CN112280689A - Culture medium capable of promoting rapid growth of osmophilic yeast in honey and preparation method thereof - Google Patents
Culture medium capable of promoting rapid growth of osmophilic yeast in honey and preparation method thereof Download PDFInfo
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- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 45
- 239000001963 growth medium Substances 0.000 title claims abstract description 39
- 235000012907 honey Nutrition 0.000 title claims abstract description 25
- 230000001737 promoting effect Effects 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 239000006286 aqueous extract Substances 0.000 claims abstract description 56
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 37
- 239000000284 extract Substances 0.000 claims abstract description 35
- 239000011941 photocatalyst Substances 0.000 claims abstract description 17
- 229920001817 Agar Polymers 0.000 claims abstract description 14
- 244000061456 Solanum tuberosum Species 0.000 claims abstract description 14
- 235000002595 Solanum tuberosum Nutrition 0.000 claims abstract description 14
- 239000008272 agar Substances 0.000 claims abstract description 14
- SESFRYSPDFLNCH-UHFFFAOYSA-N benzyl benzoate Chemical compound C=1C=CC=CC=1C(=O)OCC1=CC=CC=C1 SESFRYSPDFLNCH-UHFFFAOYSA-N 0.000 claims abstract description 14
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims abstract description 14
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims abstract description 14
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims abstract description 14
- 235000019797 dipotassium phosphate Nutrition 0.000 claims abstract description 14
- 229960001031 glucose Drugs 0.000 claims abstract description 14
- RJMUSRYZPJIFPJ-UHFFFAOYSA-N niclosamide Chemical compound OC1=CC=C(Cl)C=C1C(=O)NC1=CC=C([N+]([O-])=O)C=C1Cl RJMUSRYZPJIFPJ-UHFFFAOYSA-N 0.000 claims abstract description 14
- 229960001920 niclosamide Drugs 0.000 claims abstract description 14
- 239000000843 powder Substances 0.000 claims abstract description 14
- 108010009004 proteose-peptone Proteins 0.000 claims abstract description 14
- 239000012153 distilled water Substances 0.000 claims abstract description 8
- 240000004530 Echinacea purpurea Species 0.000 claims abstract description 7
- 235000014134 echinacea Nutrition 0.000 claims abstract description 7
- 241000490499 Cardamine Species 0.000 claims abstract description 5
- 244000248021 Vitex negundo Species 0.000 claims abstract description 3
- 235000010363 Vitex negundo Nutrition 0.000 claims abstract description 3
- 241001167107 Zornia gibbosa Species 0.000 claims description 14
- 244000208946 Arenga pinnata Species 0.000 claims description 12
- 235000006549 Arenga pinnata Nutrition 0.000 claims description 12
- 239000011259 mixed solution Substances 0.000 claims description 12
- 238000001816 cooling Methods 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 7
- 230000001954 sterilising effect Effects 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 6
- 239000008213 purified water Substances 0.000 claims description 6
- 238000009835 boiling Methods 0.000 claims description 5
- 238000004659 sterilization and disinfection Methods 0.000 claims description 5
- 241000334161 Cercis chinensis Species 0.000 claims description 4
- 241000514748 Lanceolaria Species 0.000 claims description 4
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims description 4
- XLOMVQKBTHCTTD-UHFFFAOYSA-N Zinc monoxide Chemical compound [Zn]=O XLOMVQKBTHCTTD-UHFFFAOYSA-N 0.000 claims description 4
- 229910052709 silver Inorganic materials 0.000 claims description 4
- 239000004332 silver Substances 0.000 claims description 4
- 241000007500 Acacia elongata Species 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 3
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 claims description 2
- 235000001667 Vitex agnus castus Nutrition 0.000 claims description 2
- 244000063464 Vitex agnus-castus Species 0.000 claims description 2
- 241000339989 Wolffia Species 0.000 claims description 2
- MCMNRKCIXSYSNV-UHFFFAOYSA-N ZrO2 Inorganic materials O=[Zr]=O MCMNRKCIXSYSNV-UHFFFAOYSA-N 0.000 claims description 2
- RVTZCBVAJQQJTK-UHFFFAOYSA-N oxygen(2-);zirconium(4+) Chemical compound [O-2].[O-2].[Zr+4] RVTZCBVAJQQJTK-UHFFFAOYSA-N 0.000 claims description 2
- XOLBLPGZBRYERU-UHFFFAOYSA-N tin dioxide Chemical compound O=[Sn]=O XOLBLPGZBRYERU-UHFFFAOYSA-N 0.000 claims description 2
- 229910001887 tin oxide Inorganic materials 0.000 claims description 2
- OGIDPMRJRNCKJF-UHFFFAOYSA-N titanium oxide Inorganic materials [Ti]=O OGIDPMRJRNCKJF-UHFFFAOYSA-N 0.000 claims description 2
- 239000011787 zinc oxide Substances 0.000 claims description 2
- 241000234435 Lilium Species 0.000 claims 1
- 238000004113 cell culture Methods 0.000 abstract description 2
- 239000000306 component Substances 0.000 description 14
- 238000001514 detection method Methods 0.000 description 7
- 239000002609 medium Substances 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 241000897928 Echinops latifolius Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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Abstract
The invention discloses a culture medium capable of promoting rapid growth of osmophilic yeast in honey and a preparation method thereof, and particularly belongs to the field of cell culture, wherein the culture medium comprises the following components: 0.2-0.4 g of Cardamine dichotoma aqueous extract, 0.06-0.08 g of Echinacea purpurea aqueous extract, 0.1-0.2 g of Vitex negundo aqueous extract, 1-2 g of potato extract powder, 11-13 g of anhydrous glucose, 0.7-1.4 g of potassium hydrogen phosphate, 4-6 g of casein peptone, 0.001-0.003 g of niclosamide, 0.02-0.04 g of Bengal, 0.002-0.004 g of photocatalyst and 15-20 g of agar, and the components are dissolved in 1000mL of distilled water. The culture medium has the advantage of promoting the rapid growth of osmophilic yeast.
Description
Technical Field
The invention belongs to the field of cell culture, and particularly relates to a culture medium capable of promoting rapid growth of osmophilic yeast in honey and a preparation method thereof.
Background
There are two main types of yeasts present in honey: one is osmophilic yeast, and the other is high-sugar-resistant yeast, wherein the osmophilic yeast has great influence on the quality of the honey. Therefore, new national standards increase the limit requirements for osmophilic yeast count and are also a need to further improve the safety of honey products. At present, the detection of the count of the osmophilic yeast is mainly carried out according to a method specified in appendix A of GB14963-201l national standard honey for food safety. The culture condition of the osmophilic yeast in the national standard method is that the osmophilic yeast is cultured in a constant temperature incubator at 25 +/-1 ℃ for 7 days, and the culture period is as long as 4-5 days compared with the common colony culture period.
However, when the osmophilic yeast is cultured using a conventional medium such as DG18 medium, the culture period is long and is not suitable for the improvement of the detection efficiency. Meanwhile, the color of the osmophilic yeast is similar to that of the culture medium, so that the detection technicians are very inconvenient in technology.
Disclosure of Invention
The invention provides a culture medium capable of promoting rapid growth of osmophilic yeast in honey and a preparation method thereof.
In order to achieve the purpose, the invention provides the following technical scheme:
a culture medium capable of promoting the rapid growth of osmophilic yeast in honey comprises the following components in percentage by mass: 0.2-0.4 g of Cardamine dichotoma aqueous extract, 0.06-0.08 g of Echinacea purpurea aqueous extract, 0.1-0.2 g of Vitex negundo aqueous extract, 1-2 g of potato extract powder, 11-13 g of anhydrous glucose, 0.7-1.4 g of potassium hydrogen phosphate, 4-6 g of casein peptone, 0.001-0.003 g of niclosamide, 0.02-0.04 g of Bengal, 0.002-0.004 g of photocatalyst and 15-20 g of agar, and the components are dissolved in 1000mL of distilled water.
The herba Cardui Crispi water extract, Echinops Latifolius water extract, and herba Cercis chinensis water extract have antibacterial effect, and can inhibit the growth of other microorganisms, especially mold. Since the color of the mold is the same as that of osmophilic yeast in the growth process, the only difference is the morphology, but the technical difficulty of technical detection personnel is also increased. Therefore, the fungus can be effectively killed by adding the bacteriostatic substance into the culture medium. Meanwhile, the extract contains sugar, so that the requirement of the osmophilic yeast on sugar can be met, and sufficient nutrition is provided for the rapid growth of the osmophilic yeast.
Preferably, the culture medium component further comprises any one of an aqueous extract of gomuti palm, an aqueous extract of zornia gibbosa and an aqueous extract of wolffia indica, or a mixture thereof. The water extracts of Arenga pinnata, Zornia gibbosa and radix Brassicae Rapae have antibacterial effect and can inhibit the growth of bacteria in the culture medium.
Preferably, the photocatalyst includes any one of titanium oxide, zinc oxide, tin oxide, and zirconium dioxide, or a mixture thereof. The addition of a photocatalyst may help the medium to be more effectively sterilized during the manufacturing process (uv sterilization).
Preferably, the components of the culture medium comprise the following components in percentage by mass: 0.2-0.4 g of Cardamine dichotoma aqueous extract, 0.06-0.08 g of Echinacea purpurea aqueous extract, 0.1-0.2 g of silver chaste tree aqueous extract, 0.03-0.06 g of gomuti aqueous extract, 0.01-0.04 g of zornia gibbosa aqueous extract, 1-2 g of potato extract powder, 11-13 g of anhydrous glucose, 0.7-1.4 g of potassium hydrogen phosphate, 4-6 g of casein peptone, 0.001-0.003 g of niclosamide, 0.02-0.04 g of Bengal, 0.002-0.004 g of photocatalyst and 15-20 g of agar, and the extract is dissolved in 1000mL of distilled water.
Preferably, the components of the culture medium comprise the following components in percentage by mass: 0.3g of herba Cardui Crispi aqueous extract, 0.07g of radix cynanchi Wilfordii aqueous extract, 0.15g of herba Cercis chinensis aqueous extract, 0.04g of arenga pinnata aqueous extract, 0.03g of zornia gibbosa aqueous extract, 1.6g of potato extract powder, 12g of anhydrous glucose, 0.9g of potassium hydrogen phosphate, 5g of casein peptone, 0.002g of niclosamide, 0.03g of Bengal red, 0.003g of photocatalyst and 15-20 g of agar, and the components are dissolved in 1000mL of distilled water.
The invention also provides a preparation method of the culture medium capable of promoting the rapid growth of the osmophilic yeast in the honey, and the preparation method comprises the following preparation steps:
(1) adding purified water into the container, and heating to boil.
(2) And cooling the mixture to 40-60 ℃ by boiling water, sequentially adding the Feilian water extract, the Lanceolaria water extract, the silver wattle water extract, the gomuti palm water extract, the zornia gibbosa water extract, the potato extract powder, the anhydrous glucose, the potassium hydrogen phosphate, the casein peptone, the niclosamide, the Bengal, the photocatalyst and the agar into a container, and uniformly stirring to obtain a mixed solution.
(3) And adjusting the pH of the mixed solution to 5.6-5.8 by magnesium sulfate heptahydrate, subpackaging and sealing by using conical flasks, and placing in a high-pressure steam sterilization pot for sterilization for 45-60 min to obtain the sterilized mixed solution.
(4) And (4) putting the sterilized mixed solution into a super clean bench for cooling, and obtaining the culture medium after cooling.
Preferably, in step (2), the temperature of the boiling water after cooling is 50 ℃.
Preferably, in step (3), the pH is 5.7.
Preferably, in step (3), the autoclaving time is 50 min.
Compared with the prior art, the invention has the beneficial effects that:
(1) the culture medium provided by the invention can greatly shorten the culture time of the osmophilic yeast, and is more beneficial to the detection of technical detection personnel on a large batch of samples, thereby greatly improving the experimental detection efficiency.
(2) During the culture process, the contrast between the color of the culture medium and the color of the target bacteria is bright, and the counting is easier to observe.
(3) In the formula, the components capable of inhibiting the growth of the mould are added, so that the influence of the growth of the mould on the count of the osmophilic yeast can be prevented.
Description of the drawings:
FIG. 1 is a schematic diagram showing the culture of osmophilic yeast in a medium corresponding to example 2;
FIG. 2 is a schematic diagram showing the culture of osmophilic yeast in the medium corresponding to comparative example 1.
Detailed Description
The technical solution of the present invention will be described in further detail below with reference to specific embodiments.
Example 1
A culture medium capable of promoting the rapid growth of osmophilic yeast in honey comprises the following components in percentage by mass: 0.2g of herba Cardui Crispi aqueous extract, 0.06g of Echinacea purpurea aqueous extract, 0.1g of herba Nepetae chinensis aqueous extract, 0.03g of arenga pinnata aqueous extract, 0.01g of zornia gibbosa aqueous extract, 1g of potato extract powder, 11g of anhydrous glucose, 0.7g of potassium hydrogen phosphate, 4g of casein peptone, 0.001g of niclosamide, 0.02g of Bengal, 0.002g of photocatalyst and 15-20 g of agar, and the components are dissolved in 1000mL of distilled water.
The preparation method of the culture medium comprises the following steps:
(1) adding purified water into a container, and heating to boil;
(2) and cooling the mixture to 50 ℃ by boiling water, sequentially adding the Feilian water extract, the Lanceolaria water extract, the silver wattle water extract, the gomuti palm water extract, the zornia gibbosa water extract, the potato extract powder, the anhydrous glucose, the potassium hydrogen phosphate, the casein peptone, the niclosamide, the Bengal, the photocatalyst and the agar into a container, and uniformly stirring to obtain a mixed solution.
(3) Adjusting pH of the mixed solution to 5.7 with magnesium sulfate heptahydrate, subpackaging and sealing with conical flask, and sterilizing in high pressure steam sterilizing pot for 50min to obtain sterilized mixed solution;
(4) and (4) putting the sterilized mixed solution into a super clean bench for cooling, and obtaining the culture medium after cooling.
Example 2
A culture medium capable of promoting the rapid growth of osmophilic yeast in honey comprises the following components in percentage by mass: 0.3g of herba Cardui Crispi aqueous extract, 0.07g of radix cynanchi Wilfordii aqueous extract, 0.15g of herba Cercis chinensis aqueous extract, 0.04g of arenga pinnata aqueous extract, 0.03g of zornia gibbosa aqueous extract, 1.6g of potato extract powder, 12g of anhydrous glucose, 0.9g of potassium hydrogen phosphate, 5g of casein peptone, 0.002g of niclosamide, 0.03g of Bengal red, 0.003g of photocatalyst and 15-20 g of agar, and the volume is fixed to 1L by purified water.
The preparation method of the culture medium is the same as that of example 1.
Example 3
A culture medium capable of promoting the rapid growth of osmophilic yeast in honey comprises the following components in percentage by mass: 0.36g of Cardui Crispus aqueous extract, 0.072g of Echinacea purpurea aqueous extract, 0.16g of silver negundo aqueous extract, 0.53g of arenga pinnata aqueous extract, 0.34g of zornia gibbosa aqueous extract, 1.6g of potato extract powder, 12.3g of anhydrous glucose, 0.9g of potassium hydrogen phosphate, 5.3g of casein peptone, 0.025g of niclosamide, 0.034g of Bengal, 0.003g of photocatalyst and 15-20 g of agar, and the volume is fixed to 1L by purified water.
The preparation method of the culture medium is the same as that of example 1.
Example 4
A culture medium capable of promoting the rapid growth of osmophilic yeast in honey comprises the following components in percentage by mass: 0.4g of herba Cardui Crispi aqueous extract, 0.08g of Lanceolaria dichotoma aqueous extract, 0.2g of silver negundo aqueous extract, 0.06g of arenga pinnata aqueous extract, 0.04g of zornia gibbosa aqueous extract, 2g of potato extract powder, 13g of anhydrous glucose, 1.4g of potassium hydrogen phosphate, 6g of casein peptone, 0.003g of niclosamide, 0.04g of Bengal red, 0.004g of photocatalyst and 15-20 g of agar, and the volume is fixed to 1L by purified water.
The preparation method of the culture medium is the same as that of example 1.
Comparative example 1
DG18 medium was prepared in the same manner as in example 1.
Test example 1
The present invention is now exemplified by the culture of osmophilic yeasts.
The media provided in examples 1-4 and comparative example 1 were numbered 1, 2, 3, 4, 5, respectively. And (3) respectively inoculating the osmophilic yeast separated from the honey sample to 1-5 groups, and setting 10 parallel experimental groups in each group. After inoculation, 1-5 groups and A-G groups are cultured for 7 days under the condition of low light at the temperature of 28 +/-1 ℃, and the statistics of the growth conditions of the osmophilic yeast in each culture medium are observed and compared as follows:
meanwhile, the culture media provided in example 2 and comparative example 1 were used for culturing osmophilic yeasts respectively, the sources of the osmophilic yeasts are the same, and after culturing for 7 days under the condition of low light at 28 +/-1 ℃, the growth conditions of the osmophilic yeasts are respectively shown in fig. 1 and fig. 2 (in the actual culture process, the background color of fig. 1 is red, and the fig. 1 is subjected to decolorizing treatment because the attached drawings submit standards).
TABLE 1 statistics of growth of osmophilic yeasts
Claims (9)
1. A culture medium capable of promoting the rapid growth of osmophilic yeast in honey is characterized by comprising the following components in percentage by mass: 0.2-0.4 g of Cardamine dichotoma aqueous extract, 0.06-0.08 g of Echinacea purpurea aqueous extract, 0.1-0.2 g of Vitex negundo aqueous extract, 1-2 g of potato extract powder, 11-13 g of anhydrous glucose, 0.7-1.4 g of potassium hydrogen phosphate, 4-6 g of casein peptone, 0.001-0.003 g of niclosamide, 0.02-0.04 g of Bengal, 0.002-0.004 g of photocatalyst and 15-20 g of agar, and the components are dissolved in 1000mL of distilled water.
2. The culture medium for promoting the rapid growth of osmophilic yeast in honey as claimed in claim 1, wherein the components further comprise any one of water extract of gomuti palm, water extract of zornia gibbosa and water extract of wolffia, or mixture thereof.
3. A culture medium for promoting the rapid growth of osmophilic yeast in honey as claimed in claim 1, wherein the photocatalyst comprises any one of titanium oxide, zinc oxide, tin oxide and zirconium dioxide, or a mixture thereof.
4. A culture medium capable of promoting rapid growth of osmophilic yeast in honey as claimed in any one of claims 1 to 3, which comprises the following components by mass: 0.2-0.4 g of Cardamine dichotoma aqueous extract, 0.06-0.08 g of Echinacea purpurea aqueous extract, 0.1-0.2 g of silver chaste tree aqueous extract, 0.03-0.06 g of gomuti aqueous extract, 0.01-0.04 g of zornia gibbosa aqueous extract, 1-2 g of potato extract powder, 11-13 g of anhydrous glucose, 0.7-1.4 g of potassium hydrogen phosphate, 4-6 g of casein peptone, 0.001-0.003 g of niclosamide, 0.02-0.04 g of Bengal, 0.002-0.004 g of photocatalyst and 15-20 g of agar, and the extract is dissolved in 1000mL of distilled water.
5. A culture medium capable of promoting rapid growth of osmophilic yeast in honey as claimed in claim 4, which comprises the following components by mass: 0.3g of herba Cardui Crispi aqueous extract, 0.07g of radix cynanchi Wilfordii aqueous extract, 0.15g of herba Cercis chinensis aqueous extract, 0.04g of arenga pinnata aqueous extract, 0.03g of zornia gibbosa aqueous extract, 1.6g of potato extract powder, 12g of anhydrous glucose, 0.9g of potassium hydrogen phosphate, 5g of casein peptone, 0.002g of niclosamide, 0.03g of Bengal red, 0.003g of photocatalyst and 15-20 g of agar, and the components are dissolved in 1000mL of distilled water.
6. A preparation method of a culture medium capable of promoting the rapid growth of osmophilic yeast in honey is characterized by comprising the following preparation steps:
(1) adding purified water into a container, and heating to boil;
(2) cooling with boiling water to 40-60 ℃, sequentially adding a feillow lily aqueous extract, a Lanceolaria water extract, a silver wattle aqueous extract, a gomuti palm aqueous extract, a zornia gibbosa aqueous extract, a potato extract powder, anhydrous glucose, potassium hydrogen phosphate, casein peptone, niclosamide, Bengal, a photocatalyst and agar into a container, and uniformly stirring to obtain a mixed solution;
(3) adjusting the pH of the mixed solution to 5.6-5.8 by magnesium sulfate heptahydrate, subpackaging and sealing by using conical flasks, and placing in a high-pressure steam sterilization pot for sterilization for 45-60 min to obtain a sterilized mixed solution;
(4) and (4) putting the sterilized mixed solution into a super clean bench for cooling, and obtaining the culture medium after cooling.
7. A method for preparing a culture medium for promoting the rapid growth of osmophilic yeast in honey as claimed in claim 6, wherein in step (2), the temperature of the cooled boiling water is 50 ℃.
8. A method for preparing a culture medium for promoting the rapid growth of osmophilic yeast in honey as claimed in claim 6, wherein in step (3), the pH is 5.7.
9. A method for preparing a culture medium for promoting the rapid growth of osmophilic yeast in honey as claimed in claim 6, wherein in step (3), the autoclaving time is 50 min.
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