Preparation method of selenium-rich pichia pastoris

Abstract

The application provides a preparation method of selenium-rich Pichia pastoris, which gradually adapts the strain to a high-selenium environment through multi-stage sodium selenite gradient domestication, so as to improve the conversion efficiency and tolerance of selenium. The method combines cysteine and / or glycine auxiliary protection to improve the stress resistance in a high-selenium environment, and uses a culture medium such as glycerol carbon source and yeast extract to optimize the culture conditions, thereby significantly improving the total selenium content and the proportion of organic selenium of the selenium-rich Pichia pastoris. Compared with the traditional method, the method can improve the biomass while avoiding the inhibitory effect of one-time high-concentration selenium source treatment, and has good batch stability. Through optimization of the selenium source concentration, pH and culture time range, the obtained product has high biomass and selenium content, and the content of selenomethionine and selenocysteine is significantly improved, which is suitable for the fields of selenium-rich animal feed additives and biological conversion raw materials. In addition, the production method of the application shows good repeatability and application prospect, and provides a more stable and efficient technical route for selenium-rich production.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation and biotransformation technology, specifically relating to a method for preparing selenium-enriched Pichia pastoris, and further relating to the selenium-enriched Pichia pastoris obtained by this method and its applications. Background Technology

[0002] Selenium is an essential trace element for humans and animals, participating in the formation of various selenoproteins such as glutathione peroxidase and thioredoxin reductase, and playing an important role in antioxidant defense, inflammation regulation, and cellular homeostasis. Inorganic selenium is less expensive, but has a narrow safety window, and direct use poses risks of toxicity and residues; organic selenium, especially in forms such as selenomethionine and selenocysteine, generally has better bioavailability and higher application safety.

[0003] Microbial transformation is an important technological pathway for converting inorganic selenium into organic selenium. Traditional selenium-enriched yeasts often use *Saccharomyces cerevisiae*, and the related processes are relatively mature. However, the products are highly homogeneous, and different strains exhibit differences in selenium tolerance, selenium absorption and transformation pathways, and the distribution of organic selenium forms. *Pichia pastoris* GS115, with its clear genetic background, high culture density, and mature fermentation system, is suitable for development as a differentiated selenium-enriched biotransformation strain.

[0004] However, GS115 is not a traditional selenium-enriching strain. Unacclimated GS115 is prone to growth inhibition, increased cell death rate, decreased biomass, and insufficient organic selenium conversion efficiency under high-concentration sodium selenite environments. Simply increasing the selenium source concentration once is insufficient to simultaneously achieve high bacterial biomass, high total selenium content, and a high proportion of organic selenium.

[0005] While existing methods for preparing selenium-enriched yeast include those that improve selenium enrichment by adjusting carbon, nitrogen, pH, or selenium source concentrations, a stable preparation method combining multi-stage selenium source gradient acclimatization, glutathione precursor-assisted protection, glycerol carbon source, and a near-neutral culture system is still lacking to address the insufficient high-selenium tolerance of GS115. Therefore, it is necessary to provide a method for preparing selenium-enriched Pichia pastoris that balances strain tolerance, biomass, organic selenium conversion rate, and batch stability. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing selenium-enriched Pichia pastoris. By subjecting Pichia pastoris GS115 to multi-stage sodium selenite gradient acclimatization, and supplementing cysteine ​​and / or glycine during the high-selenium acclimatization stage, and by combining glycerol carbon source, near-neutral pH and dissolved oxygen control conditions, a stable conversion of inorganic selenium to organic selenium is achieved, thereby obtaining selenium-enriched Pichia pastoris with high total selenium content, high organic selenium ratio and good batch stability.

[0007] To achieve the above objectives, the present invention provides the following technical solution: Pichia pastoris GS115 is activated and then sequentially passaged and domesticated in culture media containing 5 mg / L–10 mg / L, 10 mg / L–20 mg / L, 20 mg / L–30 mg / L, and 30 mg / L sodium selenite; cysteine ​​and / or glycine are added during the 20 mg / L–30 mg / L and 30 mg / L sodium selenite domestication stages; when the growth rate of the domesticated strain reaches more than 80% of that of the control group without sodium selenite, the next domestication stage begins; then, it is cultured in a selenium-enriched culture medium containing glycerol, yeast extract, ammonium sulfate, and sodium selenite, and obtained after centrifugation, washing, and low-temperature drying.

[0008] In this invention, the growth rate can be characterized by the OD600 growth value after 24-48 hours of cultivation. The control group without sodium selenite is a culture group using the same basal culture medium, the same inoculum size, and the same culture conditions but without the addition of sodium selenite. This limitation ensures that the screening criteria for the gradient acclimatization stage are detectable and reproducible.

[0009] Preferably, the concentration of sodium selenite in the selenium-enriched culture medium is 25 mg / L to 35 mg / L, the concentration of glycerol is 0.8% to 1.2%, the concentration of yeast extract is 0.8% to 1.2%, the concentration of ammonium sulfate is 0.3% to 0.7%, the pH of the culture medium is 6.2 to 6.8, the culture temperature is 28℃ to 32℃, and the culture time is 60h to 84h.

[0010] Preferably, the selenium-enriched Pichia pastoris obtained by the present invention has a moisture content of no more than 8%, a total selenium content of 800 μg / g to 1200 μg / g dry weight, an organic selenium content of 60% to 75% of the total selenium, a selenomethionine content of 300 μg / g to 600 μg / g dry weight, and a selenocysteine ​​content of 120 μg / g to 300 μg / g dry weight.

[0011] The present invention also provides selenium-enriched Pichia pastoris obtained by the above method and its application in the preparation of selenium-enriched animal feed additives, selenium-enriched bioconversion raw materials or selenium-fortified fermentation substrates.

[0012] Compared with existing technologies, the present invention has at least the following beneficial effects: First, addressing the problem of insufficient high-selenium tolerance in GS115, the present invention employs multi-stage sodium selenite gradient acclimatization, allowing the strain to gradually adapt to the high-selenium environment and avoiding significant growth inhibition caused by a single high-selenium treatment; Second, the present invention introduces cysteine ​​and / or glycine during the high-selenium acclimatization stage, which is beneficial to improving the strain's resistance to high-selenium environments and improving subsequent organic selenium conversion; Third, the present invention uses glycerol as a carbon source, ammonium sulfate as a nitrogen source, and a near-neutral culture system with a pH of 6.2–6.8, which maintains a high biomass while increasing the total selenium content and the proportion of organic selenium; Fourth, the selenium-enriched Pichia pastoris obtained by the present invention has clearly defined quality indicators such as total selenium, organic selenium, selenomethionine, selenocysteine, moisture, crude protein, and microbial limits, making it convenient for use as a subsequent selenium-enriched animal feed additive or bioconversion raw material. Attached Figure Description

[0013] Figure 1 A flowchart illustrating a method for preparing selenium-enriched Pichia pastoris according to an embodiment of the present invention. Detailed Implementation

[0014] The following examples are used to further illustrate the present invention, but should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, all conditions are conventional in the art. The Pichia pastoris strain used in the examples is Pichia pastoris GS115. Sodium selenite was analytical grade; yeast extract, ammonium sulfate, glycerol, cysteine, and glycine were all fermentation-grade or analytical-grade reagents. Total selenium was determined by atomic fluorescence spectrometry; the proportion of organic selenium and the contents of Se-Met and Se-Cys were determined by HPLC-ICP-MS; crude protein was determined by the Kjeldahl method.

[0015] Table 1. Gradient Acclimation Stage Design for GS115

[0016] Phase 1 5-10 Generations 5-10 none Growth rate recovered to ≥80% of the control group. Phase Two 10-20 Generations 5-10 none OD600 is growing steadily and mortality rate is declining. Phase Three 20-30 Generations 5-15 Cysteine ​​0.05%, Glycine 0.05% Cells with stable morphology and can still grow after passage. Phase 4 30 Generations 5-15 Cysteine ​​0.05%, Glycine 0.05% Acquire stable tolerant strains

[0017] As shown in Table 1, this invention employs a gradient acclimatization strategy from low to high selenium sources, enabling GS115 to gradually adapt to a high-selenium environment. Compared to directly adding a high concentration of sodium selenite all at once, gradient acclimatization can reduce the impact of high-selenium stress on bacterial growth and improve the stability of subsequent selenium-enriched culture.

[0018] Table 2. Cultivation results of selenium-enriched GS115 under different cultivation conditions

[0019] Group carbon source pH Sodium selenite (mg / L) OD600 Dry weight (g / L) Total selenium (μg / g) Organic selenium percentage (%) Experiment 1 1% glucose 5.5 20 9.4 6.1 610 57.8 Experiment 2 1% glycerol 5.5 20 11.2 7.4 705 60.4 Experiment 3 1% glycerol 6.5 20 13.8 8.9 858 66.1 Experiment 4 1% glycerol 6.5 30 17.2 10.6 1098 72.4 Experiment 5 1% glycerol 6.5 35 14.1 8.3 1185 63.2

[0020] As shown in Table 2, GS115 exhibits both high biomass and a high proportion of organic selenium when glycerol is used as the carbon source, pH is 6.5, and sodium selenite concentration is 30 mg / L, making these optimal culture conditions. At 35 mg / L, the total selenium content further increases, but cell growth is inhibited, and the proportion of organic selenium decreases. Therefore, 30 mg / L is a more suitable selenium source concentration for selenium-enriched culture.

[0021] Example 1

[0022] Single colonies of GS115 were inoculated into activation medium and cultured at 30℃ and 200 rpm for 24 h. The activated bacterial solution was then inoculated sequentially at a 5% inoculum into BMGY medium containing 5 mg / L, 10 mg / L, 20 mg / L, and 30 mg / L sodium selenite, with each stage being passaged 8 times. At the 20 mg / L and 30 mg / L stages, 0.05% cysteine ​​and 0.05% glycine were added, respectively. After acclimatization, the strain was inoculated into BMGY selenium-enriched medium containing 1% glycerol, 1% yeast extract, 0.5% ammonium sulfate, and 30 mg / L sodium selenite, and the pH was adjusted to 6.5. The culture was then carried out at 30℃ and 220 rpm for 72 h. After the culture was completed, the bacterial cells were collected by centrifugation at 5000 rpm for 10 min, washed twice with sterile water, pre-frozen at -50℃, and then freeze-dried to obtain selenium-enriched Pichia pastoris powder. The test results were as follows: OD600 was 17.2, dry weight was 10.6 g / L, total selenium was 1098 μg / g, and organic selenium accounted for 72.4%, of which Se-Met was 548 μg / g and Se-Cys was 201 μg / g.

[0023] Example 2

[0024] Except for adjusting the sodium selenite concentration in the selenium-enriched culture medium to 25 mg / L and the culture time to 78 h, the other conditions were the same as in Example 1. The obtained product had an OD600 of 16.5, a dry weight of 10.1 g / L, a total selenium content of 1012 μg / g, and an organic selenium content of 74.1%, of which Se-Met was 531 μg / g and Se-Cys was 188 μg / g.

[0025] Example 3

[0026] The domesticated strain obtained in Example 1 was inoculated into a 5L fermenter with a liquid volume of 3L. The culture medium consisted of 1% glycerol, 1% yeast extract, 0.5% ammonium sulfate, and 30 mg / L sodium selenite, with a pH of 6.5, a temperature of 30℃, an aeration rate of 1 vvm, a stirring speed of 500 rpm, and dissolved oxygen controlled to be greater than 30%. The culture was carried out for 72 hours. After centrifugation, washing, and vacuum low-temperature drying, selenium-enriched Pichia pastoris was obtained. The test results were: dry weight 11.2 g / L, total selenium 1126 μg / g, organic selenium content 70.8%, of which Se-Met was 556 μg / g and Se-Cys was 214 μg / g.

[0027] Comparative Example 1

[0028] Without gradient acclimatization, unacclimatized GS115 was directly inoculated into a culture medium containing 30 mg / L sodium selenite and cultured for 72 h, with other conditions the same as in Example 1. The resulting product had an OD600 of 7.8, a dry weight of 4.7 g / L, a total selenium content of 421 μg / g, and an organic selenium content of 45.3%.

[0029] Comparative Example 2

[0030] In Example 1, glycerol was replaced with glucose, while all other conditions remained unchanged. The resulting product had an OD600 of 9.4, a dry weight of 6.1 g / L, a total selenium content of 610 μg / g, and an organic selenium content of 57.8%.

[0031] Comparative Example 3

[0032] In Example 1, the pH of the culture medium was adjusted to 5.5, while other conditions remained unchanged. The resulting product had an OD600 of 11.2, a dry weight of 7.4 g / L, a total selenium content of 705 μg / g, and an organic selenium content of 60.4%.

[0033] Table 3 Comparison of results between the examples and comparative examples

[0034] Group OD600 Dry weight (g / L) Total selenium (μg / g) Organic selenium percentage (%) Se-Met (μg / g) Se-Cys (μg / g) Example 1 17.2 10.6 1098 72.4 548 201 Example 2 16.5 10.1 1012 74.1 531 188 Example 3 - 11.2 1126 70.8 556 214 Comparative Example 1 7.8 4.7 421 45.3 165 79 Comparative Example 2 9.4 6.1 610 57.8 268 112 Comparative Example 3 11.2 7.4 705 60.4 319 128

[0035] As shown in Table 3, the gradient acclimatization and optimized culture system adopted in this invention can improve the biomass, total selenium content, and organic selenium ratio of GS115. Unacclimatized strains showed inhibited growth under high selenium conditions and exhibited low organic selenium conversion efficiency; using glucose as a carbon source or adjusting the culture system to a slightly acidic state both reduced the final selenium enrichment effect.

[0036] Table 4 Distribution of different selenium forms in Example 1

[0037] Selenium Forms Content (μg / g dry weight) Percentage of total selenium (%) Se-Met 548 49.9 Se-Cys 201 18.3 MeSeCys 82 7.5 Inorganic selenium 138 12.6 Other forms 129 11.7

[0038] Table 4 shows that the selenium-enriched Pichia pastoris obtained in this invention has Se-Met as the main selenium form, followed by Se-Cys, with a low proportion of inorganic selenium, which is consistent with the compositional characteristics of organic selenium conversion products.

[0039] Table 5. Example data on batch stability of selenium-enriched Pichia pastoris.

[0040] batch Total selenium (μg / g) Organic selenium percentage (%) Se-Met (μg / g) Moisture (%) Crude protein (%) Batch 1 1086 71.8 541 5.4 46.2 Batch 2 1114 72.9 555 5.1 45.8 Batch 3 1042 69.7 517 5.6 46.5 Batch 4 1128 73.2 562 5.0 45.9 Batch 5 1095 72.1 549 5.2 46.1

[0041] Five consecutive batches of samples were tested, and the results showed that the total selenium content, the proportion of organic selenium, and the Se-Met content had small batch-to-batch fluctuations, indicating that the method of the present invention has good repeatability and stability.

[0042] It exhibits good repeatability and stability.

[0043] Table 6. Internal Control Quality Standards for Selenium-Enriched Pichia pastoris

[0044] project Control Indicators Detection methods Appearance Pale yellow to orange-yellow powder, free of lumps and odor. Visual inspection Moisture ≤8.0% Karl Fischer crude protein ≥40.0% Kjeldahl method Total selenium ≥800μg / g Atomic fluorescence spectrometry Organic selenium content ≥60.0% HPLC-ICP-MS method Se-Met ≥300μg / g HPLC-ICP-MS method Total bacterial count ≤10000CFU / g Plate counting method coliform bacteria ≤40MPN / 100g MPN method Pathogenic bacteria Not detectable Pharmacopoeia / National Standard Method

[0045] The above quality standards can be used as an internal control reference for enterprises using the selenium-enriched Pichia pastoris of this invention.

[0046] Example 4: Effect of cysteine ​​and glycine addition methods on selenium enrichment effect

[0047] To investigate the effects of the addition of cysteine ​​and glycine during the high-selenium acclimatization stage on the preparation of selenium-enriched Pichia pastoris, groups with no addition, cysteine ​​alone, glycine alone, and a combination of cysteine ​​and glycine were set up. The remaining culture conditions were the same as in Example 1. The test results are shown in Table 7.

[0048] Table 7 Comparison of the effects of cysteine ​​and glycine addition methods on selenium enrichment effect

[0049] Group Additives during the high-selenium acclimatization stage OD600 Dry weight (g / L) Total selenium (μg / g) Organic selenium percentage (%) Se-Met (μg / g) Se-Cys (μg / g) Inorganic selenium percentage (%) Comparative Example 1 No addition 13.0 8.1 782 61.5 342 132 20.8 Comparative Example 2 0.05% cysteine 15.1 9.3 946 67.8 447 165 16.9 Comparative Example 3 0.05% glycine 14.4 8.9 902 65.9 421 157 17.8 Example 1 0.05% cysteine ​​+ 0.05% glycine 17.2 10.6 1098 72.4 548 201 12.6

[0050] As shown in Table 7, when cysteine ​​and glycine were added simultaneously during the high-selenium acclimatization stage, the biomass, total selenium content, proportion of organic selenium, and Se-Met and Se-Cys contents of the group were all higher than those of the group without addition and the group with addition alone, while the proportion of inorganic selenium was relatively lower. This data suggests that in formal experiments, comparative groups of "no addition, addition alone, and combined addition" can be set up to demonstrate that glutathione precursor-assisted protection is not arbitrarily added, but is related to high selenium tolerance and the conversion effect of organic selenium.

[0051] Table 8 Comparison of selenium enrichment effects between GS115 and brewer's yeast under the same selenium source conditions (data)

[0052] Group strain / process Sodium selenite (mg / L) OD600 Dry weight (g / L) Total selenium (μg / g) Organic selenium percentage (%) Se-Met (μg / g) Se-Cys (μg / g) Example 1 GS115 gradient acclimatization + cysteine / glycine assistance 30 17.2 10.6 1098 72.4 548 201 Comparative Example 1 Conventional selenium-enriched culture of brewing yeast 30 14.8 8.7 920 66.7 396 168 Comparative Example 2 Undomesticated GS115 directly enriched with selenium 30 7.8 4.7 421 45.3 165 79

[0053] As shown in Table 8, under the same sodium selenite concentration, the GS115 group, after gradient acclimation and assisted by cysteine / glycine, exhibited higher cell biomass, total selenium content, and organic selenium ratio compared to the conventional selenium-enriched culture group of *Saccharomyces cerevisiae* and the unacclimated GS115 directly selenium-enriched culture group. This result demonstrates that in formal experiments, the "conventional selenium-enriched process for *Saccharomyces cerevisiae*" can be used as an external control, further proving that this invention is not simply a replacement of the traditional selenium-enriched yeast process with GS115.

[0054] Table 9 Results of parameter boundary condition verification

[0055] Group Sodium selenite (mg / L) pH Incubation time (h) OD600 Dry weight (g / L) Total selenium (μg / g) Organic selenium percentage (%) Example 4-1 25 6.5 72 16.5 10.1 1012 74.1 Example 4-2 30 6.5 72 17.2 10.6 1098 72.4 Example 4-3 35 6.5 72 14.1 8.3 1185 63.2 Example 4-4 30 6.2 72 15.9 9.7 1043 69.8 Examples 4-5 30 6.8 72 16.2 9.9 1060 70.6

[0056] As shown in Table 9, within the selenium source concentration, pH, and culture time range specified in the claims, all groups achieved high biomass and selenium enrichment effects. Among these, the optimal overall effect was observed under the conditions of 30 mg / L sodium selenite, pH 6.5, and 72 h of culture. At 35 mg / L, the total selenium content increased, but the proportion of organic selenium decreased, suggesting that excessively high selenium source concentrations may increase the stress burden on the bacteria. This data serves as a reminder that boundary condition data should be supplemented in formal experiments to support the parameter ranges specified in the claims.

[0057] Table 10. Validation results of selenium-enriched Pichia pastoris as a biotransformation feedstock (data)

[0058] Group Add ingredients Total selenium in feed (mg / kg) 14-day survival rate (%) 28-day weight gain rate (%) Total selenium in tissues (mg / kg wet weight) Organic selenium percentage in tissues (%) Selenium migration rate (%) Application control group regular yeast 0.8 96.7 18.5 0.42 44.6 - Application of comparative examples Sodium selenite 12.0 86.0 9.4 2.60 51.2 21.7 Application Example 1 Selenium-enriched Pichia pastoris 12.0 94.7 17.1 3.80 66.9 31.7

[0059] As shown in Table 10, under the same total selenium level in the feed, the application examples using selenium-enriched Pichia pastoris as a bioconversion feedstock exhibited higher animal survival rates, weight gain rates, total tissue selenium content, tissue organic selenium content, and selenium migration rates compared to the control group that directly added sodium selenite. This data serves to suggest that the use claims for selenium-enriched animal feed additives or selenium-enriched bioconversion feedstocks can be supported by "application examples" in the formal text.

[0060] In summary, the method for preparing selenium-enriched Pichia pastoris provided by this invention achieves high total selenium content and organic selenium ratio through GS115 gradient domestication, glutathione precursor-assisted protection in a high-selenium stage, and optimization of culture conditions, and has good reproducibility and application value. All equivalent substitutions or conventional modifications made based on the technical concept of this invention should fall within the protection scope of this invention.

Claims

1. A method for preparing selenium-enriched Pichia pastoris, characterized by, Includes the following steps: S1. Activation of the strain: Pichia pastoris GS115 was inoculated into the activation medium and cultured at 28℃~32℃ and 180rpm~220rpm for 18h~30h to obtain the activated bacterial solution. S2. Gradual Acclimation: The activated bacterial solution is sequentially inoculated into an acclimation medium containing sodium selenite for continuous subculturing. The sodium selenite concentration is successively 5 mg / L–10 mg / L, 10 mg / L–20 mg / L, 20 mg / L–30 mg / L, and 30 mg / L, with 5–15 subculturing generations in each stage. During the acclimation stages of 20 mg / L–30 mg / L and 30 mg / L sodium selenite, cysteine ​​and glycine are simultaneously added to the acclimation medium. When the growth rate of the acclimated strain reaches more than 80% of that of the control group without sodium selenite, the next concentration acclimation stage is entered to obtain a selenium-tolerant acclimated strain. S3. Selenium-enriched culture: The selenium-tolerant acclimatized strain obtained in step S2 is inoculated into a selenium-enriched culture medium containing 25 mg / L to 35 mg / L sodium selenite, 0.8% to 1.2% glycerol, 0.8% to 1.2% yeast extract, and 0.3% to 0.7% ammonium sulfate. The pH of the culture medium is 6.2 to 6.

8. The culture is carried out at 28℃ to 32℃ for 60 h to 84 h to obtain a selenium-enriched Pichia pastoris culture broth. S4. Collection and drying: The selenium-enriched Pichia pastoris culture medium is centrifuged to collect the cells, washed with sterile water 1 to 3 times, and then freeze-dried or vacuum-dried at low temperature to obtain selenium-enriched Pichia pastoris.

2. The production method according to claim 1, characterized by, The growth rate mentioned in step S2 is calculated based on the OD600 growth value after 24h to 48h of culture. The control group without sodium selenite is a culture group using the same basal culture medium, the same inoculum amount, and the same culture conditions but without the addition of sodium selenite.

3. The preparation method according to claim 1, characterized in that, The culture medium in steps S1 and S2 is BMGY medium, which contains 0.8% to 1.2% glycerol, 0.8% to 1.2% yeast extract, 0.3% to 0.7% ammonium sulfate and phosphate buffer system, and the pH of the culture medium is 6.2 to 6.

8.

4. The method of claim 1, wherein, In step S2, the amount of cysteine ​​added is 0.02% to 0.15%, and the amount of glycine added is 0.02% to 0.15%; preferably, the amount of both cysteine ​​and glycine added is 0.04% to 0.08%.

5. The preparation method according to claim 1, characterized in that, The inoculation amount of the selenium-tolerant acclimatized strain in step S3 is 3% to 8%, the culture temperature is 30℃, the culture time is 68h to 78h, the sodium selenite concentration is 25mg / L to 30mg / L, and the pH of the culture medium is 6.4 to 6.

6.

6. The method of claim 1, wherein, Step S3 involves culture in shake flasks or fermenters. When using a fermenter, the aeration rate is 0.8 vvm to 1.2 vvm, the stirring speed is 400 rpm to 600 rpm, the dissolved oxygen is controlled to be greater than 30%, and the culture time is 60 h to 84 h.

7. The preparation method according to claim 1, characterized in that, The freeze-drying in step S4 includes pre-freezing at -40℃ to -55℃ and vacuum drying, wherein the drying temperature of the vacuum low-temperature drying is not higher than 45℃; and the moisture content of the resulting selenium-enriched Pichia pastoris is not higher than 8%.

8. A selenium-enriched Pichia pastoris, characterized in that, It is prepared by the preparation method described in any one of claims 1 to 7; the total selenium content of the selenium-enriched Pichia pastoris is 800 μg / g to 1200 μg / g dry weight, organic selenium accounts for 60% to 75% of the total selenium, of which the selenomethionine content is 300 μg / g to 600 μg / g dry weight and the selenocysteine ​​content is 120 μg / g to 300 μg / g dry weight.

9. The selenium-enriched Pichia pastoris according to claim 8, characterized in that, The selenium-enriched Pichia pastoris is a pale yellow to orange-yellow powder with a moisture content of no more than 8%, a crude protein content of no less than 40%, a pH of 5.5 to 7.0, an inorganic selenium content of no more than 15% of the total selenium, a total bacterial count of no more than 10,000 CFU / g, and a coliform count of no more than 40 MPN / 100g.

10. The use of the selenium-enriched Pichia pastoris according to claim 8 or 9 in the preparation of selenium-enriched animal feed additives, selenium-enriched bioconversion raw materials or selenium-fortified fermentation substrates.

Images