Microbial culture medium based on alkaline mountain spring water as well as preparation method and application of microbial culture medium
By using the microbial medium prepared by alkaline mountain spring water, the problem of insufficient pH buffer in the existing technologies has been solved, and the growth and survival of microorganisms are significantly promoted, and the preparation process of the medium is simplified.
Patent Information
- Application Number
- CN202411618990.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-13
- Publication Date
- 2025-01-24
AI Technical Summary
Existing microbial culture media is difficult to effectively buffer pH, affecting the growth and survival of microorganisms.
The use of alkaline mountain spring water as the water source of the microbial medium, which is prepared by the electrolytic water mechanism, provides rich minerals to cushion the pH value of the medium, and contain nutrients such as carbon source, nitrogen sources, and inorganic salts.
Significantly promotes the growth of microorganisms, provides a stable pH environment, improves the survival rate of microorganisms, and simplifies the preparation and operation of culture media.
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Figure CN119351262A_ABST
Abstract
Description
Technical field:
[0001] The present invention relates to the field of microbial culture medium, and in particular to a microbial culture medium based on alkaline spring water and a preparation method and application thereof. Background technology:
[0002] There are many kinds of microorganisms. Although some microorganisms can cause diseases in humans, some microorganisms also play an important role in nature and human life and bring many benefits. For example, Bacillus can enhance the body's resistance, inhibit the growth of pathogens and maintain the balance of intestinal flora. It plays an important role in maintaining human health. Lactobacillus plays an important role in inhibiting putrefactive bacteria, regulating intestinal flora, promoting digestion, promoting nutrient absorption and enhancing self-immunity. Yeast contains nutrients such as protein and vitamin B and a certain amount of minerals such as selenium and chromium. It has the effects of anti-oxidation, protecting the digestive system and promoting human metabolism. In addition, yeast can be used as a fermentation agent, additive and flavoring agent, which can help digest the contents of the gastrointestinal tract, reduce the burden on the gastrointestinal tract and protect the human digestive system.
[0003] Microorganisms need six nutrients: carbon source, nitrogen source, energy, inorganic salts, growth factors and water for growth. Minerals are rich in inorganic salts and trace elements, which provide necessary nutrients for microorganisms. Microorganisms can obtain energy by oxidizing and reducing the variable elements in the mineral structure, thereby providing energy for their growth. In addition, minerals can protect microorganisms from physical damage such as strong ultraviolet rays, abrasion, temperature fluctuations, etc., and can also buffer chemical hazards such as strong acids and strong alkalis, so that they can survive in harsh environments. Summary of the invention:
[0004] The present invention provides a microbial culture medium based on alkaline spring water, and a preparation method and application thereof. Using alkaline spring water as culture medium water can not only effectively buffer the pH value of the culture medium, which is beneficial to the survival of microorganisms, but also provide a large amount of inorganic salts and energy for the growth of microorganisms, thereby significantly promoting the growth of microorganisms.
[0005] The technical solution provided by the present invention is as follows:
[0006] A microbial culture medium based on alkaline spring water comprises a carbon source, a nitrogen source, an inorganic salt and a solvent, wherein the solvent is alkaline spring water. The alkaline spring water is alkaline water prepared by an electrolytic water making machine using spring water as a raw material.
[0007] Preferably, the culture medium is medium A, comprising tryptone, yeast extract, sodium chloride, glucose, and the remainder is alkaline spring water, with a natural pH;
[0008] Or, the culture medium is medium B, including peptone, beef extract, yeast extract, glucose, Tween 80, dipotassium hydrogen phosphate, sodium acetate, triammonium citrate, magnesium sulfate, manganese sulfate, and the balance is alkaline spring water, natural pH;
[0009] Alternatively, the culture medium is C medium, including peptone, yeast extract, glucose, and the balance is alkaline spring water with natural pH.
[0010] Further preferably, the A culture medium includes 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 1 g / L glucose, and the balance is alkaline spring water, with a natural pH; the B culture medium includes 10 g / L peptone, 5 g / L beef extract, 5 g / L yeast extract, 2 g / L glucose, 1 mL / L Tween 80, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, and the balance is alkaline spring water, with a natural pH; the C culture medium includes 20 g / L peptone, 10 g / L yeast extract, 2 g / L glucose, and the balance is alkaline spring water, with a natural pH.
[0011] Preferably, the alkaline spring water is the Hydrogen-Free Alkaline Spring Water produced by Qinxing Medical Technology.
[0012] The present invention also provides a method for preparing the alkaline spring water-based microbial culture medium, which is prepared by uniformly mixing the components and contents of the culture medium and sterilizing the culture medium at a high temperature of 121° C. for 15 minutes.
[0013] The present invention also provides the use of the alkaline spring water-based microbial culture medium in microbial culture.
[0014] Preferably, the A medium is applied to Bacillus culture, the B medium is applied to Lactobacillus culture, and the C medium is applied to saccharomyces culture.
[0015] More preferably, the Bacillus is Bacillus subtilis, the Lactobacillus is Lactobacillus plantarum, and the yeast is Saccharomyces cerevisiae.
[0016] The present invention also provides a method for culturing microorganisms, which comprises inoculating the microorganisms into the above-mentioned microorganism culture medium based on alkaline spring water for culturing.
[0017] Preferably, Bacillus is inoculated into medium A for culture; Lactobacillus is inoculated into medium B for culture; and yeast is inoculated into medium C for culture.
[0018] Further preferably, OD 600 The Bacillus subtilis liquid with a concentration of 0.5 was transferred to medium A and cultured at 37°C with a shaker at 180 rpm. 600 The plant lactobacillus culture liquid with an OD of 0.5 was transferred to medium B and cultured at 30°C with a shaker at 180 rpm. 600 The 0.5% suspension of Saccharomyces cerevisiae was transferred to C medium and cultured at 30°C with a shaker at 180 rpm.
[0019] The beneficial effects of the present invention are as follows:
[0020] 1. The present invention uses alkaline spring water as the water source for the microbial culture medium. The alkaline spring water is alkaline water prepared by an electrolytic water maker using spring water as a raw material, and the minerals contained therein can effectively buffer the pH of the microbial culture medium.
[0021] 2. The alkaline spring water microbial culture medium provided by the present invention is rich in minerals, provides a large amount of inorganic salts and energy for the growth of microorganisms, and has a significant promoting effect on the growth of microorganisms.
[0022] 3. The alkaline spring water microbial culture medium provided by the present invention has abundant and natural water sources, is non-toxic and non-irritating to microorganisms, and is easy to prepare and operate, and is easy for industrial production. Description of the drawings:
[0023] Figure 1 The growth conditions of Bacillus subtilis inoculated with medium A obtained in Example 1 and Comparative Examples 1-4 are shown.
[0024] Figure 2 The growth conditions of plant lactobacillus inoculated with the B culture medium obtained in Example 1 and Comparative Examples 1-4 are shown.
[0025] Figure 3 The growth conditions of Saccharomyces cerevisiae inoculated with the C culture medium obtained in Example 1 and Comparative Examples 1-4 are shown. Specific implementation method:
[0026] It will be appreciated by those skilled in the art that the disclosed techniques in the following examples represent techniques that the inventors have found to work well in the practice of the present invention. However, many changes may be made in the disclosed specific embodiments, and still the same or similar results may be obtained without departing from the spirit and scope of the present invention. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.
[0027] Example 1
[0028] A medium, B medium, and C medium based on alkaline spring water:
[0029] A medium (1 L) was prepared with the following raw materials: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 1 g glucose, and the balance was alkaline spring water, natural pH, and sterilized for use.
[0030] Medium B (1 L) was prepared with the following raw materials: 10 g peptone, 5 g beef extract, 5 g yeast extract, 2 g glucose, 1 mL Tween 80, 2 g potassium dihydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, and the remainder was alkaline spring water, natural pH, and sterilized for later use.
[0031] C medium (1 L) was prepared with the following raw materials: 20 g peptone, 10 g yeast extract, 2 g glucose, and the balance was alkaline spring water, natural pH, and sterilized for later use.
[0032] The alkaline spring water used in the above-mentioned medium A, medium B, and medium C is the alkaline spring water of Qinxing Medical Technology (Shanxi) Co., Ltd. The sterilization condition of the culture medium is high pressure sterilization at 121°C for 15 minutes.
[0033] Bacillus subtilis NX3 (NCBI accession number OR945769) was selected as the representative of the genus Bacillus, and 100 μL of OD 600 The Bacillus subtilis bacterial liquid with a concentration of 0.5 was transferred to 100 mL A culture medium and cultured at 37°C with a shaker at 180 rpm. The bacterial concentration of Bacillus subtilis was detected at 600 nm after 18 h and 24 h of culture.
[0034] Select Lactobacillus plantarum NCMRS1 (NCBI accession number PQ569975) as the representative of the genus Lactobacillus, take 100 μL OD 600 The plant lactobacillus liquid with a concentration of 0.5 was transferred to 100 mL of B culture medium and cultured at 30°C and 180 rpm in a shaking incubator. The plant lactobacillus concentration was detected at 600 nm after 18 h and 24 h of culture.
[0035] Saccharomyces cerevisiae BY4741 (Beina Biotech) was selected as the representative of the genus Saccharomyces. 100 μL of OD 600 The 0.5% Saccharomyces cerevisiae bacterial liquid was transferred to 100 mL C medium and cultured at 30°C and 180 rpm in a shaking incubator. The concentration of Saccharomyces cerevisiae was detected at 600 nm after 18 h and 24 h of culture.
[0036] The applicant also possesses the above-mentioned Bacillus subtilis NX3 and Lactobacillus plantarum NCMRS1 and guarantees that they can be provided to the public within 20 years from the date of application.
[0037] Comparative Example 1
[0038] Refer to Example 1, except that the alkaline spring water used in the culture medium is replaced with tap water, and the pH of each culture medium is natural.
[0039] Comparative Example 2
[0040] Refer to Example 1, except that the alkaline spring water used in the culture medium is replaced with tap water, and the pH of A, B and C culture media is adjusted to be consistent with the pH of A, B and C culture media in Example 1 with 1M NaOH and 0.1M HCL.
[0041] Comparative Example 3
[0042] Refer to Example 1, except that the alkaline spring water used in the culture medium is replaced with distilled water, and the pH of each culture medium is natural.
[0043] Comparative Example 4
[0044] Refer to Example 1, except that the alkaline spring water used in the culture medium is replaced with distilled water, and the pH of A, B and C culture media is adjusted to be consistent with the pH of A, B and C culture media in Example 1 with 1M NaOH and 0.1M HCL.
[0045] The alkaline spring water selected in Example 1 is derived from the alkaline spring water of Qinxing Medical Technology Co., Ltd., and tap water and distilled water are used as the comparative water sources of alkaline spring water (Comparative Examples 1-4). The pH of alkaline spring water is 8.0, tap water is 7.5, and distilled water is 6.0. 1M NaOH and 0.1M HCL are used to adjust the pH of A, B and C culture media with tap water and distilled water as water sources to be consistent with the pH of A, B and C culture media with alkaline spring water as the water source. The pH of each culture medium is shown in Table 1.
[0046] Table 1 pH of each culture medium
[0047]
[0048] As shown in Table 1, the natural pH of culture medium A whose water sources are alkaline spring water, tap water and distilled water are pH 6.24, pH 6.50 and pH 6.17 respectively; the natural pH of culture medium B are pH 6.05, pH 6.06 and pH 6.02 respectively; the natural pH of culture medium C are pH 7.06, pH 7.05 and pH 7.04 respectively. There is no obvious difference between the pH of each culture medium whose water source is alkaline spring water and the pH of each culture medium whose water source is tap water and distilled water. This shows that the minerals in the alkaline spring water of Qinxing Medical Technology Co., Ltd. can effectively buffer the pH of the culture medium.
[0049] like Figure 1 , 2 As shown in Figure 3, when each microorganism was cultured for 24 hours, the OD of Bacillus subtilis in Example 1 (alkaline spring water, natural pH) in medium A was 600 The OD of Bacillus subtilis in Comparative Example 1 (tap water, natural pH) is 2.00. 600 The OD of Bacillus subtilis in Comparative Example 3 (distilled water, natural pH) was 1.79. 600 The OD of Lactobacillus plantarum in Example 1 (alkaline spring water, natural pH) in medium B was 1.94. 600 The OD of Lactobacillus plantarum in comparative example 1 (tap water, natural pH) was 2.29. 600 The OD of Lactobacillus plantarum in Comparative Example 3 (distilled water, natural pH) was 2.20. 600 The OD of the Saccharomyces cerevisiae in Example 1 (alkaline spring water, natural pH) in the 2.25°C medium was 600 The OD of Saccharomyces cerevisiae in Comparative Example 1 (tap water, natural pH) was 2.29. 600 The OD of Saccharomyces cerevisiae in Comparative Example 3 (distilled water, natural pH) was 2.11. 600 The above data show that under the same culture medium components and natural pH, the growth rate of microorganisms in culture medium A, B and C with alkaline spring water as the water source is significantly higher than that of microorganisms with tap water and distilled water as the water source, which indicates that the minerals in the alkaline spring water of Qinxing Medical Technology Co., Ltd. have a significant promoting effect on the growth of microorganisms.
[0050] When each microorganism was cultured for 24 h, the OD of Bacillus subtilis in Example 1 (alkaline spring water, pH 6.24) in medium A was 600 The OD of Bacillus subtilis in Comparative Example 2 (tap water, pH 6.24) was 2.00. 600 The OD of Bacillus subtilis in Comparative Example 4 (distilled water, pH 6.24) was 1.71. 600 The OD of Lactobacillus plantarum in Example 1 (alkaline spring water, pH 6.05) in medium B was 1.91.600 The OD of Lactobacillus plantarum in Comparative Example 2 (tap water, pH 6.05) was 2.29. 600 The OD of Lactobacillus plantarum in Comparative Example 4 (distilled water, pH 6.05) was 2.21. 600 The OD of the Saccharomyces cerevisiae in Example 1 (alkaline spring water, pH 7.06) in C medium was 2.26. 600 The OD of Saccharomyces cerevisiae in Comparative Example 2 (tap water, pH 7.06) was 2.29. 600 The OD of Saccharomyces cerevisiae in Comparative Example 4 (distilled water, pH 7.06) was 2.00. 600 is 2.20. The above data show that with the same culture medium components and the same pH adjustment, the growth of different microorganisms from different water sources has obvious differences, which shows that the minerals in the water source in the culture medium have a significant effect on the growth of microorganisms. In addition, when the pH of the culture medium is adjusted consistently, the growth rate of microorganisms in culture medium A, B and C whose water source is alkaline spring water is significantly higher than the growth rate of microorganisms whose water source is tap water and distilled water, which further shows that the minerals in the alkaline spring water of Qinxing Medical Technology Co., Ltd. have a significant promoting effect on the growth of microorganisms.
[0051] The above results show that the minerals in the Hehydromei alkaline spring water produced by Qinxing Medical Technology Co., Ltd. can effectively buffer the pH of the culture medium and have a significant promoting effect on the growth of microorganisms.
Claims
1. A microbial culture medium based on alkaline spring water, characterized in that: The invention comprises a carbon source, a nitrogen source, an inorganic salt and a solvent, wherein the solvent is alkaline mountain spring water.
2. The culture medium according to claim 1, characterized in that The culture medium is medium A, including tryptone, yeast extract, sodium chloride, glucose, and the balance is alkaline spring water, with natural pH; Or, the culture medium is medium B, including peptone, beef extract, yeast extract, glucose, Tween 80, dipotassium hydrogen phosphate, sodium acetate, triammonium citrate, magnesium sulfate, manganese sulfate, and the balance is alkaline spring water, natural pH; Alternatively, the culture medium is C culture medium, including peptone, yeast extract, glucose, and the balance is alkaline spring water with a natural pH.
3. The culture medium according to claim 2, characterized in that The A culture medium includes 10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride, 1g / L glucose, and the balance is alkaline spring water, with a natural pH; the B culture medium includes 10g / L peptone, 5g / L beef extract, 5g / L yeast extract, 2g / L glucose, 1mL / L Tween 80, 2g / L dipotassium hydrogen phosphate, 5g / L sodium acetate, 2g / L triammonium citrate, 0.2g / L magnesium sulfate, 0.05g / L manganese sulfate, and the balance is alkaline spring water, with a natural pH; the C culture medium includes 20g / L peptone, 10g / L yeast extract, 2g / L glucose, and the balance is alkaline spring water, with a natural pH.
4. The culture medium according to claim 2 or 3, characterized in that The alkaline spring water is Hydrogen-free alkaline spring water.
5. The method for preparing a microbial culture medium based on alkaline spring water according to any one of claims 1 to 4, characterized in that: The culture medium is mixed evenly according to its components and content, and sterilized at high temperature at 121° C. for 15 minutes.
6. Use of the microbial culture medium based on alkaline spring water as described in any one of claims 2 to 4 in microbial culture.
7. The use according to claim 6, characterized in that: The A culture medium is applied to Bacillus culture, the B culture medium is applied to Lactobacillus culture, and the C culture medium is applied to yeast culture.
8. A method for culturing microorganisms, characterized in that: The method comprises inoculating the microorganisms into the microorganism culture medium based on alkaline spring water as described in any one of claims 2 to 4 for culturing.
9. The method according to claim 8, characterized in that Bacillus was inoculated into medium A for culture; Lactobacillus was inoculated into medium B for culture; and yeast was inoculated into medium C for culture.
10. The method according to claim 9, characterized in that OD 600 The Bacillus subtilis liquid with a concentration of 0.5 was inoculated into medium A and cultured at 37°C with a shaker at 180 rpm. 600 The plant lactobacillus culture liquid with a concentration of 0.5 was inoculated into medium B and cultured at 30°C with a shaker at 180 rpm. 600 A 0.5% suspension of Saccharomyces cerevisiae was inoculated into C medium and cultured at 30°C with a shaker at 180 rpm.