WO2023029568A1 - 一种特异性的乳酸菌培养基及其培养方法和应用 - Google Patents

一种特异性的乳酸菌培养基及其培养方法和应用 Download PDF

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WO2023029568A1
WO2023029568A1 PCT/CN2022/092168 CN2022092168W WO2023029568A1 WO 2023029568 A1 WO2023029568 A1 WO 2023029568A1 CN 2022092168 W CN2022092168 W CN 2022092168W WO 2023029568 A1 WO2023029568 A1 WO 2023029568A1
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lactic acid
acid bacteria
culture
culture medium
medium
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PCT/CN2022/092168
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French (fr)
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李信
崔鹏景
张俊红
吴玲玲
陆荣松
陈雯
王芸
朱胜虎
李文婷
刘尚俊
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江苏恒顺醋业股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

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  • the invention relates to the field of biotechnology, in particular to a specific culture medium for lactic acid bacteria and its cultivation method and application.
  • microorganisms without suitable culture methods still exist in nature, and only a small part of them can be cultured using traditional bacterial culture methods.
  • the microorganisms cultivated by traditional methods are all known microorganisms, and there are few studies and reports on the medium components, isolation and cultivation methods of new microbial species. Difficulty in culturing microorganisms is related to special reasons that resist cultivation, including temperature, pH, oxygen, nutrition and many other factors.
  • the cultivation of new microbial species has many technical challenges.
  • Lactic acid bacteria generally refer to a large class of bacteria that can ferment sugars to produce a large amount of lactic acid. They are widely distributed in nature and have rich species diversity. There are currently more than 200 species. Lactic acid bacteria have various physiological functions such as regulating gastrointestinal flora, improving gastrointestinal function, increasing food digestibility and biological potency, lowering serum cholesterol, controlling endotoxin, inhibiting the growth of spoilage bacteria in the intestinal tract, and improving the body's immunity.
  • the traditional brewing of vinegar adopts open fermentation, and the raw and auxiliary materials are not sterilized.
  • the types of microorganisms in the brewing process are extremely rich.
  • Scholars such as Xu Zhenghong analyzed the microbial community structure in Zhenjiang balsamic vinegar and found that there are hundreds of species in the process.
  • Microorganisms are involved in fermentation.
  • Lactic acid bacteria commonly exist in the fermentation process of vinegar, especially in traditional solid-state brewed vinegar, where the proportion of lactic acid bacteria can reach more than 60%, which has an important impact on product quality.
  • isolation and cultivation methods and their application is of great significance to the vinegar brewing industry. Therefore, it is of great significance to innovate the medium formula and culture method to better cultivate lactic acid bacteria.
  • patent CN 202010877453.8 discloses a lactic acid bacteria isolation medium formula and cultivation method.
  • the 16S rRNA sequence of the target lactic acid bacteria and the new species of lactic acid bacteria HSLZ-75 T has 100% identity
  • the target lactic acid bacteria are listed in the paper (Lactobacillus Jinshani sp.nov., isolated from solid-state vinegar culture of Zhenjiang aromatic vinegar.Antonie van Leeuwenhoek) can't grow on the culture medium formula disclosed by lactic acid bacteria HSLZ-75 T.
  • the target lactic acid bacteria in patent CN 202010877453.8 need to be cultivated on the solid plate for 7-14 days, and the cultivation time is relatively long; There are 9 kinds; it contains wine grain extract and is used in a large amount, which may easily lead to poor stability of the medium, and it is difficult for ordinary people to obtain wine grain extract, and the overall applicability is not strong.
  • Patent CN 202011227512.3 discloses a culture medium formula and isolation method for lactic acid bacteria Z-1, but its culture time is long, and it takes 7 days to form visible colonies on a solid plate; the composition of the medium is complex, and nearly 20 species need to be added in liquid culture Substance; complex operation, need to separately prepare a solution containing a large amount of complex vitamin B, need to pass through the membrane to sterilize before use, and need two kinds of acids, lactic acid and acetic acid, to adjust the pH value; need to make bran extract solution, and the stability of the culture medium is poor.
  • the present invention provides a new type of specific lactic acid bacteria culture medium.
  • the culture medium of the present invention is mainly aimed at new types of lactic acid bacteria, and is a specific, efficient, stable and convenient new type of lactic acid bacteria culture medium
  • the formula can quickly and efficiently cultivate lactic acid bacteria.
  • the invention also provides a culture method and application of a novel culture medium of lactic acid bacteria.
  • the specific lactic acid bacteria liquid culture medium of the present invention includes the following raw materials: 8-12g of tryptone, 10-15g of beef extract, and yeast extract are added to 1L of tap water 8-12g, malt extract 30-60g, glucose 10-15g, diammonium hydrogen citrate 1.5-2.5g, dipotassium hydrogen phosphate 1.5-2.5g, manganese sulfate monohydrate 0.1-0.2g, magnesium sulfate heptahydrate 0.5- 0.7g, Tween 801 ⁇ 2mL, L-cysteine hydrochloride 1 ⁇ 2g, vitamin B 2 1 ⁇ 3mg, finally add acetic acid to adjust the total acid to 4 ⁇ 6g/100mL.
  • the lactic acid bacteria are lactic acid bacteria Acetilactobacillus jinshanensis HSLZ-75 T and Lactobacillus sp.HSCY 2073.
  • the specific lactic acid bacteria solid culture medium of the present invention includes the following raw materials in the culture medium: 8 to 12 g of tryptone, 10 to 15 g of beef extract, 8 to 12 g of yeast extract, and 30 to 30 g of malt extract are added to each 1L of tap water.
  • the lactic acid bacteria are lactic acid bacteria Acetilactobacillus jinshanensis HSLZ-75 T and Lactobacillus sp.HSCY 2073.
  • the culture method of the specific lactic acid bacteria liquid culture medium of the present invention comprises the steps:
  • the height of residual air above the liquid surface of the culture medium is ⁇ 0.5cm, and the liquid filling volume of the culture medium is more than 90%, the seal is airtight, and the culture temperature is 30-35°C.
  • the culture method of the specific lactic acid bacteria solid medium of the present invention comprises the steps:
  • the bacterial strain After the bacterial strain is inoculated in the culture medium, it is placed in an anaerobic incubator for cultivation.
  • the oxygen content in the anaerobic incubator is less than 10ppm
  • the cultivation humidity is 75-85%
  • the cultivation temperature is 30-35°C.
  • the lactic acid bacteria Acetilactobacillus jinshanensis HSLZ-75 T in the present invention is a commercial strain (CICC 6269), which is a new species of lactic acid bacteria (Lactobacillus jinshani sp.nov., isolated from solid-state vinegar culture of Zhenjiang aromatic vinegar. Antonie van Leeuwenhoek).
  • the lactic acid bacteria Lactobacillus sp.HSCY 2073 is preserved in the General Microbiology Center of the China Microbiological Culture Collection Management Committee.
  • the preservation place is the Institute of Microbiology, Chinese Academy of Sciences, No. 1, Beichen West Road, Chaoyang District, Beijing.
  • the preservation registration number is CGMCC NO. 18982, date of deposit is November 21, 2019. It is a plant with strong tolerance to acetic acid and ethanol, excellent non-volatile acid production effect, and 5 kinds of fruit aromas such as acetaldehyde, ethyl acetate, isobutyl acetate, isoamyl acetate, and ethyl 3-methylvalerate.
  • Lactic acid bacteria with high content of aroma-like substances can adapt to the harsh environment of vinegar brewing, such as high acid, high alcohol, and oligonutrients. It can improve aroma substances such as aldehydes and esters with good aroma, and finally greatly improve the overall flavor quality of vinegar products.
  • the highest tolerable total acid is 8.0g/100mL; it can tolerate 13% ethanol, and can metabolize at a low temperature of 15°C, and the optimum growth temperature is 30°C.
  • the industrial application effect of the strain HSCY 2073 in vinegar brewing is remarkable, the operation is convenient, the cost is low, and the content of non-volatile acid and aroma substances in vinegar can be significantly increased.
  • the total acid content in the fermented grains can be increased by 4.71%, the non-volatile acid content can be increased by 24.86%, the product yield can be increased by 5.14%, and the acetaldehyde content of five flavor substances with fruity aroma can be increased. increased by 9.05%, ethyl acetate content increased by 25.83%, isobutyl acetate content increased by 1.51%, isoamyl acetate content increased by 7.39%, and ethyl 3-methylvalerate content increased by 34.27%, which significantly improved the taste and taste of the product.
  • Aroma the overall flavor quality is high.
  • the use in the production of Shanxi mature vinegar can also effectively strengthen the flavor quality of Shanxi mature vinegar. Its 16S rRNA gene sequence is shown in SEQ ID NO.1.
  • Acetilactobacillus jinshanensis HSLZ-75 T and Lactobacillus sp.HSCY 2073 do not have a medium formula and culture method with strong specificity, high culture efficiency, stable culture effect, simple composition, convenient use, and low cost.
  • Acetilactobacillus jinshanensis HSLZ-75 T is a special and newly discovered new species of lactic acid bacteria. It is a technical problem to be solved urgently in this field to develop a specific, efficient, stable and convenient medium formula and culture method for commercial application.
  • the common lactic acid bacteria isolation and culture medium mostly use MRS or conventional improved MRS, but generally cannot isolate or culture Acetilactobacillus jinshanensis HSLZ-75 T and Lactobacillus sp. undiscovered cause.
  • the current MRS or improved MRS medium can isolate conventional lactic acid bacteria, but the culture effect and selectivity are poor, and the separation effect is easily interfered by other microorganisms such as lactic acid bacteria, bacillus, and yeast.
  • the specific culture medium of the present invention can quickly and efficiently cultivate specific bacterial strains Acetilactobacillus jinshanensis HSLZ-75 T and Lactobacillus sp.HSCY 2073, and has beneficial effects of specificity, high efficiency, stability and convenience.
  • the specific culture medium that the present invention sets find that conventional lactic acid bacteria do not grow on it, wherein vitamin B 2
  • the addition and amount of addition are very important.
  • the present invention specifically adds a small amount of vitamins, effectively reduces the amount of malt extract used, and adjusts the acidity, so the culture effect is obviously better.
  • the culture medium of the present invention is aimed at new-type lactic acid bacteria, is a specific, efficient, stable and convenient new-type lactic acid bacteria culture medium formula, and can quickly and efficiently cultivate lactic acid bacteria.
  • the present invention has the following advantages:
  • the medium formula and culture method of the present invention can better meet the isolation and culture requirements of Acetilactobacillus jinshanensis HSLZ-75 T and Lactobacillus sp.HSCY 2073;
  • the culture medium formula and culture method of the present invention have strong specificity, high culture efficiency, stable culture effect, simple ingredients, convenient use, low cost, Beneficial effects that are readily commercializable.
  • Fig. 1 is the thalline morphology photograph of HSCY 2073 of the present invention.
  • Figure 2 is the colony morphology of HSLZ-75 T on the solid plate of the medium of the present invention.
  • the raw materials and reagents in the present invention are commercially available unless otherwise specified.
  • model bacteria and conventional lactic acid bacteria in the present invention are all purchased from corresponding strain banks or commercially available.
  • Improved MRS medium tryptone 10g, beef extract 10g, yeast extract 5g, malt extract 150g, glucose 20g, diammonium hydrogen citrate 2g, sodium acetate 5g, dipotassium hydrogen phosphate 2g, manganese sulfate monohydrate 0.19g, Magnesium sulfate heptahydrate 0.58g, Tween 80 1.0mL, L-cysteine 1g, agar 20g, distilled water 1L, sterilize at 115°C for 25min, then add acetic acid at a final concentration of 2% (v/v).
  • MRS medium formula 1 casein peptone 10g, beef extract 10g, yeast extract 5g, glucose 5g, sodium acetate 5g, diammonium citrate 2g, dipotassium hydrogen phosphate 2g, magnesium sulfate heptahydrate 0.2g, manganese sulfate monohydrate 0.05 g, Tween 80 1g, agar 15g, distilled water 1L, pH 6.8, sterilized at 115°C for 25min.
  • MRS medium formula 2 casein peptone 10g, beef powder 8g, yeast extract 4g, glucose 20g, magnesium sulfate 0.2g, sodium acetate 5g, triammonium citrate 2g, dipotassium hydrogen phosphate 2g, manganese sulfate 0.05g, Tween 80 1g, agar 20g, distilled water 1000mL, pH 5.0, sterilized at 115°C for 25min.
  • MRS medium formula 3 400mL tomato juice, 10g glucose, 5ml salt solution A, 5ml salt solution B, 10g yeast extract, 20g agar, 600mL distilled water, pH 6.9, sterilized at 115°C for 25min.
  • Salt solution A dipotassium hydrogen phosphate 2.5g, potassium dihydrogen phosphate 2.5g, distilled water 250mL
  • salt solution B solution formula magnesium sulfate heptahydrate 10g, iron sulfate 5g, sodium chloride 5g, manganese sulfate tetrahydrate 5g, distilled water 250mL.
  • LB medium formula tryptone 10g, yeast extract 5g, sodium chloride 10g, agar 15g, water 1L, sterilized at 115°C for 25min.
  • Beef extract agar medium formula: beef extract 3g, peptone 10g, sodium chloride 5g, agar 15g, water 1L, sterilized at 115°C for 25min.
  • Medium formula 100239 tryptone 10g, beef extract 10g, yeast extract 5g, glucose 20g, Tween 80 1g, dipotassium hydrogen phosphate 2g, sodium acetate 5g, triammonium citrate 2g, magnesium sulfate heptahydrate 0.2g, a Manganese sulfate water 0.05g, agar 15g, distilled water 1L, pH 6.2-6.5, sterilized at 115°C for 20min.
  • Medium formula 20203 tryptone 10g, beef extract 10g, yeast extract 5g, glucose 20g, Tween 80 1g, dipotassium hydrogen phosphate 2g, sodium acetate 5g, triammonium citrate 2g, magnesium sulfate heptahydrate 0.2g, a Manganese sulfate water 0.05g, agar 15g, distilled water 1L, pH 6.2-6.5, sterilized at 115°C for 20min.
  • Medium formula 15814 tryptone 10g, beef extract 10g, yeast extract 15g, glucose 20g, maltose 10g, Tween 80 1g, dipotassium hydrogen phosphate 2g, sodium acetate 5g, triammonium citrate 2g, magnesium sulfate heptahydrate 0.2 g, manganese sulfate monohydrate 0.05g, agar 15g, distilled water 1L, pH 5.6, sterilized at 115°C for 20min.
  • Medium formula 20331 tryptone 10g, beef extract 10g, yeast extract 5g, glucose 20g, Tween 80 1g, dipotassium hydrogen phosphate 2g, sodium acetate 5g, triammonium citrate 2g, magnesium sulfate heptahydrate 0.2g, a Manganese sulfate water 0.05g, cysteine hydrochloride 0.5g, agar 15g, distilled water 1L, pH 5.2, sterilized at 115°C for 20min.
  • Agar is not added to all the above-mentioned media as the corresponding liquid media.
  • the HSLZ-75 T and HSCY 2073 grown to the logarithmic phase will be inserted into the sealable container containing the liquid medium of the present invention according to the inoculum amount of 10% (v/v) through the following liquid medium, and the liquid culture
  • the basic formula is as follows: tryptone 10g, beef extract 12g, yeast extract 10g, malt extract 50g, glucose 13g, diammonium hydrogen citrate 2g, dipotassium hydrogen phosphate 2g, manganese sulfate monohydrate 0.15g, magnesium sulfate heptahydrate 0.5 g.
  • Tween 80 1.5mL, L-cysteine hydrochloride 1.5g, vitamin B2 2mg , tap water 1L, sterilize at 115°C for 25min, then add acetic acid to adjust the total acid to 5g/100mL.
  • the height of residual air above the liquid level of the culture medium in the sealable container is about 0.3cm, the liquid filling volume of the culture medium is about 95%, the seal is airtight, and it is placed under the condition of 30°C for 5 days.
  • Lactobacillus caviae DSM 100239 T was cultured in a common incubator at 37°C for 2 days using the formula of medium 100239.
  • Lactobacillus fructivorans DSM 20203 T was cultured in an ordinary incubator at 30°C for 2 days using the formula of medium 20203.
  • Lactobacillus rossiae DSM 15814 T was cultured in a common incubator at 30°C for 2 days using the formula of medium 15814.
  • Pediococcus damnosus DSM 20331 T was cultured in a common incubator at 26°C for 2 days using the formula of medium 20331.
  • Lactobacillus helveticus CICC 20243 and Lactobacillus acidipiscis CICC 10822 were cultured with MRS medium formula 1 at 37°C in an ordinary incubator for 1 day.
  • Lactobacillus acetotolerans CICC10774 was cultured in MRS medium formula 2 at 37°C in a common incubator for 1 day.
  • Lactobacillus fermentum CICC 20176 was cultured in an ordinary incubator at 37°C with MRS medium formula 3 for 1 day.
  • Solid medium formula: tryptone 10g, beef extract 12g, yeast extract 10g, malt extract 50g, glucose 13g, diammonium hydrogen citrate 2g, dipotassium hydrogen phosphate 2g, manganese sulfate monohydrate 0.15g, magnesium sulfate heptahydrate 0.5g, Tween 80 1.5mL, L-cysteine hydrochloride 1.5g, vitamin B2 2mg , agar powder 22g, tap water 1L, sterilize at 115°C for 25min, cool to 50-55°C, then add acetic acid Adjust to total acid 5g/100mL.
  • the coated HSLZ-75 T group is Example 1-1
  • the colony morphology is shown in Figure 2
  • the coated HSCY 2073 is Example 1-2
  • the coated DSM 100239 T group is Example 1-3
  • the coated DSM 20203 T group is Example 1-4
  • coating DSM 15814 T group is Example 1-5
  • coating DSM 20331 T group is Example 1-6
  • coating CICC 20243 group is Example 1-7
  • coating CICC The 10822 group is Examples 1-8
  • the coated CICC10774 group is Examples 1-9
  • the coated CICC 20176 group is Examples 1-10.
  • the coated HSLZ-75 T group is Example 2-1
  • the coated HSCY 2073 is Example 2-2
  • the coated DSM 100239 T group is Example 2-3
  • the coated DSM 20203 T group is Example 2 -4
  • Coating DSM 15814 T group is Example 2-5
  • Coating DSM 20331 T group is Example 2-6
  • Coating CICC 20243 group is Example 2-7
  • Coating CICC 10822 group is Example 2 -8
  • the coated CICC10774 group is Example 2-9
  • the coated CICC 20176 group is Example 2-10.
  • the coated HSLZ-75 T group is Example 3-1
  • the coated HSCY 2073 is Example 3-2
  • the coated DSM 100239 T group is Example 3-3
  • the coated DSM 20203 T group is Example 3 -4
  • Coating DSM 15814 T group is Example 3-5
  • Coating DSM 20331 T group is Example 3-6
  • Coating CICC 20243 group is Example 3-7
  • Coating CICC 10822 group is Example 3-8
  • the coated CICC10774 group is Example 3-9
  • the coated CICC 20176 group is Example 3-10.
  • MRS medium is comparative example 4-1
  • MRS medium formula 1 is comparative example 4-2
  • MRS medium formula 2 is comparative example 4-3
  • MRS medium formula 3 is comparative example 4-4
  • LB medium formula is comparative example 4-5
  • beef extract agar medium formula is comparative example 4-6
  • medium 100239 is comparative example 4-7
  • medium 20203 For Comparative Examples 4-8, Medium 15814 for Comparative Examples 4-9, Medium 20331 for Comparative Examples 4-10.
  • MRS medium formula 1 is comparative example 5-2
  • MRS medium formula 2 is comparative example 5-3
  • MRS Medium formula 3 is comparative example 5-4
  • LB medium formula is comparative example 5-5
  • beef extract agar medium formula is comparative example 5-6
  • medium 100239 is comparative example 5-7
  • medium 20203 is comparative example Ratio 5-8
  • medium 15814 is comparative example 5-9
  • medium 20331 is comparative example 5-10.
  • the medium formulation of the present invention in Example 1 was selected, but placed in a common incubator (non-anaerobic) during cultivation, cultivated at 33° C. for 15 days, and observed the growth of colonies on the plate every day.
  • the group coated with HSLZ-75 T is comparative example 6-1
  • the group coated with HSCY 2073 is comparative example 6-2.
  • the culture medium formulation of the present invention in Example 2 was selected, but placed in an ordinary incubator during cultivation, cultivated at 33° C. for 15 days, and observed the growth of colonies on the plate every day.
  • the group coated with HSLZ-75 T is comparative example 6-3
  • the group coated with HSCY 2073 is comparative example 6-4.
  • the medium formulation of the present invention in Example 3 was selected, but placed in an ordinary incubator during cultivation, cultivated at 33° C. for 15 days, and observed the growth of colonies on the plate every day.
  • the group coated with HSLZ-75 T is comparative example 6-5
  • the group coated with HSCY 2073 is comparative example 6-6.
  • the medium formula of the present invention in Example 1 was selected, but the oxygen content in the incubator was about 20ppm during cultivation, cultivated at 33°C, and the cultivation time was 15 days, and the growth of colonies on the plate was observed every day.
  • the group coated with HSLZ-75 T is comparative example 7-1
  • the group coated with HSCY 2073 is comparative example 7-2.
  • the medium formula of the present invention in Example 2 was selected, but the oxygen content in the incubator was about 20ppm during cultivation, cultivated at 30°C, and the cultivation time was 15 days, and the growth of colonies on the plate was observed every day.
  • the group coated with HSLZ-75 T is comparative example 7-3
  • the group coated with HSCY 2073 is comparative example 7-4.
  • the medium formula of the present invention in Example 3 was selected, but the oxygen content in the incubator was about 20ppm during cultivation, cultivated at 35°C for 15 days, and observed the growth of colonies on the plate every day.
  • the group coated with HSLZ-75 T is comparative example 7-5
  • the group coated with HSCY 2073 is comparative example 7-6.
  • the above-mentioned improved MRS medium was selected, which was derived from the culture medium and culture method used for lactic acid bacteria strains in the references.
  • the references are as follows: YuYJ, LiX, Zhang JH, Chai LJ, Lu ZM, Xu ZH (2019) Lactobacillus jinshani sp.nov. , isolated from solid-state vinegar culture of Zhenjiang aromatic vinegar. 113(1):43-54.
  • the culture time was 15 days, and the growth of colonies on the plate was observed every day.
  • the group coated with HSLZ-75 T was comparative example 8-1
  • the group coated with HSCY 2073 was comparative example 8-2.
  • Solid medium formula: tryptone 10g, beef extract 12g, yeast extract 10g, malt extract 50g, glucose 13g, diammonium hydrogen citrate 2g, dipotassium hydrogen phosphate 2g, manganese sulfate monohydrate 0.15g, magnesium sulfate heptahydrate 0.5g, Tween 80 1.5mL, L-cysteine hydrochloride 1.5g, agar powder 22g, tap water 1L, sterilize at 115°C for 25min, cool to 50-55°C, then add acetic acid to adjust to 5g of total acid /100mL.
  • the coated HSLZ-75 T group is Example 9-1
  • the coated HSCY 2073 is Example 9-2.
  • Example 1 Select the formula of the medium in Example 1, prepare it as a liquid culture without adding agar, and insert the bacterial strain HSLZ-75 T that has been cultivated to the logarithmic phase according to the inoculum size of 10% (v/v), and insert it into a viable medium containing a liquid medium. in an airtight container.
  • the height of the residual air above the liquid level of the culture medium in the sealable container is 0.3cm
  • the liquid filling volume of the culture medium is 95%
  • the seal is airtight
  • it is placed at 33°C for cultivation for 3 days, which is Example 10- 1.
  • the height of the residual air above the liquid surface of the culture medium was 1.0 cm, and the liquid filling volume of the culture medium was 80%, sealed airtight, and the culture temperature was 33° C., and cultivated for 3 days, which was comparative example 10-2.
  • Example 1 Select the culture medium formula in Example 1, prepare it as a liquid culture without adding agar, and insert the strain HSCY 2073 cultivated to the logarithmic phase into a sealable container containing liquid medium according to the inoculum size of 10% (v/v) middle.
  • the height of the residual air above the liquid level of the culture medium in the sealable container is 0.3cm
  • the liquid filling volume of the culture medium is 95%
  • the seal is airtight
  • it is placed at 33°C for cultivation for 3 days, which is Example 10- 3.
  • the height of residual air above the liquid surface of the culture medium is 1.0 cm
  • the liquid filling volume of the culture medium is 80%
  • the seal is airtight.
  • the culture temperature is 33° C. and cultivated for 3 days, which is comparative example 10-4.
  • Embodiment 5 selects the culture medium formula in embodiment 2, prepares the liquid culture that does not add agar, the bacterial strain HSLZ-75 T or the bacterial strain HSCY 2073 that cultivates to logarithmic phase according to the inoculum size of 10% (v/v), inoculate into a sealable container containing liquid culture medium.
  • the height of residual air above the liquid level of the culture medium in the sealable container is 0.3 cm
  • the liquid filling volume of the culture medium is 95%
  • the seal is airtight, and it is placed at 30° C. for cultivation for 3 days.
  • Embodiment 6 selects the medium formula in Embodiment 3, and prepares it into a liquid culture without adding agar.
  • the bacterial strain HSLZ-75 T or the bacterial strain HSCY 2073 cultivated to the logarithmic phase is inoculated according to the inoculum size of 10% (v/v). into a sealable container containing liquid culture medium.
  • the height of residual air above the liquid level of the culture medium in the sealable container is 0.3 cm
  • the liquid filling volume of the culture medium is 95%
  • the seal is airtight, and it is placed at 35° C. for cultivation for 3 days.
  • Examples 1-1 to 1-10, Examples 2-1 to 2-10, and Examples 3-1 to 3-10 other conventional lactic acid bacteria cannot grow on the medium of the present invention, illustrating that the present invention
  • the medium formula has good specificity and stability.
  • Comparative Examples 6-1 to 6-6 that the target lactic acid bacteria of the present invention cannot grow under the above-mentioned conventional culture methods; it can be seen from Comparative Examples 7-1 to 7-6 that although the target lactic acid bacteria of the present invention can grow in the above-mentioned It grows under the culture method with an oxygen content of 20ppm, but its growth rate is slow, and it can only be seen by the naked eye on the 7th to 8th day, and the number of growing colonies is relatively small; combined with comparative examples 8-1 to 8-2 and comparative examples 9-1 to 9-2 It can be seen that the culture medium and culture method of the present invention have good high efficiency.
  • vitamin B2 has a great influence on the growth rate and the number of colonies of bacterial strains in the culture medium of the present invention. Also have bigger influence, and vitamin B 2 is not additionally added in general lactic acid bacteria culture medium, and the content that other substances are brought in is low, additional addition can not affect conventional bacterial strain growth, simultaneously the addition of trace vitamin among the present invention, also can The amount of malt extract used can be greatly reduced and the cultivation effect is significantly better.
  • the culture method of the specific lactic acid bacteria culture medium of the present invention has better growth suitability.
  • composition of the culture medium of the invention is stable, and the substances in the culture medium can be mixed, and water can be added to prepare a liquid or solid culture medium when ready for use, which is more convenient for application.
  • take 125g of the above mixture add it to 1L tap water, dissolve it, sterilize it at 115°C for 25min, cool it down to 50-55°C, then add acetic acid to adjust the total acid to 5g/100mL, and quickly obtain liquid culture base.
  • the time for growing visible colonies to the naked eye is 7 days, and the culture medium and culture method of the present invention (the culture medium involved in it) and culture method, solid and liquid culture medium respectively adopt embodiment 1 solid culture medium and liquid culture medium, culture method adopts the solid culture of embodiment 1 and the liquid culture of embodiment 4), grows macroscopically visible bacterium colony when adopting similar bran vinegar sample
  • the time was 3 days, and the total number of detected live bacteria was increased by 8.3%, indicating that the growth rate of the medium and the method of the present invention is faster.
  • the culture medium and culture method of the present invention have good commercial application feasibility.

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Abstract

本发明公开了一种特异性的乳酸菌培养基及其培养方法和应用，该培养基包括：每1L自来水中加入胰蛋白胨8～12g、牛肉膏10～15g、酵母提取物8～12g、麦芽提取物30～60g、葡萄糖10～15g、柠檬酸氢二铵1.5～2.5g、磷酸氢二钾1.5～2.5g、一水硫酸锰0.1～0.2g、七水硫酸镁0.5～0.7g、吐温80 1～2mL、L-半胱氨酸盐酸盐1～2g、维生素B 21～3mg，最后加入乙酸调节至4～6g/100mL。本发明的培养基配方和培养方法具有专一性强、培养效率高、培养效果稳定、成分简单、成本低廉、易于商业化，可以快速高效培养乳酸菌HSLZ-75 T和HSCY 2073。

Description

一种特异性的乳酸菌培养基及其培养方法和应用 技术领域
本发明涉及生物技术领域，具体涉及一种特异性的乳酸菌培养基及其培养方法和应用。
背景技术
研究发现自然界中尚存在90％以上未有合适培养方法的微生物，使用传统的细菌培养方法仅能培养其中的一小部分细菌。应用传统方法培养出的微生物都是已知微生物，对新的微生物物种的培养基成分、分离、培养方法的研究和报道较少。微生物培养困难是与抵制培养的特殊原因，包括温度、pH、氧气、营养等众多因素有关，微生物新种的培养具有众多的技术挑战。
乳酸菌泛指一大类能够发酵糖类产生大量乳酸的细菌，在自然界分布极为广泛，具有丰富的物种多样性，目前约有200余种。乳酸菌具有调节胃肠道菌群、改善胃肠道功能、提高食物消化率和生物效价、降低血清胆固醇、控制内毒素、抑制肠道内腐败菌生长、提高机体免疫力等多种生理功能。
传统酿造食醋采用开放式发酵、原辅料不经过灭菌处理，酿造过程中微生物种类及其丰富，许正宏等学者对镇江香醋醋醅中微生物群落结构进行解析发现该过程中有上百种微生物参与发酵。乳酸菌普遍存在于食醋发酵过程中，尤其是传统固态酿造食醋，乳酸菌占比可达到60％以上，对产品品质具有重要影响。深入研究不同类型乳酸菌培养基、分离和培养方法及其应用对食醋酿造行业具有重要意义。因此，创新培养基配方和培养方法，更好的培养乳酸菌具有重要意义。
目前，许多不同的乳酸菌对培养基和培养方法具有独特的需求，即使同一种乳酸菌的不同菌株之间，在培养基和培养方法上也具有较大的差异。如专利CN 202010877453.8公开了一种乳酸菌分离培养基配方及培养方法，虽然其所培养目的乳酸菌与新种乳酸菌HSLZ-75 T的16S rRNA序列具有100％的一致性，但其目的乳酸菌在论文(Lactobacillus jinshani sp.nov.,isolated from solid-state vinegar culture of Zhenjiang aromatic vinegar.Antonie van Leeuwenhoek)公开的乳酸菌HSLZ-75 T的培养基配方上无法生长。且专利CN 202010877453.8中目的乳酸菌在其固体平板上需培养7-14天，培养时间较长；培养基配方极其复杂、操作不便、成本高，液体培养中需要添加30余种物质，其中仅维生素就有9种；含有酒醅浸出液且用量大，易导致培养基的稳定性差，酒醅浸出液一般人员难以获得，整体应用性不强。专利CN 202011227512.3公开了一种乳酸菌Z-1的培养基配方和分离方法，但其培养时间较长，固体平板上需要7天后形成肉眼可见菌落； 培养基成分复杂，液体培养中需要添加近20种物质；操作复杂，需要单独配制含有大量复合维生素B的溶液，使用前需过膜除菌，且需要乳酸和乙酸2种酸调节pH值；需要自制麸皮浸出液，培养基稳定性差。
发明内容
发明目的：针对现有技术存在的问题，本发明提供一种新型特异性的乳酸菌培养基，本发明的培养基主要针对新型乳酸菌，是一种专一、高效、稳定、方便的新型乳酸菌培养基配方，可以快速高效培养乳酸菌。
本发明还提供了一种新型乳酸菌的培养基的培养方法和应用。
技术方案：为了实现上述目的，本发明所述的特异性的乳酸菌液体培养基，所述培养基中包括以下原料：每1L自来水中加入胰蛋白胨8～12g、牛肉膏10～15g、酵母提取物8～12g、麦芽提取物30～60g、葡萄糖10～15g、柠檬酸氢二铵1.5～2.5g、磷酸氢二钾1.5～2.5g、一水硫酸锰0.1～0.2g、七水硫酸镁0.5～0.7g、吐温801～2mL、L-半胱氨酸盐酸盐1～2g、维生素B 2 1～3mg，最后加入乙酸调节至总酸4～6g/100mL。
其中，所述乳酸菌为乳酸菌Acetilactobacillus jinshanensis HSLZ-75 T和Lactobacillus sp.HSCY 2073。
本发明所述特异性的乳酸菌固体培养基，所述培养基中包括以下原料：每1L自来水中加入胰蛋白胨8～12g、牛肉膏10～15g、酵母提取物8～12g、麦芽提取物30～60g、葡萄糖10～15g、柠檬酸氢二铵1.5～2.5g、磷酸氢二钾1.5～2.5g、一水硫酸锰0.1～0.2g、七水硫酸镁0.5～0.7g、吐温80 1～2mL、L-半胱氨酸盐酸盐1～2g、维生素B 2 1～3mg，加入琼脂粉20～25g，最后加入乙酸调节至总酸4～6g/100mL。
其中，所述乳酸菌为乳酸菌Acetilactobacillus jinshanensis HSLZ-75 T和Lactobacillus sp.HSCY 2073。
本发明所述的特异性的乳酸菌液体培养基的培养方法，包括如下步骤：
在培养基中接种菌株后，在培养基的液面上方残留空气的高度＜0.5cm，且培养基的装液量＞90％，密封不漏气，培养温度30～35℃。
本发明所述的特异性的乳酸菌固体培养基的培养方法，包括如下步骤：
在培养基中接种菌株后，置于厌氧培养箱中培养，厌氧培养箱中氧气含量＜10ppm，培养湿度75～85％，培养温度30～35℃。
本发明所述的特异性的乳酸菌液体培养基或者所述的乳酸菌的固体培养基在快速大量培养乳酸菌中的应用。
本发明中乳酸菌Acetilactobacillus jinshanensis HSLZ-75 T为商业菌株(CICC  6269)，是一株乳酸菌新种(Lactobacillus jinshani sp.nov.,isolated from solid-state vinegar culture of Zhenjiang aromatic vinegar.Antonie van Leeuwenhoek)。
乳酸菌Lactobacillus sp.HSCY 2073保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地点为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏登记入册的编号为CGMCC NO.18982，保藏日期为2019年11月21日。其是一株乙酸和乙醇耐受性强、产不挥发酸效果优、产乙醛、乙酸乙酯、乙酸异丁酯、乙酸异戊酯、3-甲基戊酸乙酯等5种果香类香气物质含量高的乳酸菌，可以适应食醋酿造的高酸、高醇、寡营养等恶劣环境，耐高酸的能力强，即能产不挥发酸以提高食醋滋味的柔和性，同时又能提高具有良好香气的醛类、酯类等香气物质，并且最终使食醋产品的整体风味品质大幅度。最高可耐受的总酸为8.0g/100mL；可耐受13％乙醇，能够在15℃的低温条件下代谢，最适生长温度为30。菌株HSCY 2073在食醋酿造的产业化应用效果显著，操作方便，成本低廉，能够显著提高食醋中不挥发酸和香气物质的含量。通过该菌株在镇江香醋酿造中的使用，可使醅卤中总酸含量提高4.71％，不挥发酸含量提高24.86％，产品出率提5.14％，具有果香味的5种风味物质乙醛含量提高9.05％、乙酸乙酯含量提高25.83％、乙酸异丁酯含量提高1.51％、乙酸异戊酯含量提高7.39％、3-甲基戊酸乙酯含量提高34.27％，显著提高了产品的滋味和香气，整体风味品质较高。同时在山西老陈醋造中的使用也可以有效强化山西老陈醋的风味品质。其16S rRNA基因序列，如SEQ ID NO.1所示。
目前，Acetilactobacillus jinshanensis HSLZ-75 T和Lactobacillus sp.HSCY 2073暂没有专一性强、培养效率高、培养效果稳定、成分简单、使用方便、成本低廉的培养基配方和培养方法。Acetilactobacillus jinshanensis HSLZ-75 T作为一个特殊的、新发现的乳酸菌新种，开发可商业化应用，专一、高效、稳定、方便的培养基配方和培养方法，是本领域亟待解决的技术难题。目前常见的乳酸菌分离和培养基多采用MRS或常规改良的MRS为主，但一般均不能对Acetilactobacillus jinshanensis HSLZ-75 T和Lactobacillus sp.HSCY 2073进行分离或培养，这也是该乳酸菌作为新种迟迟未被发现的原因。同时现行的MRS或改良的MRS培养基可对常规的乳酸菌进行分离，但培养效果和选择性较差，分离效果易受其他乳酸菌、芽孢杆菌、酵母菌等微生物的干扰。
而本发明特定的培养基对于特定的菌株Acetilactobacillus jinshanensis HSLZ-75 T和Lactobacillus sp.HSCY 2073，可以快速高效培养，具有专一、高效、稳定、方便的有益效果。
本发明中由于特定的菌株Acetilactobacillus jinshanensis HSLZ-75 T和 Lactobacillus sp.HSCY 2073需要特定的培养基，本发明设定的特定的培养基，发现常规的乳酸菌在上面是不生长的，其中维生素B 2的添加及其添加量十分重要。此外，与改良MRS培养基相比，本发明特定增加了微量的维生素，有效减少麦芽提取物的使用量，并调整酸度，培养效果明显更好。本发明的培养基针对新型乳酸菌，是一种专一、高效、稳定、方便的新型乳酸菌培养基配方，可以快速高效培养乳酸菌。
有益效果：由现有技术相比较，本发明具有以下优点：
(1)本发明的培养基配方和培养方法可较好的满足Acetilactobacillus jinshanensis HSLZ-75 T和Lactobacillus sp.HSCY 2073的分离和培养需求；
(2)本发明的培养基配方和培养方法在Acetilactobacillus jinshanensis HSLZ-75 T和Lactobacillus sp.HSCY 2073培养上具有专一性强、培养效率高、培养效果稳定、成分简单、使用方便、成本低廉、易于商业化的有益效果。
(3)本发明从多维度的综合比较实验证明，本发明的培养基配方和培养方法，具有良好的专一性、高效性和稳定性。
附图说明
图1为本发明的HSCY 2073的菌体形态照片。
图2为HSLZ-75 T在本发明培养基的固体平板上的菌落形态。
具体实施方式
以下结合附图和实施例对本发明作进一步说明。
本发明中的原料和试剂如无特殊说明，均为商业化市售。
本发明中的模式菌以及常规乳酸菌均由相应菌种库购买或者市售可得。
改良MRS培养基：胰蛋白胨10g、牛肉膏10g、酵母提取物5g、麦芽提取物150g、葡萄糖20g、柠檬酸氢二铵2g、乙酸钠5g、磷酸氢二钾2g、一水硫酸锰0.19g、七水硫酸镁0.58g、吐温80 1.0mL、L-半胱氨酸1g，琼脂20g，蒸馏水1L，115℃，25min灭菌，然后加入终浓度2％(v/v)乙酸。
MRS培养基配方1：酪蛋白胨10g，牛肉膏10g，酵母提取物5g，葡萄糖5g，乙酸钠5g，柠檬酸二铵2g，磷酸氢二钾2g，七水硫酸镁0.2g，一水硫酸锰0.05g，吐温80 1g，琼脂15g，蒸馏水1L，pH6.8，115℃，25min灭菌。
MRS培养基配方2：酪蛋白胨10g，牛肉粉8g，酵母提取物4g，葡萄糖20g，硫酸镁0.2g，乙酸钠5g，柠檬酸三铵2g，磷酸氢二钾2g，硫酸锰0.05g，吐温80 1g，琼脂20g，蒸馏水1000mL，pH 5.0，115℃，25min灭菌。
MRS培养基配方3：西红柿汁400mL，葡萄糖10g，盐溶液A 5ml，盐溶液 B 5ml，酵母膏10g，琼脂20g，蒸馏水600mL，pH 6.9，115℃，25min灭菌。盐溶液A配方：磷酸氢二钾2.5g，磷酸二氢钾2.5g，蒸馏水250mL；盐溶液B溶液配方：七水硫酸镁10g，硫酸铁5g，氯化钠5g，四水硫酸锰5g，蒸馏水250mL。
LB培养基配方：胰蛋白胨10g，酵母提取物5g，氯化钠10g，琼脂15g，水1L，115℃灭菌25min。
牛肉膏琼脂培养基配方：牛肉膏3g，蛋白胨10g，氯化钠5g，琼脂15g，水1L，115℃灭菌25min。
培养基配方100239：胰蛋白胨10g，牛肉膏10g，酵母提取物5g，葡萄糖20g，吐温80 1g，磷酸氢二钾2g，乙酸钠5g，柠檬酸三铵2g，七水硫酸镁0.2g，一水硫酸锰0.05g，琼脂15g，蒸馏水1L，pH6.2-6.5，115℃，20min灭菌。
培养基配方20203：胰蛋白胨10g，牛肉膏10g，酵母提取物5g，葡萄糖20g，吐温80 1g，磷酸氢二钾2g，乙酸钠5g，柠檬酸三铵2g，七水硫酸镁0.2g，一水硫酸锰0.05g，琼脂15g，蒸馏水1L，pH6.2-6.5，115℃，20min灭菌。
培养基配方15814：胰蛋白胨10g，牛肉膏10g，酵母提取物15g，葡萄糖20g，麦芽糖10g，吐温80 1g，磷酸氢二钾2g，乙酸钠5g，柠檬酸三铵2g，七水硫酸镁0.2g，一水硫酸锰0.05g，琼脂15g，蒸馏水1L，pH5.6，115℃，20min灭菌。
培养基配方20331：胰蛋白胨10g，牛肉膏10g，酵母提取物5g，葡萄糖20g，吐温80 1g，磷酸氢二钾2g，乙酸钠5g，柠檬酸三铵2g，七水硫酸镁0.2g，一水硫酸锰0.05g，半胱氨酸盐酸盐0.5g，琼脂15g，蒸馏水1L，pH5.2，115℃，20min灭菌。
上述所有培养基中不加入琼脂为对应的液体培养基。
1、菌株的活化培养
(1)菌株Acetilactobacillus jinshanensis HSLZ-75 T和Lactobacillus sp.HSCY 2073的活化培养。
将通过下述液体培养基将生长至对数期的HSLZ-75 T和HSCY 2073按照10％(v/v)的接种量，分别接入含有本发明液体培养基的可密封容器中，液体培养基配方如下：胰蛋白胨10g、牛肉膏12g、酵母提取物10g、麦芽提取物50g、葡萄糖13g、柠檬酸氢二铵2g、磷酸氢二钾2g、一水硫酸锰0.15g、七水硫酸镁0.5g、吐温80 1.5mL、L-半胱氨酸盐酸盐1.5g、维生素B 2 2mg，自来水1L，115℃，25min灭菌，然后加入乙酸调节至总酸5g/100mL。
可密封容器中培养基的液面上方残留空气的高度约0.3cm，培养基的装液量 约95％，密封不漏气，置于30℃条件下培养5d。
重复上述方式，将菌株活化3次，其中最后一次培养时间为5d，备用。HSCY 2073菌体形态如图1。
(2)4株16S rRNA与HSLZ-75 T相近的模式菌种的活化培养
选择相应的不添加琼脂的液体培养基进行活化3次，备用。
Lactobacillus caviae DSM 100239 T，采用培养基100239培养基配方，37℃，普通培养箱培养2天。
Lactobacillus fructivorans DSM 20203 T，采用培养基20203培养基配方，30℃，普通培养箱培养2天。
Lactobacillus rossiae DSM 15814 T，采用培养基15814培养基配方，30℃，普通培养箱培养2天。
Pediococcus damnosus DSM 20331 T，采用培养基20331培养基配方，26℃，普通培养箱培养2天。
(3)4株常规乳酸菌的活化培养
选择相应的不添加琼脂的液体培养基进行活化3次，备用。
Lactobacillus helveticus CICC 20243和Lactobacillus acidipiscis CICC 10822，用MRS培养基配方1，37℃，普通培养箱培养1天。
Lactobacillus acetotolerans CICC10774，用MRS培养基配方2，37℃，普通培养箱培养1天。
Lactobacillus fermentum CICC 20176，用MRS培养基配方3，37℃，普通培养箱培养1天。
2、菌株的涂布
取上述步骤中活化3次的各个菌株的培养液，采用无菌生理盐水进行梯度稀释，选取活菌数在1000～2000CFU/mL的稀释液0.1mL涂布于下述实施例1中相应的固体平板上。
实施例1
固体培养基配方：胰蛋白胨10g、牛肉膏12g、酵母提取物10g、麦芽提取物50g、葡萄糖13g、柠檬酸氢二铵2g、磷酸氢二钾2g、一水硫酸锰0.15g、七水硫酸镁0.5g、吐温80 1.5mL、L-半胱氨酸盐酸盐1.5g、维生素B 2 2mg，琼脂粉22g，自来水1L，115℃，25min灭菌，冷却至50～55℃，然后加入乙酸调节至总酸5g/100mL。
将上述培养基15mL倾注于直径9cm的培养皿中，待冷却后涂布上述相应稀释后的菌株菌液(1000～2000CFU/mL)0.1mL，置于厌氧培养箱中培养，厌 氧培养箱中氧气含量约8ppm，培养湿度75％，培养温度33℃，培养时间15天，每天观察平板上菌落的生长情况。
其中，涂布HSLZ-75 T组为实施例1-1，菌落形态如图2，涂布HSCY 2073为实施例1-2，涂布DSM 100239 T组为实施例1-3，涂布DSM 20203 T组为实施例1-4，涂布DSM 15814 T组为实施例1-5，涂布DSM 20331 T组为实施例1-6，涂布CICC 20243组为实施例1-7，涂布CICC 10822组为实施例1-8，涂布CICC10774组为实施例1-9，涂布CICC 20176组为实施例1-10。
实施例2
培养基配方：胰蛋白胨8g、牛肉膏10g、酵母提取物8g、麦芽提取物60g、葡萄糖10g、柠檬酸氢二铵1.5g、磷酸氢二钾2.5g、一水硫酸锰0.1g、七水硫酸镁0.5g、吐温80 1mL、L-半胱氨酸盐酸盐1g、维生素B 2 1mg，琼脂粉20g，自来水1L，115℃，25min灭菌，冷却至50～55℃，然后加入乙酸调节至总酸4g/100mL。
将上述培养基15mL倾注于直径9cm的培养皿中，待冷却后涂布上述相应稀释后的菌株菌液0.1mL，置于厌氧培养箱中培养，厌氧培养箱中氧气含量约8ppm，培养湿度85％，培养温度30℃，培养时间15天，每天观察平板上菌落的生长情况。
其中，涂布HSLZ-75 T组为实施例2-1，涂布HSCY 2073为实施例2-2，涂布DSM 100239 T组为实施例2-3，涂布DSM 20203 T组为实施例2-4，涂布DSM 15814 T组为实施例2-5，涂布DSM 20331 T组为实施例2-6，涂布CICC 20243组为实施例2-7，涂布CICC 10822组为实施例2-8，涂布CICC10774组为实施例2-9，涂布CICC 20176组为实施例2-10。
实施例3
培养基配方：胰蛋白胨12g、牛肉膏15g、酵母提取物12g、麦芽提取物30g、葡萄糖15g、柠檬酸氢二铵2.5g、磷酸氢二钾1.5g、一水硫酸锰0.2g、七水硫酸镁0.7g、吐温80 2mL、L-半胱氨酸盐酸盐2g，维生素B 2 3mg，琼脂粉25g，自来水1L，115℃，25min灭菌，冷却至50～55℃，然后加入乙酸调节至总酸6g/100mL。
将上述培养基15mL倾注于直径9cm的培养皿中，待冷却后涂布上述相应稀释后的菌株菌液0.1mL，置于厌氧培养箱中培养，厌氧培养箱中氧气含量约8ppm，培养湿度80％，培养温度35℃，培养时间15天，每天观察平板上菌落的生长情况。
其中，涂布HSLZ-75 T组为实施例3-1，涂布HSCY 2073为实施例3-2，涂 布DSM 100239 T组为实施例3-3，涂布DSM 20203 T组为实施例3-4，涂布DSM 15814 T组为实施例3-5，涂布DSM 20331 T组为实施例3-6，涂布CICC 20243组为实施例3-7，涂布CICC 10822组为实施例3-8，涂布CICC10774组为实施例3-9，涂布CICC 20176组为实施例3-10。
对比例1
在下列培养基固体平板上，涂布HSLZ-75 T，其中改良MRS培养基为对比例4-1、MRS培养基配方1为对比例4-2、MRS培养基配方2为对比例4-3、MRS培养基配方3为对比例4-4、LB培养基配方为对比例4-5、牛肉膏琼脂培养基配方为对比例4-6、培养基100239为对比例4-7、培养基20203为对比例4-8、培养基15814为对比例4-9、培养基20331为对比例4-10。
在下列培养基固体平板上，涂布HSCY 2073，其中改良MRS培养基为对比例5-1、MRS培养基配方1为对比例5-2、MRS培养基配方2为对比例5-3、MRS培养基配方3为对比例5-4、LB培养基配方为对比例5-5、牛肉膏琼脂培养基配方为对比例5-6、培养基100239为对比例5-7、培养基20203为对比例5-8、培养基15814为对比例5-9、培养基20331为对比例5-10。
将上述培养基15mL倾注于直径9cm的培养皿中，待冷却后涂布上述相应稀释后的菌株菌液0.1mL，置于厌氧培养箱中培养，厌氧培养箱中氧气含量约8ppm，培养湿度75％，培养温度33℃，培养时间15天，每天观察平板上菌落的生长情况。
对比例2
选择实施例1中的本发明培养基配方，但培养时放置于普通培养箱(非厌氧)，33℃培养，培养时间15天，每天观察平板上菌落的生长情况。其中涂布HSLZ-75 T组为对比例6-1，涂布HSCY 2073为对比例6-2。
选择实施例2中的本发明培养基配方，但培养时放置于普通培养箱，33℃培养，培养时间15天，每天观察平板上菌落的生长情况。其中涂布HSLZ-75 T组为对比例6-3，涂布HSCY 2073为对比例6-4。
选择实施例3中的本发明培养基配方，但培养时放置于普通培养箱，33℃培养，培养时间15天，每天观察平板上菌落的生长情况。其中涂布HSLZ-75 T组为对比例6-5，涂布HSCY 2073为对比例6-6。
其中涂布方式同对比例1。
对比例3
选择实施例1中的本发明培养基配方，但培养时培养箱中氧气含量约20ppm，33℃培养，培养时间15天，每天观察平板上菌落的生长情况。其中涂 布HSLZ-75 T组为对比例7-1，涂布HSCY 2073为对比例7-2。
选择实施例2中的本发明培养基配方，但培养时培养箱中氧气含量约20ppm，30℃培养，培养时间15天，每天观察平板上菌落的生长情况。其中涂布HSLZ-75 T组为对比例7-3，涂布HSCY 2073为对比例7-4。
选择实施例3中的本发明培养基配方，但培养时培养箱中氧气含量约20ppm，35℃培养，培养时间15天，每天观察平板上菌落的生长情况。其中涂布HSLZ-75 T组为对比例7-5，涂布HSCY 2073为对比例7-6。
其中涂布方式同对比例1。
对比例4
选择上述改良MRS培养基，即源于参考文献中乳酸菌菌株所用培养基和培养方法，参考文献如下：YuYJ,LiX,Zhang JH,Chai LJ,Lu ZM,Xu ZH(2019)Lactobacillus jinshani sp.nov.,isolated from solid-state vinegar culture of Zhenjiang aromatic vinegar.113(1):43-54。参照上述文献条件，培养时间15天，每天观察平板上菌落的生长情况，其中涂布HSLZ-75 T组为对比例8-1，涂布HSCY 2073为对比例8-2。
其中涂布方式同对比例1。
对比例5
固体培养基配方：胰蛋白胨10g、牛肉膏12g、酵母提取物10g、麦芽提取物50g、葡萄糖13g、柠檬酸氢二铵2g、磷酸氢二钾2g、一水硫酸锰0.15g、七水硫酸镁0.5g、吐温80 1.5mL、L-半胱氨酸盐酸盐1.5g，琼脂粉22g，自来水1L，115℃，25min灭菌，冷却至50～55℃，然后加入乙酸调节至总酸5g/100mL。
其中，涂布HSLZ-75 T组为实施例9-1，涂布HSCY 2073为实施例9-2。
其中涂布方式同对比例1。
实施例4
选择实施例1中培养基配方，制备成不添加琼脂的液体培养，将培养至对数期的菌株HSLZ-75 T按照10％(v/v)的接种量，接入含有液体培养基的可密封容器中。其中，可密封容器中培养基的液面上方残留空气的高度0.3cm，培养基的装液量95％，密封不漏气，置于33℃条件下培养，培养3天，为实施例10-1。培养基的液面上方残留空气的高度1.0cm，且培养基的装液量80％，密封不漏气，培养温度33℃，培养3天，为对比例10-2。
选择实施例1中培养基配方，制备成不添加琼脂的液体培养，将培养至对数期的菌株HSCY 2073按照10％(v/v)的接种量，接入含有液体培养基的可密封容器中。其中，可密封容器中培养基的液面上方残留空气的高度0.3cm，培养基 的装液量95％，密封不漏气，置于33℃条件下培养，培养3天，为实施例10-3。培养基的液面上方残留空气的高度1.0cm，且培养基的装液量80％，密封不漏气，培养温度33℃，培养3天，为对比例10-4。
待培养结束后，摇匀，并采用梯度稀释法，梯度稀释后采用实施例1-1的培养基和培养方法培养后进行梯度稀释后计数。
实施例5
实施例5选择实施例2中培养基配方，制备成不添加琼脂的液体培养，将培养至对数期的菌株HSLZ-75 T或者菌株HSCY 2073按照10％(v/v)的接种量，接入含有液体培养基的可密封容器中。其中，可密封容器中培养基的液面上方残留空气的高度0.3cm，培养基的装液量95％，密封不漏气，置于30℃条件下培养，培养3天。
实施例6
实施例6选择实施例3中培养基配方，制备成不添加琼脂的液体培养，将培养至对数期的菌株HSLZ-75 T或者菌株HSCY 2073按照10％(v/v)的接种量，接入含有液体培养基的可密封容器中。其中，可密封容器中培养基的液面上方残留空气的高度0.3cm，培养基的装液量95％，密封不漏气，置于35℃条件下培养，培养3天。
将上述本发明中培养基配方和培养方法的效果比较，结果如表1和2所示。
表1固体平板培养效果对比表
Figure PCTCN2022092168-appb-000001
Figure PCTCN2022092168-appb-000002
通过实施例1-1～1-10、实施例2-1～2-10、实施例3-1～3-10可以看出，其他常规乳酸菌无法在本发明的培养基上生长，说明本发明培养基配方具有良好的专一性和稳定性。
通过对比例4-1～4-10、对比例5-1～5-10可以看出，本发明的目标乳酸菌Acetilactobacillus jinshanensis HSLZ-75 T和Lactobacillus sp.HSCY 2073无法在上述常规培养基上生长，说明目标乳酸菌具有较强的特殊性。虽然目标乳酸菌可在改良MRS培养基上生长，但生长速度慢，第7天才能肉眼可见，生长菌落数偏少，说明本发明的培养基配方具有良好的高效性。
通过对比例6-1～6-6可以看出，本发明的目标乳酸菌无法在上述常规培养方法下生长；通过对比例7-1～7-6可以看出，本发明目标乳酸菌虽然可以在上述氧含量20ppm培养方法下生长，但其生长速度慢，第7-8天才能肉眼可见，同时生长菌落数偏少；结合对比例8-1～8-2、对比例9-1～9-2可以看出，本发明的培养基和培养方法，具有良好的高效性，此外对比例9-1～9-2可以看出，维生素B 2在本发明培养基中对于菌株的生长速度和菌落数也有较大的影响，而维生素B 2在一般的乳酸菌培养基中不额外添加，其他物质带入的含量低，额外添加与 否不会影响常规菌株生长，同时本发明中微量维生素的添加，还可以大量减少麦芽提取物的使用量，并且培养效果明显更好。
因此，由上述表1多维度的综合比较可以看出，本发明的特异性的乳酸菌的培养基配方和培养方法，具有良好的专一性、高效性和稳定性。
表2液体培养效果对比表
序号 活菌数CFU/mL
实施例10-1 6.3×10 8
对比例10-2 1.5×10 7
实施例10-3 7.8×10 8
对比例10-4 2.4×10 7
由上述表2可以看出，本发明的特异性的乳酸菌培养基的培养方法，具有更好的生长适宜性。
实施例7
本发明培养基成分稳定，可将培养基中的物质混合，待使用时再加水制备成液体或固体培养基，应用更方便。
将胰蛋白胨100g、牛肉膏120g、酵母提取物100g、麦芽提取物500g、葡萄糖130g、柠檬酸氢二铵20g、磷酸氢二钾20g、一水硫酸锰1.5g、七水硫酸镁5g、吐温80 15mL、L-半胱氨酸盐酸盐15g、维生素B 2 20mg混合均匀，至于-20℃冰箱中。当需要使用时，取125g上述混合物，加入1L自来水中，溶解后，115℃，25min灭菌，待冷却至50～55℃，然后加入乙酸调节至总酸5g/100mL，即快速的得到液体培养基。
将胰蛋白胨100g、牛肉膏120g、酵母提取物100g、麦芽提取物500g、葡萄糖130g、柠檬酸氢二铵20g、磷酸氢二钾20g、一水硫酸锰1.5g、七水硫酸镁5g、吐温80 15mL、L-半胱氨酸盐酸盐15g、维生素B 2 20mg，琼脂粉220g，混合均匀，至于-20℃冰箱中。当需要使用时，取125g上述混合物，加入1L自来水中，溶解后，115℃，25min灭菌，待冷却至50～55℃，然后加入乙酸调节至总酸5g/100mL，即快速的得到固体平板培养基。
采集麸醋样品，参照专利CN 202011227512.3中实施例1的麸醋样品、培养基和方法，长出肉眼可见菌落的时间为7天，而采用本发明的培养基和培养方法 (其中涉及的培养基和培养方法，固体和液体培养基分别采用实施例1固体培养基和液体培养基，培养方法采用实施例1的固体培养和实施例4的液体培养)，采用相似麸醋样品时长出肉眼可见菌落的时间为3天，且检出活菌数的总数量提高了8.3％，说明本发明的培养基和方法生长速度更快。
因此，本发明的培养基和培养方法具有良好的商业化应用可行性。
以上的实例仅仅是对本发明的实施方式进行描述，而并非对本发明的范围进行限定，对于本领域的技术人员来说，可以对上述说明进行改进，但所有的这些改进均应落入本发明的权利要求确定的保护范围。

Claims (7)

  1. 一种特异性的乳酸菌液体培养基，其特征在于，所述培养基中包括以下原料：每1L自来水中加入胰蛋白胨8～12g、牛肉膏10～15g、酵母提取物8～12g、麦芽提取物30～60g、葡萄糖10～15g、柠檬酸氢二铵1.5～2.5g、磷酸氢二钾1.5～2.5g、一水硫酸锰0.1～0.2g、七水硫酸镁0.5～0.7g、吐温80 1～2mL、L-半胱氨酸盐酸盐1～2g、维生素B 21～3mg，最后加入乙酸至4～6g/100mL。
  2. 根据权利要求1所述的特异性的乳酸菌液体培养基，其特征在于，所述乳酸菌优选为乳酸菌Acetilactobacillus jinshanensis HSLZ-75 T和Lactobacillus sp.HSCY 2073。
  3. 一种特异性的乳酸菌固体培养基，其特征在于，所述培养基中包括以下原料：每1L自来水中加入胰蛋白胨8～12g、牛肉膏10～15g、酵母提取物8～12g、麦芽提取物30～60g、葡萄糖10～15g、柠檬酸氢二铵1.5～2.5g、磷酸氢二钾1.5～2.5g、一水硫酸锰0.1～0.2g、七水硫酸镁0.5～0.7g、吐温80 1～2mL、L-半胱氨酸盐酸盐1～2g、维生素B 21～3mg，加入琼脂粉20～25g，最后加入乙酸至4～6g/100mL。
  4. 根据权利要求3所述的特异性的乳酸菌固体培养基，其特征在于，所述乳酸菌为乳酸菌Acetilactobacillus jinshanensis HSLZ-75 T和Lactobacillus sp.HSCY 2073。
  5. 一种利用权利要求1所述的特异性的乳酸菌液体培养基的培养方法，其特征在于，包括如下步骤：
    在培养基中接种菌株后，在培养基的液面上方残留空气的高度＜0.5cm，且培养基的装液量＞90％，密封不漏气，培养温度30～35℃。
  6. 一种利用权利要求3所述的特异性的乳酸菌固体培养基的培养方法，其特征在于，包括如下步骤：
    在培养基中接种菌株后，置于厌氧培养箱中培养，厌氧培养箱中氧气含量＜10ppm，培养湿度75～85％，培养温度30～35℃。
  7. 一种权利要求1所述的特异性的乳酸菌液体培养基或者权利要求3所述的乳酸菌固体培养基在快速大量培养乳酸菌中的应用。
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