Glucose-free culture medium for growth of saccharomyces boulardii as well as preparation method and application of glucose-free culture medium

Abstract

The invention discloses a glucose-free culture medium for saccharomyces boulardii growth and a preparation method and application thereof, the glucose-free culture medium comprises a component (a) and a component (b), the component (a) comprises yeast powder, peptone and cane sugar; the component (b) is a trace element solution, and the trace element is a PTM1 trace element. The method for culturing the saccharomyces boulardii through glucose-free fermentation has the advantages of being easy to operate, low in requirement for equipment and personnel, short in production period, clear in component, stable in index and high in bacterium concentration, the requirement for subsequent strain fermentation can be met, and meanwhile reference is provided for glucose-free culture of strains.

Description

Technical Field

[0001] The invention belongs to the technical field of culture medium, and in particular relates to a glucose-free culture medium for growing Saccharomyces boulardii and a preparation method and application thereof. Background Art

[0002] Saccharomyces boulardii is a non-pathogenic, probiotic yeast. In Europe, America, Africa and other countries and regions, Saccharomyces boulardii has been widely used as a microecological preparation to prevent and treat infant diarrhea, traveler diarrhea, recovery after intestinal surgery and regulation of intestinal environmental balance. Saccharomyces boulardii has been proven to be safe and effective in a large number of clinical applications. Saccharomyces boulardii tolerates various digestive enzymes and gastric acid secreted by the gastrointestinal tract, is naturally resistant to antibiotics, has an optimal growth temperature of 37°C, and can maintain a certain number of live bacteria in the gastrointestinal tract. In summary, Saccharomyces boulardii is an ideal intestinal drug delivery carrier. In the early stage of this study, Saccharomyces boulardii was genetically engineered, and the GLP-1 gene was introduced into the genome of Saccharomyces boulardii. Through the colonization of Saccharomyces boulardii in the intestine, it exerts a probiotic effect while achieving the expression of GLP-1, hoping to provide patients with a more convenient and comfortable way of administration.

[0003] Through literature research and previous studies, we found that the function of the promoter element in the Saccharomyces boulardii expression system is closely related to the expression efficiency. The promoter elements in the Saccharomyces boulardii expression system are divided into two categories, namely constitutive promoters represented by strong promoters such as pPGK1, pTEF1, pTDH3 and pADH1, and inducible promoters represented by pGAL related to galactose metabolism and pHXT related to hexose metabolism. Among them, the hexose transporter family is composed of HXT1p-HXT17p and GAL2p. The HXT transporter in Saccharomyces boulardii is a protein closely related to glucose sensing, containing 12 putative transmembrane domains, which mediates glucose transport across the plasma membrane through a facilitated diffusion mechanism. We previously constructed GLP-1 expression vectors with HXT1, HXT2, HXT4, and HXT6 promoters. The HXT1 gene was shown to encode a low-affinity glucose transporter with a glucose Km value of about 100 mM. High glucose concentrations can induce HXT1 expression up to 300 times; HXT2 and HXT4 genes are genes encoding medium-affinity glucose transporters (Km values ​​of about 10), which are maximally expressed at low glucose concentrations (1 g / L); the HXT6 gene encodes a high-affinity glucose transporter with a Km value of about 1 mM, which is highly expressed at extremely low glucose concentrations, non-fermentable carbon sources (ethanol, glycerol), maltose, and / or galactose, and is inhibited by medium and high glucose concentrations. When blood sugar in the body rises after a meal, the HXT promoter added to the engineered bacteria turns on the switch to express GLP-1, simulating the function of L cells in the body to secrete GLP-1; when blood sugar decreases, the HXT promoter is inhibited and the expression of GLP-1 stops. This oral probiotic yeast that controls the expression of GLP-1 through glucose concentration is expected to become a safer and more effective smart blood sugar lowering method.

[0004] In order to achieve full expression of GLP-1 in a glucose environment by the recombinant Saccharomyces boulardii vector expressing GLP-1 with the HXT promoter, we tried to avoid using glucose as a carbon source in the early stage of culture amplification, so that the hexose transporter family was in a dormant state. When the recombinant bacteria encountered glucose, they could quickly sense the trans-plasma membrane transport of glucose and achieve the expression of the downstream GLP-1 gene. At present, there is no glucose-free culture medium for Saccharomyces boulardii that has been widely used. Therefore, it is of great significance to develop a culture medium that can meet the growth requirements of Saccharomyces boulardii and does not contain glucose. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] One of the purposes of the present invention is to provide a glucose-free culture medium for the growth of Saccharomyces boulardii. By optimizing the composition of the culture medium, Saccharomyces boulardii can grow normally under sugar-free conditions and maintain good biological activity.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions: a glucose-free culture medium for growing Saccharomyces boulardii, comprising component (a), wherein the component (a) comprises yeast powder, peptone, and sucrose; the mass ratio of the yeast powder, peptone, and sucrose is 1:2:2-2.2; wherein the concentration of the sucrose is 20-22 g / L;

[0009] Component (b), wherein the component (b) is a trace element solution, wherein the trace element is a PTM1 trace element, and the concentration of the trace element solution is 1 to 3 mL / L.

[0010] As a preferred embodiment of the glucose-free culture medium for growing Saccharomyces boulardii of the present invention, the component (a) further comprises corn steep liquor, and the concentration of the corn steep liquor is 9-11 g / L.

[0011] As a preferred embodiment of the glucose-free culture medium for the growth of Saccharomyces boulardii of the present invention, the component (a) further comprises one or more salts selected from the group consisting of: urea, KH2PO4, (NH4)2SO4, MgSO4, ZnSO4, CaCl2.

[0012] As a preferred embodiment of the glucose-free medium for growing Saccharomyces boulardii of the present invention, the ratio of the concentration of urea to the concentration of sucrose is 5:20-22;

[0013] The ratio of the concentration of KH2PO4 to the concentration of sucrose is 2:20-22;

[0014] The ratio of the concentration of (NH4)2SO4 to the concentration of sucrose is 2:20-22;

[0015] The concentration ratio of the MgSO4 to the sucrose is 0.5:20-22;

[0016] The concentration ratio of the ZnSO4 to the sucrose is 0.3:20-22;

[0017] The ratio of the concentration of CaCl2 to the concentration of sucrose is 0.3:20-22.

[0018] Another object of the present invention is to provide a method for preparing the glucose-free medium for growing Saccharomyces boulardii as described above, comprising:

[0019] preparing component (a) and component (b);

[0020] Add component (a) into appropriate amount of distilled water, fully dissolve and mix;

[0021] The culture medium was sterilized by high temperature and high pressure at 110°C for 30 min;

[0022] After sterilization, the medium was cooled to room temperature, and component (b) was added to obtain a glucose-free medium for Saccharomyces boulardii.

[0023] Another object of the present invention is to provide the use of the glucose-free culture medium for the growth of Saccharomyces boulardii as described above, characterized in that: for the cultivation of Saccharomyces boulardii, the temperature is 28-37°C, preferably 30°C, the rotation speed is 220r / min, and the inoculation amount is 1 / 500 of the culture medium volume inoculation amount.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention provides a glucose-free culture medium for growing Saccharomyces boulardii and a preparation method thereof. The culture medium enables Saccharomyces boulardii to grow normally under sugar-free conditions and maintain good biological activity by optimizing the components. The present invention aims to provide a method for culturing Saccharomyces boulardii by glucose-free fermentation, which has the advantages of simple operation, low requirements on equipment and personnel, short production cycle, clear components, stable indicators, and high bacterial concentration, which can meet the needs of subsequent strain fermentation and also provide a reference for glucose-free culture of strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0027] Figure 1 This is a graph showing the OD value test results of each culture medium in Example 1 of the present invention.

[0028] Figure 2 This is a graph showing the OD value test results of each culture medium in Example 2 of the present invention.

[0029] Figure 3 This is a graph showing the OD value test results of each culture medium in Example 3 of the present invention. DETAILED DESCRIPTION

[0030] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0031] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0032] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0033] Unless otherwise specified, the raw materials used in the examples were purchased commercially.

[0034] The Saccharomyces boulardii used in the examples of the present invention is from Shanghai Biotechnology Collection Center (SHBCC), the strain name is Saccharomyces boulardii, and the collection number is SHBCC D25152.

[0035] Example 1

[0036] (1) Prepare the following culture medium formula and dissolve it in an appropriate amount of distilled water or deionized water. Use a magnetic stirrer or stir manually to ensure that all ingredients are fully dissolved and mixed evenly.

[0037] Medium A: YPD medium: yeast powder 10 g / L, peptone 20 g / L, glucose 20 g / L;

[0038] Medium B: yeast powder 10g / L, peptone 20g / L, phosphate 100mM, glycerol 10g / L; amino-free yeast nitrogen source (YNB) 13.4g / L, Biotin (D-biotin) 0.4mg / L (0.22um filtration sterilization, added after high temperature sterilization of the culture medium);

[0039] Medium C: yeast powder 10g / L, peptone 20g / L, phosphate 100mM, sucrose 10.8g / L; amino-free yeast nitrogen source (YNB) 13.4g / L, Biotin (D-biotin) 0.4mg / L (0.22um filtration sterilization, added after high temperature sterilization of the culture medium);

[0040] Culture medium D: yeast powder 10g / L, peptone 20g / L, phosphate 100mM, sucrose 10.8g / L, corn steep liquor 10g / L, urea 5g / L, KH2PO42g / L, (NH4)2SO42g / L, MgSO40.5g / L, ZnSO40.3g / L, CaCl20.3g / L; amino-free yeast nitrogen source (YNB) 13.4g / L, Biotin (D-biotin) 0.4mg / L, PTM1 trace elements 2mL / L (0.22um filtration sterilization, added after high temperature sterilization of the culture medium).

[0041] (2) The prepared culture medium is divided into high temperature and high pressure resistant glass bottles, ensuring that the amount of the culture medium does not exceed 1 / 3 of the container to leave enough space for steam penetration and circulation; the culture medium is placed in a high pressure steam sterilizer for high temperature and high pressure sterilization at 110°C for 30 min; after the high pressure sterilization is completed, it is naturally cooled to room temperature to obtain each culture medium; if long-term storage is required, the culture medium can be placed in a refrigerator at 4°C for refrigeration.

[0042] Inoculate Saccharomyces boulardii at 1 / 500 of the culture medium volume and culture in a shaker at 30°C and 220 rpm. Use a spectrophotometer to detect the OD value. Fill the cuvette with sterile water and adjust it to zero as a blank control. Add the diluted yeast culture solution to the cuvette, turn on the spectrophotometer, and detect its OD value at the OD600 wavelength. Measure OD600 every 2 to 8 hours. The test results of culture medium A to D are as follows: Figure 1 shown.

[0043] Depend on Figure 1 It can be seen that the effects of different carbon sources on the growth of Saccharomyces boulardii were explored in the culture medium. Equal amounts of glucose, glycerol, and sucrose were used as carbon sources to culture Saccharomyces boulardii. The results showed that Saccharomyces boulardii grew best in culture medium D and could be maintained for a long time after reaching the stable period. Sucrose as a carbon source, while adding corn syrup and corresponding trace elements can effectively promote the growth of Saccharomyces boulardii.

[0044] Example 2

[0045] (1) Prepare the following culture medium formula, dissolve it in an appropriate amount of distilled water or deionized water, and use a magnetic stirrer or stir manually to ensure that all ingredients are fully dissolved and mixed evenly;

[0046] Medium A: YPD medium: yeast powder 10 g / L, peptone 20 g / L, glucose 20 g / L;

[0047] Culture medium E: phosphoric acid (H3PO4) 25ml / L, calcium sulfate dihydrate (CaSO4·2H2O) 0.93g / L, potassium sulfate (K2SO4) 18.2g / L, magnesium sulfate (MgSO4) 7.27g / L, trisodium citrate (C6H5Na3O7·2H2O) 0.4g / L, potassium hydroxide (KOH) 10.6g / L, PTM1 trace elements 2ml / L, glycerol 50ml / L;

[0048] Medium F: yeast powder 10 g / L, peptone 20 g / L, maltodextrin 20 g / L;

[0049] Culture medium G: yeast powder 10 g / L, peptone 20 g / L, sucrose 21 g / L.

[0050] (2) The prepared culture medium is divided into high temperature and high pressure resistant glass bottles, ensuring that the amount of the culture medium does not exceed 1 / 3 of the container to leave enough space for steam penetration and circulation; the culture medium is placed in a high pressure steam sterilizer for high temperature and high pressure sterilization at 110°C for 30 min; after the high pressure sterilization is completed, it is naturally cooled to room temperature to obtain each culture medium; if long-term storage is required, the culture medium can be placed in a refrigerator at 4°C for refrigeration.

[0051] Inoculate Saccharomyces boulardii at 1 / 500 of the culture medium volume and culture in a shaker at 30°C and 220 rpm. Detect the OD value with a spectrophotometer. Fill the cuvette with sterile water and adjust to zero as a blank control. Add the diluted yeast culture solution to the cuvette, turn on the spectrophotometer, and detect its OD value at the OD600 wavelength. Measure OD600 every 2 to 8 hours. The test results of culture medium A to G are as follows: Figure 2 shown.

[0052] Depend on Figure 2 It can be seen that the effects of different carbon sources on the growth of Saccharomyces boulardii were explored in the culture medium. Equal amounts of glucose, glycerol, maltodextrin and sucrose were used as carbon sources to culture Saccharomyces boulardii. The results showed that Saccharomyces boulardii grew well in culture medium G and could be maintained for a long time after reaching the stable period. It was further determined that sucrose as a carbon source can effectively promote the growth of Saccharomyces boulardii.

[0053] Example 3

[0054] (1) Prepare the following culture medium formula, dissolve it in an appropriate amount of distilled water or deionized water, and use a magnetic stirrer or stir manually to ensure that all ingredients are fully dissolved and mixed evenly;

[0055] Medium G: yeast powder 10g / L, peptone 20g / L, sucrose 21g / L;

[0056] Medium H: yeast powder 10 g / L, peptone 20 g / L, sucrose 21 g / L, PTM1 trace elements 2 mL / L (added before inoculation);

[0057] Medium I: yeast powder 10 g / L, peptone 20 g / L, sucrose 21 g / L, corn steep liquor 10 g / L, PTM1 trace elements 2 mL / L (added before inoculation);

[0058] Culture medium J: yeast powder 10 g / L, peptone 20 g / L, sucrose 21 g / L, corn steep liquor 10 g / L, urea 5 g / L, KH2PO4 2 g / L, (NH4)2SO4 2 g / L, MgSO4 0.5 g / L, ZnSO4 0.3 g / L, CaCl2 0.3 g / L, PTM1 trace elements 2 mL / L (added before inoculation).

[0059] (2) The prepared culture medium is divided into high temperature and high pressure resistant glass bottles, ensuring that the amount of the culture medium does not exceed 1 / 3 of the container to leave enough space for steam penetration and circulation; the culture medium is placed in a high pressure steam sterilizer for high temperature and high pressure sterilization at 110°C for 30 min; after the high pressure sterilization is completed, it is naturally cooled to room temperature to obtain each culture medium; if long-term storage is required, the culture medium can be placed in a refrigerator at 4°C for refrigeration.

[0060] Inoculate Saccharomyces boulardii at 1 / 500 of the culture medium volume and culture in a shaker at 30°C and 220 rpm. Detect the OD value with a spectrophotometer. Fill the cuvette with sterile water and adjust to zero as a blank control. Add the diluted yeast culture solution to the cuvette, turn on the spectrophotometer, and detect its OD value at the OD600 wavelength. Measure OD600 every 2 to 8 hours. The test results of culture media G to J are as follows: Figure 3 shown.

[0061] Depend on Figure 3 It can be seen that based on the results of using A~G culture medium for Saccharomyces boulardii, there is no glucose carbon source in the culture system, which makes the hexose transporter family in the Saccharomyces boulardii strain in a dormant state. At the same time, the addition of sucrose can replace glucose as a carbon source, so that Saccharomyces boulardii can grow and expand rapidly. According to the results of D, H, I and J culture media, the addition of corn steep liquor and corresponding trace elements to the culture medium can effectively improve the growth efficiency of Saccharomyces boulardii, and can maintain its stable period for a longer time.

[0062] The present invention provides a glucose-free culture medium for growing Saccharomyces boulardii. The culture medium enables Saccharomyces boulardii to grow normally under sugar-free conditions and maintain good biological activity by optimizing the composition. The present invention aims to provide a method for culturing Saccharomyces boulardii by glucose-free fermentation. The method has the advantages of simple operation, low requirements on equipment and personnel, short production cycle, clear composition, stable indicators, and high bacterial concentration, which can meet the needs of subsequent strain fermentation and also provide a reference for glucose-free culture of strains.

[0063] 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 preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A glucose-free medium for growing Saccharomyces boulardii, characterized in that: The invention comprises a component (a), wherein the component (a) comprises yeast powder, peptone and sucrose; the mass ratio of the yeast powder, peptone and sucrose is 1:2:2-2.2; wherein the concentration of the sucrose is 20-22 g / L; Component (b), wherein the component (b) is a trace element solution, wherein the trace element is a PTM1 trace element, and the concentration of the trace element solution is 1 to 3 mL / L.

2. The glucose-free medium for growing Saccharomyces boulardii as claimed in claim 1, characterized in that: The component (a) further comprises corn steep liquor, and the concentration of the corn steep liquor is 9-11 g / L.

3. The glucose-free medium for growing Saccharomyces boulardii according to claim 1 or 2, characterized in that: The component (a) further comprises one or more salts selected from the group consisting of: urea, KH2PO4, (NH4)2SO4, MgSO4, ZnSO4, CaCl2.

4. The glucose-free medium for growing Saccharomyces boulardii as claimed in claim 3, characterized in that: The ratio of the concentration of the urea to the concentration of the sucrose is 5:20-22; The ratio of the concentration of KH2PO4 to the concentration of sucrose is 2:20-22; The ratio of the concentration of (NH4)2SO4 to the concentration of sucrose is 2:20-22; The concentration ratio of the MgSO4 to the sucrose is 0.5:20-22; The concentration ratio of the ZnSO4 to the sucrose is 0.3:20-22; The concentration ratio of the CaCl2 to the sucrose is 0.3:20-22.

5. The method for preparing a glucose-free medium for growing Saccharomyces boulardii according to any one of claims 1 to 4, characterized in that: include, preparing component (a) and component (b); Add component (a) into appropriate amount of distilled water, fully dissolve and mix; The culture medium was sterilized by high temperature and high pressure at 110°C for 30 min; After sterilization, the medium was cooled to room temperature, and component (b) was added to obtain a glucose-free medium for Saccharomyces boulardii.

6. Use of a glucose-free medium for growing Saccharomyces boulardii according to any one of claims 1 to 4, characterized in that: For the culture of Saccharomyces boulardii, the temperature is 28-37°C, the rotation speed is 220r / min, and the inoculation amount is 1 / 500 of the culture medium volume.

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