bottle respectively, and perform headspace solid-phase microextraction-gas chromatography-mass spectrometry detection.

[0079] GC conditions: Chromatographic column: InretCap (column length 30 m, inner diameter 0.25 mm, film thickness 0.25 μm); carrier gas: high-purity He, column flow rate: 1.0 mL / min, split ratio 1:1; injection port temperature: 250 °C; temperature programming: starting at 40 °C, holding for 3 min, rising to 130 °C at 2.5 °C / min, then rising to 240 °C at 7 °C / min, holding for 5 min; post-run temperature 250 °C, post-run for 5 min. MS conditions: transfer line temperature 280 °C, connecting rod temperature 150 °C; ion source: EI electron impact ionization source, electron energy 70 eV, ion source temperature 230 °C, mass scan range 33 - 450 m / z; ACQ mode Scan.

[0080] The detection results of volatile compounds adopt SPME-GC-MS technology, and the detected peak chart is as Figure 3 shown. The detection results show that the fermentation of strain LLMJ-DNXJ-01 can produce characteristic volatile aroma substances of Daqu such as isopentanol, isopentenol, caryophyllene, phenethyl alcohol, 4-vinylguaiacol, etc., and the proportions are 55.51%, 14.6%, 7.34%, 6.17%, 4.83% respectively.

[0081] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and decorations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A strain of Klebsiella sp. LLMJ-DNXJ-01, characterized in that: It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on November 25, 2024, with the deposit number CGMCC No.32792.

2. A microbial preparation containing the Klebsiella LLMJ-DNXJ-01 according to claim 1.

3. The microbial preparation according to claim 2, characterized in that The bacterial concentration of Klebsiella LLMJ-DNXJ-01 in the microbial preparation is at least 1×10 6 CFU / mL or 1×10 6 CFU / g.

4. A method for producing tannase, characterized in that: The method comprises fermenting and producing tannase using the Klebsiella LLMJ-DNXJ-01 described in claim 1 or the microbial preparation described in claim 2 or 3.

5. The method according to claim 4, characterized in that The method comprises inoculating the Klebsiella LLMJ-DNXJ-01 described in claim 1 or the microbial preparation described in claim 2 or 3 into an enzyme production system, and culturing at 28-35° C. for 100-150 hours.

6. The method according to claim 5, characterized in that In the enzyme production system, the inoculation amount of the Klebsiella LLMJ-DNXJ-01 is 5%-10% (v / v).

7. Use of the Klebsiella LLMJ-DNXJ-01 according to claim 1 or the microbial preparation according to claim 2 or 3 in degrading tannin in sorghum.

8. A method for producing characteristic volatile aroma substances of Daqu, characterized in that: The method uses sorghum as a raw material, utilizes the Klebsiella LLMJ-DNXJ-01 described in claim 1 or the microbial preparation described in claim 2 or 3 to form a fermentation system, and ferments and produces characteristic volatile aroma substances of Daqu; the characteristic volatile aroma substances of Daqu include isopentanol, isopentenol, caryophyllene, phenylethyl alcohol, and 4-vinylguaiacol.

9. The method according to claim 8, characterized in that In the fermentation system, the inoculation amount of the Klebsiella LLMJ-DNXJ-01 is 5%-10% (v / v).

10. Use of the Klebsiella LLMJ-DNXJ-01 according to claim 1 or the microbial preparation according to claim 2 or 3 in the field of winemaking.

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