Isolation culture medium and culture method of lactobacillus acidophilus

Abstract

The invention belongs to the technical field of biology, and relates to an isolation culture medium and a culture method of lactobacillus acidophilus. The culture medium comprises peptone, beef extract, yeast extract, glucose, anhydrous sodium acetate, tween-80, monopotassium phosphate, dipotassium phosphate, diammonium hydrogen citrate, magnesium sulfate heptahydrate, manganese sulfate heptahydrate, ferrous sulfate heptahydrate, lactic acid, acetic acid, cysteine and fermented grain extract. According to the culture medium formula and the culture method disclosed by the invention, efficient enrichment and separation of lactobacillus acidophilus are realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a Lactobacillus acetotolerans Jinshanensis separation culture medium and a culture method. BACKGROUND

[0002] With the rapid development of the traditional fermented food industry, the research on food microorganisms and their functions has become a focus of attention. Functional microorganisms determine the flavor, quality and stability of products, and therefore the separation and culture of specific functional strains has become a key breakthrough for optimizing fermentation processes and improving product quality. Lactobacillus acetotolerans Jinshanensis is a kind of lactic acid bacteria discovered in traditional fermented foods (liquor and vinegar) in recent years. It plays a key role in acid production, flavor substance formation and microbial community stability during the fermentation process of traditional foods. However, there are few studies on the separation, purification and culture methods of this strain. The existing technology cannot efficiently and stably obtain high-purity Lactobacillus acetotolerans Jinshanensis strains, which limits its further functional research and application.

[0003] Currently, the conventional lactic acid bacteria separation and culture method has certain limitations. The general culture medium (such as LB, MRS, etc.) has insufficient selectivity and relatively single nutritional components, which makes it difficult to effectively enrich target strains in special microenvironments (such as high-acid niches, etc.). In particular, Lactobacillus acetotolerans Jinshanensis has special requirements for pH, organic acids, temperature and growth factors, which makes it difficult to meet the separation and culture conditions with conventional culture methods. Therefore, establishing an efficient separation and optimal culture system for this strain has important value for in-depth study of the biological characteristics, metabolic functions and application in fermented foods of Lactobacillus acetotolerans Jinshanensis. SUMMARY

[0004] In some embodiments, the present application provides a Lactobacillus acetotolerans Jinshanensis separation culture medium, which comprises the following components: proteose peptone, beef extract, yeast extract, glucose, anhydrous sodium acetate, Tween-80, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, diammonium hydrogen citrate, magnesium sulfate heptahydrate, manganese sulfate heptahydrate, ferrous sulfate heptahydrate, lactic acid, acetic acid, cysteine and fermented grains extract.

[0005] In some embodiments, the present application provides a Lactobacillus acidophilus isolation medium, comprising the following components: peptone 1.0-50.0 g / L, beef extract 0.5-20.0 g / L, yeast extract 1.0-30.0 g / L, glucose 1.0-100.0 g / L, sodium acetate anhydrous 1.0-100.0 g / L, Tween-80 0.1-10.0 g / L, potassium dihydrogen phosphate 0.1-10.0 g / L, dipotassium hydrogen phosphate 0.1-10.0 g / L, diammonium hydrogen citrate 0.5-20.0 g / L, magnesium sulfate heptahydrate 0.05-5.0 g / L, manganese sulfate heptahydrate 0.0015-0.0175 g / L, ferrous sulfate heptahydrate 0.001-0.1 g / L, lactic acid 0.10%-6.0% v / v, acetic acid 0.10%-5.0% v / v, cysteine 0.1-10.0 g / L, fermented grains extract 5.0%-60.0% v / v, and the balance is water.

[0006] In some embodiments, the present application provides a Lactobacillus acidophilus isolation medium, comprising the following components: peptone 2.5-25.0 g / L, beef extract 1.0-15.0 g / L, yeast extract 2.0-20.0 g / L, glucose 5.0-80.0 g / L, sodium acetate anhydrous 5.0-50.0 g / L, Tween-80 0.2-8.0 g / L, potassium dihydrogen phosphate 0.1-8.0 g / L, dipotassium hydrogen phosphate 0.1-8.0 g / L, diammonium hydrogen citrate 1.0-15.0 g / L, magnesium sulfate heptahydrate 0.08-2.0 g / L, manganese sulfate heptahydrate 0.0025-0.0125 g / L, ferrous sulfate heptahydrate 0.002-0.08 g / L, lactic acid 0.25%-3.5% v / v, acetic acid 0.25%-2.5% v / v, cysteine 0.2-8.0 g / L, fermented grains extract 10.0%-50.0% v / v, and the balance is water.

[0007] In some embodiments, the present application provides a Lactobacillus acidophilus isolation medium, comprising the following components: peptone 5.0-15.0 g / L, beef extract 1.5-8.0 g / L, yeast extract 3.0-10.0 g / L, glucose 10.0-50.0 g / L, sodium acetate anhydrous 10.0-30.0 g / L, Tween-80 0.5-6.0 g / L, potassium dihydrogen phosphate 0.2-6.0 g / L, dipotassium hydrogen phosphate 0.2-6.0 g / L, diammonium hydrogen citrate 1.5-8.0 g / L, magnesium sulfate heptahydrate 0.1-1.0 g / L, manganese sulfate heptahydrate 0.005-0.01 g / L, ferrous sulfate heptahydrate 0.005-0.05 g / L, lactic acid 0.5%-2.5% v / v, acetic acid 0.4%-2.0% v / v, cysteine 0.5-6.0 g / L, fermented grains extract 20.0%-41.0% v / v, and the balance is water.

[0008] In some embodiments, the present invention provides a *Lactobacillus aceticus* isolation culture medium, comprising the following components: peptone 10.0 g / L, beef extract 2.0 g / L, yeast extract 5.0 g / L, glucose 20.0 g / L, anhydrous sodium acetate 20.0 g / L, Tween-80 1.08 g / L, potassium dihydrogen phosphate 0.5 g / L, dipotassium hydrogen phosphate 0.5 g / L, diammonium citrate 2.0 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate heptahydrate 0.0075 g / L, ferrous sulfate heptahydrate 0.01 g / L, lactic acid 1.5% v / v, acetic acid 0.5% v / v, cysteine ​​1.0 g / L, fermented mash extract 38.0% v / v, with the balance being water.

[0009] In some embodiments, the pH of the culture medium is 3.0 to 4.5.

[0010] In some embodiments, the pH of the culture medium is 3.2 to 4.2.

[0011] In some embodiments, the pH of the culture medium is 3.5 to 4.0.

[0012] In some implementations, the culture medium also includes agar.

[0013] In some implementations, the content of the agar is 5-50 g / L.

[0014] In some implementations, the content of the agar is 8-30 g / L.

[0015] In some implementations, the content of the agar is 10-20 g / L.

[0016] In some embodiments, the steps of the method for preparing the mash extract include: mixing mash with water, centrifuging, taking the supernatant, and obtaining the mash extract.

[0017] In some implementations, the mixing and centrifugation processes may further include oscillation, ultrasonication, or filtration.

[0018] In some implementation schemes, the fermented mash is selected from grains that have been steamed and fermented during the production of baijiu (Chinese liquor).

[0019] In some implementation schemes, the fermented mash is selected from sorghum that has been steamed and fermented during the production of soy sauce-flavored liquor.

[0020] In some implementation schemes, the fermented mash is selected from the first to seventh batches of fermented mash produced during the production of Maotai-flavor liquor.

[0021] In some implementations, the mash is selected from one or more of stacked mash, fermented mash in cellars, and fermented mash in cellars.

[0022] In some implementations, the mash is selected from mash stored in a cellar.

[0023] In some implementations, the mash is selected from mash from the fourth fermentation batch.

[0024] In some embodiments, in the method for preparing the fermented mash extract, the mass ratio of the fermented mash to the water is 1:(0.1-10).

[0025] In some embodiments, in the method for preparing the fermented mash extract, the mass ratio of the fermented mash to the water is 1:(0.2-5).

[0026] In some embodiments, in the method for preparing the fermented mash extract, the mass ratio of the fermented mash to the water is 1:(0.5-2).

[0027] In some implementations, the oscillation time is 2-60 minutes.

[0028] In some implementations, the oscillation time is 5-40 minutes.

[0029] In some implementations, the oscillation time is 8-30 minutes.

[0030] In some implementations, the ultrasound duration is 2-60 minutes.

[0031] In some implementations, the ultrasound duration is 5-40 minutes.

[0032] In some implementations, the ultrasound duration is 8-30 minutes.

[0033] In some implementations, the filtration is performed using gauze.

[0034] In some implementations, the centrifugation speed is 2000-20000 rpm.

[0035] In some implementations, the centrifugation speed is 4000-16000 rpm.

[0036] In some implementations, the centrifugation speed is 6000-12000 rpm.

[0037] In some implementations, the centrifugation time is 5-60 minutes.

[0038] In some implementations, the centrifugation time is 8-40 minutes.

[0039] In some implementations, the centrifugation time is 10-30 minutes.

[0040] In some embodiments, the present invention provides a method for preparing the Lactobacillus acetic acid isolation culture medium, the method comprising the following steps:

[0041] (1) Mix peptone, beef extract, yeast extract, glucose, anhydrous sodium acetate, Tween-80, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, diammonium citrate, magnesium sulfate heptahydrate, manganese sulfate heptahydrate, ferrous sulfate heptahydrate, cysteine ​​and mash extract with water.

[0042] (2) Add acetic acid and lactic acid and mix well.

[0043] In some implementations, in step (1), the mixture needs to be sterilized.

[0044] In some implementations, the components to be mixed with water in step (1) also include agar.

[0045] In some implementations, the sterilization is performed at 121°C for 15-20 minutes.

[0046] In some embodiments, the present invention provides the application of the Lactobacillus aceticus isolation medium in the isolation, screening, or amplification of Lactobacillus aceticus.

[0047] In some implementation schemes, the *Lactobacillus acetic acid* is derived from fermented mash or daqu (a type of starter culture).

[0048] In some implementations, the mash is selected from any stage of the brewing process.

[0049] In some implementations, the mash is selected from one or more of stacked mash, fermented mash in cellars, and fermented mash in cellars.

[0050] In some embodiments, the present invention provides a method for isolating or screening Lactobacillus jinshanensis using the culture medium, wherein the isolation or screening of Lactobacillus jinshanensis employs a dilution plating method.

[0051] In some implementation schemes, the *Lactobacillus acetic acid* is derived from fermented mash or daqu (a type of starter culture).

[0052] In some implementations, the temperature for isolating or screening Lactobacillus acetic acid in Jinshan is 20-45°C.

[0053] In some implementations, the temperature for isolating or screening Lactobacillus acetic acid in Jinshan is 25-40°C.

[0054] In some implementations, the temperature for isolating or screening Lactobacillus acetic acid in Jinshan is 25-35°C. Attached Figure Description

[0055] Figure 1This shows the plate isolation and culture results of Lactobacillus acetic acid from Jinshan.

[0056] Figure 2 Scanning electron microscopy images of Lactobacillus acetic acid from Jinshan.

[0057] Figure 3 The effect of different amounts of acetic acid added on the growth of Lactobacillus acetic acid.

[0058] Figure 4 The effect of different amounts of sodium acetate on the growth of Lactobacillus acetic acid.

[0059] Figure 5 The effect of different amounts of lactic acid added on the growth of Lactobacillus acetic acid in Jinshan.

[0060] Figure 6 The effect of different amounts of fermented mash extract added on the growth of Lactobacillus acetic acid from Jinshan.

[0061] Figure 7 The effect of different temperatures on the growth of Lactobacillus acetic acid in Jinshan. Detailed Implementation

[0062] The following specific embodiments further illustrate the technical solution of the present invention. These specific embodiments do not represent a limitation on the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0063] In this article, the identification results of different strains are based on their Latin scientific names.

[0064] The materials and reagents used in this study were sourced from the following sources: peptone (Beijing Aoboxing), yeast extract (Oxoid), beef extract powder (Beijing Aoboxing), yeast extract (Oxoid), Tween-80 (Phygene), malt extract (Beijing Aoboxing), tryptone (Beijing Aoboxing), beef extract (Beijing Aoboxing), acetic acid (Xilong Scientific, analytical grade), and lactic acid (Comio, analytical grade).

[0065] In this article, experimental methods without specific conditions were performed using conventional techniques in this technical field or according to the conditions recommended by the manufacturer; reagents or instruments without specified manufacturers were all commercially available products.

[0066] In this article, "Daqu" refers to the Daqu used in the production process of Maotai-flavor liquor. "Jiu mai" refers to the fermented mai in the cellar during the production process of Maotai-flavor liquor, specifically samples of mai undergoing anaerobic fermentation in the cellar.

[0067] The culture media involved in this article are as follows:

[0068] (1) LB solid culture medium formula (content of each component per liter of culture medium):

[0069] 10.0g peptone, 5.0g yeast extract, 10.0g sodium chloride, 15.0g agar.

[0070] (2) MRS solid culture medium formulation (content of each component per liter of culture medium):

[0071] 10.0g peptone, 10.0g beef extract, 5.0g yeast extract, 20.0g glucose, 1.08g Tween-80, 2.0g dipotassium hydrogen phosphate, 5.0g sodium acetate, 2.0g triammonium citrate, 0.1g magnesium sulfate, 0.05g manganese sulfate, 15.0g agar.

[0072] (3) MRSM solid culture medium formulation (content of each component per liter of culture medium):

[0073] 10.0g peptone, 10.0g beef extract, 5.0g yeast extract, 20.0g glucose, 1.08g Tween-80, 2.0g dipotassium hydrogen phosphate, 5.0g sodium acetate, 2.0g triammonium citrate, 0.1g magnesium sulfate (MgSO4·7H2O), 0.05g manganese sulfate (MnSO4·4H2O), 100.0g malt extract, 15.0g agar.

[0074] (4) MRSD solid culture medium formulation (content of each component per liter of culture medium):

[0075] 12.0g tryptone, 15.0g beef extract, 12.0g yeast extract, 60.0g malt extract, 15.0g glucose, 2.5g diammonium citrate, 2.5g dipotassium hydrogen phosphate, 0.2g manganese sulfate monohydrate, 0.7g magnesium sulfate heptahydrate, 2.16g Tween-80, 2.0g L-cysteine ​​hydrochloride, 3.0mg vitamin B2, 15.0g agar, and finally, acetic acid was added to adjust the total acid to 60.0g.

[0076] (5) Lac solid culture medium formulation (content of each component per liter of culture medium):

[0077] The ingredients are: 12.0g peptone, 9.6g beef extract, 4.8g yeast extract, 24.0g glucose, 2.4g dipotassium hydrogen phosphate, 2.4g diammonium hydrogen citrate, 6.0g sodium acetate, 0.24g magnesium sulfate, 0.048g manganese sulfate, 1.2g Tween-80, 100.0mL Daqu-fermented mash extract, and 15.0g agar, with water as the solvent.

[0078] (6) MYM culture medium formulation (content of each component per liter of culture medium):

[0079] 10.0g peptone, 2.0g beef extract, 5.0g yeast extract, 20.0g glucose, 20.0g anhydrous sodium acetate, 1.08g Tween-80, 0.5g potassium dihydrogen phosphate, 0.5g dipotassium hydrogen phosphate, 2.0g diammonium citrate, 0.2g magnesium sulfate heptahydrate, 0.0075g manganese sulfate heptahydrate, 0.01g ferrous sulfate heptahydrate, 10.0mL lactic acid, 10.0mL acetic acid, 1.0g cysteine, 300.0mL fermented mash extract.

[0080] (7) MYM solid culture medium formulation (content of each component per liter of culture medium):

[0081] 10.0g peptone, 2.0g beef extract, 5.0g yeast extract, 20.0g glucose, 20.0g anhydrous sodium acetate, 1.08g Tween-80, 0.5g potassium dihydrogen phosphate, 0.5g dipotassium hydrogen phosphate, 2.0g diammonium citrate, 0.2g magnesium sulfate heptahydrate, 0.0075g manganese sulfate heptahydrate, 0.01g ferrous sulfate heptahydrate, 10.0mL lactic acid, 10.0mL acetic acid, 1.0g cysteine, 15.0g agar, 300.0mL fermented mash extract.

[0082] The solvent used in the culture media mentioned above in this article is water.

[0083] The method for preparing MYM culture medium provided in this invention is as follows: According to the formula, peptone, beef extract, yeast extract, glucose, anhydrous sodium acetate, Tween-80, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, diammonium citrate, magnesium sulfate heptahydrate, manganese sulfate heptahydrate, ferrous sulfate heptahydrate, cysteine, and fermented mash extract are dissolved and mixed in water, and the volume is adjusted to 1L. The mixture is then autoclaved at 121℃ for 20 minutes. After sterilization, the culture medium is cooled to 60℃, and lactic acid and acetic acid are added and mixed thoroughly to obtain MYM culture medium.

[0084] The method for preparing MYM culture medium provided in this invention is as follows: According to the formula, peptone, beef extract, yeast extract, glucose, anhydrous sodium acetate, Tween-80, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, diammonium citrate, magnesium sulfate heptahydrate, manganese sulfate heptahydrate, ferrous sulfate heptahydrate, cysteine, agar, and wine mash extract are dissolved and mixed in water, and the volume is adjusted to 1L. The mixture is then autoclaved at 121℃ for 20 minutes. After sterilization, the culture medium is cooled to 60℃, and lactic acid and acetic acid are added and mixed thoroughly to obtain MYM solid culture medium.

[0085] Unless otherwise specified, the culture medium is prepared as follows: Dissolve and mix the culture medium components in water according to the formula, bring the volume to 1L, and autoclave at 121℃ for 20min.

[0086] Among the above-mentioned culture media, the pH of LB solid medium, MRS solid medium, MRSM solid medium, Lac solid medium, MYM medium and MYM solid medium were not adjusted and were at their natural pH.

[0087] In this article, the preparation method of fermented mash extract is as follows: Fresh fermented mash from the fourth fermentation in the cellar is taken, water is added and mixed thoroughly (mash mass: water mass = 1:2), shaken for 10 minutes, sonicated for 10 minutes, filtered through gauze, centrifuged at 10,000 rpm for 15 minutes, and the supernatant is taken and frozen for later use.

[0088] The preparation method of Daqu-fermented mash extract in this article includes the following steps:

[0089] (1) Weigh out the Daqu and the fermented mash, with a mass ratio of Daqu to fermented mash of 1:5, and mix them together;

[0090] (2) Add pure water to the Daqu-fermentation mash mixture and boil for 20 minutes. The mass ratio of the Daqu-fermentation mash mixture to pure water is 1:5.

[0091] (3) After cooling, filter the solution using four layers of gauze to obtain the filtrate for later use.

[0092] (4) After freezing the filtrate overnight, take it out to thaw, centrifuge it and take the supernatant for later use.

[0093] The Daqu-fermented mash extract contains Daqu selected from high-temperature Daqu used in the production of sauce-flavored Baijiu; the fermented mash in the Daqu-fermented mash extract contains fermented mash from the third round of fermentation in the cellar during the production of sauce-flavored Baijiu.

[0094] Example 1

[0095] Take 2g of fermented mash sample from the fourth fermentation in the cellar and place it in a sterile centrifuge tube. Add 10mL of sterile physiological saline and shake thoroughly for 5 minutes to prepare the initial bacterial suspension. Then perform a series of serial dilutions: add 1mL of the initial bacterial suspension to 9mL of sterile physiological saline and mix well to obtain a 10⁻² dilution; repeat this process to prepare 10⁻² dilutions. -3 10 -4 10 -5 10 -6 Gradient dilutions.

[0096] Take 100 μL of bacterial culture for each dilution and spread it on MYM solid medium plates. Set up 3 replicates for each dilution, and then incubate in an anaerobic incubator at 30℃ for 14 days. Observe the colony growth daily and record the colony morphology, quantity and distribution of different dilution plates.

[0097] After culturing, select single colonies with typical morphology and good dispersion, pick them with a sterile inoculation loop, and streak them onto fresh MYM solid medium plates for isolation. Incubate under anaerobic conditions at 30°C for 5 days. Repeat the above streak purification steps until a pure culture is obtained.

[0098] Pure strains were identified by 16S rRNA gene sequencing and compared with existing data in the NCBI database. Those with a sequence similarity of more than 97% were defined as the same species.

[0099] The results are shown in Table 1. The dilution coating method was used to apply the coatings from 10⁻³ and 10⁻³. -4 10 -5 10 - 6 A total of 68 strains were isolated from serially diluted MYM solid medium. Among them, there were 38 strains of *Acetilactobacillus jinshanensis*, 29 strains of acid-fast *Lactobacillus acetotolerans*, and 1 strain of *Fructilactobacillus fructivorans*.

[0100] Morphological observation revealed that *Lactobacillus acetic acid* var. *jinshanensis* exhibited regular morphology on MYM solid agar plates, with round colonies, smooth and regular edges, and opaque colonies. Figure 1 As shown. Further observation using scanning electron microscopy revealed that *Lactobacillus jinshanensis* has a rod-shaped morphology, as... Figure 2 As shown.

[0101] The above results indicate that MYM solid culture medium has a selective enrichment effect on Lactobacillus jinshanensis, achieving efficient screening and isolation of Lactobacillus jinshanensis.

[0102] Table 1. Microorganisms isolated from fermented mash samples on MYM solid medium.

[0103]

[0104]

[0105]

[0106]

[0107] Comparative Example 1

[0108] The experiment was conducted according to Example 1, except that the MYM solid medium was replaced with the LB solid medium, and the remaining experimental steps were the same as in Example 1.

[0109] As shown in Table 2, the results showed that a total of 35 strains were isolated from LB solid medium by the dilution plating method, including 3 strains of Roseomonas gilardii, 2 strains of Klebsiella pneumoniae, 1 strain of Bacillus safensis, 2 strains of Bacillus subtilis, 1 strain of Enterobacter hormaechei, 16 strains of Bacillus velezensis, 1 strain of Bacillus sonorensis, 6 strains of Bacillus licheniformis, 1 strain of Exiguobacterium mexicanum, and 2 strains of Escherichia coli.

[0110] In summary, LB solid medium does not have a selective enrichment effect on Lactobacillus acetic acid.

[0111] Table 2 Microorganisms isolated from fermented mash samples on LB solid medium

[0112] Strain identification results Strain number Roseomonas gilardii 3 Klebsiella pneumoniae 2 Bacillus safensis 1 Bacillus subtilis 2 Enterobacter hormaechei 1 Bacillus velezensis 16 Bacillus sonorensis 1 Bacillus licheniformis 6 Exiguobacterium mexicanum 1 Escherichia coli 2

[0113] Comparative Example 2

[0114] The experiment was conducted according to Example 1, except that the MYM solid medium was replaced with the MRS solid medium, and the remaining experimental steps were the same as in Example 1.

[0115] As shown in Table 3, a total of 60 strains were isolated from MRS solid medium by the dilution plating method. Among them, there were 11 strains of *Fructilactobacillus fructivorans*, 5 strains of *Bacillus siamensis*, 15 strains of *Bacillus velezensis*, 3 strains of *Delftia tsuruhatensis*, 2 strains of *Lentilactobacillus buchneri*, 2 strains of *Lactiplantibacillus plantarum*, 2 strains of *Pediococcus acidilactici*, 14 strains of *Enterococcus faecium*, and 6 strains of *Lactobacillus casei*.

[0116] The above results indicate that MRS solid culture medium does not have a selective enrichment effect on Lactobacillus acetic acid.

[0117] Table 3 Microorganisms isolated from fermented mash samples on MRS solid medium

[0118] Strain identification results Strain number Fructilactobacillus fructivorans 11 Bacillus siamensis 5 Bacillus velezensis 15 Delftia tsuruhatensis 3 Lentilactobacillus buchneri 2 Lactiplantibacillus plantarum 2 Pediococcus acidilactici 2 Enterococcus faecium 14 Lacticaseibacillus casei 6

[0119] Comparative Example 3

[0120] The experiment was conducted according to Example 1, except that the MYM solid medium was replaced with the MRSM solid medium, and the remaining experimental steps are the same as in Example 1.

[0121] As shown in Table 4, a total of 35 strains were isolated from MRSM solid medium by the dilution plating method. Among them, there were 15 strains of *Fructilactobacillus fructivorans*, 6 strains of *Lactobacillus acetotolerans*, 1 strain of *Bacillus velezensis*, and 13 strains of *Lactiplantibacillus plantarum*.

[0122] The above results indicate that MRSM solid medium does not have a selective enrichment effect on Lactobacillus acetic acid.

[0123] Table 4. Microorganisms isolated from fermented mash samples on MRSM solid medium.

[0124] Strain identification results Strain number Fructilactobacillus fructivorans 15 Lactobacillus acetotolerans 6 Bacillus velezensis 1 Lactiplantibacillus plantarum 13

[0125] Comparative Example 4

[0126] The experiment was conducted according to Example 1, except that the MYM solid medium was replaced with the MRSD solid medium, and the remaining experimental steps were the same as in Example 1.

[0127] As shown in Table 5, a total of 47 strains were isolated from MRSD solid medium by the dilution plating method. Among them, there were 4 strains of *Fructilactobacillus fructivorans*, 11 strains of *Lactobacillus acetotolerans*, 3 strains of *Lentilactobacillus buchneri*, 3 strains of *Lentibacillus daqui*, 8 strains of *Lactiplantibacillus plantarum*, 9 strains of *Lactobacillus panis*, 7 strains of *Enterococcus faecalis*, and 2 strains of *Acetilactobacillus jinshanensis*.

[0128] The above results indicate that MRSD solid medium is less effective than MYM solid medium in selectively enriching Lactobacillus jinshanensis.

[0129] Table 5 Microorganisms isolated from fermented mash samples on MRSD solid medium

[0130] Strain identification results Strain number Fructilactobacillus fructivorans 4 Lactobacillus acetotolerans 11 Lentilactobacillus buchneri 3 Lentibacillus daqui 3 Lactiplantibacillus plantarum 8 Lactobacillus panis 9 Enterococcus faecalis 7 Acetilactobacillus jinshanensis 2

[0131] Comparative Example 5

[0132] The experiment was conducted according to Example 1, except that the MYM solid medium was replaced with the Lac solid medium, and the remaining experimental steps were the same as in Example 1.

[0133] As shown in Table 6, a total of 49 strains were isolated from Lac solid medium by dilution plating method, but Lactobacillus jinshanensis was not isolated, indicating that Lac medium does not have a selective enrichment effect on Lactobacillus jinshanensis.

[0134] Table 6 Microorganisms isolated from fermented mash samples on Lac solid medium

[0135] Strain identification results Strain number Loigolactobacillus coryniformis 3 Lactobacillus acetotolerans 19 Lentibacillus daqui 7 Lactiplantibacillus plantarum 9 Lactobacillus panis 11

[0136] Note: In the table, Loigolactobacillus coryniformis is Lactobacillus coryniformis; Lactobacillus acetotolerans is Lactobacillus acetotolerans; Lentibacillus daqui is Lactobacillus daqui; Lactiplantibacillus plantarum is Lactobacillus plantarum; and Lactobacillus panis is Lactobacillus breading.

[0137] Comparative Example 6

[0138] The experiment was conducted according to Example 1, except that lactic acid was removed from the MYM solid culture medium in Example 1. The composition of the culture medium is as follows (content of each component per liter of culture medium):

[0139] 10.0g peptone, 2.0g beef extract, 5.0g yeast extract, 20.0g glucose, 20.0g anhydrous sodium acetate, 1.08g Tween-80, 0.5g potassium dihydrogen phosphate, 0.5g dipotassium hydrogen phosphate, 2.0g diammonium citrate, 0.2g magnesium sulfate heptahydrate, 0.0075g manganese sulfate heptahydrate, 0.01g ferrous sulfate heptahydrate, 10.0mL acetic acid, 1.0g cysteine, 300.0mL fermented mash extract, 15.0g agar.

[0140] The remaining experimental steps are described in Example 1.

[0141] Results: No Lactobacillus acetic acid was isolated (see Table 7).

[0142] Comparative Example 7

[0143] The experiment was conducted according to Example 1, except that the mash extract in the MYM solid culture medium of Example 1 was removed. The composition of the culture medium is as follows (content of each component per liter of culture medium):

[0144] Peptone 10.0g, beef extract 2.0g, yeast extract 5.0g, glucose 20.0g, anhydrous sodium acetate 20.0g, Tween-80 1.08g, potassium dihydrogen phosphate 0.5g, dipotassium hydrogen phosphate 0.5g, diammonium citrate 2.0g, magnesium sulfate heptahydrate 0.2g, manganese sulfate heptahydrate 0.0075g, ferrous sulfate heptahydrate 0.01g, lactic acid 10.0mL, acetic acid 10.0mL, cysteine ​​1.0g, agar 15.0g.

[0145] The remaining experimental steps are described in Example 1.

[0146] Results: No Lactobacillus acetic acid was isolated (see Table 7).

[0147] Comparative Example 8

[0148] The experiment was conducted according to Example 1, except that the acetic acid in the MYM solid culture medium of Example 1 was removed. The composition of the culture medium is as follows (content of each component per liter of culture medium):

[0149] 10.0g peptone, 2.0g beef extract, 5.0g yeast extract, 20.0g glucose, 20.0g anhydrous sodium acetate, 1.08g Tween-80, 0.5g potassium dihydrogen phosphate, 0.5g dipotassium hydrogen phosphate, 2.0g diammonium citrate, 0.2g magnesium sulfate heptahydrate, 0.0075g manganese sulfate heptahydrate, 0.01g ferrous sulfate heptahydrate, 10.0mL lactic acid, 1.0g cysteine, 300.0mL fermented mash extract, 15.0g agar.

[0150] The remaining experimental steps are described in Example 1.

[0151] Results: No Lactobacillus acetic acid was isolated (see Table 7).

[0152] Comparative Example 9

[0153] The experiment was conducted according to Example 1, but the mash extract component in the MYM solid culture medium of Example 1 was replaced with malt extract. The composition of the culture medium is as follows (content of each component per liter of culture medium):

[0154] Peptone 10.0g, beef extract 2.0g, yeast extract 5.0g, glucose 20.0g, anhydrous sodium acetate 20.0g, Tween-80 1.08g, potassium dihydrogen phosphate 0.5g, dipotassium hydrogen phosphate 0.5g, diammonium hydrogen citrate 2.0g, magnesium sulfate heptahydrate 0.2g, manganese sulfate heptahydrate 0.0075g, ferrous sulfate heptahydrate 0.01g, lactic acid 10.0mL, acetic acid 10.0mL, cysteine ​​1.0g, malt extract (Beijing Aoboxing) 60.0g, agar 15.0g.

[0155] The remaining experimental steps are described in Example 1.

[0156] Results: One strain of Lactobacillus acetic acid was isolated (see Table 7).

[0157] Comparative Example 10

[0158] The experiment was conducted according to Example 1, except that the component of the MYM solid culture medium, the mash extract, was replaced with the Daqu-mash extract. The composition of the culture medium is as follows (content of each component per liter of culture medium):

[0159] 10.0g peptone, 2.0g beef extract, 5.0g yeast extract, 20.0g glucose, 20.0g anhydrous sodium acetate, 1.08g Tween-80, 0.5g potassium dihydrogen phosphate, 0.5g dipotassium hydrogen phosphate, 2.0g diammonium citrate, 0.2g magnesium sulfate heptahydrate, 0.0075g manganese sulfate heptahydrate, 0.01g ferrous sulfate heptahydrate, 10.0mL lactic acid, 10.0mL acetic acid, 1.0g cysteine, 300.0mL Daqu-fermentation mash extract, 15.0g agar.

[0160] The remaining experimental steps are described in Example 1.

[0161] Results: Two strains of Lactobacillus acetic acid were isolated (see Table 7).

[0162] Table 7. Microbial isolation results from different experimental groups

[0163]

[0164] Note: In the table, *Acetilactobacillus jinshanensis* is *Lactobacillus jinshanensis*; *Fructilactobacillus fructivorans* is *Lactobacillus fructivorans*; *Lactobacillus acetotolerans* is *Lactobacillus acidophilus*; *Lentilactobacillus buchneri* is *Lactobacillus buchneri*; *Acetobacter pasteurianus* is *Acetobacter pasteurianus*; *Clostridium moniliforme* is *Clostridium moniliforme*; *Enterococcus faecalis* is *Enterococcus faecalis*; *Fusobacterium necrogenes* is *Clostridium necrogenes*; *Acetobacter pasteurianus subsp. pasteurianus* is *Acetobacter pasteurianus* subsp. *pasteurianus*; *Lactiplantibacillus plantarum* is *Lactobacillus plantarum*; *Pediococcus acidilactici* is *Pediococcus acidilactici*; *Enterococcus faecium* is *Enterococcus faecium*; and *Bacillus velezensis* is *Bacillus belezensis*.

[0165] Example 2

[0166] Preparation method of seed culture: Using a sterile inoculation loop, pick up the *Lactobacillus aceticus* colonies purified by streaking in Example 1 and inoculate them into 10 mL of MYM medium. Incubate at 30°C under anaerobic conditions for 72 h to obtain a bacterial suspension. Take 1 mL of the bacterial suspension and inoculate it into 9 mL of MYM medium. Incubate at 30°C under anaerobic conditions for 72 h to obtain the seed culture.

[0167] To reveal the optimal culture medium composition for Lactobacillus acetic acid, the amount of acetic acid added to MYM medium was adjusted, with acetic acid additions of 1 mL, 5 mL, 10 mL, 15 mL, 20 mL, and 25 mL set, while other components and their contents remained consistent with MYM medium, to prepare liquid culture medium.

[0168] The seed culture was inoculated at 10% of the total volume of the culture system. After inoculation, the culture was carried out in an anaerobic incubator at 30℃ for 72 hours. All other conditions were kept constant. The bacterial OD600 was measured to characterize the growth of the strain.

[0169] like Figure 3 As shown, both high and low acetic acid concentrations inhibit the growth of *Lactobacillus jinshanensis*. In summary, the culture medium prepared with 5 mL of acetic acid in a 1 L culture medium system exhibits the best growth of *Lactobacillus jinshanensis*, indicating that 0.5% v / v acetic acid is the optimal concentration.

[0170] Example 3

[0171] Based on the MYM medium, the amount of acetic acid added was adjusted to the optimal amount in Example 2 to prepare a liquid medium. The amount of sodium acetate added in the medium was adjusted to 1.0g, 5.0g, 10.0g, 20.0g, 30.0g, 50.0g, and 100.0g.

[0172] The seed culture was inoculated at 10% of the total volume of the culture system. The seed culture was obtained in the same way as in Example 2. After inoculation, the culture was carried out in an anaerobic incubator at 30°C for 72 hours. All other conditions were kept the same. The bacterial cell OD600 was used to characterize the growth of the strain.

[0173] like Figure 4 As shown, both higher and lower sodium acetate concentrations inhibit the growth of *Lactobacillus jinshanensis*. In summary, the culture medium prepared with 20.0 g of sodium acetate in a 1 L culture medium system exhibits the best growth of *Lactobacillus jinshanensis*, indicating that 2.0% w / v sodium acetate is the optimal concentration.

[0174] Example 4

[0175] Based on MYM medium, the amount of acetic acid added was adjusted to the optimal amount in Example 2, and the amount of sodium acetate added was adjusted to the optimal amount in Example 3. Liquid medium was prepared, and the amount of lactic acid added in the medium was adjusted to 5 mL, 10 mL, 15 mL, 20 mL, 25 mL, and 30 mL.

[0176] The seed culture was inoculated at 10% of the total volume of the culture system. The seed culture was obtained in the same way as in Example 2. After inoculation, the culture was carried out in an anaerobic incubator at 30°C for 72 hours. All other conditions were kept the same. The bacterial cell OD600 was used to characterize the growth of the strain.

[0177] like Figure 5As shown, both high and low lactic acid concentrations inhibit the growth of *Lactobacillus aceticus*. In summary, the culture medium prepared with 15 mL of lactic acid in a 1 L culture medium system exhibits the best growth of *Lactobacillus aceticus*, indicating that 1.5% v / v lactic acid is the optimal concentration.

[0178] Example 5

[0179] Based on MYM medium, the amount of acetic acid added was adjusted to the optimal amount in Example 2, the amount of sodium acetate added was adjusted to the optimal amount in Example 3, and the amount of lactic acid added was adjusted to the optimal amount in Example 4. The effect of the amount of mash extract added on the growth of the strain was further investigated. Liquid culture medium was prepared with mash extract added at amounts of 410 mL, 380 mL, 350 mL, 320 mL, 300 mL, 290 mL, 260 mL, 230 mL, and 200 mL.

[0180] The seed culture was inoculated at 10% of the total volume of the culture system. The seed culture was obtained in the same way as in Example 2. After inoculation, the culture was carried out in an anaerobic incubator at 30°C for 72 hours. All other conditions were kept the same. The bacterial cell OD600 was used to characterize the growth of the strain.

[0181] like Figure 6 As shown, without the addition of mash extract, the strain hardly grows. In summary, the culture medium prepared with 380 mL of mash extract in a 1 L culture medium system exhibits the best growth for Lactobacillus jinshanensis, indicating that 38.0% v / v mash extract is the optimal concentration.

[0182] Example 6

[0183] Based on MYM medium, the amount of acetic acid added was adjusted to the optimal amount in Example 2, the amount of sodium acetate added was adjusted to the optimal amount in Example 3, the amount of lactic acid added was adjusted to the optimal amount in Example 4, and the amount of mash extract added was adjusted to the optimal amount in Example 5. The effect of culture temperature on the growth of the strain was further investigated, and culture temperatures of 20℃, 25℃, 30℃, 35℃, 40℃, and 45℃ were set.

[0184] The seed culture was inoculated at 10% of the total volume of the culture system. The seed culture was obtained in the same way as in Example 2. After inoculation, the culture was carried out in an anaerobic incubator for 72 hours. All other conditions were kept the same. The bacterial cell OD600 was measured to characterize the growth of the strain.

[0185] like Figure 7 As shown, the strains exhibited different growth characteristics at different culture temperatures, with the cell density (OD) at 30℃ being the highest. 600The value reached a maximum of 2.97, indicating that 30℃ is the optimal culture temperature for Lactobacillus acetic acid.

[0186] In summary, the optimal culture medium composition and culture conditions determined through system optimization are as follows:

[0187] The optimal concentrations for acetic acid and sodium acetate were 0.5% v / v, 2.0% w / v, 1.5% v / v, and 38.0% v / v for mash extract, with an incubation temperature of 30℃.

[0188] The optimal culture medium formula is as follows (content of each component per liter of culture medium):

[0189] Peptone 10.0g, beef extract 2.0g, yeast extract 5.0g, glucose 20.0g, anhydrous sodium acetate 20.0g, Tween-80 1.08g, potassium dihydrogen phosphate 0.5g, dipotassium hydrogen phosphate 0.5g, diammonium citrate 2.0g, magnesium sulfate heptahydrate 0.2g, manganese sulfate heptahydrate 0.0075g, ferrous sulfate heptahydrate 0.01g, lactic acid 1.5% v / v, acetic acid 0.5% v / v, cysteine ​​1.0g, fermented mash extract 38.0% v / v.

[0190] The above embodiments are only for further elaboration and explanation of the technical solutions of the present invention, so that those skilled in the art can more accurately understand the inventive concept and operation scheme of the present invention, and are not intended to further limit the present invention. Any non-prominent substantive features and non-significant improvements made by those skilled in the art on this basis shall fall within the protection scope of the present invention.

Claims

1. A culture medium for isolating Lactobacillus acetic acid, characterized in that, It includes the following components: peptone, beef extract, yeast extract, glucose, anhydrous sodium acetate, Tween-80, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, diammonium citrate, magnesium sulfate heptahydrate, manganese sulfate heptahydrate, ferrous sulfate heptahydrate, lactic acid, acetic acid, cysteine, and mash extract.

2. The Lactobacillus acetic acid isolation culture medium as described in claim 1, characterized in that, Includes the following components: Peptone 1.0-50.0 g / L, Beef extract 0.5-20.0g / L, Yeast extract 1.0-30.0 g / L, Glucose 1.0-100.0 g / L Anhydrous sodium acetate 1.0-100.0 g / L Tween-80 0.1-10.0 g / L Potassium dihydrogen phosphate 0.1-10.0 g / L, Dipotassium hydrogen phosphate 0.1-10.0 g / L Diammonium hydrogen citrate 0.5-20.0 g / L, Magnesium sulfate heptahydrate 0.05-5.0 g / L, Manganese sulfate heptahydrate: 0.0015-0.0175 g / L Ferrous sulfate heptahydrate 0.001-0.1 g / L Lactic acid 0.10%-6.0% v / v Acetic acid 0.10%-5.0% v / v Cysteine ​​0.1-10.0 g / L, Distillery mash extract 5.0%-60.0% v / v The remainder is water.

3. The Lactobacillus acetic acid isolation culture medium as described in claim 1, characterized in that, Includes the following components: Peptone 2.5-25.0 g / L, Beef extract 1.0-15.0g / L, Yeast extract 2.0-20.0 g / L, Glucose 5.0-80.0 g / L Anhydrous sodium acetate 5.0-50.0 g / L, Tween-80 0.2-8.0 g / L Potassium dihydrogen phosphate 0.1-8.0 g / L, Dipotassium hydrogen phosphate 0.1-8.0 g / L Diammonium hydrogen citrate 1.0-15.0 g / L Magnesium sulfate heptahydrate 0.08-2.0 g / L, Manganese sulfate heptahydrate 0.0025-0.0125 g / L Ferrous sulfate heptahydrate 0.002-0.08 g / L Lactic acid 0.25%-3.5% v / v Acetic acid 0.25%-2.5% v / v Cysteine ​​0.2-8.0 g / L, Distillery mash extract 10.0%-50.0% v / v The remainder is water.

4. The Lactobacillus acetic acid isolation culture medium as described in claim 1, characterized in that, Includes the following components: Peptone 5.0-15.0 g / L, Beef extract 1.5-8.0g / L, Yeast extract 3.0-10.0 g / L, Glucose 10.0-50.0 g / L Anhydrous sodium acetate 10.0-30.0 g / L, Tween-80 0.5-6.0 g / L Potassium dihydrogen phosphate 0.2-6.0 g / L, Dipotassium hydrogen phosphate 0.2-6.0 g / L Diammonium hydrogen citrate 1.5-8.0 g / L, Magnesium sulfate heptahydrate 0.1-1.0 g / L, Manganese sulfate heptahydrate 0.005-0.01 g / L Ferrous sulfate heptahydrate 0.005-0.05 g / L Lactic acid 0.5%-2.5% v / v Acetic acid 0.4%-2.0% v / v Cysteine ​​0.5-6.0 g / L, Distillery mash extract 20.0%-41.0% v / v The remainder is water.

5. The Lactobacillus acetic acid isolation culture medium as described in claim 1, characterized in that, Includes the following components: Peptone 10.0 g / L Beef extract 2.0g / L Yeast extract 5.0 g / L, Glucose 20.0 g / L Anhydrous sodium acetate 20.0 g / L, Tween-80 1.08 g / L Potassium dihydrogen phosphate 0.5 g / L, Dipotassium hydrogen phosphate 0.5 g / L Diammonium hydrogen citrate 2.0 g / L Magnesium sulfate heptahydrate 0.2 g / L Manganese sulfate heptahydrate 0.0075 g / L Ferrous sulfate heptahydrate 0.01 g / L Lactic acid 1.5% v / v Acetic acid 0.5% v / v Cysteine ​​1.0 g / L Distilled mash extract 38.0% v / v The remainder is water.

6. The Lactobacillus acetic acid isolation culture medium as described in claim 1, characterized in that, The pH of the culture medium is 3.0–4.5; Preferably, the pH of the culture medium is 3.2 to 4.2; Preferably, the pH of the culture medium is 3.5 to 4.0; Preferably, the culture medium further includes agar; Preferably, the content of the agar is 5-50 g / L; Preferably, the content of the agar is 8-30 g / L; Preferably, the content of the agar is 10-20 g / L.

7. The Lactobacillus acetic acid isolation culture medium as described in claim 1, characterized in that, The steps of the method for preparing the fermented mash extract include: mixing the fermented mash with water, centrifuging, taking the supernatant, and obtaining the fermented mash extract; Preferably, the mixing and centrifugation process further includes shaking, ultrasonication, or filtration; Preferably, the fermented mash is selected from grains that have been steamed and fermented during the production of baijiu (Chinese liquor); Preferably, the fermented mash is selected from sorghum that has been steamed and cooked during the production of soy sauce-flavored liquor; Preferably, the fermented mash is selected from the first to seventh batches of fermented mash produced during the production of soy sauce-flavored baijiu; Preferably, the fermented mash is selected from one or more of the following: stacked fermented mash, fermented mash placed in a cellar, and fermented mash already in the cellar. Preferably, the fermented mash is selected from fermented mash stored in a cellar; Preferably, the mash is selected from mash from the fourth fermentation batch; Preferably, in the method for preparing the fermented mash extract, the mass ratio of the fermented mash to the water is 1:(0.1-10); Preferably, in the method for preparing the fermented mash extract, the mass ratio of the fermented mash to the water is 1:(0.2-5); Preferably, in the method for preparing the fermented mash extract, the mass ratio of the fermented mash to the water is 1:(0.5-2); Preferably, the oscillation time is 2-60 minutes; Preferably, the oscillation time is 5-40 minutes; Preferably, the oscillation time is 8-30 minutes; Preferably, the ultrasound duration is 2-60 minutes; Preferably, the ultrasound duration is 5-40 minutes; Preferably, the ultrasound duration is 8-30 minutes; Preferably, the filtration is performed using gauze. Preferably, the centrifugation speed is 2000-20000 rpm; Preferably, the centrifugation speed is 4000-16000 rpm; Preferably, the centrifugation speed is 6000-12000 rpm; Preferably, the centrifugation time is 5-60 minutes; Preferably, the centrifugation time is 8-40 minutes; Preferably, the centrifugation time is 10-30 minutes.

8. A method for preparing the *Lactobacillus acetic acid* isolation culture medium according to any one of claims 1-7, characterized in that, The method includes the following steps: (1) Mix peptone, beef extract, yeast extract, glucose, anhydrous sodium acetate, Tween-80, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, diammonium hydrogen citrate, magnesium sulfate heptahydrate, manganese sulfate heptahydrate, ferrous sulfate heptahydrate, cysteine ​​and mash extract with water. (2) Add acetic acid and lactic acid, and mix well; Preferably, in step (1), the mixture needs to be sterilized; Preferably, the components to be mixed with water in step (1) further include agar; Preferably, the sterilization is performed at 121°C for 15-20 minutes.

9. The application of the Lactobacillus acetic acid isolation culture medium according to any one of claims 1-7 in the isolation, screening, or amplification of Lactobacillus acetic acid; Preferably, the *Lactobacillus acetic acid* is derived from fermented mash or daqu (a type of starter culture). Preferably, the mash is selected from any stage of the brewing process; Preferably, the mash is selected from one or more of the following: stacked mash, mash placed in a cellar, and mash already in the cellar.

10. A method for isolating or screening *Lactobacillus jinshanensis* using any one of the culture media described in claims 1-7, characterized in that, The isolation or screening of Lactobacillus acetic acid in Jinshan was performed using the dilution plating method; Preferably, the *Lactobacillus acetic acid* is derived from fermented mash or daqu (a type of starter culture). Preferably, in the method, the temperature for isolating or screening Lactobacillus acetic acid is 20-45℃; Preferably, in the method, the temperature for isolating or screening Lactobacillus acetic acid is 25-40℃; Preferably, in the method, the temperature for isolating or screening Lactobacillus acetic acid is 25-35℃.

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