Culture medium for culturing eurotium cristatum and application

Abstract

The invention discloses a culture medium for culturing eurotium cristatum and application. Based on each liter of water, the culture medium is prepared from the following components: 2 to 20g of corn steep liquor dry powder, 2 to 10g of beta-cyclodextrin, 5 to 20g of glucose, 5 to 30g of soluble starch, 0.1 to 1.5 g of magnesium sulfate and 0.1 to 1.5 g of monopotassium phosphate. A method for producing Fuzhuan tea through fermentation comprises the following steps: A, strain culture: inoculating eurotium cristatum SHBCC D15081 strain in the culture medium, and performing shake-flask culture to obtain a fermentation culture solution; and B, fermenting the tea leaves: uniformly inoculating the fermentation culture solution obtained in the step A onto the tea leaves, and fermenting to produce the Fuzhuan tea. According to the culture medium, the growth speed of eurotium cristatum can be increased, and the adaptability of eurotium cristatum in the Fuzhuan tea fermentation production process is improved.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation engineering, and in particular to a culture medium for culturing Eurotium cristatum and an application thereof. Background Art

[0002] Aspergillus cristatus is an edible and medicinal fungus that is extremely important in the fermentation process of dark tea, especially Fuzhuan tea. It is generally believed that the quality and quantity of Aspergillus cristatus growth is one of the important indicators for judging the quality of dark tea such as Fuzhuan tea.

[0003] In the traditional production of Fuzhuan tea, the natural fermentation of the Eurycoma coronata in the environment is carried out as a strain. It usually takes a long period (usually 10-15 days) to reach the ideal growth density, which will limit the production capacity and production efficiency in large-scale industrial production. In addition, natural fermentation has the following disadvantages: as the external environment changes, when the content of other microorganisms around is high, the Eurycoma coronata cannot become the dominant organism, resulting in failure of flowering and mildew of the product; microorganisms involved in fermentation brought into the natural environment can produce fungal toxins, posing a food safety hazard; the composition of fermented microorganisms is complex, resulting in uncontrollable product quality, unstable ingredients, and the efficacy and sensory quality cannot be guaranteed; accordingly, the production cycle is not fixed, resulting in waste of production capacity.

[0004] To solve the above problems, existing research has begun to turn to the isolation and purification of the Eurotium cristatum strains, and introduce specific purified strains into the fermentation process of tea to improve product quality. However, the current scientific research on the existing Eurotium cristatum fermentation process is relatively small compared with other widely used fermentation fungi. If the applied strain grows slowly and has poor environmental adaptability, not only will the early strain cultivation cause a large amount of resource waste, but the industrial production cycle of tea fermentation will be extended, resulting in an increase in production costs.

[0005] Therefore, the development of culture medium suitable for the rapid cultivation of E. coronaria and supporting standardized fermentation processes is of great significance to improving production efficiency and reducing product production costs. Summary of the invention

[0006] In order to solve the above technical problems, the present invention first proposes a culture medium for culturing Eurotium cristatum, which can accelerate the growth rate of Eurotium cristatum and improve its adaptability in the fermentation process of producing Fuzhuan tea.

[0007] The invention also provides a method for producing Fuzhuan tea by fermentation, which can significantly shorten the fermentation period of tea leaves while reducing the inoculation amount of Eurotium cristatum species, thereby being beneficial to improving economic benefits.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows: Based on the first aspect of the present invention, a culture medium for culturing Eurotium cristatum is first provided, which comprises the following components based on per liter of water: Corn syrup powder 2-20g, β-cyclodextrin 2-10g, glucose 5-20g, soluble starch 5-30g, magnesium sulfate 0.1-1.5g, potassium dihydrogen phosphate 0.1-1.5g.

[0009] In some preferred examples, the pH of the culture medium is 4.0-7.0.

[0010] In some preferred examples, the pH of the culture medium is 5.0-6.5.

[0011] In some preferred examples, the culture medium is a solid culture medium or a liquid culture medium; when the culture medium is adjusted to a solid culture medium, the culture medium composition further includes 10-40 g of agar.

[0012] The inventors have found in their ongoing research that culturing the Aspergillus niger with the aforementioned culture medium can accelerate the growth of the fungus, especially when the obtained fermentation culture solution is mixed with tea leaves for fermentation, the fermentation process of the tea leaves can be significantly promoted, and Fuzhuan tea can be efficiently fermented and produced at a lower inoculation rate.

[0013] It is worth noting that, among the aforementioned culture medium components of the present invention, corn slurry powder can provide a rich and high-quality nitrogen source for Aspergillus niger, and it also contains nutrients such as various vitamins (such as B vitamins, etc.) and growth factors. It is speculated that it has a special activation and promotion effect on certain key metabolic pathways of Aspergillus niger, thereby helping to improve its growth ability in the complex environment of tea. However, based on per liter of water, the amount of corn slurry powder should not be higher than 20g, otherwise it will show a trend of inhibiting the growth of Aspergillus niger. In addition, β-cyclodextrin has a unique cyclic structure and has the ability to selectively form inclusion complexes with small molecules. This inclusion complex is speculated to change the release rate and existence form of nutrients in the culture medium, making it more beneficial to the absorption and utilization of nutrients by Aspergillus niger during tea fermentation. At the same time, it has an interfering effect on the nutrient acquisition of other bacteria and inhibits the growth of bacteria. However, studies have found that the amount of β-cyclodextrin is not suitable to be higher than 10g. In addition, glucose, as the main carbon source, provides energy for the growth of Aspergillus niger; soluble starch, as a supplementary carbon source, can promote the growth and reproduction of Aspergillus niger. The magnesium ions provided by magnesium sulfate are activators of various enzymes of Aspergillus niger (such as some enzymes involved in RNA polymerization). During the fermentation of tea leaves, the presence of magnesium ions can ensure the efficient operation of the enzyme system of Aspergillus niger, so that Aspergillus niger can better decompose the components in tea leaves to obtain nutrients, thereby accelerating the growth rate. Potassium dihydrogen phosphate mainly plays the role of regulating the pH value in the culture medium, so that the pH value of the culture medium is maintained in a relatively stable range suitable for the growth of Aspergillus niger. The synergistic combination of the above formulas can produce a specific selective enrichment effect on Aspergillus niger, shorten the strain culture cycle, and improve the growth promotion effect on Aspergillus niger during the fermentation of tea leaves, thereby shortening the flowering cycle and improving the quality of tea leaves.

[0014] Based on the second aspect of the present invention, a method for producing Fuzhuan tea by fermentation is also provided, comprising the following steps: A. Strain culture: inoculate the strain of Eurotium cristatum SHBCC D15081 in the culture medium described above, and culture in a shake flask to obtain a fermentation culture solution; B. Tea fermentation: The fermentation culture solution obtained in step A is evenly inoculated onto tea leaves to ferment and produce Fuzhuan tea.

[0015] As a preferred process condition, during the shake flask culture in step A, the colony count in the obtained fermentation broth is controlled to be 70-90 CFU / ml.

[0016] As a preferred process condition, the conditions for the shake flask culture in step A are: temperature 25-28°C, shaker speed 180-210 rpm / min, and culture 80-120h.

[0017] As a preferred process condition, the bacterial culture in step A includes at least one liquid shake flask culture. The bacterial culture can be directly inoculated into a liquid culture medium for shake flask culture, or a conventional solid plate culture is first used, and then inoculated into a liquid culture medium for shake flask culture. No further restrictions are made on this. When solid plate culture is required, the solid culture medium used is preferably a culture medium described above with 10-40 g of agar added to it, based on per liter of water.

[0018] As a preferred process condition, in step B, the fermentation broth obtained in step A is mixed with tea leaves for fermentation at an inoculation rate of 500-800 CFU / g tea leaves.

[0019] As a preferred process condition, the specific process of tea fermentation includes: S1, rehydration process: soak the tea leaves in pure water for 1-2 hours to allow them to fully absorb water; the ambient temperature of this process is preferably controlled at 25-30°C; S2, pre-fermentation process: the rehydrated tea leaves are placed in a fermentation room, the temperature is controlled at 25-30°C, the humidity is adjusted to 70-80%, and the pre-fermentation is carried out for 6-8 hours; S3, sterilization process: use high temperature steam to sterilize the tea leaves, the temperature is set to 100-120℃, and the treatment time is 15-20min; S4, steaming process: steam the tea leaves in a steamer for 15-20 minutes at a steaming temperature of 100-110°C; S5, inoculation process: inoculate the fermentation culture liquid of Eurotium cristatum onto the tea leaves and mix them evenly; cool the tea leaves to below 40° C. before inoculation; S6, brick pressing process: pressing the mixed tea leaves into tea bricks through a mold; S7, film wrapping process: Use food-grade plastic wrap to wrap the tea brick tightly; S8, first blooming process: put the wrapped tea bricks into the blooming room, keep the temperature at 28-30℃, adjust the humidity to 80-85%, and ferment for 2-4 days; S9, Secondary Flowering Process: The tea bricks that have undergone the first flowering are transferred to another flowering room, the temperature is lowered to 26-28℃, the humidity is maintained at 75-80%, and fermented for another 2-3 days; through two flowering processes with different control conditions, not only the rapid growth and reproduction of Eurotium cristatum is promoted, so that golden "golden flowers" are formed on the surface of the tea leaves, but also the astringent catechins in the dark tea are decomposed and converted into substances such as theabrownin with a mellower flavor, thereby improving the quality of the tea leaves; S10, drying process: drying the fermented tea leaves at 50-60°C until the moisture content of the tea leaves is 5-8%.

[0020] As a preferred process condition, the tea leaves are dark tea.

[0021] By using the fermentation method of the present invention, 1100×10 3 The number of golden flowers is CFU / g tea and above, the total number of other molds and bacteria is less than 120 CFU / g tea, the catechin content is lower than 5.2 mg / g, and the total amount of theabrownin, theaflavins and thearubigins is higher than 12.5 mg / g. DETAILED DESCRIPTION

[0022] The present invention is further described below by means of specific examples. The examples described in the present invention are only used to illustrate the present invention and do not limit the scope of the present invention.

[0023] Unless otherwise specified, the raw materials and reagents in the following examples of the present invention can be purchased through commercial channels.

[0024] The analytical testing methods used in the following embodiments of the present invention are: (1) Number of strains Plate count method: 1 ml of fermentation culture medium is diluted with sterile saline to an appropriate volume multiple (e.g. 10 -1 , 10 -2 , 10 -3 etc.), to ensure that the number of colonies on the subsequent plates is moderate and easy to count; take a quantitative dilution and evenly spread it on the solid culture medium provided by the present invention, place it at a constant temperature of 28°C, and count it after the golden colonies are formed. At least three parallel experiments were performed for each group of samples, and the final results were expressed in CFU / ml. The experimental strain was SHBCC D15081 (from Shanghai Collection of Microorganisms).

[0025] (2) Detection of the number of golden flowers Referring to the method in standard GB / T 4789.15-2016 “National Food Safety Standard - Microbiological Examination of Foods - Mould and Yeast Counts”, the number of golden colonies in the centre of the Fuzhuan brick tea was tested (unit: CFU / g tea).

[0026] (3) Other mold and bacteria count tests Referring to the method in the standard GB 4789.2-2022 "National Food Safety Standard Food Microbiological Examination - Determination of Total Colony Count", the total number of colonies in the center of the Fu brick tea (unit CFU / g tea) is tested, and the number of golden colonies in the same unit is subtracted to obtain the total number of other molds and bacteria.

[0027] (4) Catechin content test The Fuzhuan tea samples were ground into powder and dissolved with ethanol as solvent. The catechin content was analyzed by high performance liquid chromatography.

[0028] (5) Tea pigment content test The Fuzhuan tea samples were ground into powder and dissolved in boiling water to obtain tea soup, and the total contents of theabrownin, thearubigins and theaflavins were analyzed by spectrophotometry.

[0029] Different culture media were prepared according to the formulas in the following Examples 1-5 and Comparative Examples 1-3: [Example 1] To 1 L of water, 10 g of corn steep liquor powder, 5 g of β-cyclodextrin, 20 g of glucose, 20 g of soluble starch, 0.5 g of magnesium sulfate, and 0.5 g of potassium dihydrogen phosphate were added, and the pH was adjusted to 5.5 to obtain culture medium A.

[0030] [Example 2] 5 g of corn steep liquor powder, 10 g of β-cyclodextrin, 5 g of glucose, 30 g of soluble starch, 0.1 g of magnesium sulfate and 1.5 g of potassium dihydrogen phosphate were added to 1 L of water, and the pH was adjusted to 6.5 to obtain culture medium B.

[0031] [Example 3] 2 g corn steep liquor powder, 10 g β-cyclodextrin, 20 g glucose, 5 g soluble starch, 1.5 g magnesium sulfate and 0.1 g potassium dihydrogen phosphate were added to 1 L water, and the pH was adjusted to 5.0 to obtain culture medium C.

[0032] [Example 4] To 1 L of water, 15 g of corn steep liquor powder, 4 g of β-cyclodextrin, 10 g of glucose, 15 g of soluble starch, 1.0 g of magnesium sulfate, and 0.5 g of potassium dihydrogen phosphate were added, and the pH was adjusted to 6.0 to obtain a culture medium D.

[0033] [Example 5] Per 1 L of water, 20 g of corn steep liquor powder, 2 g of β-cyclodextrin, 15 g of glucose, 25 g of soluble starch, 0.5 g of magnesium sulfate and 1.0 g of potassium dihydrogen phosphate were added, and the pH was adjusted to 5.5 to obtain medium E.

[0034] [Comparative Example 1] The medium F was prepared according to the formula substantially the same as that in Example 1, except that no corn steep liquor powder was added.

[0035] [Comparative Example 2] The medium G was prepared according to a formula substantially the same as that in Example 1, except that β-cyclodextrin was not added.

[0036] [Comparative Example 3] Prepare liquid medium H according to the following recipe: For every 1L of water, add 20g of glucose, 20g of maltose, and 1g of yeast extract, and adjust the pH to 5.5.

[0037] The tea leaves were fermented according to the methods in the following application examples 1-5 and comparative application examples 1-3:

Application Example 1

[0038]

Application Example 2

[0039]

Application Example 3

[0040]

Application example 4

[0041]

Application Example 5

[0042]

Comparative Application Example 1

[0043]

Comparative Application Example 2

[0044]

Comparative Application Example 3

[0045] The tea leaves obtained by fermentation in each application example were analyzed for the number of golden flowers, the total number of other molds and bacteria, the content of catechins, and the total amount of theabrownin, theaflavins and thearubigins. The results are shown in Table 1.

[0046] Table 1. Analysis results Golden flower quantity CFU / g Total number of other molds and bacteria CFU / g Catechin content mg / g The total amount of theabrownin, theaflavins and thearubigins mg / g Application Example 1 <![CDATA[1200×10 3 ]]> <100 5.2 12.5 Application Example 2 <![CDATA[1180×10 3 ]]> <120 4.8 13.0 Application Example 3 <![CDATA[1150×10 3 ]]> <110 5.0 12.6 Application Example 4 <![CDATA[1250×10 3 ]]> <80 4.9 13.2 Application Example 5 <![CDATA[1100×10 3 ]]> <90 5.1 12.7 Comparative application example 1 <![CDATA[400×10 3 ]]> 350 8.2 9.0 Comparative Application Example 2 <![CDATA[500×10 3 ]]> 320 7.9 8.7 Comparative Application Example 3 <![CDATA[180×10 3 ]]> 200 8.7 7.1

[0047] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A culture medium for culturing Eurotium cristatum, characterized in that: Based on each liter of water, it includes the following components: Corn syrup powder 2-20g, β-cyclodextrin 2-10g, glucose 5-20g, soluble starch 5-30g, magnesium sulfate 0.1-1.5g, potassium dihydrogen phosphate 0.1-1.5g.

2. The culture medium for culturing Eurotium cristatum according to claim 1, characterized in that The pH of the culture medium is 4.0-7.

0.

3. The culture medium for culturing Eurotium cristatum according to claim 1, characterized in that The pH of the culture medium is 5.0-6.

5.

4. The culture medium for culturing Eurotium cristatum according to any one of claims 1 to 3, characterized in that The culture medium is a solid culture medium or a liquid culture medium; when the culture medium is adjusted to be a solid culture medium, the culture medium composition also includes 10-40g of agar.

5. A method for producing Fuzhuan tea by fermentation, characterized in that: The following steps are involved: A. Bacterial culture: inoculating the strain of Eurotium cristatum SHBCC D15081 in the culture medium according to any one of claims 1 to 3, and culturing in a shake flask to obtain a fermentation culture solution; B. Tea fermentation: The fermentation culture solution obtained in step A is evenly inoculated onto tea leaves to ferment and produce Fuzhuan tea.

6. The method for producing Fuzhuan tea by fermentation according to claim 5, characterized in that: During the shake flask culture process in step A, the colony count in the obtained fermentation culture solution is controlled to be 70-90 CFU / ml.

7. The method for producing Fuzhuan tea by fermentation according to claim 6, characterized in that: The conditions for the shake flask culture in step A are: temperature 25-28° C., shaker speed 180-210 rpm / min, and culture 80-120 h.

8. The method for producing Fuzhuan tea by fermentation according to any one of claims 5 to 7, characterized in that: In step B, the fermentation culture solution obtained in step A is mixed with tea leaves at an inoculation amount of 500-800 CFU / g tea leaves for fermentation.

9. The method for producing Fuzhuan tea by fermentation according to claim 8, characterized in that: The specific process of tea fermentation includes: S1, rehydration process: soak the tea leaves in pure water for 1-2 hours to allow them to fully absorb water; S2, pre-fermentation process: the rehydrated tea leaves are placed in a fermentation room, the temperature is controlled at 25-30°C, the humidity is adjusted to 70-80%, and the pre-fermentation is carried out for 6-8 hours; S3, sterilization process: use high temperature steam to sterilize the tea leaves, the temperature is set to 100-120℃, and the treatment time is 15-20min; S4, steaming process: steam the tea leaves in a steamer for 15-20 minutes at a steaming temperature of 100-110°C; S5, inoculation step: inoculating the fermented culture solution of Eurotium cristatum onto the tea leaves and mixing them evenly; S6, brick pressing process: pressing the mixed tea leaves into tea bricks through a mold; S7, film wrapping process: Use food-grade plastic wrap to wrap the tea brick tightly; S8, first blooming process: put the wrapped tea bricks into the blooming room, keep the temperature at 28-30℃, adjust the humidity to 80-85%, and ferment for 2-4 days; S9, secondary blooming process: transfer the tea bricks that have undergone the first blooming to another blooming room, lower the temperature to 26-28℃, maintain the humidity at 75-80%, and ferment for another 2-3 days; S10, drying process: drying the fermented tea leaves at 50-60°C until the moisture content of the tea leaves is 5-8%.

10. The method for producing Fuzhuan tea by fermentation according to any one of claims 5 to 7, characterized in that: The tea leaves are dark tea.