A method for producing selenium in k. marxianus

Abstract

The application belongs to the technical field of fermentation engineering, and specifically provides a production method of selenium of Kluyveromyces marxianus. A Kluyveromyces marxianus strain with good tolerance to sodium selenite, high selenium conversion rate and good growth is screened through domestication, fermentation production is carried out by using the strain, and fermentation process and post-treatment drying process are optimized, so that the selenium content of the selenium product of the yeast is greatly improved to 5200 mg / kg, and the organic selenium content is greater than or equal to 98.0%. The method has simple process and is easy to mass produce, the strain grows vigorously, the cycle is short, and high-quality industrialized production conversion is realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of fermentation engineering, and particularly relates to a production method of selenium of Kluyveromyces marxianus. BACKGROUND

[0002] Selenium is an indispensable trace element for animal physiology. It has been proved that selenium has a wide range of biological effects, and a series of diseases such as cardiovascular disease, liver and pancreas disease, hypertension, diabetes, anemia, Keshan disease, etc. will occur due to selenium deficiency. The domestic selenium supplement sources are mainly inorganic compounds such as sodium selenite. Such products have the disadvantages of low absorption rate, high toxicity, etc. Therefore, adding inorganic selenium not only cannot achieve the ideal effect of selenium supplement, but also pollutes the environment and has potential harm to animals. Yeast selenium, as a high-quality organic selenium source, has a high absorption rate, and can be stored in the animal body after meeting the physiological needs of animals for selenium, thereby avoiding selenium deficiency in a short period of time.

[0003] At present, the selenium-rich yeast produced in China mainly uses Saccharomyces cerevisiae as the strain, and the selenium content is generally 1000-2500 mg / kg (i.e. 1000-2500 ppm). The selenium content is low, the organic selenium content is lower, and some are only simple yeast and selenium adsorption, which are not suitable for industrial production and have poor repeatability. Some yeasts can be produced, but the efficiency is very low. To obtain high-content yeast selenium, the most important thing is the breeding of strains and the improvement of fermentation process. Therefore, it is crucial to select strains that grow well and are tolerant to selenium, and to optimize the fermentation process, in order to improve the selenium content of selenium-rich yeast. Therefore, the present application provides a method, which selects Kluyveromyces marxianus with good tolerance to sodium selenite, and optimizes the fermentation process, thereby greatly improving the selenium content of yeast selenium products. SUMMARY

[0004] The purpose of the present application is to overcome the problems of low production efficiency and low yeast selenium content of the prior art selenium-rich yeast.

[0005] Therefore, the present application provides a production method of selenium of Kluyveromyces marxianus, which comprises the following steps:

[0006] (1) selecting Kluyveromyces marxianus with tolerance to 500-800 ppm sodium selenite to obtain a target strain;

[0007] (2) sequentially performing primary seed culture and secondary seed culture on the target strain, and then inoculating a fermenter for fermentation in the fermenter;

[0008] (3) when the wet weight of fermentation reaches 10-50 g / L, start feeding A, when the wet weight of fermentation liquid reaches 30-200 g / L, start feeding B, stop feeding B after 10-15 h, reduce the feeding speed of A, stop feeding A after 5-10 h, and end the fermentation after 1-3 h.

[0009] The feed A comprises 100-500 g / L anhydrous glucose, 100-500 g / L sucrose, 50-200 g / L glycerol, 2-8 mg / L biotin, 10-100 mg / L myo-inositol, 10-100 mg / L p-aminobenzoic acid, 10-50 mg / L choline, 80-150 mg / L nicotinic acid and 80-150 mg / L calcium pantothenate; the feed B comprises 50-100 g / L sodium selenite;

[0010] (4) After the fermentation is completed, the fermentation liquor is centrifuged and dried to obtain the selenium-containing Kluyveromyces marxianus.

[0011] Specifically, the step (1) comprises the following steps:

[0012] S1, screening Kluyveromyces marxianus resistant to 50-300 ppm sodium selenite to obtain a primary screening strain;

[0013] S2, inoculating the primary screening strain into a culture medium containing 300-500 ppm sodium selenite and continuously culturing;

[0014] S3, inoculating the strain obtained by the continuous culture in S2 into a culture medium containing 500-800 ppm sodium selenite and continuously culturing to obtain a target strain resistant to 500-800 ppm sodium selenite.

[0015] Specifically, the step (1) is specifically:

[0016] S1, inoculating Kluyveromyces marxianus seed into YPD liquid medium for activation, the activation temperature is 28-35℃, the activation time is 15-30 h, and the activated seed liquid is diluted and coated on YPD screening plates containing 50, 100, 200 and 300 ppm sodium selenite respectively, and then incubated at 28-35℃ for 24-48 h to obtain a primary screening strain;

[0017] S2, inoculating the primary screening strain into YPD liquid medium containing 300-500 ppm sodium selenite at an inoculation amount of 5-10%, and then incubating at 28-35℃ and 200-300 r / min for 18-24 h, and then inoculating into YPD liquid medium containing 300-500 ppm sodium selenite at an inoculation amount of 5-10%, and then incubating at 28-35℃ and 200-300 r / min for 18-24 h, and repeating the process, and continuously culturing for 10-15 generations;

[0018] S3, inoculate the strain obtained by continuous culture in S2 into YPD liquid medium containing 500-800 ppm sodium selenite at an inoculation amount of 5-10%, and culture at 28-35°C and 200-300 r / min for 18-24 h, repeat the process, and continuously culture for 10-15 generations to obtain a target strain resistant to 500-800 ppm sodium selenite.

[0019] Specifically, the primary seed culture in step (2) is as follows: inoculate the target strain into a primary seed liquid culture medium, the initial pH value is 5.0-5.5, the culture temperature is 28-32°C, the shaking speed is 150-300 rpm / min, and the culture time is 18-25 h to obtain the primary seed liquid.

[0020] Specifically, the primary seed liquid culture medium comprises 0.5-3% anhydrous glucose, 0.5-3% sucrose, 0.5-3% glycerol, 0.2-1% (NH4)2SO4, 0.01-0.1% MgSO4·7H2O, 0.05-0.2% CaSO4·2H2O, 0.1-1% KH2PO4, 0.1-0.5% 500x trace element stock solution, and 0.1-0.5% 1000x vitamin stock solution; the 500x trace element stock solution comprises 1-10 g / L KCl, 5-10 g / L EDTA, 1-5 g / L ZnSO4·7H2O, 0.1-1 g / L MnCl2·7H2O, 0.1-0.5 g / L CoCl2·6H2O, 0.1-0.5 g / L CuSO4·5H2O, 0.1-0.5 g / L Na2MoO4·2H2O, 1-5 g / L CaCl2·2H2O, 1-5 g / L FeSO4·7H2O, 0.1-1 g / L H3BO3, and 0.01-0.1 g / L KI; and the 1000x vitamin stock solution comprises 0.05-0.2 g / L biotin, 0.5-2 mg / L niacin, 10-100 mg / L inositol, 0.5-2 mg / L thiamine, 0.5-2 mg / L pyridoxal, 10-100 mg / L p-aminobenzoic acid, 10-50 mg / L choline, 1-20 mg / L riboflavin, 0.05-0.2 g / L calcium pantothenate, and 0.05-0.2 g / L nicotinic acid.

[0021] Specifically, the secondary seed culture in step (2) is as follows: the product of the primary seed culture is inoculated into a seed fermentation tank, the inoculation amount is preferably 2%-15%, more preferably 5%-8%, the fermentation temperature is 28-32°C, the rotation speed is 200-400 rpm / min, the filling pressure is 0.02-0.05 Mpa, the initial pH value is 5.0-5.5, the pH value is adjusted to 5.0-5.5 by ammonia water during the whole fermentation process, the dissolved oxygen is controlled to be more than 20% during the fermentation process, the fermentation is ended when the wet weight of the fermentation broth is 20-100 g / L, and the seed fermentation broth is obtained.

[0022] Specifically, the fermentation in the fermentation tank in step (2) is as follows: the product after the secondary seed culture is inoculated into a fermentation tank containing a culture medium, the fermentation temperature is 28-38°C, the rotation speed is 200-650 rpm / min, the filling pressure is 0.02-0.05 Mpa, the initial pH value is 4.0-5.5, the pH value is adjusted to 4.0-6.0 by ammonia water in stages during the whole fermentation process, and the dissolved oxygen is controlled to be more than 40%.

[0023] Specifically, the inoculation amount of the product after the secondary seed culture is 2-15%, preferably 8-10%.

[0024] Specifically, the above-mentioned culture medium comprises: 0.5-2% anhydrous glucose, 0.5-2% sucrose, 0.5-2% glycerol, 0.1-0.5% yeast extract powder, 0.5-2% corn syrup, 0.1-2% MgSO4·7H2O, 0.1-2% KH2PO4, 0.1-2% NH4H2PO4, 0.01-0.1% FeSO4·7H2O, 0.01-0.1% ZnSO4·7H2O, 0.1-1% CaCl2, 0.1-1% KCl, 0.1-1% 500x trace element stock solution, 0.1-0.5% 1000x vitamin stock solution and 0.01-0.1% defoamer; the 500x trace element stock solution comprises 1-10 g / L KCl, 5-10 g / L EDTA, 1-5 g / L ZnSO4·7H2O, 0.1-1 g / L MnCl2·7H2O, 0.1-0.5 g / L CoC12·6H2O, 0.1-0.5 g / L CuSO4·5H2O, 0.1-0.5 g / L Na2MoO4·2H2O, 1 g-5 g / L CaC12·2H2O, 1-5 g / L FeSO4·7H2O, 0.1-1 g / L H3BO3 and 0.01-0.1 g / L KI; the 1000x vitamin stock solution comprises 0.05-0.2 g / L biotin, 0.5-2 mg / L niacin, 10-100 mg / L inositol, 0.5-2 mg / L thiamine, 0.5-2 mg / L pyridoxine, 10-100 mg / L p-aminobenzoic acid, 10-50 mg / L choline, 1-20 mg / L riboflavin, 0.05-0.2 g / L calcium pantothenate and 0.05-0.2 g / L nicotinic acid.

[0025] Specifically, the step (4) is specifically as follows: after the fermentation is completed, the fermentation liquor is centrifuged and washed for 2-4 times, the supernatant is removed, the precipitate is heated to 70-80℃, and kept for half an hour, then the precipitate is atomized by high-speed centrifugation and then is subjected to spray drying to obtain the powder-like selenium-containing Kluyveromyces marxianus.

[0026] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0027] The production method of the selenium-containing Kluyveromyces marxianus provided by the present application has the advantages of good tolerance to sodium selenite, high selenium conversion rate and good growth of the strain, the fermentation production is carried out by using the strain, the fermentation process and the post-treatment drying process are optimized, the selenium content of the yeast selenium product is greatly improved to reach 5200 mg / kg (i.e. 5200 ppm), and the organic selenium content is ≥98.0%. The method has simple process and is easy for mass production, the strain grows vigorously, the cycle is short, and the high-quality industrialized production conversion is realized. DETAILED DESCRIPTION

[0028] The technical solutions in the present application will be clearly and completely described below with examples. Obviously, the described examples are only some of the embodiments of the present application, but not all the embodiments. Although the representative embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the present application without departing from the scope of the present application. Therefore, the scope of the present application should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.

[0029] The effect of the method for producing selenium in Kluyveromyces marxianus according to the present application is studied below through specific examples.

[0030] The Kluyveromyces marxianus strain used in the embodiments of the present application is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 10621.

[0031] Example 1

[0032] The present embodiment provides a method for producing selenium in Kluyveromyces marxianus, and the specific steps are as follows.

[0033] (1) Screening of target strain

[0034] S1, inoculate the seed of Kluyveromyces marxianus in YPD liquid medium for activation, the activation temperature is 32℃, the activation time is 20h, dilute and spread the activated seed liquid on the screening YPD plates containing 50, 100, 200, 300ppm sodium selenite respectively, and incubate at 32℃ for 30h, select single colonies with better growth and slightly red or no red color, and store them in slant YPD medium, which is the primary screening strain.

[0035] S2, inoculate the primary screening strain in YPD liquid medium containing 400ppm sodium selenite at an inoculation amount of 10%, and incubate in a constant temperature shaker at 32℃ and 250r / min for 20h. Inoculate the obtained bacterial liquid in YPD liquid medium containing 400ppm sodium selenite at an inoculation amount of 10%, and incubate at 32℃ and 250r / min for 20h, repeat the process, and continuously culture for 10 generations;

[0036] S3, inoculate the bacterial liquid obtained by continuous culture in S2 in YPD liquid medium containing 700ppm sodium selenite at an inoculation amount of 10%, and incubate in a constant temperature shaker at 32℃ and 250r / min for 20h, repeat the process, and continuously culture for 10 generations, finally measure the OD value of the bacterial liquid 600 25 and the selenium enrichment amount reaches 5.35mg / g, and the organic selenium content reaches 5.25mg / g, and the target strain with high selenium conversion rate is screened.

[0037] (2) Primary seed culture

[0038] The target strain is inoculated into the first-stage seed liquid culture medium, the initial pH value is 5.0, the culture temperature is 32°C, the shaking speed is 200 rpm / min, and the culture is performed for 20 h to obtain the first-stage seed liquid.

[0039] The first-stage seed liquid culture medium comprises 1% anhydrous glucose, 1% sucrose, 1% glycerol, 0.5% (NH4)2SO4, 0.05% MgSO4·7H2O, 0.1% CaSO4·2H2O, 0.3% KH2PO4, 0.2% 500x trace element stock solution, and 0.1% 1000x vitamin stock solution, and the medium is sterilized at 115°C for 20 min after being dispensed.

[0040] The 500x trace element stock solution comprises 5 g / L KCl, 7.5 g / L EDTA, 2.25 g / L ZnSO4·7H2O, 0.5 g / L MnCl2·7H2O, 0.15 g / L CoCl2·6H2O, 0.15 g / L CuSO4·5H2O, 0.2 g / L Na2MoO4·2H2O, 2.25 g / L CaCl2·2H2O, 1.5 g / L FeSO4·7H2O, 0.5 g / L H3BO3, and 0.05 g / L KI.

[0041] The 1000x vitamin stock solution comprises 0.1 g / L biotin, 1 mg / L nicotinic acid, 25 mg / L myo-inositol, 1 mg / L thiamine, 1 mg / L pyridoxal, 25 mg / L p-aminobenzoic acid, 15 mg / L choline, 5 mg / L riboflavin, 0.1 g / L calcium pantothenate, and 0.1 g / L nicotinic acid.

[0042] (3) Second-stage seed culture

[0043] The first-stage seed liquid is inoculated into the seed fermentation tank at a 10% inoculation amount, the fermentation temperature is 32°C, the rotation speed is 400 rpm / min, the filling pressure is 0.03 MPa, the initial pH value is 5.0, the pH value is adjusted to 5.0 by using ammonia water during the whole fermentation process, the rotation speed and the aeration amount are adjusted during the fermentation process to control the dissolved oxygen to be higher than 20%, and the fermentation is ended when the wet weight of the fermentation liquid is 30 g / L after about 12 h of fermentation to obtain the seed fermentation liquid, and the seed transfer is started.

[0044] The culture medium used in the second-stage seed culture is the same as the first-stage seed liquid culture medium.

[0045] (4) Fermentation tank fermentation

[0046] The seed fermentation broth is inoculated into a 30L fermenter containing culture medium at an inoculation amount of 10%, and the fermentation medium volume is 12L; the fermentation temperature is 32°C, the initial rotation speed is 200rpm / min, and then the rotation speed is gradually increased to 650rpm / min as the dissolved oxygen decreases; the pressure is 0.03Mpa; the initial pH value is 4.5, and the pH value is adjusted to 4.5-5.5 by ammonia water during the whole fermentation process, the pH value is increased to 4.8 after 4h of fermentation, the pH value is increased to 5.2 after 8h of fermentation, and the pH value is increased to 5.5 after 11h of fermentation; the rotation speed and the aeration amount are alternately adjusted to control the dissolved oxygen to be above 40% during the first 10h of fermentation.

[0047] The fermentation is performed for about 4h, and when the fermentation wet weight reaches 15g / L, the feeding A is started at an initial feeding speed of 100ml / h, and the feeding speed is increased by 20ml per hour; the fermentation is performed for 9h, and then the feeding A speed is adjusted to 250ml / h, and then the feeding speed is increased by 50ml per hour, the fermentation is performed for 17h, at this time the feeding A speed is 650ml / h, and the constant feeding speed is maintained for fermentation for 24h, the feeding A speed is reduced to 400ml / h at the 25th hour, and then the feeding speed is reduced by 100ml per hour, and the feeding A is stopped at the 29th hour.

[0048] The fermentation is performed for about 11h, and when the fermentation broth wet weight reaches 80g / L, the feeding B is started at an initial speed of 5ml / h, and the feeding speed is increased by 2ml per hour, the fermentation is performed for 17h, at this time the feeding B speed is 17ml / h, and then the constant feeding speed is maintained for fermentation for 24h, and the feeding B is stopped.

[0049] After the feeding A is stopped, the fermentation is continued for 1h, and then the fermentation is ended, and the whole fermentation cycle is 30h. By controlling the feeding A and B, the growth of the fermentation broth biomass is regulated, and the final obtained selenium-rich yeast fermentation broth biomass is 300g / L.

[0050] The culture medium comprises: 1% anhydrous glucose, 1.5% sucrose, 0.5% glycerol, 0.1% yeast extract powder, 0.5% corn syrup, 0.5% MgSO4·7H2O, 0.15% KH2PO4, 0.2% NH4H2PO4, 0.015% FeSO4·7H2O, 0.015% ZnSO4·7H2O, 0.15% CaCl2, 0.3% KCl, 0.7% 500x trace element stock solution, 0.2% 1000x vitamin stock solution and 0.05% diquat; the 500x trace element stock solution comprises 5g / L KCl, 7.5g / L EDTA, 2.25g / L ZnSO4·7H2O, 0.5g / L MnCl2·7H2O, 0.15g / L CoCl2·6H2O, 0.15g / L CuSO4·5H2O, 0.2g / L Na2MoO4·2H2O, 2.25g / L CaCl2·2H2O, 1.5g / L FeSO4·7H2O, 0.5g / L H3BO3 and 0.05g / L KI; the 1000x vitamin stock solution comprises 0.1g / L biotin, 1mg / L nicotinic acid, 25mg / L myo-inositol, 1mg / L thiamine, 1mg / L pyridoxal, 25mg / L p-aminobenzoic acid, 15mg / L choline, 5mg / L riboflavin, 0.1g / L calcium pantothenate and 0.1g / L nicotinic acid.

[0051] The feed A comprises: 100g / L anhydrous glucose, 400g / L sucrose, 50g / L glycerol, 5mg / L biotin, 25mg / L myo-inositol, 25mg / L p-aminobenzoic acid, 15mg / L choline, 100mg / L nicotinic acid and 100mg / L calcium pantothenate, sterilized at 110°C for 20min, wherein the biotin, myo-inositol, p-aminobenzoic acid, choline, nicotinic acid and calcium pantothenate are filter-sterilized and then added into the sterilized and cooled feed. The feed B is 50g / L sodium selenite.

[0052] (5) Drying of the fermented liquid after treatment

[0053] Centrifugal washing: after the fermentation is completed, the selenium-rich yeast milk is obtained by centrifugation and washing twice to remove inorganic selenium by washing with water and removing the supernatant.

[0054] Warming: the selenium-rich yeast milk is warmed to 75°C and kept for half an hour.

[0055] Drying: the selenium-rich yeast milk is atomized by high-speed centrifugation, enters a spray drying tower, contacts with dry hot air, instantaneously evaporates water and is dried into powder-like yeast selenium; the spray drying tower has a spray inlet air temperature of 220°C, an outlet air temperature of 115°C and a feeding frequency of 6-7Hz.

[0056] Packaging and storage: packaged with inner polyethylene film composite woven bag, inner bag is tied tightly, outer package is sewn, and stored at room temperature in warehouse.

[0057] Comparative Example 1:

[0058] The same production method of selenium yeast of Kluyveromyces marxianus as Example 1 was adopted in this comparative example, the difference being that the Kluyveromyces marxianus used was not subjected to the domestication and screening of step (1).

[0059] Comparative Example 2:

[0060] The same production method of selenium yeast of Kluyveromyces marxianus as Example 1 was adopted in this comparative example, the difference being that the YPD liquid medium in S2 of step (1) contained 200 ppm sodium selenite, and the YPD liquid medium in S3 contained 400 ppm sodium selenite.

[0061] Comparative Example 3:

[0062] The same production method of selenium yeast of Kluyveromyces marxianus as Example 1 was adopted in this comparative example, the difference being that the ammonia was used to control the fermentation PH to be constant at 5.0 during the fermentation in the fermenter.

[0063] Comparative Example 4:

[0064] The same production method of selenium yeast of Kluyveromyces marxianus as Example 1 was adopted in this comparative example, the difference being that after the completion of feeding B, feeding A was not slowed down, and the feeding A speed was 650 ml / h, and the feeding A was stopped at 29 h of fermentation.

[0065] Comparative Example 5:

[0066] The same production method of selenium yeast of Kluyveromyces marxianus as Example 1 was adopted in this comparative example, the difference being that during the fermentation in the fermenter, feeding B was continuously fed at a flow rate of 14 ml / h until the end of fermentation, and the total amount of feeding B in the whole fermentation process was the same as that of Example 1.

[0067] Example 2:

[0068] The total selenium content, organic selenium content and conversion rate of the selenium yeast products obtained in each example and comparative example were measured respectively, and the results are shown in Table 1.

[0069] Table 1 Total selenium content, organic selenium content and conversion rate of the selenium yeast product

[0070]

[0071]

[0072] As can be seen from the data in Table 1, in the fermentation of the selenium-rich yeast using the strain of Kluyveromyces marxianus as the selenium-rich yeast, by suitable domestication of the strain, selection of suitable fermentation culture conditions, feeding during the fermentation process, and by the stage control of feeding, the growth rate of the yeast cells and the conversion and utilization of sodium selenite can be precisely adjusted, so that the selenium content in the selenium product of the yeast can be greatly increased.

[0073] The above examples are only illustrative of the present application and do not constitute a limitation on the protection scope of the present application, and any design identical or similar to the present application falls within the protection scope of the present application.

Claims

1. A method for producing selenium from Kluyveromyces martensii, characterized in that, Includes the following steps: (1) The target strain was obtained by screening for strains of Kluyveromyces roxburghii that are tolerant to 500-800 ppm sodium selenite through domestication. (2) The target strain was cultured in a primary seed culture and a secondary seed culture in sequence, and then inoculated into a fermenter for fermentation. (3) When the wet weight of the fermentation liquid reaches 15 g / L, start feeding A with an initial feeding rate of 100 ml / h, and increase the feeding rate by 20 ml per hour; after 9 hours of fermentation, adjust the feeding rate of feeding A to 250 ml / h, and then increase the feeding rate by 50 ml per hour. After 17 hours of fermentation, maintain this constant feeding rate until 24 hours of fermentation. At 25 hours, reduce the feeding rate of feeding A to 400 ml / h, and then reduce the feeding rate by 100 ml per hour. Stop feeding A at 29 hours. When the wet weight of the fermentation liquid reaches 80 g / L, start feeding B with an initial feeding rate of 5 ml / h, and increase the feeding rate by 2 ml per hour. After 17 hours of fermentation, the feeding rate of feeding B is 17 ml / h. Then maintain this constant feeding rate until 24 hours of fermentation, and stop feeding B. Stop feeding A 1-3 hours after stopping feeding A. The supplement A comprises 100 g / L anhydrous glucose, 400 g / L sucrose, 50 g / L glycerol, 5 mg / L biotin, 25 mg / L inositol, 25 mg / L para-aminobenzoic acid, 15 mg / L choline, 100 mg / L niacin, and 100 mg / L calcium pantothenate; the supplement B is 50 g / L sodium selenite. (4) After fermentation, the fermentation liquid was centrifuged and dried to obtain Max Kluyveromycin selenium.

2. The method for producing selenium from Kluyveromyces martensii as described in claim 1, characterized in that, Step (1) includes the following steps: S1. Screening for Kluyveromyces maculae that are tolerant to 50-300 ppm sodium selenite to obtain the initial screening strains; S2. Inoculate the initially screened yeast strain into a culture medium containing 300-500 ppm sodium selenite and culture continuously. S3. The strain obtained from continuous culture in S2 is inoculated into a culture medium containing 500-800 ppm sodium selenite and cultured continuously to obtain the target strain tolerant to 500-800 ppm sodium selenite.

3. The method for producing selenium from Kluyveromyces martensii as described in claim 2, characterized in that: The specific steps (1) are as follows: S1. Inoculate the Kluyveromyces martensii seed into YPD liquid medium and activate it at 28-35℃ for 15-30h. Dilute the activated seed solution and spread it on YPD screening plates containing 50, 100, 200, and 300 ppm sodium selenite, respectively. Incubate at 28-35℃ for 24-48h to obtain the initial screening strain. S2. Inoculate the initially screened strains at a rate of 5-10% into YPD liquid medium containing 300-500 ppm sodium selenite, and incubate at 28-35℃ and 200-300 r / min for 18-24 h. Then, inoculate the strains at a rate of 5-10% into YPD liquid medium containing 300-500 ppm sodium selenite, and incubate at 28-35℃ and 200-300 r / min for 18-24 h. Repeat this process for 10-15 generations. S3. Inoculate the strain obtained from continuous culture in S2 at an inoculum of 5-10% into YPD liquid medium containing 500-800 ppm sodium selenite, and culture at 28-35℃ and 200-300 r / min for 18-24 h. Repeat this process for 10-15 generations to obtain the target strain tolerant to 500-800 ppm sodium selenite.

4. The method for producing selenium from Kluyveromyces martensii as described in claim 1, characterized in that, The primary seed culture in step (2) is as follows: the target strain is inoculated into the primary seed culture medium with an initial pH of 5.0-5.5, a culture temperature of 28-32℃, a shaking speed of 150-300 rpm / min, and cultured for 18-25 hours to obtain the primary seed culture.

5. The method for producing selenium from Kluyveromyces martensii as described in claim 4, characterized in that, The primary seed culture medium comprises: 0.5-3% anhydrous glucose, 0.5-3% sucrose, 0.5-3% glycerol, 0.2-1% (NH4)2SO4, 0.01-0.1% MgSO4·7H2O, 0.05-0.2% CaSO4·2H2O, 0.1-1% KH2PO4, 0.1-0.5% 500x micronutrient stock solution, and 0.1-0.5% 1000x vitamin stock solution; the 500x micronutrient stock solution comprises 1-10 g / L KCl, 5-10 g / L EDTA, 1-5 g / L ZnSO4·7H2O, 0.1-1 g / L MnCl2·7H2O, 0.1-0.5 g / L CoCl2·6H2O, 0.1-0.5 g / L CuSO4·5H2O, and 0.1-0.5 g / L... The 1000x vitamin stock solution comprises: Na2MoO4·2H2O, 1g-5g / L CaCl2·2H2O, 1-5g / L FeSO4·7H2O, 0.1-1g / L H3BO3, and 0.01-0.1g / L KI; the 1000x vitamin stock solution includes: 0.05-0.2g / L biotin, 0.5-2mg / L nicotinic acid, 10-100mg / L inositol, 0.5-2mg / L thiamine, 0.5-2mg / L pyridoxine, 10-100mg / L para-aminobenzoic acid, 10-50mg / L choline, 1-20mg / L riboflavin, 0.05-0.2g / L calcium pantothenate, and 0.05-0.2g / L niacin.

6. The method for producing selenium from Kluyveromyces martensii as described in claim 1, characterized in that, The secondary seed culture in step (2) is as follows: the primary seed culture product is inoculated into a seed fermentation tank, the fermentation temperature is 28-32℃, the rotation speed is 200-400rpm / min, the injection pressure is 0.02-0.05Mpa, the initial pH value is 5.0-5.5, the pH is adjusted to 5.0-5.5 with ammonia water throughout the fermentation process, the dissolved oxygen is controlled to be above 20% during the fermentation process, and the fermentation is stopped when the wet weight of the fermentation liquid is 20-100 g / L, and the seed fermentation liquid is obtained.

7. The method for producing selenium from Kluyveromyces martensii as described in claim 1, characterized in that, The fermentation in step (2) specifically involves: inoculating the product after secondary seed culture into a fermenter containing culture medium, fermenting at a temperature of 28-38℃, rotating at 200-650 rpm / min, pressurizing at 0.02-0.05 MPa, with an initial pH of 4.0-5.5, and adjusting the pH to 4.0-6.0 in stages with ammonia water throughout the fermentation process, while controlling dissolved oxygen to be above 40%.

8. The method for producing selenium from Kluyveromyces martensii as described in claim 7, characterized in that: The inoculum size of the product after secondary seed culture is 2-15%.

9. The method for producing selenium from Kluyveromyces martensii as described in claim 7, characterized in that, The culture medium comprises: 0.5-2% anhydrous glucose, 0.5-2% sucrose, 0.5-2% glycerol, 0.1-0.5% yeast extract, 0.5-2% corn steep liquor, 0.1-2% MgSO4·7H2O, 0.1-2% KH2PO4, 0.1-2% NH4H2PO4, 0.01-0.1% FeSO4·7H2O, 0.01-0.1% ZnSO4·7H2O, 0.1-1% CaCl2, 0.1-1% KCl, 0.1-1% 500x micronutrient stock solution, 0.1-0.5% 1000x vitamin stock solution, and 0.01-0.1% foaming agent; the 500x micronutrient stock solution comprises 1-10 g / L KCl, 5-10 g / L EDTA, and 1-5 g / L... ZnSO4·7H2O, 0.1-1g / L MnCl2·7H2O, 0.1-0.5g / L CoC12·6H2O, 0.1-0.5g / L CuSO4·5H2O, 0.1-0.5g / L Na2MoO4·2H2O, 1-5g / L CaC12·2H2O, 1-5g / L FeSO4·7 H2O, 0.1-1g / LH3BO3 and 0.01-0.1g / L KI; The 1000x vitamin stock solution comprises 0.05-0.2 g / L biotin, 0.5-2 mg / L nicotinic acid, 10-100 mg / L inositol, 0.5-2 mg / L thiamine, 0.5-2 mg / L pyridoxine, 10-100 mg / L para-aminobenzoic acid, 10-50 mg / L choline, 1-20 mg / L riboflavin, 0.05-0.2 g / L calcium pantothenate, and 0.05-0.2 g / L niacin.

10. The method for producing selenium from Kluyveromyces martensii as described in claim 1, characterized in that, The specific steps (4) are as follows: after fermentation, the fermentation liquid is centrifuged and washed 2-4 times. After removing the supernatant, the precipitate is heated to 70-80℃ and kept at that temperature for half an hour. After high-speed centrifugation and atomization, it is spray-dried to obtain powdered Max Kluyveromycin selenium.

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