A culture medium and method for high-throughput screening of probiotics from traditional Chinese medicine fermentation using microfluidic technology

Abstract

The present invention provides a culture medium and method for high-throughput screening of probiotics from fermented Chinese medicine by microfluidic technology, which relates to the field of microbial technology, wherein Tween-80 is removed from the culture medium. The droplet spectrum baseline formed is flat, and the proportion of positive bacteria screened is significantly increased. Single bacteria from a sample colony are embedded in a droplet containing the above-mentioned culture medium through a microfluidic system. A single run experiment of droplet microfluidic technology can process about 5,000 droplets. The droplets are cultured and screened through a set target OD value range to obtain positive droplets, and the positive droplets are placed in a functional screening culture medium to culture and obtain potential probiotics. It was found through experimental verification that the number and abundance of potential probiotics screened by microfluidic technology are superior to traditional screening methods, greatly reducing the time and labor costs of screening.

Description

Technical Field

[0001] The invention relates to the field of microbial technology, and in particular to a culture medium and a method for high-throughput screening of probiotics from traditional Chinese medicine fermentation using microfluidic technology. Background Art

[0002] Probiotics are one of the microecological preparations currently being studied. They refer to living microorganisms that produce health benefits to the host when ingested in sufficient quantities. Traditional Chinese medicine fermentation has existed since ancient times and is a processing process. Using probiotics to ferment traditional Chinese medicine can not only promote the dissolution of effective substances in traditional Chinese medicine, decompose macromolecular substances to improve efficacy or produce new effective substances, but also reduce the toxic side effects of traditional Chinese medicine. For example, probiotics that produce pectinase and cellulase can increase the release of effective substances in traditional Chinese medicine; strains that produce β-glucosidase can convert major ginsenosides into rare ginsenosides, increase the utilization rate of ginsenosides and enhance efficacy. The purposeful fermentation of traditional Chinese medicine with probiotics has become a current research hotspot. In the "List of Bacteria that Can Be Used in Food" and the "List of Bacteria that Can Be Used in Infant Food", these probiotics in the catalog can be used as food raw materials. The research on fermented traditional Chinese medicine probiotics is based on the construction of a strain library, and how to quickly establish a target strain library is the key.

[0003] Probiotics come from a wide range of sources. Traditional fermented foods such as traditional fermented fruits and vegetables, fermented dairy products, and fermented meats are good sources of probiotics. The existing method for isolating probiotics is to spread the sample through gradient dilution, obtain a single bacterium after one or more plate streaking purification, and then perform in vitro functional evaluation on the single bacterium to screen out probiotics with the potential to ferment Chinese medicine. The plate spreading method allows strains with low bacterial populations in the sample to grow on plates with local dilutions, but due to the dense growth of high-abundance strains in low-dilution plates, single bacteria cannot be separated. Therefore, this method can only isolate high-abundance strains in the sample. This method has the problems of high time cost and high labor cost. Droplet microfluidics refers to a means of controlling fluids in a space at the micron level, which can achieve high-throughput analysis and sorting of biological functions at the single-cell level. At present, the bacterial probiotics in the "List of Bacteria that Can Be Used in Food", "List of Bacteria that Can Be Used in Infant Food", and "List of Microbial Species for Traditional Fermented Foods in China" are mostly microaerobic, facultative aerobic, and obligate aerobic bacteria that produce acid and can live in a low pH environment. Low-pH SL medium was used as the screening medium. However, in actual applications, it was found that the screening medium in the prior art affected the embedding efficiency and strain growth of the droplets when a single bacterium was embedded in a droplet containing the screening medium through a microfluidic system. The number of strains and species screened was low, and it was not suitable for microfluidic high-throughput screening of Chinese medicine probiotics, which in turn limited the application of microfluidic technology in high-throughput screening of Chinese medicine fermented probiotics.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] One of the purposes of the present invention is to provide a culture medium for high-throughput screening of probiotics from traditional Chinese medicine fermentation using microfluidic technology, so as to at least solve one of the technical problems existing in the prior art.

[0006] The second object of the present invention is to provide the application of the above culture medium in high-throughput screening of traditional Chinese medicine fermentation probiotics using microfluidic technology.

[0007] The third object of the present invention is to provide a method for high-throughput screening of probiotics from traditional Chinese medicine fermentation using microfluidic technology.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0009] In a first aspect, the present invention provides a culture medium for high-throughput screening of Chinese medicine fermentation probiotics using microfluidic technology, which is composed of the following components in concentrations: 8-12 g / L tryptone, 4-6 g / L yeast extract, 5-7 g / L dipotassium hydrogen phosphate, 1-3 g / L triammonium citrate, 15-27 g / L sodium acetate, 18-25 g / L glucose, 0.4-0.6 g / L magnesium sulfate heptahydrate, 0.1-0.2 g / L manganese sulfate tetrahydrate, 0.01-0.1 g / L ferric sulfate heptahydrate and 0.05-0.2 g / L cycloheximide.

[0010] Furthermore, the pH of the culture medium is 4.0-5.0.

[0011] Further, the culture medium is composed of the following components in concentrations: 10 g / L tryptone, 5 g / L yeast extract, 6 g / L potassium dihydrogen phosphate, 2 g / L ammonium citrate, 25 g / L sodium acetate, 20 g / L glucose, 0.58 g / L magnesium sulfate heptahydrate, 0.15 g / L manganese sulfate tetrahydrate, 0.03 g / L ferric sulfate heptahydrate and 0.1 g / L cycloheximide.

[0012] In a second aspect, the present invention provides the use of the above culture medium in high-throughput screening of probiotics from traditional Chinese medicine fermentation using microfluidic technology.

[0013] In a third aspect, the present invention provides a method for high-throughput screening of probiotics fermented from traditional Chinese medicine using microfluidic technology, comprising the following steps:

[0014] A. Prepare microdroplets and microbubbles of bacteria in the sample through a microfluidic chip;

[0015] B. Anaerobic culture of the droplets in step A, screening the droplets within the target OD value range as positive droplets;

[0016] C. Performing functional screening on the positive droplets obtained by screening in step B to obtain potential probiotics;

[0017] The droplets are composed of the above-mentioned culture medium;

[0018] The target OD value is 1.20~2.50.

[0019] Furthermore, before setting the target OD value, the step also includes removing 100±30 unstable droplets;

[0020] The number of colonies in the sample is 50-100 cfu / mL;

[0021] The culture conditions of the droplets include anaerobic culture at 30-42°C;

[0022] The culture time of the droplets is 1 to 3 days.

[0023] Furthermore, the functional screening includes culturing the positive droplets in functional screening culture medium respectively.

[0024] Furthermore, the functional screening includes placing the positive droplets in a 96-well plate containing a functional screening medium for culturing, and detecting the OD value of each well after culturing. If the difference between the OD value of the functional screening medium after culturing and the OD value of the functional screening medium before culturing is at least 0.5, a color reaction is considered to have occurred, and the probiotics in the corresponding positive droplets are potential probiotics.

[0025] Further, the functional screening medium includes the following components in concentrations: 1-10 g / L functional component, 0.5-2.5 g / L color development component, 8-10 g / L bacteriological peptone, 9-11 g / L beef extract, 3-5 g / L yeast extract, 1-2 g / L anhydrous citric acid, 4-6 g / L sodium acetate, 2-4 g / L glucose, 0.1-0.3 g / L anhydrous magnesium sulfate, 0.04-0.05 g / L manganese sulfate monohydrate, 1-3 g / L dipotassium hydrogen phosphate and 1-1.5 g / L Tween-80;

[0026] The functional component comprises any one of sodium carboxymethyl cellulose, pectin or aesculin.

[0027] The color developing component includes Congo red or ammonium ferric citrate.

[0028] Further, when the functional component is sodium carboxymethyl cellulose, the functional screening medium includes the following components at concentrations: 2 g / L functional component, 2 g / L Congo red, 10 g / L bacteriological peptone, 10 g / L beef extract, 4 g / L yeast extract, 1 g / L anhydrous citric acid, 5 g / L sodium acetate, 3 g / L glucose, 0.2 g / L anhydrous magnesium sulfate, 0.05 g / L manganese sulfate monohydrate, 2 g / L dipotassium hydrogen phosphate and 1.08 g / L Tween-80;

[0029] When the functional component is pectin, the functional screening medium includes the following components in concentrations: 1 g / L functional component, 2 g / L Congo red, 10 g / L bacteriological peptone, 10 g / L beef extract, 4 g / L yeast extract, 1 g / L anhydrous citric acid, 5 g / L sodium acetate, 3 g / L glucose, 0.2 g / L anhydrous magnesium sulfate, 0.05 g / L manganese sulfate monohydrate, 2 g / L dipotassium hydrogen phosphate and 1.08 g / L Tween-80;

[0030] When the functional component is aesculin, the functional screening culture medium includes components with the following concentrations: 2 g / L functional component, 0.5 g / L ammonium ferric citrate, 10 g / L bacteriological peptone, 10 g / L beef extract, 4 g / L yeast extract powder, 1 g / L anhydrous citric acid, 5 g / L sodium acetate, 3 g / L glucose, 0.2 g / L anhydrous magnesium sulfate, 0.05 g / L manganese sulfate monohydrate, 2 g / L dipotassium hydrogen phosphate and 1.08 g / L Tween-80.

[0031] The present invention provides a culture medium for high-throughput screening of Chinese medicine fermented probiotics by microfluidic technology, and the droplet spectrum baseline formed by the culture medium is flat, and the proportion of positive bacteria screened is significantly increased. The technical problem of the lack of a culture medium suitable for high-throughput screening of Chinese medicine fermented probiotics by microfluidic technology in the prior art is solved. On the other hand, the present invention provides a method for high-throughput screening of Chinese medicine fermented probiotics by microfluidic technology, in which a single bacterium from a sample colony is embedded in a droplet containing the above-mentioned culture medium by a microfluidic system, and a single run experiment of droplet microfluidic technology can process about 5000 droplets, and the droplets are cultured and screened by setting a target OD value range to obtain positive droplets, and the positive droplets are placed in a functional screening culture medium to culture and obtain potential probiotics. It was verified by experiments that the number and abundance of potential probiotics screened by microfluidic technology are better than those of traditional screening methods, greatly reducing the time and labor costs of screening bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0033] Figure 1 The droplet integrity test result of the culture medium for high-throughput screening of probiotics from traditional Chinese medicine fermentation using the microfluidic technology provided in Example 1 of the present invention;

[0034] Figure 2 A flow chart comparing a method for high-throughput screening of probiotics from fermented Chinese medicine using microfluidics technology provided by the present invention and a traditional method for screening probiotics from fermented Chinese medicine. DETAILED DESCRIPTION

[0035] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0036] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0037] On the one hand, the present invention provides a culture medium for high-throughput screening of traditional Chinese medicine fermentation probiotics using microfluidic technology, which is composed of the following components in concentrations: 8-12 g / L tryptone, 4-6 g / L yeast extract, 5-7 g / L dipotassium hydrogen phosphate, 1-3 g / L triammonium citrate, 15-27 g / L sodium acetate, 18-25 g / L glucose, 0.4-0.6 g / L magnesium sulfate heptahydrate, 0.1-0.2 g / L manganese sulfate tetrahydrate, 0.01-0.1 g / L ferric sulfate heptahydrate and 0.05-0.2 g / L cycloheximide.

[0038] The experiment proved that the droplet spectrum baseline formed by the culture medium containing Tween-80 was uneven, with many broken droplets, which affected the integrity of the droplets. The droplet spectrum baseline was flat after removing Tween-80, and the droplet flatness was good; the proportion of positive bacteria screened by the culture medium after removing Tween-80 increased significantly. The technical problem of the lack of culture medium suitable for high-throughput screening of traditional Chinese medicine fermentation probiotics by microfluidic technology in the prior art was solved.

[0039] The concentration of tryptone may be, but is not limited to, 8 g / L, 8.5 g / L, 9 g / L, 9.5 g / L, 10 g / L, 10.5 g / L, 11 g / L, 11.5 g / L or 12 g / L, or any value between 8 and 12 g / L, preferably 10 g / L.

[0040] The concentration of the yeast extract may be, but is not limited to, 4 g / L, 4.2 g / L, 4.4 g / L, 4.6 g / L, 4.8 g / L, 5 g / L, 5.2 g / L, 5.4 g / L, 5.6 g / L, 5.8 g / L or 6 g / L, and may be any value between 4 and 6 g / L, preferably 5 g / L.

[0041] The concentration of the dipotassium hydrogen phosphate may be, but is not limited to, 5 g / L, 5.2 g / L, 5.4 g / L, 5.6 g / L, 5.8 g / L, 6 g / L, 6.2 g / L, 6.4 g / L, 6.6 g / L, 6.8 g / L or 7 g / L, and may be any value between 5 and 7 g / L, preferably 6 g / L.

[0042] The concentration of the triammonium citrate may be, but is not limited to, 1 g / L, 1.2 g / L, 1.4 g / L, 1.6 g / L, 1.8 g / L, 2 g / L, 2.2 g / L, 2.4 g / L, 2.6 g / L, 2.8 g / L or 3 g / L, and may be any value between 1 and 3 g / L, preferably 2 g / L.

[0043] The concentration of the sodium acetate can be, but is not limited to, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L or 27 g / L, and can be any value between 15 and 27 g / L, and is preferably 25 g / L.

[0044] The concentration of glucose can be but is not limited to 18 g / L, 18.5 g / L, 19 g / L, 19.5 g / L, 20 g / L, 20.5 g / L, 21 g / L, 21.5 g / L, 22 g / L, 22.5 g / L, 23 g / L, 23.5 g / L, 24 g / L, 24.5 g / L or 25 g / L, and can be any value between 18 and 25 g / L, and is preferably 20 g / L.

[0045] The concentration of the magnesium sulfate heptahydrate may be, but is not limited to, 0.4 g / L, 0.42 g / L, 0.44 g / L, 0.46 g / L, 0.48 g / L, 0.5 g / L, 0.52 g / L, 0.54 g / L, 0.56 g / L, 0.58 g / L or 0.6 g / L, and may be any value between 0.4 and 0.6 g / L, preferably 0.58 g / L.

[0046] The concentration of manganese sulfate tetrahydrate may be, but is not limited to, 0.1 g / L, 0.12 g / L, 0.14 g / L, 0.16 g / L, 0.18 g / L or 0.2 g / L, and may be any value between 0.1 and 0.2 g / L, preferably 0.15 g / L.

[0047] The concentration of the ferric sulfate heptahydrate may be, but is not limited to, 0.01 g / L, 0.02 g / L, 0.03 g / L, 0.04 g / L, 0.05 g / L, 0.06 g / L, 0.07 g / L, 0.08 g / L, 0.09 g / L or 0.1 g / L, and may be any value between 0.01 and 0.1 g / L, preferably 0.03 g / L.

[0048] The concentration of cycloheximide can be, but is not limited to, 0.05 g / L, 0.07 g / L, 0.09 g / L, 0.1 g / L, 0.1 g / L, 0.1 g / L, 0.1 g / L, 0.1 g / L, 0.1 g / L or 0.2 g / L, and can also be any value between 0.05 and 0.2 g / L, preferably 0.1 g / L.

[0049] In some specific embodiments, the pH of the culture medium is 4.0 to 4.5. By further limiting the pH of the culture medium to 4.0 to 5.0, the positive strain acquisition rate is increased.

[0050] The pH of the culture medium may be, but is not limited to, 4.0, 4.1, 4.3, 4.5, 4.7 or 5.0, or any value between 4.0 and 5.0, preferably 4.5.

[0051] Wherein, when the screening target does not include Lactobacillus rhamnosus, Bifidobacterium animalis and Bifidobacterium longum, the pH of the culture medium is 4.0. For example, when the pH is 4.0, the obtained potential probiotics do not include Lactobacillus rhamnosus, Bifidobacterium animalis and Bifidobacterium longum.

[0052] In some specific embodiments, the culture medium consists of components with the following concentrations: 10 g / L tryptone, 5 g / L yeast extract, 6 g / L potassium dihydrogen phosphate, 2 g / L ammonium citrate, 25 g / L sodium acetate, 20 g / L glucose, 0.58 g / L magnesium sulfate heptahydrate, 0.15 g / L manganese sulfate tetrahydrate, 0.03 g / L ferric sulfate heptahydrate and 0.1 g / L cycloheximide.

[0053] The culture medium for high-throughput screening of Chinese medicine fermented probiotics by microfluidic technology provided by the present invention can form complete droplets, increase the proportion of positive bacteria, and is suitable for high-throughput screening of Chinese medicine fermented probiotics by microfluidic technology. According to another aspect of the present invention, the application of the above culture medium in high-throughput screening of Chinese medicine fermented probiotics by microfluidic technology is also provided.

[0054] According to another aspect of the present invention, a method for high-throughput screening of probiotics from traditional Chinese medicine fermentation using microfluidic technology is provided, comprising the following steps:

[0055] A. Prepare microdroplets and microbubbles of bacteria in the sample through a microfluidic chip;

[0056] B. Anaerobic culture of the droplets in step A, screening the droplets within the target OD value range as positive droplets;

[0057] C. Performing functional screening on the positive droplets obtained by screening in step B to obtain potential probiotics;

[0058] The droplets are composed of the above-mentioned culture medium;

[0059] The target OD value is 1.20~2.50.

[0060] The single bacteria from the sample colony are embedded in the droplets containing the above culture medium through the microfluidic system. The droplet microfluidic technology can process about 5,000 droplets in a single run experiment. The droplets are cultured and screened through the set target OD value range to obtain positive droplets. The positive droplets are placed in the functional screening culture medium to culture potential probiotics. Through experimental verification, it was found that the number and abundance of potential probiotics screened by microfluidic technology high-throughput are better than traditional screening methods, greatly reducing the time and labor costs of screening.

[0061] The target OD value specifically selects a stable droplet OD value in the range of 1.20 to 2.50, and the stable droplet is a droplet with an obvious and stable baseline state.

[0062] In some specific embodiments, the step of setting the target OD value further includes removing 100±30 unstable droplets to increase the positive rate of obtaining positive droplets, preferably 100 unstable droplets.

[0063] In some specific embodiments, the number of colonies in the sample is 50-100 cfu / mL.

[0064] In some specific embodiments, the culture conditions include anaerobic culture at 30-42°C.

[0065] In some specific embodiments, the culturing time is 1 to 3 days;

[0066] In some specific embodiments, the functional screening comprises culturing the positive droplets separately in functional screening culture medium.

[0067] In some specific embodiments, the functional screening includes placing the positive droplets in a 96-well plate containing a functional screening medium for culturing, and detecting the OD value of each well after culturing. If the difference between the OD value of the functional screening medium after culturing and the OD value of the functional screening medium before culturing is at least 0.5, a color reaction is considered to have occurred, and the probiotics in the corresponding positive droplets are potential probiotics.

[0068] In some specific embodiments, the functional screening medium includes components with the following concentrations: 1~10 g / L functional component, 0.5-2.5 g / L color development component, 8~10 g / L bacteriological peptone, 9~11 g / L beef extract, 3~5 g / L yeast extract, 1~2 g / L anhydrous citric acid, 4~6 g / L sodium acetate, 2~4 g / L glucose, 0.1~0.3 g / L anhydrous magnesium sulfate, 0.04~0.05 g / L manganese sulfate monohydrate, 1~3 g / L dipotassium hydrogen phosphate and 1~1.5 g / L Tween-80.

[0069] In some specific embodiments, the functional component includes any one of sodium carboxymethyl cellulose, pectin or aesculin.

[0070] In some specific embodiments, the color developing component includes one of Congo red and / or ammonium ferric citrate.

[0071] In some specific embodiments, when the functional component is sodium carboxymethyl cellulose, the functional screening medium comprises the following components in concentrations: 2 g / L functional component, 2 g / L Congo red, 10 g / L bacteriological peptone, 10 g / L beef extract, 4 g / L yeast extract, 1 g / L anhydrous citric acid, 5 g / L sodium acetate, 3 g / L glucose, 0.2 g / L anhydrous magnesium sulfate, 0.05 g / L manganese sulfate monohydrate, 2 g / L dipotassium hydrogen phosphate and 1.08 g / L Tween-80; when the functional component is pectin, the functional screening medium comprises the following components in concentrations: 1 g / L functional component, 2 g / L Congo red, 10 g / L bacteriological peptone, 10 g / L beef extract, 4 g / L yeast extract, 1 g / L anhydrous citric acid, 5 g / L sodium acetate, 3 g / L glucose, 0.2 g / L anhydrous magnesium sulfate, 0.05 g / L manganese sulfate monohydrate, 2 g / L potassium dihydrogen phosphate and 1.08 g / L Tween-80; when the functional component is aesculin, the functional screening medium includes components with the following concentrations: 2 g / L functional component, 0.5 g / L ammonium ferric citrate, 10 g / L bacteriological peptone, 10 g / L beef extract, 4 g / L yeast extract powder, 1 g / L anhydrous citric acid, 5 g / L sodium acetate, 3 g / L glucose, 0.2 g / L anhydrous magnesium sulfate, 0.05 g / L manganese sulfate monohydrate, 2 g / L potassium dihydrogen phosphate and 1.08 g / L Tween-80.

[0072] In some specific embodiments, the volume of the functional screening medium in each well of the 96-well plate is 100-200 μL.

[0073] When the functional component is sodium carboxymethyl cellulose or pectin, the volume of the culture medium in each well is 100 μL; when the functional component is aesculin, the volume of the culture medium in each well is 200 μL.

[0074] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0075] Fermented sauerkraut samples: 10 samples of fermented sauerkraut from Guangxi were collected using sterile centrifuge tubes or sample bags. After obtaining the samples, liquid and semi-solid samples were mixed with 50% glycerol at a ratio of 1:1 as soon as possible, and then stored in a -80℃ refrigerator. Each sample had 2 copies. Solid samples were mixed with 25% glycerol at a ratio of 1:9 as soon as possible, and then stored in a -80℃ refrigerator. Each sample had 2 copies.

[0076] Lactobacillus plantarum, Lactobacillus acidophilus, Pediococcus acidilactici, Bifidobacterium animalis, Bifidobacterium longum, Lactobacillus fermentum, Lactobacillus johnsonii, Lactobacillus rhamnosus and Lactobacillus delbrueckii subspecies bulgaricus were all isolated from the fermented sauerkraut sample using the traditional method of Comparative Example 4.

[0077] MRS solid culture medium: peptone 10g / L, beef extract 10g / L, yeast extract 5g / L, dipotassium hydrogen phosphate 2g / L, triammonium citrate 2g / L, sodium acetate 5g / L, glucose 20g / L, Tween-801g / L, magnesium sulfate heptahydrate 0.58g / L, manganese sulfate tetrahydrate 0.25g / L, agar 15g / L.

[0078] The components of SL medium include: tryptone 10g / L, yeast extract 5g / L, potassium dihydrogen phosphate 6g / L, ammonium citrate trihydrate 2g / L, sodium acetate 25g / L, glucose 20g / L, Tween-80 1g / L, magnesium sulfate heptahydrate 0.58g / L, manganese sulfate tetrahydrate 0.15g / L, iron sulfate heptahydrate 0.03g / L, cycloheximide 0.1g / L, pH 5.40.

[0079] Example 1

[0080] A culture medium for high-throughput screening of traditional Chinese medicine fermentation probiotics using microfluidic technology, comprising the following components: 10 g / L tryptone, 5 g / L yeast extract, 6 g / L dipotassium hydrogen phosphate, 2 g / L triammonium citrate, 25 g / L sodium acetate, 20 g / L glucose, 0.58 g / L magnesium sulfate heptahydrate, 0.15 g / L manganese sulfate tetrahydrate, 0.03 g / L iron sulfate heptahydrate, 0.1 g / L cycloheximide, and pH 4.5.

[0081] Example 2

[0082] A culture medium for high-throughput screening of traditional Chinese medicine fermentation probiotics using microfluidic technology, comprising the following components: 8 g / L tryptone, 6 g / L yeast extract, 7 g / L dipotassium hydrogen phosphate, 1 g / L triammonium citrate, 15 g / L sodium acetate, 25 g / L glucose, 0.6 g / L magnesium sulfate heptahydrate, 0.2 g / L manganese sulfate tetrahydrate, 0.01 g / L iron sulfate heptahydrate, 0.2 g / L cycloheximide, and pH 4.0.

[0083] Example 3

[0084] A culture medium for high-throughput screening of traditional Chinese medicine fermentation probiotics using microfluidic technology, comprising the following components: 12 g / L tryptone, 4 g / L yeast extract, 5 g / L dipotassium hydrogen phosphate, 3 g / L triammonium citrate, 27 g / L sodium acetate, 18 g / L glucose, 0.4 g / L magnesium sulfate heptahydrate, 0.1 g / L manganese sulfate tetrahydrate, 0.1 g / L iron sulfate heptahydrate, 0.05 g / L cycloheximide, and pH 5.0.

[0085] Example 4

[0086] The difference from Example 1 is that the pH is 4.0. It is used for the screening task that the screening target does not contain Lactobacillus rhamnosus, Bifidobacterium animalis and Bifidobacterium longum.

[0087] Comparative Example 1

[0088] The difference from Example 1 is that 1 g / L Tween-80 is also included.

[0089] Comparative Example 2

[0090] The difference from Example 1 is that 0.5 g / L Tween-80 is also included.

[0091] Test 1 Droplet integrity

[0092] The culture media of Example 1 and Comparative Examples 1-2 were respectively selected, and pure water was used instead of the culture media as a control, and they were respectively put into sample bottles. After the droplets were prepared on the machine, the droplets were sorted, and the integrity of the droplets was judged by the droplet spectrum.

[0093] Specific results such as Figure 1 The left side shows the spectrum of droplets at a wavelength of 600 nm, and the right side shows the spectrum of droplets at a wavelength of 507 nm. Comparing the groups, it was found that the droplet spectrum baseline of SL culture medium without Tween-80 was the flattest and the droplet integrity was the best. The higher the Tween-80 content, the more uneven the droplet spectrum baseline was and the more broken droplets there were.

[0094] Experiment 2: Accuracy of droplet sorting

[0095] The culture medium of Example 1 and Comparative Example 2 was selected. After culturing plant lactobacillus, it was inoculated in the culture medium of Example 1 and Comparative Example 2, respectively, and the bacterial concentration was adjusted to 100 cfu / mL, loaded into a sample bottle, and droplets were prepared on the machine, and anaerobically cultured for 24 hours, and then droplets were sorted by OD value. That is, according to the baseline situation, after a stable baseline appeared, the first about 100 unstable droplet data were removed, and according to the OD value distribution of the droplets, the OD value range of the collected droplets was set to 1.20~2.50, and the positive droplets were collected.

[0096] A 96-well plate containing MRS solid medium containing bromocresol green was used to collect droplets within the target OD value to verify the positive strain. The collected 96-well plate was cultured at 37°C in an anaerobic environment for 2 days, and the growth of the strain in each positive well and the acid production and discoloration of the strain were observed, and the acquisition rate of the positive strain (potential probiotics) was compared. The specific results are shown in Table 1.

[0097] Table 1 Effect of Tween-80 content on the accuracy of droplet sorting

[0098]

[0099] As shown in Table 1, although the number of positive droplets collected by SL medium with a Tween-80 content of 0.5 g / L was large, the number of positive droplets with growing bacteria was small, and the final positive strain acquisition rate was 35.50%, which was significantly lower than the positive strain acquisition rate of 96.20% of SL medium without Tween-80.

[0100] Experiment 3 Activity of target strain after culture in SL medium without Tween-80

[0101] The classic bacterial probiotics recorded in the "List of Bacteria that can be used for food": Lactobacillus plantarum, Lactobacillus acidophilus, Pediococcus acidilactici, Bifidobacterium animalis, Bifidobacterium longum, Lactobacillus fermentum, Lactobacillus johnsonii, Lactobacillus rhamnosus and Lactobacillus delbrueckii subspecies bulgaricus were inoculated into the culture medium of Example 1, anaerobically cultured for 24 hours, and the absorbance value of the bacterial solution at a wavelength of 610nm was detected. It was found that all strains could grow. It shows that removing Tween-80 does not affect the growth of the strains, and can be used as a microfluidic high-throughput screening probiotic.

[0102] Experiment 4: Optimization of pH of culture medium

[0103] Based on the culture medium containing Example 1, the culture medium with pH values ​​of 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5 and 3.0 was set respectively. Lactobacillus plantarum, Lactobacillus acidophilus, Pediococcus acidilactici, Bifidobacterium animalis, Bifidobacterium longum, Lactobacillus fermentum, Lactobacillus johnsonii, Lactobacillus rhamnosus and Lactobacillus delbrueckii subspecies bulgaricus and an acid-intolerant negative bacteria were inoculated in culture mediums of different pH values ​​respectively, and cultured anaerobically for 24 h, and the OD value change of the bacterial liquid before and after the culture was detected by a spectrophotometer.

[0104] The results showed that although the number of strains screened in the medium above pH 5.0 was large, further testing found that the proportion of non-target strains increased. In the medium at pH 4.5, all positive bacteria were able to grow, and negative strains did not grow. In the medium at pH 4.0, Lactobacillus rhamnosus, Bifidobacterium animalis, and Bifidobacterium longum grew poorly. When the pH was further reduced, it was found that the number of target strains grown in the medium at pH 3.5 and pH 3.0 was greatly reduced. Therefore, the medium pH was selected to be 4.0-5.0.

[0105] Example 5

[0106] A method for high-throughput screening of probiotics from fermented Chinese medicine using microfluidics technology, wherein the sample is diluted to a colony concentration of 50-100 cfu / mL using a culture medium for screening probiotics from fermented Chinese medicine, and the specific operation is as follows:

[0107] 1) Load the diluted sample into the microfluidic injection vial. According to the process of the microfluidic system (MISS cell cultureomics), self-check, install consumables, initialize, and then prepare droplets. After preparation, perform anaerobic culture at 37°C for 2 days.

[0108] 2) Perform droplet sorting according to the process of the microfluidic system. After removing the 100 initially unstable droplets, set the target OD value range to 1.20-2.50 for droplet collection based on the OD value distribution of the droplets. Collect positive droplets within the target OD value range.

[0109] 3) Place the positive droplets on a 96-well plate for cellulase colorimetric screening, a 96-well plate for pectinase colorimetric screening, and a 96-well plate for β-glucosidase colorimetric screening. The collected 96-well plates were cultured at 37°C in an aerobic environment for 2 days, and the color changes of the plates and the growth of the strains were observed. Strains with corresponding colorimetric reactions are potential strains (potential probiotics). After the strains were purified, morphological observations and 16s identification were performed. The strains were stored, with 2 tubes for each strain.

[0110] Among them, each well of the 96-well plate for cellulase colorimetric screening and the 96-well plate for pectinase colorimetric screening contains 100 uL of functional screening medium, and each well of the 96-well plate for β-glucosidase colorimetric screening contains 150 uL of functional screening medium.

[0111] Cellulase screening medium: 2 g / L sodium carboxymethyl cellulose, 2 g / L Congo red, 10 g / L bacteriological peptone, 10 g / L beef extract, 4 g / L yeast extract powder, 1 g / L anhydrous citric acid, 5 g / L sodium acetate, 3 g / L glucose, 0.2 g / L anhydrous magnesium sulfate, 0.05 g / L manganese sulfate monohydrate, 2 g / L dipotassium hydrogen phosphate and 1.08 g / L Tween-80.

[0112] Pectinase screening medium: 1 g / L pectin, 2 g / L Congo red, 10 g / L bacteriological peptone, 10 g / L beef extract, 4 g / L yeast extract powder, 1 g / L anhydrous citric acid, 5 g / L sodium acetate, 3 g / L glucose, 0.2 g / L anhydrous magnesium sulfate, 0.05 g / L manganese sulfate monohydrate, 2 g / L dipotassium hydrogen phosphate and 1.08 g / L Tween-80.

[0113] β-glucosidase production screening medium: 2 g / L aesculin, 0.5 g / L ammonium ferric citrate, 10 g / L bacteriological peptone, 10 g / L beef extract, 4 g / L yeast extract, 1 g / L anhydrous citric acid, 5 g / L sodium acetate, 3 g / L glucose, 0.2 g / L anhydrous magnesium sulfate, 0.05 g / L manganese sulfate monohydrate, 2 g / L potassium dihydrogen phosphate and 1.08 g / L Tween-80. Test 5 Positive bacteria verify the accuracy of the high-throughput screening method of probiotics using microfluidic technology

[0114] The culture medium of Example 1 was used to dilute the positive bacteria (Lactobacillus plantarum) to a colony concentration of 40 cfu / mL and 100 cfu / mL, respectively, and the positive bacteria + negative bacteria (Bacillus citrifolius) (mixed at a live cell count ratio of 1:1) were diluted to a colony concentration of 40 cfu / mL, and used as samples for screening according to the method provided in Example 5.

[0115] The positive droplets obtained by diluting the samples at different concentrations in Example 5 were placed in a 96-well plate (100uL / well) containing an MRS medium with a bromocresol green indicator, and cultured at 37°C in an anaerobic environment for 2 days, and the color change of the well plate and the growth of the strain were observed. The acid-producing strain will cause the MRS medium to change from green to yellow. Among them, the 96-well plate containing the MRS medium with a bromocresol green indicator was sterilized by ultraviolet in advance. The details are shown in Table 2.

[0116] Table 2 Positive droplet verification results

[0117]

[0118] As can be seen from Table 2, the accuracy of the positive droplets collected by the method provided in Example 5 reached above 94.64.

[0119] Embodiment 6~9

[0120] Fermented food samples were selected as samples, and the culture media of Examples 1 to 4 were used to dilute the fermented food samples until the colony count was 100 cfu / mL, and the samples were screened according to the method provided in Example 5.

[0121] Example 10

[0122] A fermented food sample was selected as a sample, and the culture medium of Example 1 was used to dilute the fermented food sample to a colony count of 100 cfu / mL and screened according to the method provided in Example 5. The difference in the screening process was that 100 unstable droplets were not removed before setting the target OD value, and the OD value distribution range of all droplets was set to 1.20~2.50 as the target OD value.

[0123] Comparative Example 3

[0124] A fermented food sample was selected as a sample, and the culture medium of Example 1 was used to dilute the fermented food sample to a colony count of 100 cfu / mL, and screening was performed according to the method provided in Example 5. The difference in the screening process was that the OD value was set in the range of 0.30-3.50.

[0125] Comparative Example 4

[0126] Screening of fermented food samples using traditional methods follows these steps:

[0127] Prepare SL solid medium containing 0.1% L-cysteine ​​hydrochloride and bromocresol green and prepare plates. Dilute the sample to 10~10 with physiological saline. -6 times, spread on a solid plate, culture for 24-48 hours, pick the acid-producing strains for purification as positive strains, and perform 16S identification on the purified strains. And save the strains, 2 tubes for each strain.

[0128] Test 6: Positive rate of positive droplets

[0129] According to the method of Experiment 5, the positive rates of the positive droplets obtained in Examples 6 to 11 and Comparative Example 3 were tested respectively. The specific results are shown in Table 3.

[0130] Table 3 Positive rate of positive droplets under different treatments

[0131]

[0132] Experiment 7 Optimization of cellulase screening medium

[0133] The solid functional screening medium containing sodium carboxymethyl cellulose (CMC-Na) with concentrations of 2g / L, 5g / L, and 10g / L, respectively, and the other components are the same, namely 2g Congo red, 10g bacteriological peptone, 10g beef extract, 4g yeast extract powder, 1g anhydrous citric acid, 5g sodium acetate, 3g glucose, 0.2g anhydrous magnesium sulfate, 0.05g manganese sulfate monohydrate, 2g dipotassium hydrogen phosphate, 1.08g Tween-80, 15g agar, and 1L pure water was prepared. After high-pressure sterilization at 121℃ for 20min, the temperature was lowered to about 75℃. Before the functional screening medium solidified, the functional screening medium was dispensed into a 96-well plate using a dispenser on a clean bench, with 100uL, 150uL, and 200uL per well, respectively. Two positive strains of cellulase-producing bacteria, Lactobacillus plantarum and one negative strain of Lactobacillus acidophilus, were activated and 2uL of bacterial solution was taken. The bacterial concentration was 1×10 9 cfu / mL, inoculated in the cellulase colorimetric 96-well plate culture medium at 37°C, cultured in an anaerobic environment for 3 days, and observed the degree of transparency of the functional screening culture medium. The absorbance of the 96-well plate at a wavelength of 540nm was tested by an ELISA instrument. The specific results are shown in Table 4. The optimal carboxymethyl cellulose sodium concentration was compared with the optimal volume of the 96-well plate. The experimental results showed that when the positive strain producing cellulase was added to each well at a CMC-Na concentration of 2g / L and 100uL was added, the difference between the absorbance value of the positive well and the blank group was greater than 0.500, and the blank OD value was below 3.000, and the OD value of the negative well was the smallest difference from the blank well. It is easiest to judge the enzyme production situation from the absorbance value.

[0134] Table 4 Cellulase screening medium optimization data

[0135]

[0136] OVRFLW means the detected OD value exceeds the upper limit of detection; #VALUE! means it cannot be calculated due to invalid data.

[0137] Experiment 8 Optimization of pectinase screening medium

[0138] The solid functional screening medium containing pectin concentrations of 1g / L, 2g / L, and 5g / L was prepared, and the other components were the same, namely, 2g Congo red, 10g bacteriological peptone, 10g beef extract, 4g yeast extract powder, 1g anhydrous citric acid, 5g sodium acetate, 3g glucose, 0.2g anhydrous magnesium sulfate, 0.05g manganese sulfate monohydrate, 2g dipotassium hydrogen phosphate, 1.08g Tween-80, 15g agar, and 1L pure water. After high-pressure sterilization at 121℃ for 20min, the temperature was lowered to about 75℃. Before the functional screening medium solidified, the functional screening medium was dispensed into 96-well plates using a discharge gun on the clean bench, respectively, in 100uL, 150uL, and 200uL. Two positive bacteria plantarum lactobacillus and one negative bacteria fermentation mucus lactobacillus that produced pectinase in the laboratory were activated, and 2uL of bacterial solution was taken, and the bacterial concentration was 1×10 9 cfu / mL, inoculated in pectinase colorimetric 96-well plate culture medium at 37°C, cultured in anaerobic environment for 3 days, and observed the degree of transparency of the functional screening culture medium. The absorbance of the 96-well plate at a wavelength of 540nm was tested by an ELISA instrument. The specific results are shown in Table 5. The optimal pectin concentration and the optimal volume of the 96-well plate were compared. The experimental results showed that when the pectin concentration of the positive strain producing pectinase was 1g / L and 100uL was added to each well, the difference between the absorbance value of the positive well and the blank group was greater than 0.500, and the blank OD value was below 3.000, and the OD value of the negative well was the smallest difference from the blank well. It is easiest to judge the enzyme production situation from the absorbance value.

[0139] Table 5 Optimization data of pectinase screening medium

[0140]

[0141] Note: OVRFLW means the detected OD value exceeds the upper limit of detection; #VALUE! means #VALUE! cannot be calculated due to invalid data.

[0142] Experiment 9 Optimization of screening medium for β-glucosidase production

[0143] The solid functional screening medium containing esculin at concentrations of 2 g / L, 4 g / L, and 6 g / L was prepared, and the other components were the same, namely 0.5 g / L ammonium ferric citrate, 10 g bacteriological peptone, 10 g beef extract, 4 g yeast extract powder, 1 g anhydrous citric acid, 5 g sodium acetate, 3 g glucose, 0.2 g anhydrous magnesium sulfate, 0.05 g manganese sulfate monohydrate, 2 g dipotassium hydrogen phosphate, 1.08 g Tween-80 and 15 g agar, and 1 L of pure water. After high-pressure sterilization at 121 ° C for 20 min, the temperature was lowered to about 75 ° C. Before the functional screening medium solidified, the functional screening medium was dispensed into a 96-well plate using a discharge gun on the clean bench, with 150 uL per well. Two positive bacteria plantarum and one negative bacteria Lactobacillus acidophilus that produced β-glucosidase in the laboratory were activated, and 2 uL of bacterial solution was taken, with a bacterial concentration of 1×10 9 cfu / mL, inoculated in β-glucosidase colorimetric 96-well plate culture medium at 37°C, cultured in an anaerobic environment for 3 days, and observed the degree of black circle in the culture medium. The absorbance of the 96-well plate at a wavelength of 540nm was tested by an ELISA instrument. The specific results are shown in Table 6. The optimal concentration of aesculin and the optimal volume of the 96-well plate were compared. The experimental results showed that when the positive strain producing β-glucosidase was under the condition of aesculin concentration of 2g / L and 150uL added to each well, the OD value of the positive bacteria was <3.000, the difference between the positive well and the blank OD value was large, and the difference was >0.500; the OD value of the negative well was small compared with the blank well, and the enzyme production was most easily judged from the absorbance value.

[0144] Table 6 β-glucosidase culture medium optimization data

[0145]

[0146] Test 10 Functional screening

[0147] Functional screening was performed on the positive droplets obtained in Example 6 and the positive strains screened in Comparative Example 4, and the screening results are shown in Table 7.

[0148] Table 7 Function screening results

[0149]

[0150] As shown in Table 7, the number and abundance of probiotics that can be used for Chinese medicine fermentation obtained by high-throughput screening of probiotics from Chinese medicine fermentation using the microfluidics technology provided in Example 6 are higher than those obtained using the traditional screening method. Figure 2 Compared with the traditional screening method, the microfluidic technology high-throughput screening method for Chinese medicine fermentation probiotics provided in Example 6 not only screens a large number of positive strains and a large abundance of strains, but also has simpler steps and is more efficient than the traditional screening method.

[0151] Test 11 Enzyme activity detection

[0152] In order to further illustrate the effectiveness of the obtained probiotics, the enzyme activities of the probiotics with different functions screened in Example 6 were tested. Specifically, the following steps were followed:

[0153] The strains producing cellulase, pectinase and β-glucosidase were inoculated into MRS liquid medium containing 0.1% L-cysteine ​​hydrochloride, and the cellulase activity, pectinase activity and β-glucosidase activity were measured after culturing for 24 hours. The specific results are shown in Tables 8 to 10. It can be seen that the strains producing cellulase, pectinase and β-glucosidase screened by this method have corresponding enzyme activities and can be used as strains for subsequent Chinese medicine fermentation.

[0154] Table 8 Cellulase activity

[0155]

[0156] Table 9 Pectinase activity

[0157]

[0158] Table 10 β-glucosidase activity

[0159]

[0160] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A culture medium for high-throughput screening of probiotics from traditional Chinese medicine fermentation using microfluidic technology, characterized in that: The composition is composed of the following components: 8-12 g / L tryptone, 4-6 g / L yeast extract, 5-7 g / L potassium dihydrogen phosphate, 1-3 g / L ammonium citrate, 15-27 g / L sodium acetate, 18-25 g / L glucose, 0.4-0.6 g / L magnesium sulfate heptahydrate, 0.1-0.2 g / L manganese sulfate tetrahydrate, 0.01-0.1 g / L iron sulfate heptahydrate and 0.05-0.2 g / L cycloheximide; The probiotics are selected from at least one of Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus curvatum, Lactobacillus acidophilus, Pediococcus acidilactici, Pediococcus pentosaceus, Bifidobacterium animalis, Bifidobacterium longum, Lactobacillus fermentum, Lactobacillus johnsonii, Lactobacillus rhamnosus or Lactobacillus delbrueckii subspecies bulgaricus.

2. The culture medium according to claim 1, characterized in that The pH of the culture medium is 4.0-5.

0.

3. The culture medium according to claim 1 or 2, characterized in that The culture medium is composed of the following components: 10 g / L tryptone, 5 g / L yeast extract, 6 g / L potassium dihydrogen phosphate, 2 g / L ammonium citrate, 25 g / L sodium acetate, 20 g / L glucose, 0.58 g / L magnesium sulfate heptahydrate, 0.15 g / L manganese sulfate tetrahydrate, 0.03 g / L iron sulfate heptahydrate and 0.1 g / L cycloheximide.

4. Use of the culture medium according to any one of claims 1 to 3 in high-throughput screening of traditional Chinese medicine fermentation probiotics using microfluidic technology; The probiotics are selected from at least one of Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus curvatum, Lactobacillus acidophilus, Pediococcus acidilactici, Pediococcus pentosaceus, Bifidobacterium animalis, Bifidobacterium longum, Lactobacillus fermentum, Lactobacillus johnsonii, Lactobacillus rhamnosus or Lactobacillus delbrueckii subspecies bulgaricus.

5. A method for high-throughput screening of probiotics from traditional Chinese medicine fermentation using microfluidic technology, characterized in that: The following steps are involved: A. Prepare microdroplets and microbubbles of bacteria in the sample through a microfluidic chip; B. Anaerobic culture of the droplets in step A, screening the droplets within the target OD value range as positive droplets; C. Performing functional screening on the positive droplets obtained by screening in step B to obtain potential probiotics; The droplets are composed of the culture medium according to any one of claims 1 to 3; The target OD value is 1.20-2.50; The potential probiotics are selected from at least one of Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus curvatum, Lactobacillus acidophilus, Pediococcus acidilactici, Pediococcus pentosaceus, Bifidobacterium animalis, Bifidobacterium longum, Lactobacillus fermentum, Lactobacillus johnsonii, Lactobacillus rhamnosus or Lactobacillus delbrueckii subspecies bulgaricus.

6. The method according to claim 5, characterized in that The target OD value setting also includes removing 100±30 unstable droplets; The number of colonies in the sample is 50-100 cfu / mL; The culture conditions of the droplets include anaerobic culture at 30-42°C; The culture time of the droplets is 1 to 3 days.

7. The method according to claim 5, characterized in that The functional screening comprises placing the positive droplets in functional screening culture medium for culture respectively.

8. The method according to claim 7, characterized in that The functional screening includes placing the positive droplets in a 96-well plate containing a functional screening medium for culturing, and detecting the OD value of each well after culturing. If the difference between the OD value of the functional screening medium after culturing and the OD value of the functional screening medium before culturing is at least 0.5, a color reaction is considered to have occurred, and the probiotics in the corresponding positive droplets are potential probiotics.

9. The method according to claim 8, characterized in that The functional screening medium comprises the following components: 1-10 g / L functional component, 0.5-2.5 g / L color development component, 8-10 g / L bacteriological peptone, 9-11 g / L beef extract, 3-5 g / L yeast extract, 1-2 g / L anhydrous citric acid, 4-6 g / L sodium acetate, 2-4 g / L glucose, 0.1-0.3 g / L anhydrous magnesium sulfate, 0.04-0.05 g / L manganese sulfate monohydrate, 1-3 g / L dipotassium hydrogen phosphate and 1-1.5 g / L Tween-80; The functional component is selected from any one of sodium carboxymethyl cellulose, pectin or aesculin; The color developing component is selected from Congo red or ammonium ferric citrate.

10. The method according to claim 9, characterized in that When the functional component is sodium carboxymethyl cellulose, the functional screening medium comprises the following components in concentrations: 2 g / L functional component, 2 g / L Congo red, 10 g / L bacteriological peptone, 10 g / L beef extract, 4 g / L yeast extract, 1 g / L anhydrous citric acid, 5 g / L sodium acetate, 3 g / L glucose, 0.2 g / L anhydrous magnesium sulfate, 0.05 g / L manganese sulfate monohydrate, 2 g / L dipotassium hydrogen phosphate and 1.08 g / L Tween-80; When the functional component is pectin, the functional screening medium includes the following components in concentrations: 1 g / L functional component, 2 g / L Congo red, 10 g / L bacteriological peptone, 10 g / L beef extract, 4 g / L yeast extract, 1 g / L anhydrous citric acid, 5 g / L sodium acetate, 3 g / L glucose, 0.2 g / L anhydrous magnesium sulfate, 0.05 g / L manganese sulfate monohydrate, 2 g / L dipotassium hydrogen phosphate and 1.08 g / L Tween-80; When the functional component is aesculin, the functional screening culture medium includes components with the following concentrations: 2 g / L functional component, 0.5 g / L ammonium ferric citrate, 10 g / L bacteriological peptone, 10 g / L beef extract, 4 g / L yeast extract powder, 1 g / L anhydrous citric acid, 5 g / L sodium acetate, 3 g / L glucose, 0.2 g / L anhydrous magnesium sulfate, 0.05 g / L manganese sulfate monohydrate, 2 g / L dipotassium hydrogen phosphate and 1.08 g / L Tween-80.

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