Organic selenium culture medium and application thereof in preparation of selenium-enriched yeast

Abstract

The invention discloses an organic selenium culture medium and application thereof in preparation of selenium-enriched yeast. The initial culture medium is prepared from 6mL / L of hydrolyzed sugar solution, 0.5 mL / L of stronger ammonia water, 0.2 g / L of ammonium dihydrogen phosphate, 2g / L of magnesium sulfate, 0.25 g / L of zinc sulfate, 15mg / L of vitamin B1, 3mg / L of vitamin B6, 5mg / L of calcium pantothenate, 1.5 mg / L of biotin and 18mg / L of selenoglucose, and the pH is adjusted to 5.5 by sulfuric acid. A supplemented culture medium is an organic selenium solution with the selenium content of 5% (W / W), culture is conducted for 27 h at the temperature of 30 DEG C, and dissolved oxygen is controlled to be not lower than 20% in the culture process. Wherein selenoglucose is adopted as a selenium source, compared with traditional inorganic selenium such as sodium selenite and the like, the yeast is easier to absorb and convert, the selenium content in the obtained selenium-enriched yeast exceeds 4000 ppm, the conversion rate of selenium in a culture medium is larger than 99%, and the environment-friendly treatment pressure of the selenium element in the fermentation wastewater is effectively reduced while the selenium-enriched yeast with the high selenium content is prepared.

Description

Technical Field

[0001] The invention belongs to the technical field of fermentation industry, and in particular relates to an organic selenium culture medium and application thereof in preparing selenium-enriched yeast. Background Art

[0002] Selenium is one of the essential trace elements for the human body. It has the functions of protecting cell membranes, preventing atherosclerosis, reducing the occurrence of thrombosis and myocardial infarction, etc. Selenium is also involved in physiological activities such as regulating the body's immunity and antagonizing toxic elements. Selenium deficiency can lead to the occurrence of many diseases such as Keshan disease and Kashin-Beck disease. In 1973, the World Health Organization classified selenium as an essential trace element for humans and animals, and recommended that adults supplement 200 μg of selenium per day to effectively prevent diseases caused by selenium deficiency. In the same year, the US FDA also approved sodium selenite and sodium selenate as feed additives for use in animal husbandry.

[0003] Selenium in nature mainly exists in the form of inorganic selenium, which is mainly absorbed by simple diffusion and has low bioavailability. It will promote the occurrence of oxidation reactions under high food intake and has certain biological toxicity. It is also easy to pollute the environment. Organic selenium has the advantages of high food safety and strong bioavailability, so it has become the first choice for selenium supplementation. In 2009, my country's agricultural authorities approved selenium-enriched yeast (yeast selenium) as a feed additive for aquaculture. Selenium-enriched yeast can convert inorganic selenium into organic selenium, and mainly exists in the form of selenomethionine and selenocysteine. It is actively absorbed through the amino acid transport system with a high absorption rate. After meeting the physiological needs of animals for selenium, it can also be stored in the animal body to give full play to the physiological function of selenium. Selenium-enriched yeast is currently mainly a microbial fermentation product obtained by adding inorganic selenium such as sodium selenite during the yeast culture process to convert it into organic selenium and enrich it in selenium-enriched yeast. The evaluation of the production level of selenium-enriched yeast mainly depends on its unit organic selenium content and biomass. The higher the unit organic selenium content and biomass, the higher the level. The type of selenium and the way of addition are important factors affecting the production of selenium-enriched yeast. However, inorganic selenium has an inhibitory effect on yeast growth. If the amount of selenium added during the culture process is insufficient, the organic selenium content per yeast unit is low. If too much inorganic selenium is added, the biomass will be reduced. At the same time, the post-treatment of the inorganic selenium remaining in the culture medium is also more troublesome, making the environmental protection cost very high.

[0004] Therefore, developing new selenium-enriched yeast culture media and preparing high-quality selenium-enriched yeast rich in organic selenium have become important research directions for selenium-enriched yeast. Summary of the invention

[0005] 1. Technical issues to be resolved In view of the deficiencies in the prior art, the present invention provides an organic selenium culture medium and application thereof in the preparation of selenium-enriched yeast, thereby overcoming the deficiencies in the prior art and providing an organic selenium culture medium.

[0006] (II) Technical solution To achieve the above object, the present invention provides the following technical solutions: an organic selenium culture medium and its use in preparing selenium-enriched yeast, an organic selenium culture medium, comprising the following components by weight: Initial culture medium: hydrolyzed sugar solution 6 mL / L, concentrated ammonia 0.5 mL / L, diammonium phosphate 0.2 g / L, magnesium sulfate 2 g / L, zinc sulfate 0.25 g / L, vitamin B 1 15mg / L, Vitamin B 6 3mg / L, calcium pantothenate 5mg / L, biotin 1.5mg / L, organic selenium 18mg / L, sulfuric acid to adjust pH to 5.5; Feed medium: Selenium content is 5% (W / W) organic selenium solution.

[0007] Preferably, the organic selenium in the initial culture medium is selenoglucose.

[0008] Preferably, the hydrolyzed sugar solution in the initial culture medium is prepared by grinding rice and then hydrolyzing it with amylase and saccharifying enzyme, and its sugar concentration after dilution is 30%.

[0009] Preferably, the organic selenium culture medium is used in the preparation of selenium-enriched yeast.

[0010] Preferably, the preparation method of the selenium-enriched yeast cell is: The culture medium containing selenoglucose was used as the initial culture medium for selenium-enriched yeast fermentation. The yeast strain was inoculated at a rate of 10% (V / V) and cultured at 30°C for 4 hours. Then, a selenoglucose solution with a selenium content of 5% (W / W) and a hydrolyzed sugar feed medium were added. The addition was completed after 12 hours. Ammonia water was used to control the pH value to 5.5 during the culture process. After 27 hours of fermentation, the tank was released and the selenium-enriched yeast body was isolated. The selenium content in the selenium-enriched yeast was detected to be more than 4000ppm, and the conversion rate of selenium in the culture medium was greater than 99%.

[0011] 3. Beneficial technical effects The present invention provides an organic selenium culture medium, in which selenoglucose is used as a selenium source. Compared with traditional inorganic selenium such as sodium selenite, yeast is easier to absorb and convert. The selenium content in the obtained selenium-enriched yeast exceeds 4000ppm, and the conversion rate of selenium in the culture medium is greater than 99%. While preparing selenium-enriched yeast with a high selenium content, the environmental protection treatment pressure of selenium in fermentation wastewater is effectively reduced. DETAILED DESCRIPTION

[0012] Example 1

[0013] Preparation of Broken Rice Hydrolyzed Sugar Solution Weigh 6.5 kg of broken rice, crush it into powder, add 20 kg of hot water while stirring, stir evenly, adjust the pH to 5.6, heat to 95℃, add 1g of high-temperature amylase (calculated as 0.15kg enzyme per ton of broken rice), and keep it at 95℃ for liquefaction for 1.5h. Cool down to about 60℃, adjust the pH to 4.1, add about 4g of saccharifying enzyme, keep it at 60℃ for 32h. Determine the sugar content in the hydrolyzate, and dilute the broken rice hydrolyzate to a solution with a sugar content of 30% according to the sugar content for fermentation.

[0014] Example 2

[0015] Preparation of selenium-enriched yeast (seleno-glucose fed culture) (1) Activation of strains: Take a glycerol tube of brewer's yeast (commercially available) stored at -80°C and activate it on a YPD plate. After constant temperature cultivation at 30°C for 48 h, store it in a refrigerator at 4°C for later use. (2) Seed preparation: Use an inoculation loop to transfer a single colony to seed culture medium (10% sucrose, 2% yeast powder, 0.1% magnesium sulfate, 0.1% potassium dihydrogen phosphate, pH 4.8), culture at 250 rpm, 30°C, and shake for 12 h or more (based on the alcohol concentration reaching 2% and the wet weight reaching 25 g / L), and no abnormalities are found under microscopic examination; (3) Cultivation of selenium-enriched yeast. Inoculate the seeds with 10% (V / V) inoculation volume into a medium containing initial culture medium (6 mL / L hydrolyzed sugar solution, 0.5 mL / L concentrated ammonia water, 0.2 g / L diammonium phosphate, 2 g / L magnesium sulfate, 0.25 g / L zinc sulfate, vitamin B 1 15mg / L, Vitamin B 6 3mg / L, calcium pantothenate 5mg / L, biotin 1.5mg / L, selenoglucose 18mg / L, sulfuric acid to adjust pH to 5.5) in a fermenter, cultured at 30℃, 30% sugar content hydrolyzed sugar solution and 15% ammonia solution were added as carbon and nitrogen sources during the culture process, selenoglucose solution was added after 4 hours of culture, and the addition was completed after 10 hours. The dissolved oxygen was controlled to be not less than 20% during the culture process, and the tank was separated after 27 hours of fermentation. The yeast mud was taken, washed twice with distilled water, and dried to obtain selenium-enriched yeast. The dry weight of selenium-enriched yeast, selenium content and selenium content in the fermentation supernatant were tested respectively, and the conversion rate of selenium was calculated.

[0016] Example 3

[0017] Preparation of selenium-enriched yeast (selenoglucose is added once): (1) Activation of strains: Take a glycerol tube of brewer's yeast (commercially available) stored at -80°C and activate it on a YPD plate. After constant temperature cultivation at 30°C for 48 h, store it in a refrigerator at 4°C for later use. (2) Seed preparation. Use an inoculation loop to transfer a single colony to a seed culture medium (10% sucrose, 2% yeast powder, 0.1% magnesium sulfate, 0.1% potassium dihydrogen phosphate, pH 4.8), culture at 250 rpm, 30°C, and shake for 12 hours or more (based on the alcohol concentration reaching 2% and the wet weight reaching 25 g / L), and no abnormalities are found under microscopic examination; (3) Cultivation of selenium-enriched yeast. Inoculate the seeds with 10% (V / V) inoculation volume into a medium containing initial culture medium (6 mL / L hydrolyzed sugar solution, 0.5 mL / L concentrated ammonia water, 0.2 g / L diammonium phosphate, 2 g / L magnesium sulfate, 0.25 g / L zinc sulfate, vitamin B 1 15mg / L, Vitamin B 6 3mg / L, 5mg / L pantothenate calcium, 1.5mg / L biotin, 0.3 g / L selenoglucose, and sulfuric acid to adjust pH to 5.5. ) was cultured at 30℃. During the culture process, hydrolyzed sugar solution with a sugar content of 30% and 15% ammonia solution were added as carbon and nitrogen sources. During the culture process, the dissolved oxygen was controlled to be not less than 20%. After fermentation for 27h, the tank was separated, the yeast mud was taken, washed twice with distilled water, and dried to obtain selenium-enriched yeast. The dry weight of selenium-enriched yeast, selenium content and selenium content in the fermentation supernatant were tested respectively, and the conversion rate of selenium was calculated.

[0018] Example 4

[0019] Preparation of selenium-enriched yeast (sodium selenite fed culture) (1) Activation of strains. Take a glycerol tube of brewer's yeast (commercially available) stored at -80°C and activate it on a YPD plate. After constant temperature cultivation at 30°C for 48 h, store it in a refrigerator at 4°C for later use. (2) Seed preparation: Use an inoculation loop to transfer a single colony to seed culture medium (10% sucrose, 2% yeast powder, 0.1% magnesium sulfate, 0.1% potassium dihydrogen phosphate, pH 4.8), culture at 250 rpm, 30°C, and shake for 12 h or more (based on the alcohol concentration reaching 2% and the wet weight reaching 25 g / L), and no abnormalities are found under microscopic examination; (3) Cultivation of selenium-enriched yeast: Inoculate the seeds with an inoculum of 10% (V / V) into a medium containing initial culture medium (6 mL / L hydrolyzed sugar solution, 0.5 mL / L concentrated ammonia water, 0.2 g / L diammonium phosphate, 2 g / L magnesium sulfate, 0.25 g / L zinc sulfate, vitamin B 1 15mg / L, Vitamin B 63mg / L, calcium pantothenate 5mg / L, biotin 1.5mg / L, sulfuric acid to adjust pH to 5.5) in a fermenter, cultured at 30℃, 30% sugar content hydrolyzed sugar solution and 15% ammonia solution were added as carbon and nitrogen sources during the culture process, and 15% (W / W) sodium selenite solution was added after 4h of culture, and the addition was completed in 13h. The dissolved oxygen was controlled to be not less than 20% during the culture process. After 27h of fermentation, the tank was separated, the yeast mud was taken, washed twice with distilled water, and dried to obtain selenium-enriched yeast. The dry weight of selenium-enriched yeast, selenium content and selenium content in the fermentation supernatant were tested respectively, and the conversion rate of selenium was calculated.

[0020] Table 1 Effect of selenium-enriched yeast culture Yeast dry weight Selenium content in yeast Selenium content in fermentation supernatant Selenium conversion rate Example 2 84g / L 4130 ppm ND >99% Example 3 75g / L 4058 ppm ND >99% Example 4 56g / L 2685 ppm 86 mg / L 68% It can be seen that compared with adding a large amount of selenoglucose at one time (Example 3), adding a small amount of selenoglucose to the initial culture medium and then adding an appropriate amount of selenoglucose through feeding (Example 2), after culturing for 27 hours, the yeast dry weight per unit fermentation broth and the selenium content per unit yeast are both higher, indicating that adding selenoglucose in small amounts multiple times is more conducive to the cultivation of selenium-enriched yeast. Compared with using organic selenium as a selenium source (Examples 2 and 3), using traditional sodium selenite to cultivate selenium-enriched yeast (Example 4), the yeast dry weight per unit fermentation broth and the selenium content per unit yeast are significantly lower, indicating that selenoglucose is more conducive to the absorption and growth of selenium-enriched yeast than sodium selenite.

[0021] In summary, the present invention provides an organic selenium culture medium, in which selenoglucose is used as a selenium source. Compared with traditional inorganic selenium such as sodium selenite, yeast is easier to absorb and convert. The selenium content in the obtained selenium-enriched yeast exceeds 4000ppm, and the conversion rate of selenium in the culture medium is greater than 99%. While preparing selenium-enriched yeast with a high selenium content, the environmental protection treatment pressure of selenium in fermentation wastewater is effectively reduced.

[0022] 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. An organic selenium culture medium, characterized in that It consists of the following components by weight: Initial culture medium: 6 mL / L hydrolyzed sugar solution, 0.5 mL / L concentrated ammonia water, 0.2 g / L diammonium phosphate, 2 g / L magnesium sulfate, 0.25 g / L zinc sulfate, 15 mg / L vitamin B1, 3 mg / L vitamin B6, 5 mg / L calcium pantothenate, 1.5 mg / L biotin, 18 mg / L organic selenium, and sulfuric acid to adjust pH to 5.5; Feed medium: Selenium content is 5% (W / W) organic selenium solution.

2. The organic selenium culture medium according to claim 1, characterized in that The organic selenium in the initial culture medium is selenoglucose.

3. The organic selenium culture medium according to claim 1, characterized in that The hydrolyzed sugar solution in the initial culture medium is prepared by grinding rice and then hydrolyzing it with amylase and saccharifying enzyme, and its sugar concentration is 30% after dilution.

4. The organic selenium culture medium according to claim 1, characterized in that The application of the organic selenium culture medium in preparing selenium-enriched yeast.

5. Use of the organic selenium culture medium according to claim 4 in preparing selenium-enriched yeast, characterized in that: The preparation method of the selenium-enriched yeast cell: The culture medium containing selenoglucose was used as the initial culture medium for selenium-enriched yeast fermentation. The yeast strain was inoculated at a rate of 10% (V / V) and cultured at 30°C for 4 hours. Then, selenoglucose solution with a selenium content of 5% (W / W) and hydrolyzed sugar feed medium were added. The addition lasted for 12 hours. Ammonia water was used to control the pH value to 5.5 during the culture process. After 27 hours of fermentation, the tank was released and the selenium-enriched yeast cells were isolated.