Culture medium containing ergothioneine and application thereof in promoting growth of probiotics

Abstract

The invention provides a culture medium containing ergothioneine and application of the culture medium in promoting growth of probiotics, and particularly relates to the technical field of microorganisms. The invention finds that the ergothioneine can promote the growth of the probiotics for the first time, and the verification finds that the ergothioneine has the effects of shortening the growth time, improving the bacterial growth rate, increasing the bacterial quantity and promoting the strain proliferation on various probiotics.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and in particular to a culture medium containing ergothioneine and an application thereof in promoting the growth of probiotics. Background Art

[0002] Ergothioneine (EGT) is a natural amino acid that comes from plants but can be accumulated in animals. Studies have shown that EGT has a strong antioxidant effect and plays a cell protection function in the body.

[0003] Probiotics refer to a class of active microorganisms that can produce health benefits for the host. A large number of studies have shown that probiotics have the functions of promoting digestion and absorption of the host, improving the body's immune ability, and protecting the intestinal barrier, and play an important role in human health. Lactobacillus and Bifidobacterium are the most studied probiotics.

[0004] Although both ergothioneine and probiotics themselves can exert beneficial effects on the host, the effect of ergothioneine on the growth of probiotics or the intestinal flora is still unclear. Summary of the invention

[0005] The application unexpectedly finds that thioneine can promote probiotic growth, particularly can improve probiotic growth rate and / or promote probiotic proliferation.On this basis, the application further finds that along with the reduction of thioneine concentration, the growth rate of probiotic is further improved instead, thus completes the present invention.

[0006] Specifically, this application involves the following aspects:

[0007] On the one hand, the present application provides the use of ergothioneine in promoting the growth of probiotics.

[0008] Furthermore, the promoting the growth of probiotics includes increasing the growth rate of probiotics and / or promoting the proliferation of probiotics.

[0009] Furthermore, the increasing the growth rate of probiotics includes shortening the growth generation time of probiotics, and the promoting the proliferation of probiotics includes increasing the number of live probiotics.

[0010] Preferably, the probiotics include intestinal probiotics.

[0011] Preferably, the probiotics include Lactobacillus, Lacticaseibacillus, Limosilactobacillus, Lactiplantibacillus, Ligilactobacillus, Latilactobacillus, Streptococcus, Lactococcus, Propionibacterium, Acidipropionibacterium, Leuconostoc, Pediococcus, Weizmannia, Mammaliicoccus, Staphylococcus, Kluyveromyces, and Bifidobacterium.

[0012] The Lactobacillus genus includes: Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii, Lactobacillus helveticus, Lactobacillus gasseri, Lactobacillus johnsonii, and Lactobacillus kefiranofaciens.

[0013] The genus Lactobacillus includes: Lacticaseibacillus casei, Lacticaseibacillus paracasei, and Lacticaseibacillus rhamnosus.

[0014] The genus Lactobacillus includes: Limosilactobacillus fermentum and Limosilactobacillus reuteri.

[0015] The Lactobacillus genus includes: Lactiplantibacillus plantarum.

[0016] The genus Lactobacillus includes: Ligilactobacillus salivarius.

[0017] The genus Latilactobacillus includes: Latilactobacillus curvatus and Latilactobacillus sakei.

[0018] The Streptococcus genus includes: Streptococcus salivarius subsp. thermophilus.

[0019] The Lactococcus genus includes: Lactococcus lactis and Lactococcus cremoris.

[0020] The Propionibacterium genus includes: Propionibacterium freudenreichii subsp. shermanii.

[0021] The genus Propionic acid bacteria includes: Propionic acid-producing bacteria (Acidipropionibacteriumacidipropionici).

[0022] The genus Leuconostoc includes: Leuconostoc mesenteroides subsp. mesenteroides.

[0023] The genus Pediococcus includes: Pediococcus acidilactici and Pediococcus pentosaceus.

[0024] The genus Weizmannia includes: Weizmannia coagulans.

[0025] The genus Zoococcus includes: Mammaliicoccus vitulinus.

[0026] The Staphylococcus genus includes: Staphylococcus xylosus and Staphylococcus carnosus.

[0027] The genus Kluyveromyces includes: Kluyveromyces marxianus.

[0028] The genus Bifidobacterium includes: Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, and Bifidobacterium longum.

[0029] Preferably, the probiotics include Lactobacillus probiotics and Bifidobacterium probiotics.

[0030] More preferably, the probiotics include Lactobacillus rhamnosus, Lactobacillus paracasei, and Bifidobacterium animalis.

[0031] In a preferred embodiment, the Lactobacillus rhamnosus is Lactobacillus rhamnosus CCFM1259, the Lactobacillus paracasei is Lactobacillus paracasei CCFM1157, and the Bifidobacterium animalis is Bifidobacterium animalis subsp. lactis CCFM1160.

[0032] Ergothioneine is used for the first time in the present application to promote the growth of probiotics. It has been verified that ergothioneine has the effects of shortening the growth generation time, increasing the amount of bacteria, and promoting the proliferation of bacteria on a variety of probiotics (such as Lactobacillus hamnosus, Lactobacillus paracasei, and Bifidobacterium animalis).

[0033] On the other hand, the present application also provides a culture medium for promoting the growth of probiotics, wherein the culture medium comprises ergothioneine.

[0034] Further, in terms of mass percentage of the culture medium, the concentration of the ergothioneine is ≤10%; for cost-saving considerations, preferably, the concentration of the ergothioneine is ≤1%; for considerations of both cost and efficient cultivation of probiotics, more preferably, the concentration of the ergothioneine is <0.2%.

[0035] In a preferred embodiment, the concentration of described ergothioneine is 0.0001%, 0.0005%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%.

[0036] Preferably, the culture medium may also include known nutrients or functional components required for culturing probiotics.

[0037] In a preferred embodiment, the culture medium may further include ingredients such as peptone, beef extract, glucose, sodium acetate, yeast powder, diammonium hydrogen citrate, dipotassium hydrogen phosphate, magnesium sulfate, manganese sulfate, Tween 80, and agar.

[0038] On the other hand, the present application also provides a method for promoting the growth of probiotics, the method comprising: applying ergothioneine to the probiotics.

[0039] Further, in percentage by mass, the concentration of the ergothioneine applied to the probiotics is ≤10%; for cost savings, preferably, the concentration of the ergothioneine applied to the probiotics is ≤1%; for consideration of both cost and efficient cultivation of probiotics, more preferably, the concentration of the ergothioneine applied to the probiotics is <0.2%.

[0040] In a preferred embodiment, the concentration of ergothioneine applied to the probiotics is 0.0001%, 0.0005%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04% , 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%.

[0041] In a preferred embodiment, a method for promoting the growth of probiotics comprises: adding ergothioneine at a concentration of less than 0.2% into a probiotic culture medium, and culturing the probiotics.

[0042] Preferably, the inoculation amount of the probiotics is 1%-5%; more preferably, the inoculation amount of the probiotics is 2%.

[0043] On the other hand, the present application also provides the use of the culture medium or the method as described above in promoting the growth of probiotics.

[0044] The present invention has the following beneficial effects:

[0045] It is discovered for the first time in the present application that ergothioneine can promote the growth of probiotics. After verification, it is found that ergothioneine has the effect of shortening the growth generation time and increasing the bacterial amount on a variety of probiotics, which suggests that ergothioneine has the effect of increasing the growth rate of probiotics and promoting the proliferation of bacterial species.

[0046] Furthermore, the present application finds that as the concentration of ergothioneine decreases, the effect of promoting the growth of probiotics can still be maintained or even enhanced.

[0047] Further, the application finds that along with the reduction of thioneine concentration, shows the effect of enhancing the probiotic growth rate, which not only greatly shortens the time required for bacterial strain to reach stable phase, but also saves the use cost of thioneine. Therefore, the application provides new ideas for improving the production efficiency, energy conservation and cost of probiotic culture. DETAILED DESCRIPTION

[0048] Technical terms:

[0049] Growth generation time: refers to the time required for a cell to divide once, also known as generation time or amplification time.

[0050] In order to more clearly illustrate the overall concept of the application, the following is described in detail by way of embodiment. In the following description, a large amount of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0051] If no specific conditions are specified in the examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer.

[0052] Among them, the ergothioneine involved in the following embodiments is provided by Huaxi Biotechnology Co., Ltd.; the reagents used in the MRS culture medium involved in the following embodiments are all purchased from Sinopharm Group; the rhamnosus lactobacillus CCFM1259 involved in the following embodiments is disclosed in the clinical efficacy evaluation of hyaluronic acid and rhamnosus lactobacillus CCFM1259 compound antagonistic Helicobacter pylori infection (Wang Botao, Zhang Huayue, Cui Shumao, et al. Clinical efficacy evaluation of hyaluronic acid and rhamnosus lactobacillus CCFM1259 compound antagonistic Helicobacter pylori infection [J / OL]. Food and Fermentation Industries: 1-9) paper, and the deposit number is GDMCC No: 62419; the paracasei lactobacillus CCFM1157 involved in the following embodiments is recorded in the patent application text with publication number CN113956993A, and the deposit number is GDMCC No: 61497; the animal Bifidobacterium lactis subspecies CCFM1160 involved in the following embodiments is recorded in the publication number CN In the patent application text of 113897300A, the deposit number is GDMCC No: 61500.

[0053] The culture medium involved in the following examples is as follows:

[0054] MRS solid culture medium (g / L): peptone 10g / L, beef extract 10g / L, glucose 20g / L, sodium acetate 2g / L, yeast powder 5g / L, diammonium hydrogen citrate 2g / L, K2PO4·3H2O 2.6g / L, MgSO4·7H2O 0.1g / L, MnSO4 0.05g / L, Tween 80 1mL / L, agar 20g / L; Sterilization temperature: 115℃ for 20min.

[0055] MRS liquid culture medium (g / L): peptone 10g / L, beef extract 10g / L, glucose 20g / L, sodium acetate 2g / L, yeast powder 5g / L, diammonium hydrogen citrate 2g / L, K2PO4·3H2O 2.6g / L, MgSO4·7H2O 0.1g / L, MnSO4 0.05g / L, Tween 80 1mL / L; Sterilization temperature: 115℃ for 20min.

[0056] mMRS solid culture medium: peptone 10g / L, beef extract 10g / L, glucose 20g / L, sodium acetate 2g / L, yeast powder 5g / L, diammonium hydrogen citrate 2g / L, K2HPO4·3H2O 2.6g / L, MgSO4·7H2O0.1g / L, MnSO4 0.05g / L, Tween 80 1mL / L, agar 20g / L, cysteine ​​salt 1g / L; sterilization temperature: 115℃ for 20min.

[0057] mMRS liquid culture medium: peptone 10g / L, beef extract 10g / L, glucose 20g / L, sodium acetate 2g / L, yeast powder 5g / L, diammonium hydrogen citrate 2g / L, K2HPO4·3H2O 2.6g / L, MgSO4·7H2O0.1g / L, MnSO4 0.05g / L, Tween 80 1mL / L, cysteine ​​salt 1g / L; sterilization temperature: 115℃ for 20min.

[0058] Unless otherwise specified, in the following embodiments, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0059] The detection method of the viable count of probiotics involved in the following embodiments is as follows: refer to the national standard "GB 4789.35-2016 National Food Safety Standard Food Microbiology Detection Lactic Acid Bacteria Detection".

[0060] The calculation formula for probiotic growth generation time is:

[0061]

[0062] Among them, t1 represents the initial time of the logarithmic growth phase of probiotics, t2 represents the final time of the logarithmic growth phase of probiotics, and W1 is the OD measured at the beginning of the logarithmic phase. 600 , W2 is the OD measured at the end of the logarithmic phase 600 Example 1 Effect of different concentrations of ergothioneine on the growth of Lactobacillus rhamnosus CCFM1259 Example 1.1 Effect of different concentrations of ergothioneine on the growth time of Lactobacillus rhamnosus CCFM1259

[0063] (1) Strain activation: a glycerol tube of Lactobacillus rhamnosus CCFM1259 stored at -80°C was thawed at 4°C, and the Lactobacillus rhamnosus CCFM1259 in the tube was streaked on an MRS solid culture medium using an inoculation loop, and the tube was placed in a constant temperature and humidity incubator at 37°C for 24 hours to obtain a single colony; a single colony was selected and inoculated into an MRS liquid culture medium, and the culture was carried out in a constant temperature and humidity incubator at 37°C for 12-24 hours; a bacterial solution was inoculated into a fresh MRS liquid culture medium at an inoculum amount of 2% (v / v), and the culture was carried out at 37°C for 12-16 hours to obtain an activated third-generation bacterial solution.

[0064] (2) Determination of the growth generation time of Lactobacillus rhamnosus CCFM1259: The activated three-generation bacterial solution was inoculated into MRS medium containing 1%, 0.50%, 0.25%, 0.05%, 0.01%, and 0.001% ergothioneine at a 2% (v / v) inoculation amount, and cultured at 37°C. The absorbance of the bacterial solution at 600nm for 0-20h was continuously measured using a fully automatic microbial growth curve analyzer, and the growth curve of Lactobacillus rhamnosus CCFM1259 under the culture conditions was measured, and the growth generation time of Lactobacillus rhamnosus CCFM1259 under the culture conditions was calculated. The calculation results are shown in Table 1, and the *, **, ***, and **** in the table indicate that the blank group and the experimental group have significant differences, respectively p<0.05, p<0.01, p<0.001, and p<0.0001.

[0065] Table 1 Growth time of Lactobacillus rhamnosus CCFM1259 in different concentrations of ergothioneine

[0066]

[0067] As shown in Table 1, the concentration of thioneine added in the culture medium is different to the growth of Lactobacillus rhamnosus CCFM1259. Data show that in the culture medium with thioneine added, the growth of Lactobacillus rhamnosus CCFM1259 has been significantly reduced, indicating that adding thioneine can shorten the time required for the somatic cell division of Lactobacillus rhamnosus CCFM1259, improve its growth rate, especially in the culture medium containing thioneine below 0.25%, the growth of Lactobacillus rhamnosus CCFM1259 is significantly shortened. For example, compared with the growth generation time of about 1.25h in the blank medium without ergothioneine, in the medium with 0.05% ergothioneine, the growth generation time of rhamnosus lactobacillus CCFM1259 was shortened to about 0.96h, indicating that the time required for each cell division was reduced by nearly 18 minutes; in the medium with 0.01% ergothioneine, the growth generation time of rhamnosus lactobacillus CCFM1259 was shortened to about 1.00h, indicating that the time required for each cell division was reduced by nearly 15 minutes; in the medium with 0.001% ergothioneine, the growth generation time of rhamnosus lactobacillus CCFM1259 was shortened to about 1.08h, indicating that the time required for each cell division was reduced by nearly 11 minutes. This shows that the addition of a specific amount of ergothioneine to the culture medium will greatly increase the growth rate of the strain and greatly shorten the time required for the strain to reach the stable phase.

[0068] Example 1.2 Effect of different concentrations of ergothioneine on the viable count of Lactobacillus rhamnosus CCFM1259

[0069] (1) Strain activation: a glycerol tube of Lactobacillus rhamnosus CCFM1259 stored at -80°C was thawed at 4°C, and the Lactobacillus rhamnosus CCFM1259 in the tube was streaked on an MRS solid culture medium using an inoculation loop, and the tube was placed in a constant temperature and humidity incubator at 37°C for 24 hours to obtain a single colony; a single colony was selected and inoculated into an MRS liquid culture medium, and the culture was carried out in a constant temperature and humidity incubator at 37°C for 12-24 hours; a bacterial solution was inoculated into a fresh MRS liquid culture medium at an inoculum amount of 2% (v / v), and the culture was carried out at 37°C for 12-16 hours to obtain an activated third-generation bacterial solution.

[0070] (2) Determination of the number of viable bacteria of Lactobacillus rhamnosus CCFM1259: The activated third-generation bacterial solution was inoculated with an inoculum of 2% (v / v) into MRS medium containing 1%, 0.50%, 0.25%, 0.05%, 0.01%, and 0.001% ergothioneine, and the number of viable bacteria of Lactobacillus rhamnosus CCFM1259 was determined according to the national standard "GB 4789.35-2016 National Food Safety Standard Food Microbiology Detection Lactic Acid Bacteria Detection". In the table, *, **, ***, and **** indicate that there are significant differences between the blank group and the experimental group, respectively, p<0.05, p<0.01, p<0.001, and p<0.0001.

[0071] Table 2 The number of viable bacteria of Lactobacillus rhamnosus CCFM1259 in different concentrations of ergothioneine

[0072]

[0073] Table 2 shows the viable counts of Lactobacillus rhamnosus CCFM1259 after 12 h of culture. As shown in Table 2, after 12 h of culture, the viable counts of Lactobacillus rhamnosus CCFM1259 in all culture media reached 10 8 CFU / mL, among which the viable count of Lactobacillus rhamnosus CCFM1259 in blank (no ergothioneine added) medium was (3.79±0.06)×10 8 CFU / mL, and in the culture medium supplemented with ergothioneine, the number of viable bacteria of rhamnosus Lactobacillus CCFM1259 increased significantly, indicating that adding ergothioneine to the culture medium can significantly increase the amount of rhamnosus Lactobacillus and promote the proliferation of rhamnosus Lactobacillus. Among them, after growing for 12 hours in the culture medium supplemented with 0.01% ergothioneine, the number of viable bacteria of rhamnosus Lactobacillus CCFM1259 was (7.37±0.12)×10 8CFU / mL, which was 1.94 times higher than that of the blank medium. In the medium containing 0.05% ergothioneine, the number of viable bacteria of Lactobacillus rhamnosus CCFM1259 reached (7.83±0.05)×10 after 12 h of growth. 8 CFU / mL, which was 2.07 times higher than that of the blank medium. In the medium containing 0.001% ergothioneine, the viable count of Lactobacillus rhamnosus CCFM1259 reached (6.91±0.07)×10 after 12 h of growth. 8 CFU / mL, compared with the number of live bacteria in the blank medium increased by 1.82 times. Example 2 The effect of different concentrations of ergothioneine on the growth of Lactobacillus paracasei CCFM1157 Example 2.1 Effect of different concentrations of ergothioneine on the growth of Lactobacillus paracasei CCFM1157

[0074] (1) Activation of strains: a glycerol tube of Lactobacillus paracasei CCFM1157 stored at -80°C was thawed at 4°C, an inoculation loop was used to pick up Lactobacillus paracasei CCFM1157 in the tube and streaked on an MRS solid culture medium, and the tube was placed in a constant temperature and humidity incubator at 37°C for 24 hours to obtain a single colony; a single colony was selected and inoculated into an MRS liquid culture medium, and the culture was carried out in a constant temperature and humidity incubator at 37°C for 12-24 hours; a bacterial solution was inoculated into a fresh MRS liquid culture medium at an inoculum amount of 2% (v / v), and the culture was carried out at 37°C for 12-16 hours to obtain an activated third-generation bacterial solution.

[0075] (2) Growth generation time of Lactobacillus paracasei CCFM1157: The activated three-generation bacterial liquid was inoculated into MRS medium containing 1%, 0.50%, 0.25%, 0.05%, 0.01%, and 0.001% ergothioneine with 2% (v / v) inoculum, and cultured at 37°C. The absorbance of the bacterial liquid at 600nm was continuously measured at 0-20h using a fully automatic microbial growth curve analyzer, and the growth curve of Lactobacillus paracasei CCFM1157 under the culture conditions was measured, and the growth generation time of Lactobacillus paracasei CCFM1157 under the culture conditions was calculated. The calculation results are shown in Table 3, and in the table, *, **, ***, and **** indicate that the blank group is significantly different from the experimental group, and are p<0.05, p<0.01, p<0.001, and p<0.0001, respectively.

[0076] Table 3 Growth time of Lactobacillus paracasei CCFM1157 in different concentrations of ergothioneine

[0077]

[0078] As shown in Table 3, the concentration of the thioneine added in the culture medium is different on the growth of Lactobacillus paracasei CCFM1157. The impact of generation time is also different. Data show, in the culture medium with the addition of thioneine, the growth of Lactobacillus paracasei CCFM1157 has been significantly reduced, indicating that adding thioneine can shorten the time needed for the frequent division of Lactobacillus paracasei CCFM1157 somatic cells, improves its growth rate, especially in the culture medium containing thioneine below 0.25%, the growth of Lactobacillus paracasei CCFM1157 is shortened significantly. For example, compared to the growth generation time of about 1.31h in the blank medium without ergothioneine, in the medium with 0.05% ergothioneine, the growth generation time of Lactobacillus paracasei CCFM1157 was shortened to about 1.12h, indicating that the time required for each cell division was reduced by nearly 12 minutes; in the medium with 0.01% ergothioneine, the growth generation time of Lactobacillus paracasei CCFM1157 was shortened to about 1.14h, indicating that the time required for each cell division was reduced by nearly 11 minutes; in the medium with 0.001% ergothioneine, the growth generation time of Lactobacillus paracasei CCFM1157 was shortened to about 1.17h, indicating that the time required for each cell division was reduced by nearly 9 minutes. This shows that the addition of a specific amount of ergothioneine to the culture medium will greatly increase the growth rate of the strain and greatly shorten the time required for the strain to reach the stable phase.

[0079] Example 2.2 Effect of different concentrations of ergothioneine on the viable count of Lactobacillus paracasei CCFM1157

[0080] (1) Activation of strains: a glycerol tube of Lactobacillus paracasei CCFM1157 stored at -80°C was thawed at 4°C, an inoculation loop was used to pick up Lactobacillus paracasei CCFM1157 in the tube and streaked on an MRS solid culture medium, and the tube was placed in a constant temperature and humidity incubator at 37°C for 24 hours to obtain a single colony; a single colony was selected and inoculated into an MRS liquid culture medium, and the culture was carried out in a constant temperature and humidity incubator at 37°C for 12-24 hours; a bacterial solution was inoculated into a fresh MRS liquid culture medium at an inoculum amount of 2% (v / v), and the culture was carried out at 37°C for 12-16 hours to obtain an activated third-generation bacterial solution.

[0081] (2) Determination of the number of viable bacteria of Lactobacillus paracasei CCFM1157: The activated third-generation bacterial solution was inoculated with an inoculum of 2% (v / v) into MRS medium containing 1%, 0.50%, 0.25%, 0.05%, 0.01%, and 0.001% ergothioneine, and the number of viable bacteria of Lactobacillus paracasei CCFM1157 was determined according to the national standard "GB 4789.35-2016 National Food Safety Standard Food Microbiology Detection Lactic Acid Bacteria Detection". In the table, **, ***, and **** indicate that the blank group and the experimental group are significantly different, respectively, p<0.01, p<0.001, and p<0.0001.

[0082] Table 4 The number of viable bacteria of Lactobacillus paracasei CCFM1157 in different concentrations of ergothioneine

[0083]

[0084] Table 4 shows the viable counts of Lactobacillus paracasei CCFM1157 after 12 h of culture. As shown in Table 4, after 12 h of culture, the viable counts of Lactobacillus paracasei CCFM1157 in all culture media reached 10 8 CFU / mL, among which the viable count of Lactobacillus paracasei CCFM1157 in blank (no ergothioneine added) medium was (2.58±0.07)×10 8 CFU / mL, and in the culture medium supplemented with ergothioneine, the number of viable bacteria of Lactobacillus paracasei CCFM1157 increased significantly, indicating that the addition of ergothioneine in the culture medium can significantly increase the amount of Lactobacillus paracasei and promote the proliferation of Lactobacillus paracasei. Among them, after Lactobacillus paracasei CCFM1157 grew in the culture medium supplemented with 0.01% ergothioneine for 12 hours, the number of viable bacteria was (4.38±0.16)×10 8 CFU / mL, which was 1.70 times higher than that of the blank medium. In the medium containing 0.05% ergothioneine, the number of viable bacteria of Lactobacillus paracasei reached (4.72±0.13)×10 after 12 h of growth. 8 CFU / mL, which increased by 1.83 times compared with the blank culture medium; in the culture medium containing 0.001% ergothioneine, the number of viable bacteria of Lactobacillus paracasei reached (3.58±0.14)×10 after 12h of growth. 8 CFU / mL increased by 1.39 times compared with the blank culture medium.

[0085] Example 3 Effect of different concentrations of ergothioneine on the growth of animal Bifidobacterium lactis subspecies CCFM1160 Example 3.1 Effect of different concentrations of ergothioneine on the growth time of animal Bifidobacterium lactis subspecies CCFM1160

[0086] (1) Strain activation: a glycerol tube of animal Bifidobacterium lactis subsp. CCFM1160 stored at -80°C was thawed at 4°C, and an inoculation loop was used to streak the animal Bifidobacterium lactis subsp. CCFM1160 in the tube on an mMRS solid culture medium, and the tube was incubated anaerobically at 37°C for 36-48 hours to obtain a single colony; a single colony was selected and inoculated into an mMRS liquid culture medium, and the tube was incubated anaerobically at 37°C for 12-24 hours; a bacterial solution was inoculated into a fresh mMRS liquid culture medium at an inoculum size of 2% (v / v), and the tube was incubated anaerobically at 37°C for 12-16 hours to obtain an activated third-generation bacterial solution.

[0087] (2) Animal Bifidobacterium lactis subspecies CCFM1160 growth generation time determination: The activated three-generation bacterial liquid was inoculated into the mMRS medium containing 1%, 0.50%, 0.25%, 0.05%, 0.01%, and 0.001% ergothioneine with a 2% (v / v) inoculum, and cultured anaerobically at 37°C. The absorbance of the bacterial liquid at 600nm was continuously measured for 0-24h using a fully automatic microbial growth curve analyzer, and the growth curve of animal Bifidobacterium lactis subspecies CCFM1160 under the culture conditions was determined, and the growth generation time of animal Bifidobacterium lactis subspecies CCFM1160 under the culture conditions was calculated. The calculation results are shown in Table 5, wherein **, ***, and **** indicate that the blank group and the experimental group are significantly different, and are p<0.01, p<0.001, and p<0.0001, respectively.

[0088] Table 5 Growth time of Bifidobacterium animalis subsp. lactis CCFM1160 in different concentrations of ergothioneine

[0089]

[0090] As shown in Table 5, the concentration of thioneine added in the culture medium is different on the growth generation of animal bifidobacterium lactis CCFM1160.Data show, in the culture medium with thioneine added, the growth generation of animal bifidobacterium lactis CCFM1160 has been significantly decreased, indicating that adding thioneine can shorten the time required for the every division of animal bifidobacterium lactis CCFM1160 somatic cells, improve its growth rate.Especially in the culture medium containing 0.05%, 0.01%, 0.001% thioneine, compared to the growth generation of about 1.77h in the blank culture medium without thioneine, in the culture medium with 0.05%, 0.01%, 0.001% thioneine added respectively, the growth generation of animal bifidobacterium lactis CCFM1160 was shortened to 1.43h, 1.41h, 1.47h or so respectively, indicating that the time required for the every division of cells was reduced by at least 18 minutes or even more than 20 minutes. This shows that adding a specific amount of ergothioneine to the culture medium will greatly increase the growth rate of the strain and greatly shorten the time required for the strain to reach the stable phase.

[0091] Example 3.2 Effect of different concentrations of ergothioneine on the viable count of animal Bifidobacterium lactis subspecies CCFM1160

[0092] (1) Strain activation: a glycerol tube of animal Bifidobacterium lactis subsp. CCFM1160 stored at -80°C was thawed at 4°C, and an inoculation loop was used to streak the animal Bifidobacterium lactis subsp. CCFM1160 in the tube on an mMRS solid culture medium, and the tube was incubated anaerobically at 37°C for 36-48 hours to obtain a single colony; a single colony was selected and inoculated into an mMRS liquid culture medium, and the tube was incubated anaerobically at 37°C for 12-24 hours; a bacterial solution was inoculated into a fresh mMRS liquid culture medium at an inoculum size of 2% (v / v), and the tube was incubated anaerobically at 37°C for 12-16 hours to obtain an activated third-generation bacterial solution.

[0093] (2) Determination of the number of live bacteria of animal Bifidobacterium lactis subsp. CCFM1160: The activated third-generation bacterial solution was inoculated with an inoculum of 2% (v / v) into mMRS medium containing 1%, 0.50%, 0.25%, 0.05%, 0.01%, and 0.001% ergothioneine, and the number of live bacteria of animal Bifidobacterium lactis subsp. CCFM1160 was determined according to the national standard "GB 4789.35-2016 National Food Safety Standard Food Microbiology Detection Lactic Acid Bacteria Detection" after anaerobic culture at 37°C for 14 hours. The results of the determination of the number of live bacteria are shown in Table 6, where **** indicates that there is a very significant difference between the blank group and the experimental group, p<0.0001.

[0094] Table 6 The number of viable cells of Bifidobacterium animalis subspecies lactis CCFM1160 in different concentrations of ergothioneine

[0095]

[0096] Table 6 shows the viable counts of Bifidobacterium animalis subspecies CCFM1160 after 14 h of culture. The results show that after 14 h of culture, the viable counts of Bifidobacterium animalis subspecies CCFM1160 in all culture media reached 10 8 CFU / mL, among which the viable count of animal Bifidobacterium lactis subsp. CCFM1160 in blank (no ergothioneine added) medium was (3.82±0.13)×10 8 CFU / mL, and in the culture medium with ergothioneine added, the number of live bacteria of animal Bifidobacterium lactis subspecies CCFM1160 was significantly increased, indicating that adding ergothioneine to the culture medium can significantly increase the amount of animal Bifidobacterium and promote the proliferation of animal Bifidobacterium. Among them, after growing for 14 hours in the culture medium with 0.05% ergothioneine, the number of live bacteria of animal Bifidobacterium lactis subspecies CCFM1160 was (5.96±0.11)×10 8 CFU / mL, which was 1.56 times higher than that of the blank medium. In the medium containing 0.01% ergothioneine, the viable count of animal Bifidobacterium lactis subsp. CCFM1160 reached (6.13±0.11)×10 after 14 h of growth. 8 CFU / mL, which increased by 1.60 times compared with the blank culture medium; in the culture medium containing 0.001% ergothioneine, the viable count of animal Bifidobacterium lactis subsp. CCFM1160 reached (5.11±0.11)×10 after 14h of growth. 8 CFU / mL increased by 1.34 times compared with the blank culture medium.

[0097] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. Application of ergothioneine in promoting the growth of probiotics.

2. The use according to claim 1, characterized in that: The promoting the growth of probiotics includes increasing the growth rate of probiotics and / or promoting the proliferation of probiotics.

3. A culture medium for promoting the growth of probiotics, characterized in that: The culture medium includes ergothioneine.

4. The culture medium according to claim 3, characterized in that In terms of mass percentage of the culture medium, the concentration of the ergothioneine is ≤10%; preferably, the concentration of the ergothioneine is ≤1%; more preferably, the concentration of the ergothioneine is <0.2%.

5. A method for promoting the growth of probiotics, characterized in that: The method comprises administering ergothioneine to the probiotic bacteria.

6. The method according to claim 5, characterized in that In percentage by mass, the concentration of the ergothioneine applied to the probiotics is ≤10%; preferably, the concentration of the ergothioneine applied to the probiotics is ≤1%; more preferably, the concentration of the ergothioneine applied to the probiotics is <0.2%.

7. Use of the culture medium as claimed in claim 3 or 4 or the method as claimed in claim 5 or 6 in promoting the growth of probiotics.

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