Selenium-enriched yeast strain with high inorganic selenium tolerance and application thereof

Abstract

The invention relates to a selenium-enriched yeast strain with high inorganic selenium tolerance and application thereof, and belongs to the technical field of bioengineering. The name of the strain is S.cerevisiae TSE 001, the strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.34111, and the strain has the advantages of strong selenium resistance, high organic selenium conversion rate and genetic stability. The strain can still normally grow in a high-concentration inorganic selenium environment, the biomass can reach 2.5 g / L, the organic selenium content exceeds 1600 micrograms / g, and the organic selenium conversion rate reaches 55% or above. The strain can be applied to production of selenium-enriched yeast, is suitable for large-scale production of organic selenium functional foods, health-care products and feed additives, and has a wide industrial application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and in particular relates to a selenium-rich yeast with high inorganic selenium tolerance constructed by utilizing adaptive evolution technology and an application thereof. Technical Background

[0002] Selenium is an essential trace element for humans and animals, possessing numerous important physiological functions, including immune enhancement, antioxidant protection, cardiovascular disease prevention, and anti-tumor properties. As a nutritional fortifier, selenium intake plays a crucial role in maintaining normal physiological metabolism and resisting disease. Organic selenium, with its high bioactivity and low toxicity, is an ideal additive currently used in functional foods, health supplements, and feed additives.

[0003] Although inorganic selenium (such as sodium selenite and sodium selenate) is relatively abundant in nature and inexpensive, its bioavailability is low, and excessive intake can easily lead to acute or chronic toxic reactions, making it unsuitable as a direct source of nutritional supplements. In contrast, organic selenium such as selenomethionine and selenocystine are more easily absorbed and utilized by the human body or animals, with low toxicity and high safety. Selenium-enriched yeast is an effective way to convert inorganic selenium into organic selenium through microbial metabolism, and is often fermented using brewer's yeast as the host. However, the traditional selenium-enriched yeast preparation process still has multiple technical bottlenecks, including poor tolerance of the strain to high-concentration selenium sources, low selenium conversion efficiency, low biomass, and insufficient genetic stability.

[0004] Therefore, it is urgent to construct a yeast strain with high selenium tolerance, high organic selenium conversion rate and high biomass, and establish a corresponding efficient preparation process to improve the quality and application value of selenium-enriched yeast products. Summary of the Invention

[0005] The present invention aims to provide a high-selenium-tolerant, selenium-enriched yeast strain and a method for its preparation, addressing technical issues such as limited growth, low organic selenium conversion, insufficient biomass, and genetic instability of existing selenium-enriched yeast strains in high-selenium environments. The yeast strain obtained by the technical means described in the present invention is capable of normal growth and efficient selenium enrichment in the presence of relatively high concentrations of inorganic selenium. The preparation method is simple, stable, and reliable, with promising prospects for industrial application.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] (1) Using food-grade brewer's yeast S. cerevisiae as the starting strain, gene mutation was induced by ultraviolet mutagenesis, and the mutant strain S. cerevisiae Y1 with a certain selenium resistance was screened on sodium selenite gradient plates.

[0008] (2) The obtained mutagenic strain S.cerevisiae Y1 was used as the initial strain and subjected to laboratory adaptive evolution at 25% and 50% sodium selenite inhibition concentrations, respectively. Through continuous subculture, its tolerance to inorganic selenium was gradually enhanced.

[0009] (3) During the adaptive evolution process, the biomass and organic selenium content of the induced strain S.cerevisiae Y1 were detected in real time, and the evolved strain with good growth performance and selenium enrichment ability under high selenium stress conditions was screened out and named S.cerevisiae TSE 001, the full name of which is Saccharomyces cerevisiae TSE 001.

[0010] (4) Further verification of genetic stability confirmed that the strain can still maintain its growth characteristics and selenium-enriching ability in continuous multi-generation culture, ensuring its stability and consistency in industrial applications.

[0011] Based on the constructed strain S.cerevisiae TSE 001, the present invention provides a method for producing selenium-enriched yeast using S.cerevisiae TSE 001, comprising the following steps:

[0012] (1) S. cerevisiae TSE 001 was inoculated into a liquid synthetic culture medium rich in carbon source, nitrogen source, and an appropriate amount of inorganic selenium;

[0013] (2) Cultivate at 30°C and 150 rpm to the target biomass;

[0014] (3) Selenium-enriched yeast powder is prepared after freeze-drying.

[0015] The organic selenium content of the obtained selenium-enriched yeast powder can reach above 1600 μg / g.

[0016] As a preferred embodiment, in the above method, the culture medium components include: 20 g / L anhydrous glucose, 2 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, 1 g / L ammonium sulfate, 1 g / L yeast extract powder, and 88-133.8 mg / L inorganic selenium salt.

[0017] The inorganic selenium salt can be sodium selenite, potassium selenite or other soluble inorganic selenium salts.

[0018] The evolved strain S. cerevisiae TSE 001, obtained using the method of the present invention, maintains excellent growth even under culture conditions with high selenium concentrations, achieving biomass up to 2.5 g / L, organic selenium content of 1600 μg / g or more, and an organic selenium conversion rate exceeding 55%. This strain exhibits strong tolerance, high enrichment capacity, and genetic stability, demonstrating strong potential for industrial application.

[0019] The beneficial effects of the present invention are:

[0020] (1) Using adaptive evolution technology to improve the tolerance of yeast strains to inorganic selenium, solving the problem that traditional strains are susceptible to selenium toxicity;

[0021] (2) Using adaptive evolution technology to improve the conversion efficiency of yeast to inorganic selenium and significantly increase the accumulation of organic selenium;

[0022] (3) The strains obtained using adaptive evolution technology have high biomass and stable genetic traits, making them suitable for large-scale production;

[0023] (4) The process flow of high selenium-tolerant selenium-enriched yeast constructed using adaptive evolution technology is simple, easy to operate and industrially scale up. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a comparison chart of the growth of the starting strain S.cerevisiae and the mutant strain S.cerevisiae Y1 in the present invention in a liquid culture medium without sodium selenite and in a liquid culture medium containing 100 μg / mL sodium selenite;

[0025] Figure 2 Schematic diagram of the changes in biomass and enriched organic selenium content of the mutagenic strain S.cerevisiae Y1 during the continuous adaptive evolution process at 25% and 50% sodium selenite inhibition concentrations in the present invention;

[0026] Figure 3 Schematic diagram of the growth of the starting strain S. cerevisiae, the adaptively evolved strain S. cerevisiae TSE 001 and the genetically stable verified strain S. cerevisiae X60 in the present invention in a liquid synthetic culture medium with a 50% sodium selenite inhibition concentration;

[0027] Figure 4 Schematic diagram of the biomass, organic selenium content and organic selenium conversion rate of the starting strain S. cerevisiae, the adaptively evolved strain S. cerevisiae TSE 001 and the genetically stable verified strain S. cerevisiae X60 in the present invention.

[0028] Deposit information: Strain name: S.cerevisiae TSE 001, classified as: Saccharomyces cerevisiae, deposited on April 7, 2025 in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC No. 34111, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Postal Code 100101. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described in detail below through specific embodiments.

[0030] Example 1: Construction of a selenium-tolerant mutant strain S. cerevisiae Y1

[0031] This embodiment includes the following steps:

[0032] (1) Activation and preservation of bacterial strains

[0033] Weigh 0.02g of Angel Yeast dry powder and add it to 20mL / 100mL of sterile liquid synthetic culture medium. Incubate the culture in a shaker at 30℃ and 150rpm for 18h. Pipette 100μl of the bacterial solution and perform a 10-fold gradient dilution in a clean bench. Spread solid culture medium for each gradient. Place the solid culture medium in a 30℃ constant temperature incubator and invert and culture for 48h to grow single colonies.

[0034] A single colony was picked and transferred to a 50 mL / 250 mL liquid synthetic culture medium for shake flask culture (30°C, 150 rpm). The absorbance at an absorption wavelength of 600 nm (OD600) was measured using an ultraviolet spectrophotometer. When the OD600 reached 8, 1 mL of the bacterial solution was mixed with the same volume of 60% (v / v) sterile glycerol, and the mixture was transferred to a sterile cryovial and stored in a -80°C refrigerator. This strain is the starting strain S. cerevisiae of the present invention.

[0035] The liquid synthetic culture medium is prepared as follows: 20 g / L anhydrous glucose, 2 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, 1 g / L ammonium sulfate, and 10 g / L yeast extract powder, at 115° C. for 20 min.

[0036] The solid culture medium is prepared by adding 1.5% agar to the liquid culture medium, while other conditions remain unchanged.

[0037] The operation method of the 10-fold gradient dilution is as follows: 100 μL of bacterial solution is transferred to 900 μL of sterile liquid synthetic culture medium, which is 10 -1; After mixing, take 100 μL of bacterial solution and transfer it to 900 μL of sterile liquid synthetic culture medium, which is 10 -2 . Gradual dilution to 10 -5 .

[0038] (2) UV mutagenesis and selenium-resistant strain screening

[0039] Pick the preserved starting strain S.cerevisiae and inoculate it on a solid culture medium. Invert and culture at 30℃ for 48h until a single colony grows. Pick a single colony and inoculate it into a 50mL / 250mL liquid culture medium and shake it in a flask for 18h (30℃, 150rpm). Take 1mL of bacterial solution, centrifuge it and wash it with sterile saline. Resuspend it in sterile saline to prepare a bacterial suspension. Place the bacterial suspension in a sterile culture dish, irradiate it at a distance of 30cm from a 15W ultraviolet lamp for 10min, and then keep it away from light. Dilute the above bacterial suspension 10 times in a gradient to 10 under dark conditions. -3 , 10 -4 , 10 -5 100 μL of each concentration was spread onto a solid medium containing 100 μg / mL sodium selenite and incubated at 30°C in the dark until a single colony emerged. Surviving strains with excellent growth traits were selected for repeated UV mutagenesis treatment. Strains with excellent traits were selected for preservation and designated S. cerevisiae Y1.

[0040] The solid culture medium containing 100 μg / mL sodium selenite is prepared by adding sodium selenite to the solid culture medium to a final concentration of 100 μg / mL.

[0041] (3) Determination of growth curve

[0042] Take the strain stored at -80℃ and inoculate it into 50mL / 250mL liquid synthetic culture medium for activation culture (30℃, 150rpm, 18h), transfer the activated strain into fresh 50mL / 250mL liquid synthetic culture medium (without sodium selenite, containing 100μg / mL sodium selenite) with an inoculum size of 10% and continue to culture (30℃, 150rpm), take 1mL sample into a centrifuge tube at 0h, 3h, 6h, 9h, 12h, 15h and 24h respectively, centrifuge at 10000rpm for 5min, remove the supernatant, add an equal amount of distilled water to resuspend, use a UV spectrophotometer to measure OD600, and draw a growth curve. The growth of the starting strain S.cerevisiae and the mutant strain S.cerevisiae Y1 under selenium-free and selenium-containing conditions is shown in Figure 2. Figure 1 The growth of the induced strain S.cerevisiae Y1 was better than that of the starting strain S.cerevisiae under both conditions.

[0043] The preparation method of the liquid synthetic culture medium containing 100 μg / mL sodium selenite is as follows: sodium selenite is added to the liquid synthetic culture medium to a final concentration of 100 μg / mL.

[0044] Example 2: Construction of high selenium-tolerant selenium-enriched yeast using adaptive evolution technology

[0045] (1) Adaptive evolution of the mutant strain S. cerevisiae Y1

[0046] The mutagenic strain S.cerevisiae Y1 was activated (liquid synthetic medium, 30°C, 150rpm, 18h), and the activated bacterial liquid was transferred to 50mL / 250mL of fresh 25% inhibitory concentration sodium selenite liquid synthetic medium at a 10% inoculation volume. After each 12h of culture, it was transferred to a fresh 25% inhibitory concentration sodium selenite liquid synthetic medium at a 10% inoculation volume until the growth of the strain was significantly improved. This process involved 60 batches of transfer culture. It was then transferred to a fresh 50% inhibitory concentration sodium selenite liquid synthetic medium for continued transfer culture. After 60 transfer cultures, an adaptive evolution strain was obtained, named S.cerevisiae TSE 001, and preserved in the General Microbiology Center of the China Microbiological Culture Collection Administration. The biomass of the strain during the subculture process was detected and the organic selenium content of the strain was randomly measured. The test results are shown in the table. Figure 2 .

[0047] The preparation method of the 25% inhibition concentration sodium selenite liquid synthetic culture medium is as follows: 20g / L anhydrous glucose, 2g / L potassium dihydrogen phosphate, 1g / L magnesium sulfate, 1g / L ammonium sulfate, 1g / L yeast extract powder, 88mg / L sodium selenite, 115°C, 20min.

[0048] The preparation method of the 50% inhibition concentration sodium selenite liquid synthetic culture medium is as follows: 20 g / L anhydrous glucose, 2 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, 1 g / L ammonium sulfate, 1 g / L yeast extract powder, 133.8 mg / L sodium selenite, 115° C., 20 min.

[0049] The strain biomass detection method is as follows: 50 mL of bacterial liquid after each generation of transfer culture is taken, centrifuged at 8000 rpm for 5 minutes, the supernatant is removed, and the mixture is washed three times with sterile saline, freeze-dried to prepare a powder, and the weight is weighed to obtain the strain biomass.

[0050] The method for determining the organic selenium content of the strain is as follows: the determination is performed according to the fluorescence spectrophotometry method in the national standard GB5009.93-2017, wherein the setting parameters of the fluorescence spectrophotometer are: fixed excitation wavelength; chopping rate: 40; excitation wavelength: 376nm; emission wavelength: 520nm; excitation unit slit: 2.5nm; emission unit slit: 1.0nm; phototube negative high voltage: 700V.

[0051] (2) Comparison of fermentation performance between the evolved strain and the starting strain

[0052] The starting strain S. cerevisiae and the evolved strain S. cerevisiae TSE 001 were activated and cultured respectively, and fermentation parameters such as growth curve, organic selenium content and organic selenium conversion rate were detected according to the above-mentioned method. The growth curve results are shown in Figure 3 , organic selenium content and conversion rate see Figure 4 The results showed that the biomass, organic selenium content and organic selenium conversion rate of the adaptively evolved strain S.cerevisiae TSE 001 were significantly higher than those of the starting strain S.cerevisiae, which were 11.5 times, 4.71 times and 11.6 times higher, respectively, indicating that it had a stronger fermentation performance advantage.

[0053] Example 3: Genetic stability test of high selenium tolerance selenium-enriched yeast

[0054] The S.cerevisiae TSE 001 strain was activated and subcultured for 6 generations at 12h intervals. The resulting strain was named S.cerevisiae X60, and its growth curve and organic selenium content were tested. Figure 3 , organic selenium content and conversion rate results refer to Figure 4 The results showed that the biomass, organic selenium content and conversion rate of S.cerevisiae X60 increased by 8.7%, decreased by 6.1% and increased by 3.4% respectively compared with the evolved strain S.cerevisiae TSE 001. The change range was within 10%, indicating that there was a certain stability between the strains.

[0055] This study, using a combination of mutagenesis breeding and laboratory adaptive evolution, has successfully constructed a strain of Saccharomyces cerevisiae (S. cerevisiae TSE 001) with high selenium tolerance, strong selenium enrichment capacity, and stable genetic traits. This research provides reliable technical support for the safe, efficient, and large-scale production of organic selenium products. It effectively addresses core technical bottlenecks in the production of traditional selenium-enriched yeast, such as poor strain tolerance, low selenium conversion efficiency, and genetic instability. It has broad industrial application value and potential for widespread promotion.

Claims

1. A selenium-enriched yeast strain with high inorganic selenium tolerance, characterized by: The yeast is named S. cerevisiae TSE 001 and is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No. 34111.

2. A method for producing selenium-enriched yeast using the S.cerevisiae TSE 001 strain according to claim 1, characterized in that The following steps are involved: (1) S. cerevisiae TSE 001 was inoculated into a liquid synthetic culture medium rich in carbon source, nitrogen source, and an appropriate amount of inorganic selenium; (2) Cultivate at 30°C and 150 rpm to the target biomass; (3) Selenium-enriched yeast powder is prepared after freeze-drying.

3. The method according to claim 2, wherein the components of the culture medium include: Anhydrous glucose 20g / L, potassium dihydrogen phosphate 2g / L, magnesium sulfate 1g / L, ammonium sulfate 1g / L, yeast extract powder 1g / L, inorganic selenium salt 88-133.8mg / L.

4. The method according to claim 3, characterized in that The inorganic selenium salt is sodium selenite or potassium selenite.

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