Culture medium composition for separating prokaryotes from yeast for making hard liquor as well as preparation method and application of culture medium composition

Abstract

The invention belongs to the technical field of wine brewing, and particularly relates to a culture medium composition for separating prokaryotes from yeast for making hard liquor as well as a preparation method and application of the culture medium composition. The culture medium composition comprises a culture medium containing wheat flour, a culture medium containing starch and a culture medium containing nystatin; aiming at a complex microbial community structure in yeast for making hard liquor, wheat flour and starch are added into a culture medium to simulate in-situ environment conditions, so that a culture medium formula is designed and developed, and nystatin is added to inhibit growth of non-target microbial groups; compared with a conventional culture medium, prokaryotes separated by the culture medium disclosed by the invention are more in variety and better in effect, and the culture medium disclosed by the invention has important value and significance on exploration of culturability of wine brewing microorganisms and mining of microorganism resources.

Description

Technical Field

[0001] The present application belongs to the field of winemaking technology, and specifically relates to a culture medium combination for separating prokaryotes from Daqu, and a preparation method and application thereof. Background Art

[0002] Chinese baijiu (Chinese liquor) can be categorized into four major flavor types: Luzhou-flavor, Maotai-flavor, Qingxiang, and Mixiang. Maotai-flavor baijiu has the longest production history. During the baijiu brewing process, koji (dice yeast) plays the role of a saccharifying agent, fermenting agent, and flavoring agent, decisively influencing the yield, quality, and flavor of the liquor. High-temperature koji, the saccharifying and fermenting agent for Maotai-flavor baijiu, contains a rich variety of microorganisms and the enzymes they produce, which are key to producing the Maotai-flavor flavor.

[0003] Currently, culture-free techniques such as metagenomics, metatranscriptomics, and fluorescence in situ hybridization are important means of studying the structure and function of microbial communities. Related studies have shown that bacteria are the most abundant microbial group in high-temperature Daqu and the main contributor to the production of sauce-flavor substances; molds are second in abundance and mainly play a saccharification role; yeasts are relatively few in number and are the source of fermentation power. However, obtaining pure culture strains of microorganisms is particularly important for directly analyzing their metabolic characteristics and physiological functions, and is also the basis for the development and application of microbial resources. Traditional isolation and culture methods are limited by the differences in nutrient levels, temperature, humidity, etc. under laboratory conditions and the original environmental conditions, resulting in a small number of isolated microorganisms.

[0004] Due to the complex koji-making process and open fermentation environment, high-temperature daqu harbors a complex microbial community. In-depth research into the diversity of culturable microorganisms in high-temperature daqu and the isolation and purification of functional microorganisms are crucial for understanding the flavor of sauce-flavored baijiu, improving its quality, and advancing baijiu brewing techniques. Summary of the Invention

[0005] In one aspect, the present application provides a culture medium combination for isolating prokaryotes from high-temperature Daqu, wherein the culture medium combination comprises a culture medium containing wheat flour, a culture medium containing starch, and a culture medium containing nystatin.

[0006] In some specific embodiments, the culture medium composition includes a culture medium containing wheat flour infusion.

[0007] In some embodiments, the culture medium containing wheat flour infusion is a nutrient agar medium containing wheat flour.

[0008] In some specific embodiments, the culture medium combination includes a culture medium containing rice straw infusion;

[0009] In some specific embodiments, the culture medium containing rice straw infusion is a nutrient agar medium containing rice straw infusion.

[0010] In some embodiments, the culture medium combination includes culture medium A, B and C;

[0011] The culture medium A is a nutrient agar medium containing starch;

[0012] The culture medium B is a nutrient agar medium containing wheat flour;

[0013] The culture medium C is a culture medium containing at least one of rice straw extract and wheat flour extract;

[0014] In some specific embodiments, the culture medium C is a culture medium containing wheat flour infusion;

[0015] In some specific embodiments, the culture medium C is a culture medium containing wheat flour infusion and rice straw infusion.

[0016] In some specific embodiments, the rice straw infusion is prepared by the following preparation method: water is added according to a wheat flour to water mass volume ratio of 25:1 in g:L, heated and boiled for 8 to 12 minutes, filtered, and then diluted to the original volume of water to obtain the desired infusion.

[0017] The wheat flour extract is prepared by the following method: adding 50 g of wheat flour to 1 L of water, heating and boiling the wheat flour for 10 minutes, filtering the wheat flour with gauze, collecting the obtained filtrate and adjusting the volume to 1 L to obtain the wheat flour extract.

[0018] The rice straw infusion is prepared by the following method: rice straw is cut into small segments of 3 to 5 cm in length, water is added according to a straw to water mass volume ratio of 25:1 (in g:L), the mixture is heated and boiled for 8 to 12 minutes, filtered, and then the volume is adjusted to the original volume of the added water to obtain the desired infusion.

[0019] The rice straw extract is prepared by the following method: cutting rice straw into small segments of 3 to 5 cm in length, heating and boiling the rice straw water at a ratio of 25 g of rice straw to 1 L of water for 10 minutes, filtering the rice straw with gauze, and collecting the filtrate and adjusting the volume to 1 L to obtain the rice straw extract / wheat flour extract.

[0020] In some specific embodiments, the culture medium A is a culture medium containing beef extract, peptone, soluble starch, sodium chloride, trace salt solution, vitamin solution and agar.

[0021] In some specific embodiments, the mass concentration ratio of beef extract, peptone, soluble starch, sodium chloride and agar in the culture medium A is: 2-4:4-6:12-17:4-6:12-17.

[0022] In some specific embodiments, the mass concentration ratio of beef extract, peptone, soluble starch, sodium chloride and agar in the culture medium A is: 3:5:15:5:15.

[0023] In some specific embodiments, the culture medium A contains: 2-4 g / L beef extract, 4-6 g / L peptone, 10 g / L-20 g / L soluble starch, 4-6 g / L sodium chloride, 10-20 g / L agar, 8-12 mL / L trace salt solution, and 1-3 mL / L vitamin solution.

[0024] In some specific embodiments, the culture medium A comprises: 3.0 g / L beef extract, 5.0 g / L peptone, 15.0 g / L soluble starch, 5.0 g / L sodium chloride, 10.0 mL / L trace salt solution, 2.0 mL / L vitamin solution, 15 g / L agar, and a culture medium with a nearly neutral pH value.

[0025] In some specific embodiments, the culture medium B is a culture medium containing beef extract, peptone, wheat flour, sodium chloride, trace salt solution, vitamin solution and agar;

[0026] In some specific embodiments, the mass concentration ratio of beef extract, peptone, wheat flour, sodium chloride and agar in the culture medium B is: 2-4:4-6:12-17:4-6:12-17.

[0027] In some specific embodiments, the culture medium B contains: 2-4 g / L beef extract, 4-6 g / L peptone, 10-20 g / L wheat flour, 4-6 g / L sodium chloride, 10-20 g / L agar, 8-12 mL / L trace salt solution, and 1-3 mL / L vitamin solution.

[0028] In some specific embodiments, the culture medium B comprises: 3.0 g / L beef extract, 5.0 g / L peptone, 15.0 g / L wheat flour, 5.0 g / L sodium chloride, 10.0 mL / L trace salt solution, 2.0 mL / L vitamin solution, 15 g / L agar, and a pH value close to neutral.

[0029] In some specific embodiments, the culture medium C comprises: a culture medium containing wheat flour extract, rice straw extract, sodium acetate, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, trace salt solution, vitamin solution and agar.

[0030] In some specific embodiments, the mass concentration ratio of wheat flour extract, rice straw extract, sodium acetate, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride and agar in the culture medium C is: 700-750: 300-400: 3-5: 15-25: 3-5200-250.

[0031] In some specific embodiments, the mass concentration ratio of wheat flour extract, rice straw extract, sodium acetate, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride and agar in the culture medium C is: 714:357:4:19:4:214.

[0032] In some specific embodiments, the culture medium C includes: 40 g / L to 60 g / L wheat flour extract, 20 g / L to 30 g / L rice straw extract, 0.2 g / L to 0.4 g / L sodium acetate, 1.0 g / L to 1.6 g / L ammonium sulfate, 0.2 g / L to 0.3 g / L potassium dihydrogen phosphate, 0.2 g / L to 0.3 g / L magnesium sulfate heptahydrate, 0.06 g / L to 0.08 g / L calcium chloride dihydrate, 10 to 20 g / L agar, 8 to 12 mL / L trace salt solution, and 1 to 3 mL / L vitamin solution.

[0033] In some specific embodiments, the culture medium C includes: 50 g / L wheat flour extract, 25 g / L rice straw extract, 0.3 g / L sodium acetate, 1.3 g / L ammonium sulfate, 0.28 g / L potassium dihydrogen phosphate, 0.25 g / L magnesium sulfate heptahydrate, 0.07 g / L calcium chloride dihydrate, 10.0 mL / L trace salt solution, 2.0 mL / L vitamin solution, 15 g / L agar, and the pH value is nearly neutral.

[0034] In some specific embodiments, the culture medium composition includes a culture medium containing a trace amount of salt solution,

[0035] In some specific embodiments, the trace salts include: nitrilotriacetic acid, magnesium sulfate, manganese sulfate, sodium chloride, ferrous sulfate, cobalt chloride, calcium chloride, zinc sulfate, copper sulfate, potassium aluminum sulfate, boric acid and sodium molybdate.

[0036] In some specific embodiments, the mass concentration ratio of the nitrilotriacetic acid, magnesium sulfate, manganese sulfate, sodium chloride, ferrous sulfate, cobalt chloride, calcium chloride, zinc sulfate, copper sulfate, potassium aluminum sulfate, boric acid and sodium molybdate is: (120-170): (250-350): (40-60): (80-120): (8-12): (8-12): (8-12): (8-12): 1:1:1:1.

[0037] In some specific embodiments, the mass concentration ratio of the nitrilotriacetic acid, magnesium sulfate, manganese sulfate, sodium chloride, ferrous sulfate, cobalt chloride, calcium chloride, zinc sulfate, copper sulfate, potassium aluminum sulfate, boric acid and sodium molybdate is: (120-170): (250-350): (40-60): (80-120): (8-12): (8-12): (8-12): (8-12): (8-12): 1:1:1:1, and the concentration of the nitrilotriacetic acid is 1-2 g / L.

[0038] In some specific embodiments, the trace salts include: nitrilotriacetic acid 1-2 g / L, magnesium sulfate 2-4 g / L, manganese sulfate dihydrate 0.3-0.8 g / L, sodium chloride 0.8-1.2 g / L, ferrous sulfate 0.08-0.12 g / L, cobalt chloride 0.08-0.12 g / L, calcium chloride 0.08-0.12 g / L, zinc sulfate 0.08-0.12 g / L, copper sulfate pentahydrate 0.008-0.012 g / L, potassium aluminum sulfate 0.008-0.012 g / L, boric acid 0.008-0.012 g / L, sodium molybdate 0.008-0.012 g / L;

[0039] In some specific embodiments, the trace salts include: nitrilotriacetic acid 1.5 g / L, magnesium sulfate 3 g / L, manganese sulfate dihydrate 0.5 g / L, sodium chloride 1 g / L, ferrous sulfate 0.1 g / L, cobalt chloride 0.1 g / L, calcium chloride 0.1 g / L, zinc sulfate 0.1 g / L, copper sulfate pentahydrate 0.01 g / L, potassium aluminum sulfate 0.01 g / L, boric acid 0.01 g / L, sodium molybdate dihydrate 0.01 g / L;

[0040] In some specific embodiments, the culture medium composition includes a culture medium containing a vitamin solution,

[0041] The vitamins include: biotin, folic acid, pyridoxine hydrochloride, riboflavin, thiamine, niacin, pantothenic acid, vitamin, lipoic acid, and para-aminobenzoic acid.

[0042] In some specific embodiments, the mass concentration ratio of biotin, folic acid, pyridoxine hydrochloride, riboflavin, thiamine, niacin, pantothenic acid, vitamins, lipoic acid, and p-aminobenzoic acid is: 1-3:1-3:8-12:4-6:4-6:4-6:4-6:4-6:4-6:4-6:4-6:4-6:4-6.

[0043] In some specific embodiments, the mass concentration ratio of biotin, folic acid, pyridoxine hydrochloride, riboflavin, thiamine, niacin, pantothenic acid, vitamins, lipoic acid, and para-aminobenzoic acid is: 1-3:1-3:8-12:4-6:4-6:4-6:4-6:4-6:4-6:4-6:4-6, and the mass concentration of biotin is 3-8 mg / L.

[0044] In some specific embodiments, the mass concentration ratio of biotin, folic acid, pyridoxine hydrochloride, riboflavin, thiamine, niacin, pantothenic acid, vitamins, lipoic acid, and p-aminobenzoic acid is 2:2:10:5:5:5:5:5:5:5:5.

[0045] In some specific embodiments, the vitamins include: biotin 3mg / L~8mg / L, folic acid 3mg / L~8mg / L, pyridoxine hydrochloride 20mg / L~30mg / L, riboflavin 10mg / L~15mg / L, thiamine 10mg / L~15mg / L, niacin 10mg / L~15mg / L, pantothenic acid 10mg / L~15mg / L, vitamin B12 10mg / L~15mg / L, lipoic acid 10mg / L~15mg / L, and p-aminobenzoic acid 10mg / L~15mg / L.

[0046] In some specific embodiments, the vitamins include: biotin 5 mg / L, folic acid 5 mg / L, pyridoxine hydrochloride 25 mg / L, riboflavin 12.5 mg / L, thiamine 12.5 mg / L, niacin 12.5 mg / L, pantothenic acid 12.5 mg / L, vitamin B12 12.5 mg / L, lipoic acid 12.5 mg / L, and para-aminobenzoic acid 12.5 mg / L.

[0047] In the present application, the addition of trace salts and vitamins can provide essential nutritional factors for microorganisms, thereby increasing the number of individuals of some rare prokaryotic groups and improving the probability of their isolation.

[0048] In some specific embodiments, the final concentration of nystatin added to the culture medium in the culture medium combination is 40 mg / L to 60 mg / L.

[0049] The second aspect of the present application provides a method for preparing a culture medium combination for separating prokaryotes from high-temperature Daqu as described in the first aspect, comprising the following steps:

[0050] (1) After preparing the culture medium matrix, sterilize it by high pressure;

[0051] (2) preparing a trace salt solution and a vitamin solution, filtering and sterilizing the trace salt solution and the vitamin solution to obtain a sterilized trace salt solution and vitamin solution;

[0052] (3) preparing a nystatin solution, filtering and sterilizing the nystatin solution to obtain a nystatin mother solution;

[0053] (4) After the autoclaved culture medium combination has cooled, add a small amount of filtered sterilized trace salt solution and vitamin solution, and add nystatin mother solution to make the final concentration of nystatin 45 mg / L to 55 mg / L. Mix well and pour into a plate.

[0054] In some specific embodiments, the ratio of culture medium matrix: trace salt solution: vitamin solution added to the filtered and sterilized trace salt solution and vitamin solution is 500:5:1.

[0055] In some specific embodiments, the filtration sterilization in step (2) includes: filtration sterilization using a 0.22 μm filter membrane.

[0056] In some specific embodiments, the high-pressure sterilization conditions in step (1) are: high-pressure sterilization for 10 minutes to 20 minutes.

[0057] In some specific embodiments, the high-pressure sterilization conditions in step (1) are: high-pressure sterilization at 110° C. to 130° C. for 10 to 20 minutes.

[0058] In some specific embodiments, the high-pressure sterilization conditions in step (1) are: high-pressure sterilization at 121° C. for 15 minutes.

[0059] In some specific embodiments, the step (1) of adding the filtered sterilized trace salt solution, vitamin solution, and nystatin mother solution in proportion after the culture medium combination to be sterilized by high pressure is cooled comprises: adding the filtered sterilized trace salt solution, vitamin solution, and nystatin mother solution in proportion after the culture medium combination to be sterilized by high pressure is cooled until the temperature drops to 55° C.

[0060] In some specific embodiments, the high-pressure sterilization conditions in step (4) are: high-pressure sterilization at 121° C. for 15 minutes.

[0061] In some specific embodiments, the preparation of the trace salt solution and the vitamin solution in step (1) includes: preparing the trace salt solution and the vitamin solution using distilled water as a solvent.

[0062] In some specific embodiments, the step (3) of preparing nystatin at a concentration of 40 to 60 mg / mL comprises: dissolving nystatin in dimethyl sulfoxide to prepare nystatin at a concentration of 40 to 60 mg / mL.

[0063] A third aspect of the present application provides a method for isolating prokaryotes from Daqu, comprising the following steps: diluting a Daqu sample and applying the sample to the culture medium combination described in the first aspect, and then placing the culture medium combination in an incubator for culturing;

[0064] In some specific embodiments, the dilution of the Daqu sample comprises: weighing the high-temperature Daqu sample, adding sterile water and shaking it thoroughly to prepare a suspension, and recording 1 mL of the suspension as a dilution of 10 -1 , dilute the suspension to 10 -5 ~10 -3 Then, 80 to 120 μL is applied to the culture medium combination; wherein, in terms of g:mL, the material-liquid ratio of the high-temperature Daqu to sterile water is 1:10;

[0065] In some specific embodiments, placing the culture medium combination in an incubator for culturing includes: culturing each culture medium combination at 27°C to 29°C, 36°C to 38°C, 44°C to 46°C, and 54°C to 56°C for 7 to 14 days.

[0066] In some specific embodiments, placing the culture medium combination in an incubator for culturing includes: culturing each culture medium combination at 28° C., 37° C., 45° C., and 55° C. for 7 to 14 days.

[0067] In some specific embodiments, the final concentration of nystatin added to the culture medium is 50 mg / L.

[0068] In some specific embodiments, the isolated prokaryotes are classified and identified by amplifying and sequencing 16S rRNA gene universal primers.

[0069] Compared with the prior art, the present invention has the following beneficial effects:

[0070] The present invention targets the complex microbial community structure in Daqu. By adding wheat flour and starch to the culture medium to simulate in-situ environmental conditions, a culture medium formula is designed and developed. Meanwhile, the addition of nystatin can inhibit the growth of non-target microbial groups. Compared with conventional culture media, the culture medium of the present invention isolates more types of prokaryotes and has better effects. This has important value and significance for exploring the culturability of brewing microorganisms and exploring microbial resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] The accompanying drawings are provided to further understand the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention.

[0072] Figure 1 The following are pictures of the elements and biomes cultured on the three culture media in the examples;

[0073] Figure 2 The percentage of the number of strains isolated in the examples and comparative examples;

[0074] Figure 3 The figure shows the quantitative distribution of the strains isolated in the examples and comparative examples at the family level. DETAILED DESCRIPTION

[0075] In order to better illustrate the purpose, technical solutions, and advantages of this application, the specific implementation methods of the technical solutions of this application are further described below through examples. Obviously, the described examples are only part of the examples of this application, not all of the examples. Based on the examples in this application, all other examples obtained by ordinary technicians in this field based on this application are within the scope of protection of this application.

[0076] Unless otherwise specified, the raw materials used in the examples of this application can be purchased on the market.

[0077] The present application provides a culture medium combination for separating prokaryotes from high-temperature Daqu, wherein the culture medium combination comprises a culture medium containing wheat flour, a culture medium containing starch, and a culture medium containing nystatin.

[0078] In some specific embodiments, the culture medium composition includes a culture medium containing wheat flour infusion.

[0079] In some embodiments, the culture medium containing wheat flour infusion is a nutrient agar medium containing wheat flour.

[0080] In some specific embodiments, the culture medium combination includes a culture medium containing rice straw infusion;

[0081] In some specific embodiments, the culture medium containing rice straw extract is a nutrient agar medium containing rice straw extract;

[0082] In some embodiments, the culture medium combination includes culture medium A, B and C;

[0083] The culture medium A is a nutrient agar medium containing starch;

[0084] The culture medium B is a nutrient agar medium containing wheat flour;

[0085] The culture medium C is a culture medium containing at least one of rice straw extract and wheat flour extract;

[0086] In some specific embodiments, the culture medium C is a culture medium containing wheat flour infusion;

[0087] In some specific embodiments, the culture medium C is a culture medium containing wheat flour extract and rice straw extract;

[0088] In some specific embodiments, the culture medium A is a culture medium containing beef extract, peptone, soluble starch, sodium chloride, trace salt solution, vitamin solution and agar;

[0089] In some embodiments, the mass concentration ratio of beef extract, peptone, soluble starch, sodium chloride and agar in the culture medium A is 2-4:4-6:12-17:4-6:12-17.

[0090] In some embodiments, the mass concentration ratio of beef extract, peptone, soluble starch, sodium chloride and agar in the culture medium A is 3:5:15:5:15.

[0091] In some embodiments, the culture medium A comprises beef extract 2-4 g / L, peptone 4-6 g / L, soluble starch 10 g / L-20 g / L, sodium chloride 4-6 g / L agar 10-20 g / L, trace salt solution 8-12 mL / L, vitamin solution 1-3 mL / L.

[0092] In some embodiments, the culture medium A comprises beef extract 3.0 g / L, peptone 5.0 g / L, soluble starch 15.0 g / L, sodium chloride 5.0 g / L, trace salt solution 10.0 mL / L, vitamin solution 2.0 mL / L, agar 15 g / L, and a medium with near neutral pH.

[0093] In some embodiments, the culture medium B is a culture medium containing beef extract, peptone, wheat powder, sodium chloride, trace salt solution, vitamin solution and agar.

[0094] In some embodiments, the mass concentration ratio of beef extract, peptone, wheat powder, sodium chloride and agar in the culture medium B is 2-4:4-6:12-17:4-6:12-17.

[0095] In some embodiments, the culture medium B comprises beef extract 2-4 g / L, peptone 4-6 g / L, wheat powder 10-20 g / L, sodium chloride 4-6 g / L agar 10-20 g / L, trace salt solution 8-12 mL / L, vitamin solution 1-3 mL / L.

[0096] In some embodiments, the culture medium B comprises beef extract 3.0 g / L, peptone 5.0 g / L, wheat powder 15.0 g / L, sodium chloride 5.0 g / L, trace salt solution 10.0 mL / L, vitamin solution 2.0 mL / L, agar 15 g / L, and a medium with near neutral pH.

[0097] In some embodiments, the culture medium C comprises a culture medium containing wheat powder extract, rice straw extract, sodium acetate, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, trace salt solution, vitamin solution and agar.

[0098] In some specific embodiments, the mass concentration ratio of wheat flour extract, rice straw extract, sodium acetate, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride and agar in the culture medium C is: 700-750: 300-400: 3-5: 15-25: 3-5200-250.

[0099] In some specific embodiments, the mass concentration ratio of wheat flour extract, rice straw extract, sodium acetate, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride and agar in the culture medium C is: 714:357:4:19:4:214.

[0100] In some specific embodiments, the culture medium C includes: 40 g / L to 60 g / L wheat flour extract, 20 g / L to 30 g / L rice straw extract, 0.2 g / L to 0.4 g / L sodium acetate, 1.0 g / L to 1.6 g / L ammonium sulfate, 0.2 g / L to 0.3 g / L potassium dihydrogen phosphate, 0.2 g / L to 0.3 g / L magnesium sulfate heptahydrate, 0.06 g / L to 0.08 g / L calcium chloride dihydrate, 10 to 20 g / L agar, 8 to 12 mL / L trace salt solution, and 1 to 3 mL / L vitamin solution.

[0101] In some specific embodiments, the culture medium C includes: 50 g / L wheat flour extract, 25 g / L rice straw extract, 0.3 g / L sodium acetate, 1.3 g / L ammonium sulfate, 0.28 g / L potassium dihydrogen phosphate, 0.25 g / L magnesium sulfate heptahydrate, 0.07 g / L calcium chloride dihydrate, 10.0 mL / L trace salt solution, 2.0 mL / L vitamin solution, 15 g / L agar, and the pH value is nearly neutral.

[0102] In some specific embodiments, the culture medium composition includes a culture medium containing a trace amount of salt solution,

[0103] In some specific embodiments, the trace salts include: nitrilotriacetic acid, magnesium sulfate, manganese sulfate, sodium chloride, ferrous sulfate, cobalt chloride, calcium chloride, zinc sulfate, copper sulfate, potassium aluminum sulfate, boric acid and sodium molybdate.

[0104] In some specific embodiments, the mass concentration ratio of the nitrilotriacetic acid, magnesium sulfate, manganese sulfate, sodium chloride, ferrous sulfate, cobalt chloride, calcium chloride, zinc sulfate, copper sulfate, potassium aluminum sulfate, boric acid and sodium molybdate is: (120-170): (250-350): (40-60): (80-120): (8-12): (8-12): (8-12): (8-12): (8-12): 1:1:1:1.

[0105] In some embodiments, the mass concentration ratio of the nitrilotriacetic acid, magnesium sulfate, manganese sulfate, sodium chloride, ferrous sulfate, cobalt chloride, calcium chloride, zinc sulfate, copper sulfate, potassium aluminum sulfate, boric acid and sodium molybdate is (120-170):(250-350):(40-60):(80-120):(8-12):(8-12):(8-12):(8-12):1:1:1:1, and the concentration of the nitrilotriacetic acid is 1-2 g / L.

[0106] In some embodiments, the trace salt includes: nitrilotriacetic acid 1-2 g / L, magnesium sulfate 2-4 g / L, manganese sulfate dihydrate 0.3-0.8 g / L, sodium chloride 0.8-1.2 g / L, ferrous sulfate 0.08-0.12 g / L, cobalt chloride 0.08-0.12 g / L, calcium chloride 0.08-0.12 g / L, zinc sulfate 0.08-0.12 g / L, copper sulfate pentahydrate 0.008-0.012 g / L, potassium aluminum sulfate 0.008-0.012 g / L, boric acid 0.008-0.012 g / L, and sodium molybdate 0.008-0.012 g / L.

[0107] In some embodiments, the trace salt includes: nitrilotriacetic acid 1.5 g / L, magnesium sulfate 3 g / L, manganese sulfate dihydrate 0.5 g / L, sodium chloride 1 g / L, ferrous sulfate 0.1 g / L, cobalt chloride 0.1 g / L, calcium chloride 0.1 g / L, zinc sulfate 0.1 g / L, copper sulfate pentahydrate 0.01 g / L, potassium aluminum sulfate 0.01 g / L, boric acid 0.01 g / L, and sodium molybdate dihydrate 0.01 g / L.

[0108] In some embodiments, the medium combination includes a medium containing a vitamin solution,

[0109] The vitamins include: biotin, folic acid, pyridoxine hydrochloride, riboflavin, thiamine, nicotinic acid, pantothenic acid, vitamin, lipoic acid, and p-aminobenzoic acid.

[0110] In some embodiments, the mass concentration ratio of the biotin, folic acid, pyridoxine hydrochloride, riboflavin, thiamine, nicotinic acid, pantothenic acid, vitamin, lipoic acid, and p-aminobenzoic acid is 1-3:1-3:8-12:4-6:4-6:4-6:4-6:4-6:4-6:4-6:4-6.

[0111] In some embodiments, the mass concentration ratio of the biotin, folic acid, pyridoxine hydrochloride, riboflavin, thiamine, nicotinic acid, pantothenic acid, vitamin, lipoic acid, and p-aminobenzoic acid is 1-3:1-3:8-12:4-6:4-6:4-6:4-6:4-6:4-6:4-6:4-6, and the mass concentration of the biotin is 3-8 mg / L.

[0112] In some specific embodiments, the mass concentration ratio of biotin, folic acid, pyridoxine hydrochloride, riboflavin, thiamine, niacin, pantothenic acid, vitamins, lipoic acid, and p-aminobenzoic acid is 2:2:10:5:5:5:5:5:5:5:5.

[0113] In some specific embodiments, the vitamins include: biotin 3-8 mg / L, folic acid 3-8 mg / L, pyridoxine hydrochloride 20-30 mg / L, riboflavin 10-15 mg / L, thiamine 10-15 mg / L, niacin 10-15 mg / L, pantothenic acid 10-15 mg / L, vitamin B12 10-15 mg / L, lipoic acid 10-15 mg / L, and p-aminobenzoic acid 10-15 mg / L.

[0114] In some specific embodiments, the vitamins include: biotin 5 mg / L, folic acid 5 mg / L, pyridoxine hydrochloride 25 mg / L, riboflavin 12.5 mg / L, thiamine 12.5 mg / L, niacin 12.5 mg / L, pantothenic acid 12.5 mg / L, vitamin B12 12.5 mg / L, lipoic acid 12.5 mg / L, and para-aminobenzoic acid 12.5 mg / L.

[0115] In the present application, the addition of trace salts and vitamins can provide essential nutritional factors for microorganisms, thereby increasing the number of individuals of some rare prokaryotic groups and improving the probability of their isolation.

[0116] In some specific embodiments, the final concentration of nystatin added to the culture medium in the culture medium combination is 40-60 mg / L.

[0117] The second aspect of the present application provides a method for preparing a culture medium combination for separating prokaryotes from high-temperature Daqu as described in the first aspect, comprising the following steps:

[0118] (1) After preparing the culture medium matrix, sterilize it by high pressure;

[0119] (2) preparing a trace salt solution and a vitamin solution, filtering and sterilizing the trace salt solution and the vitamin solution to obtain a sterilized trace salt solution and vitamin solution;

[0120] (3) preparing a nystatin solution, filtering and sterilizing the nystatin solution to obtain a nystatin mother solution;

[0121] (4) After the autoclaved culture medium combination has cooled, add a small amount of filtered sterilized trace salt solution and vitamin solution, and add nystatin mother solution to make the final concentration of nystatin 45 mg / L to 55 mg / L. Mix well and pour into the plate.

[0122] In some specific embodiments, the filtration sterilization in step (2) includes: filtration sterilization using a 0.22 μm filter membrane.

[0123] In some specific embodiments, the high-pressure sterilization conditions in step (1) are: high-pressure sterilization for 10 minutes to 20 minutes.

[0124] In some specific embodiments, the high-pressure sterilization conditions in step (1) are: high-pressure sterilization at 110° C. to 130° C. for 10 to 20 minutes.

[0125] In some specific embodiments, the high-pressure sterilization conditions in step (1) are: high-pressure sterilization at 121° C. for 15 minutes.

[0126] In some specific embodiments, the step (1) of adding the filtered sterilized trace salt solution, vitamin solution, and nystatin mother solution in proportion after the culture medium combination to be sterilized by high pressure is cooled comprises: adding the filtered sterilized trace salt solution, vitamin solution, and nystatin mother solution in proportion after the culture medium combination to be sterilized by high pressure is cooled until the temperature drops to 55° C.

[0127] In some specific embodiments, the high-pressure sterilization conditions in step (4) are: high-pressure sterilization at 121° C. for 15 minutes.

[0128] In some specific embodiments, the preparation of the trace salt solution and the vitamin solution in step (1) includes: preparing the trace salt solution and the vitamin solution using distilled water as a solvent.

[0129] In some specific embodiments, the step (3) of preparing nystatin at a concentration of 40 to 60 mg / mL comprises: dissolving nystatin in dimethyl sulfoxide to prepare nystatin at a concentration of 40 to 60 mg / mL.

[0130] The third aspect of the present application provides a method for isolating prokaryotes from Daqu, comprising the following steps: diluting the Daqu sample and applying it to the culture medium combination described in the first aspect of the present application, and then placing the culture medium combination in an incubator for culturing.

[0131] In some specific embodiments, the dilution of the Daqu sample comprises: weighing the high-temperature Daqu sample, adding sterile water and shaking it thoroughly to prepare a suspension, and recording 1 mL of the suspension as a dilution of 10 -1 , dilute the suspension to 10 -5 ~10 -3 Then, 80 to 120 μL is taken and applied to the culture medium combination; wherein, in terms of g:mL, the material-liquid ratio of the high-temperature Daqu to sterile water is 1:10.

[0132] In some specific embodiments, placing the culture medium combination in an incubator for culturing includes: culturing each culture medium combination at 27°C to 29°C, 36°C to 38°C, 44°C to 46°C, and 54°C to 56°C for 7 to 14 days.

[0133] In some specific embodiments, the culture medium is combined with the addition of nystatin. Final concentration It is 50mg / L.

[0134] In some specific embodiments, the isolated prokaryotes are classified and identified by amplifying and sequencing 16S rRNA gene universal primers.

[0135] Example 1: High-temperature Daqu prokaryotic separation culture medium and preparation method thereof

[0136] This embodiment provides a culture medium combination for isolating prokaryotes from high-temperature Daqu and a preparation method thereof. The components of each culture medium are shown in Tables 1 to 3:

[0137] Table 1: Components of NA+starch medium (i.e., medium A, the same below)

[0138]

[0139] Table 2: Components of NA+wheat flour medium (i.e., medium B, the same below)

[0140]

[0141] Table 3: Components of wheat flour medium (i.e., medium C, the same below)

[0142]

[0143] The trace salt solutions in Tables 1 to 3 above contain the following components: nitrilotriacetic acid 1.5 g / L, magnesium sulfate heptahydrate 3 g / L, manganese sulfate dihydrate 0.5 g / L, sodium chloride 1 g / L, ferrous sulfate heptahydrate 0.1 g / L, cobalt chloride 0.1 g / L, calcium chloride dihydrate 0.1 g / L, zinc sulfate 0.1 g / L, copper sulfate pentahydrate 0.01 g / L, potassium aluminum sulfate 0.01 g / L, boric acid 0.01 g / L, and sodium molybdate dihydrate 0.01 g / L.

[0144] The vitamin solutions in Tables 1 to 3 above contain the following components: biotin 0.005 g / L, folic acid 0.005 g / L, pyridoxine hydrochloride 0.025 g / L, riboflavin 0.0125 g / L, thiamine 0.0125 g / L, niacin 0.0125 g / L, pantothenic acid 0.0125 g / L, vitamin B12 0.0125 g / L, lipoic acid 0.0125 g / L, and p-aminobenzoic acid 0.0125 g / L.

[0145] The specific steps of the preparation method of the culture medium combination for isolating prokaryotes from high-temperature Daqu are as follows:

[0146] (1) According to the formulas in Tables 1 to 3, weigh all the components except the trace salt solution and vitamin solution to prepare NA+starch medium, NA+wheat flour medium, and wheat flour medium. After filling and sealing the bottles, adjust the pH to neutral and sterilize them by autoclaving at 121°C for 15 min.

[0147] (2) Prepare trace salt solution and vitamin solution according to the formula and filter and sterilize.

[0148] (3) Nystatin was dissolved in dimethyl sulfoxide to prepare a solution with a concentration of 50 mg / mL, and the solution was sterilized by filtration to obtain a nystatin stock solution.

[0149] (4) After the sterilized culture medium combination in step (1) is cooled to 55°C, a small amount of filtered sterilized trace salt solution, vitamin solution and nystatin mother solution are added in proportion, mixed evenly and poured into a plate.

[0150] Example 2: High-temperature Daqu prokaryotic separation method

[0151] This embodiment provides a method for isolating prokaryotes from high-temperature Daqu, using the three culture media of Example 1 of the present invention to isolate and culture prokaryotes, specifically comprising the following steps:

[0152] Weigh 5g of high-temperature Daqu sample and add it to a sterile conical flask containing 50mL of sterile water and 10 sterile glass beads. Shake thoroughly to make a suspension. 1mL of this solution is recorded as 10 -1 and diluted to 10 using sterile water. -5 Then take 10 -5 , 10 -4 , 10 -3 100 μL of each dilution was evenly spread on the three culture media mentioned above, and then the plates were placed in incubators at 28°C, 37°C, 45°C and 55°C for 7 to 14 days, and single colonies were picked for culture.

[0153] Comparative Example 1: A solid culture medium for separating prokaryotes and its preparation method

[0154] This comparative example provides a solid culture medium (i.e., NA culture medium) for isolating prokaryotes, the components of which are as follows:

[0155] Beef extract 3.0 g / L, peptone 5.0 g / L, trace salt solution 10.0 mL / L, vitamin solution 2.0 mL / L, agar 15.0 g / L.

[0156] The above-prepared raw materials were mixed and diluted to 1 L with water, the pH was adjusted to neutral, and the mixture was sterilized by high pressure at 121° C. for 15 minutes.

[0157] Comparative Example 2: A solid culture medium for separating prokaryotes and its preparation method

[0158] This comparative example provides a solid culture medium (i.e., R2A culture medium) for isolating prokaryotes, the components of which are as follows:

[0159] Yeast extract 0.5 g / L, tryptone 0.5 g / L, casein 0.5 g / L, glucose 0.5 g / L, soluble starch 0.5 g / L, dipotassium hydrogen phosphate 0.3 g / L, magnesium sulfate heptahydrate 0.05 g / L, sodium pyruvate 0.3 g / L, trace salt solution 10.0 mL / L, vitamin solution 2.0 mL / L, agar 15.0 g / L.

[0160] The above-prepared raw materials were mixed and diluted to 1 L with water, the pH was adjusted to neutral, and the mixture was sterilized by high pressure at 121° C. for 15 minutes.

[0161] Comparative Example 3: A solid culture medium for separating prokaryotes and its preparation method

[0162] This comparative example provides a solid culture medium (i.e., GYM medium) for isolating prokaryotes, the components of which are as follows:

[0163] Yeast extract 4.0 g / L, glucose 4.0 g / L, malt extract 10.0 g / L, calcium carbonate 10.0 g / L, trace salt solution 10.0 mL / L, vitamin solution 2.0 mL / L, agar 15.0 g / L.

[0164] The above-prepared raw materials were mixed and diluted to 1 L with water, the pH was adjusted to neutral, and the mixture was sterilized by high pressure at 121° C. for 15 minutes.

[0165] Comparative Example 4: A solid culture medium for separating prokaryotes and its preparation method

[0166] This comparative example provides a solid culture medium (i.e., DSM 88 culture medium) for isolating prokaryotes, the components of which are as follows:

[0167] Yeast extract 1.0 g / L, ammonium sulfate 1.3 g / L, dipotassium hydrogen phosphate 0.28 g / L, magnesium sulfate heptahydrate 0.25 g / L, calcium chloride dihydrate 0.07 g / L, ferric chloride hexahydrate 0.02 g / L, trace salt solution 10.0 mL / L, vitamin solution 2.0 mL / L, agar 15.0 g / L.

[0168] The above-prepared raw materials were mixed and diluted to 1 L with water, the pH was adjusted to 7.6±0.2, and the mixture was sterilized by high pressure at 121°C for 15 minutes.

[0169] Comparative Example 5: A solid culture medium for separating prokaryotes and its preparation method

[0170] This comparative example provides a solid culture medium (i.e., SY culture medium) for isolating prokaryotes, the components of which are as follows:

[0171] Soluble starch 15.0 g / L, yeast extract 10.0 g / L, anhydrous magnesium sulfate 0.5 g / L, trace salt solution 10.0 mL / L, vitamin solution 2.0 mL / L, agar 15.0 g / L.

[0172] The above-prepared raw materials were mixed and diluted to 1 L with water, the pH was adjusted to neutral, and the mixture was sterilized by high pressure at 121° C. for 15 minutes.

[0173] Comparative Example 6: A solid culture medium for separating prokaryotes and its preparation method

[0174] This comparative example provides a solid culture medium (i.e., TSA culture medium) for isolating prokaryotes, the components of which are as follows:

[0175] Tryptone 15.0 g / L, soy peptone 5.0 g / L, sodium chloride 5.0 g / L, trace salt solution 10.0 mL / L, vitamin solution 2.0 mL / L, agar 15.0 g / L.

[0176] The above-prepared raw materials were mixed and diluted to 1 L with water, the pH was adjusted to 7.3±0.2, and the mixture was sterilized by high pressure at 121°C for 15 minutes.

[0177] Comparative Example 7: A solid culture medium for separating prokaryotes and its preparation method

[0178] This comparative example provides a solid culture medium (i.e., MRS culture medium) for separating prokaryotes, the components of which are as follows:

[0179] Peptone 10.0 g / L, beef extract 5.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, Tween-80 1.08 g / L, dipotassium hydrogen phosphate 2.0 g / L, sodium acetate 5.0 g / L, triammonium citrate 2.0 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate tetrahydrate 0.05 g / L, trace salt solution 10.0 mL / L, vitamin solution 2.0 mL / L, agar 15.0 g / L.

[0180] The above-prepared raw materials were mixed and diluted to 1 L with water, the pH was adjusted to 6.2±0.2, and the mixture was sterilized by high pressure at 121°C for 15 minutes.

[0181] Example 3

[0182] This application example uses high-temperature Daqu provided by Kweichow Moutai Co., Ltd. as a sample, and uses the culture media described in Example 1 and Comparative Examples 1 to 7 as separation media. The isolation and culture of high-temperature Daqu prokaryotes were carried out in the Microbial Resources and Ecology Laboratory of the School of Life Sciences at Sun Yat-sen University. The specific isolation method is as follows:

[0183] First, the culture medium was prepared according to the formula of Example 1 and Comparative Examples 1 to 6 and poured into plates for later use.

[0184] Then weigh 5g of high-temperature Daqu sample and add it to a sterile conical flask containing 50mL of sterile water and 10 sterile glass beads. After shaking thoroughly, a suspension is prepared. 1mL of this solution is recorded as 10 -1 and diluted to 10 using sterile water. -5 Then take 10 -5 , 10 -4 , 10 -3 100 μL of each dilution was evenly spread on the above solid culture medium, and then the plates were placed in incubators at 28°C, 37°C, 45°C, and 55°C for 7 to 14 days.

[0185] After the culture was completed, single colonies were picked and identified by 16S rRNA gene sequencing to count the types and numbers of pure cultured bacteria in the high-temperature Daqu samples.

[0186] The separation effect diagram of the high temperature Daqu prokaryotic separation culture medium combination of Example 1 of the present invention is as follows Figure 1 As shown, the colonies on the separation plate are diverse in morphology and abundant in number, with good separation effect.

[0187] Figure 1 The three small pictures in the middle are, from left to right, the culture results of NA+starch medium, NA+wheat flour medium, and wheat flour medium.

[0188] The statistical results of the number of pure cultured bacteria isolated from high-temperature Daqu using the culture medium described in Example 1 and Comparative Examples 1 to 7 of the present invention are as follows: Figure 2 and Figure 3 As shown, a total of 833 bacterial strains were isolated.

[0189] The results showed that the high-temperature Daqu prokaryotic separation medium combination of Example 1 of the present invention had the best separation effect, containing 467 bacterial strains distributed in 3 phyla, 5 classes, 12 orders, 22 families and 35 genera (detailed genus names are shown in Table 4 below).

[0190] A total of 366 bacterial strains were isolated using the culture media of Comparative Examples 1 to 7, from 3 phyla, 3 classes, 4 orders, 10 families and 16 genera (see Table 5 below for detailed genus names).

[0191] Based on the statistical results of the number of pure cultured bacteria isolated from high-temperature Daqu samples using different culture media, it can be analyzed that compared with conventional separation culture media, the separation culture medium combination for separating prokaryotes in high-temperature Daqu of the present invention can separate more prokaryotic species and has a better separation effect, which is of great significance for exploring the diversity of culturable microorganisms in high-temperature Daqu and realizing the separation and purification of functional microorganisms.

[0192] It is understood that the present application is described by way of some embodiments, and it is understood by 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 application. In addition, under the guidance of the present application, these features and embodiments may be modified to adapt to specific circumstances and materials without departing from the scope of the present application. Therefore, the present application is not limited to the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of the present application are within the scope of protection of the present application.

[0193] Table 4

[0194]

[0195]

[0196] Table 5

[0197]

Claims

1. A culture medium combination for separating prokaryotes from high-temperature Daqu, characterized in that: The culture medium combination comprises a culture medium containing wheat flour, a culture medium containing starch and a culture medium containing nystatin.

2. The culture medium combination according to claim 1, wherein The culture medium combination includes a culture medium containing wheat flour infusion.

3. The culture medium combination according to claim 1, wherein The culture medium combination includes a culture medium containing rice straw infusion.

4. The culture medium combination according to claim 1, wherein The culture medium combination includes culture medium A, B and C; The culture medium A is a nutrient agar medium containing starch; The culture medium B is a nutrient agar medium containing wheat flour; The culture medium C is a culture medium containing at least one of rice straw extract and wheat flour extract; Preferably, the culture medium A is a culture medium containing beef extract, peptone, soluble starch, sodium chloride, trace salt solution, vitamin solution and agar; Preferably, the culture medium B is a culture medium containing beef extract, peptone, wheat flour, sodium chloride, trace salt solution, vitamin solution and agar; Preferably, the culture medium C is a culture medium containing wheat flour extract, rice straw extract, sodium acetate, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, trace salt solution, vitamin solution and agar.

5. The culture medium combination according to any one of claims 1 to 4, characterized in that The culture medium combination includes a culture medium containing a trace amount of salt solution; The trace salts include: nitrilotriacetic acid, magnesium sulfate, manganese sulfate, sodium chloride, ferrous sulfate, cobalt chloride, calcium chloride, zinc sulfate, copper sulfate, potassium aluminum sulfate, boric acid and sodium molybdate.

6. The culture medium combination according to any one of claims 1 to 4, characterized in that The culture medium combination includes a culture medium containing a vitamin solution; The vitamins include: biotin, folic acid, pyridoxine hydrochloride, riboflavin, thiamine, niacin, pantothenic acid, vitamin, lipoic acid, and para-aminobenzoic acid.

7. The culture medium combination according to any one of claims 1 to 4, characterized in that The final concentration of nystatin added to the culture medium in the culture medium combination is 40 mg / L to 60 mg / L.

8. A method for preparing a culture medium combination for separating prokaryotes from high-temperature Daqu according to any one of claims 1 to 7, characterized in that: The steps include: (1) After preparing the culture medium matrix, sterilize it by high pressure; (2) preparing a trace salt solution and a vitamin solution, filtering and sterilizing the trace salt solution and the vitamin solution to obtain a sterilized trace salt solution and vitamin solution; (3) preparing a nystatin solution, filtering and sterilizing the nystatin solution to obtain a nystatin mother solution; (4) After the autoclaved culture medium combination has cooled, add a small amount of filtered sterilized trace salt solution and vitamin solution, and add nystatin mother solution to make the final concentration of nystatin 45 mg / L to 55 mg / L. Mix well and pour into the plate.

9. A method for isolating prokaryotes from Daqu, characterized in that: The Daqu sample is diluted and applied to the culture medium combination according to any one of claims 1 to 7, and then the culture medium combination is placed in an incubator for culture.

10. The culture medium combination according to claim 1, wherein Placing the culture medium combination in an incubator for culturing includes: culturing each culture medium combination at 27°C to 29°C, 36°C to 38°C, 44°C to 46°C, and 54°C to 56°C for 7 to 14 days.

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