A selenium-rich saccharomyces cerevisiae strain, a screening method and application thereof

Abstract

The application discloses a kind of selenium-rich saccharomyces cerevisiae strains and its screening method and application, it is related to microbial fermentation technical field, the strain includes at least one of saccharomyces cerevisiae ZKCM-1252, ZKCM-1251 and ZKCM-33074;The screening method is obtained by gradient resistance plate preliminary screening, ion beam mutagenesis and adaptive evolution combination strategy, and excellent strain with high selenium tolerance and high selenium-rich capacity, wherein preferably strain ZKCM-1252 is prepared Yeast selenium total selenium content can reach 5266.59 μg / g, and organic selenium proportion reaches 95.51%;The organic selenium proportion of ZKCM-33074 is as high as 99.46%.The strain screened in the application has excellent selenium enrichment performance, and the selenium peptide prepared has the dual biological activity of selenium and peptide, and can be widely applied in food, feed and health products and other fields.

Description

Technical Field

[0001] This invention relates to the fields of microbial fermentation and bioactive peptide technology, and in particular to a selenium-enriched Saccharomyces cerevisiae strain and its screening method and application. Background Technology

[0002] Currently, selenium supplements are mainly divided into two categories: inorganic selenium and organic selenium. Inorganic selenium (such as sodium selenite, chemical formula: ...) Although it is inexpensive and high in selenium, its absorption depends on liver conversion, and it has a narrow toxicity threshold, posing safety risks with long-term or excessive intake. In contrast, selenium-enriched yeast, as an important source of organic selenium, has significant advantages such as high absorption and utilization rate, low toxicity, and good safety, and is considered an ideal selenium supplement.

[0003] However, existing selenium-enriched yeast production technologies still have significant shortcomings: On the one hand, conventional brewing yeast strains generally have low tolerance and conversion efficiency to inorganic selenium, and the organic selenium content in the fermented yeast is usually only 2000~3000μg / g, which is insufficient to meet the market demand for high-selenium-enriched products; on the other hand, existing strain screening methods mostly employ single chemical mutagenesis or domestication strategies, resulting in low screening efficiency, long cycles, and poor genetic stability of the obtained strains, making it difficult to achieve stable industrial production. Furthermore, using selenium-enriched yeast directly as a selenium supplement presents problems such as selenium existing in the form of large protein molecules, and the need to improve human digestion and absorption efficiency. Further enzymatically hydrolyzing selenium-enriched yeast protein into low-molecular-weight selenopeptides can combine the dual biological activities of selenium and peptides, but currently there are few reports on systematic preparation methods for selenium-enriched yeast peptides, lacking a complete technical solution from strain screening to selenopeptide preparation.

[0004] To address the above problems, this invention proposes a selenium-enriched Saccharomyces cerevisiae strain, its screening method, and its application. Summary of the Invention

[0005] The purpose of this invention is to provide a selenium-enriched Saccharomyces cerevisiae strain and its screening method and application, in order to solve the technical problems of low selenium conversion efficiency, poor growth performance at high selenium concentrations, long screening cycle and low throughput, and low organic selenium content in existing selenium-enriched Saccharomyces cerevisiae strains.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: A selenium-enriched Saccharomyces cerevisiae strain, wherein the strain is at least one of the following strains: Saccharomyces cerevisiae with accession number CGMCC No. 34511 Saccharomyces cerevisiae ZKCM-1252; Saccharomyces cerevisiae ZKCM-1251 with accession number CGMCC No.38811; The brewing yeast ZKCM-33074 with accession number CGMCC No.38812.

[0007] The deposit date of ZKCM-1252 is May 12, 2025; The deposit date for ZKCM-1251 and ZKCM-33074 is May 21, 2026; All of the above strains were deposited at the China General Microbiological Culture Collection Center (CGMCC) prior to the application date, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0008] A method for screening selenium-enriched Saccharomyces cerevisiae strains includes the following steps: Step S1: Inoculate the yeast strain to be screened onto MEB solid medium and culture at 30°C until colonies grow. Then transfer it to MEB liquid medium and culture at 30°C and 180-250 r / min for 16-24 h to obtain activated yeast culture. Step S2: The solution was filtered and sterilized, then added to MEB solid culture medium that remained liquid even after sterilization. The final concentrations were set in a gradient of 100 μg / mL, 200 μg / mL, 400 μg / mL, 800 μg / mL, 1600 μg / mL, and 3200 μg / mL. After mixing, the mixture was poured into plates to prepare selenium-containing resistance plates. Step S3: Spread the activated yeast culture evenly on selenium-resistant plates and selenium-free control plates, incubate at 30°C, and screen for strains that can grow in the medium with the highest relative selenium concentration and colony diameter ≥2mm to obtain the initial screening strains. Step S4: The primary screening strains obtained in step S3 were transferred to a solution containing 200 μg / mL of [a specific solution / concentration]. In MEB liquid medium, after shaking culture at 30℃, 220r / min and 48h, the biomass and total intracellular selenium content were measured. The dominant strains were selected with biomass ≥5g / L and total intracellular selenium content ≥800μg / g as the standard. Step S5: Prepare bacterial suspensions from the dominant strains selected in Step S4, and perform ion beam mutagenesis treatment with different injection doses. Spread the mutagenized bacterial suspensions at each dose onto a substrate containing 200 μg / mL of ion beam mutagenesis solution. On YEPD solid medium plates, the culture was carried out at 30°C. Single colonies with fast growth rate and colony diameter ≥2mm were selected to obtain positive mutant strains. Step S6: Inoculate the positive mutant strain obtained in step S5 with a solution containing 200 μg / mL After culturing in YEPD liquid medium at 30℃ and 220 r / min for 24 h, the culture was transferred to fresh selenium-containing YEPD medium at a volume ratio of 5%. The inoculum was then increased every 20 generations. The concentration was increased by 100 μg / mL, and the transfer operation was repeated continuously to obtain a selenium-enriched Saccharomyces cerevisiae strain.

[0009] Preferably, the MEB liquid culture medium in step S1 comprises malt extract, maltose, glucose and yeast extract, and the pH of the culture medium is 4.7±0.2.

[0010] Preferably, in step S5, ion beam induction is performed. Ion implantation, implantation energy of 10 keV, implantation doses are as follows: , and .

[0011] Preferably, the number of repeated switching operations in step S6 is 80 to 100 times, with 90 times being optimal.

[0012] The application of a selenium-enriched Saccharomyces cerevisiae strain as described above in the preparation of yeast selenium includes the following steps: Step L1: The above-mentioned selenium-enriched Saccharomyces cerevisiae strain was inoculated into MEB medium for activation and cultured to the logarithmic phase to obtain the activated bacterial solution; Step L2: Inoculate the activated bacterial solution at a volume ratio of 5% into MEB liquid medium, and culture with shaking at 30℃ and 220r / min. Step L3: After culturing for 6 hours, add [the following solution] to a final concentration of 25 μg / mL. The solution was cultured for another 48 hours to obtain a yeast culture medium. Step L4: Collect yeast cells by centrifugation from yeast culture medium, wash with deionized water by centrifugation, and dry to constant weight to obtain yeast selenium.

[0013] Preferably, the MEB liquid culture medium contains malt extract, maltose, glucose and yeast extract, and the pH of the culture medium is 4.7±0.2.

[0014] The application of a selenium-enriched Saccharomyces cerevisiae strain as described above in the preparation of selenium-enriched yeast selenopeptides includes the following steps: Step M1: Inoculate the selenium-enriched Saccharomyces cerevisiae strain into MEB medium for activation culture; Step M2: Inoculate the activated bacterial solution into MEB liquid medium at a volume ratio of 10% and culture at 30℃ and 220r / min with shaking. Step M3: When the bacterial solution... Add to a final mass concentration of 25 μg / mL. The solution was incubated for another 40 hours. Step M4: Collect bacterial cells by centrifugation at 8000 r / min, 4℃, and 10 min, and wash with pre-cooled PBS buffer to remove residual culture medium and external impurities; Step M5: Resuspend the washed cells in pre-cooled cell disruption buffer and mix thoroughly; Step M6: Place the cell suspension in a high-pressure homogenizer for pressure cell disruption; Step M7: Centrifuge the cell wall disruption solution to remove unbroken cell debris and large particulate impurities, and collect the supernatant; Step M8: Adjust the pH of the supernatant to 8.0, add trypsin, mix well, and incubate at 37°C with shaking for enzymatic hydrolysis; Step M9: Centrifuge to remove undissolved residue, collect the clarified liquid, perform ultrafiltration using an ultrafiltration device, collect the permeate, and obtain the product rich in low molecular weight selenium peptides.

[0015] Preferably, the cell disruption buffer in step M5 is 50mM Tris-HCl, 1mM EDTA, pH 8.0; the pressure of the high-pressure homogenizer in step M6 is 1200 bar, the cell disruption time is 20 min, and the cycle is 2-3 times.

[0016] Preferably, in step M8, the mass ratio of trypsin to substrate is 1:50, and the enzymatic hydrolysis time is 4-6 hours; in step M9, the ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 3 kDa.

[0017] Compared with the prior art, the beneficial technical effects of the present invention are as follows: The selenium tolerance of the strain obtained in this invention is significantly improved. Through high-concentration gradient screening combined with ion beam mutagenesis and adaptive evolution strategy, the obtained selenium-enriched Saccharomyces cerevisiae strain can still grow normally at a high selenium concentration of 3200 μg / mL, and its selenium tolerance is greatly improved compared with the starting strain.

[0018] The present invention has a high intracellular selenium content. The total intracellular selenium content and the proportion of organic selenium in the selenium-enriched Saccharomyces cerevisiae strain obtained through screening are significantly better than those of the existing selenium-enriched yeast strains, which can effectively meet the production needs of selenium-enriched foods and feed additives.

[0019] The screening method of this invention is highly efficient and reliable. It adopts a multi-stage screening strategy of "gradient resistance plate primary screening → liquid secondary screening → ion beam mutagenesis → long-term adaptive evolution". The process system is complete, with high screening throughput, short cycle and good repeatability and stability.

[0020] This invention utilizes products with high organic selenopeptide content. By applying selenium-enriched brewer's yeast strains to the preparation of yeast selenium and selenopeptide products, and through optimized fermentation conditions and enzymatic hydrolysis processes, a product rich in low molecular weight selenopeptides is obtained. The product has high selenopeptide content, small molecular weight, and good bioavailability.

[0021] This invention has broad industrial application prospects. The selenium-enriched brewer's yeast strain and its screening and application methods are suitable for large-scale industrial production and can be widely used in selenium-enriched foods, selenium-enriched feed additives, and selenium-enriched functional raw materials. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of a screening method for selenium-enriched brewer's yeast strains provided in an embodiment of the present invention; Figure 2 This is a schematic diagram illustrating the application process of a selenium-enriched Saccharomyces cerevisiae strain in the preparation of yeast selenium, provided in an embodiment of the present invention. Figure 3 This is a schematic diagram illustrating the application process of a selenium-enriched Saccharomyces cerevisiae strain in the preparation of selenium-enriched yeast selenopeptides, provided in an embodiment of the present invention. Figure 4 This is a linear fitting curve of selenium content and absorbance provided in an embodiment of the present invention; Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1: Screening and performance determination of selenium-enriched Saccharomyces cerevisiae strains Strain source: The starting strain was the Saccharomyces cerevisiae strain isolated from a certain soy sauce-flavored liquor koji sample (referring to the original wild-type strain without any mutagenesis or screening treatment), which was confirmed by 18S rDNA molecular biological identification.

[0025] The specific culture medium formula is as follows: 1L MEB solid medium: 20g malt extract, 15g maltose, 10g glucose, 3g yeast extract, 20g agar. Adjust the pH to 4.7±0.2 with 1mol / L hydrochloric acid (HCl) or sodium hydroxide (NaOH), and sterilize at 121℃ for 15min.

[0026] MEB liquid culture medium: The above formula does not include agar, but the rest is the same.

[0027] 1. LYEPD medium: 10g yeast extract, 20g peptone, 20g glucose, pH 5.5~6.0, no adjustment required, sterilize at 121℃ for 15min.

[0028] Selenium-containing culture medium: After sterilization of MEB solid medium and cooling to approximately 55°C, add selenium-containing medium that has been sterilized by filtration through a 0.22 μm membrane. The mother liquor was mixed and poured into plates to achieve final selenium concentrations of 100 μg / mL, 200 μg / mL, 400 μg / mL, 800 μg / mL, 1600 μg / mL, and 3200 μg / mL.

[0029] like Figure 1 As shown, the screening steps for selenium-enriched Saccharomyces cerevisiae strains are as follows: Step S1: Strain activation. The activated yeast strain was inoculated onto MEB solid medium and cultured at 30°C for 48 hours until single colonies appeared. Single colonies were then picked and inoculated onto MEB liquid medium and cultured at 30°C and 220 rpm for 18 hours on a shaker to obtain the activated yeast culture. From approximately 1.0.

[0030] Step S2: Prepare selenium-containing resistant plates. The solution was sterilized by filtration through a 0.22 μm filter membrane and added to MEB solid culture medium, which was still in liquid state after sterilization, to achieve final selenium concentrations of 100 μg / mL, 200 μg / mL, 400 μg / mL, 800 μg / mL, 1600 μg / mL, and 3200 μg / mL, respectively. After mixing, the solution was poured into plates to prepare selenium-containing resistance plates with different concentration gradients.

[0031] Step S3: Initial screening. Take 100 μL of the activated bacterial solution from Step S1 and spread it onto the solutions prepared in Step S2 containing different concentrations of bacteria. Three replicates were prepared for each concentration on MEB-resistant plates and selenium-free control plates, and the plates were incubated statically at 30°C for 72 hours. Colony growth on each plate was observed, and colonies with a diameter ≥2 mm that could still grow on plates with the highest relative selenium concentration were screened out. A total of 12 initial screening strains were obtained, with 800 μg / mL used in this example.

[0032] Step S4: Secondary screening – Selecting dominant strains. The 12 primary screening strains obtained in step S3 were inoculated separately into a solution containing 200 μg / mL... The cells were cultured in MEB liquid medium at 30℃ and 220 r / min for 48 h. The cells were collected by centrifugation, washed three times with deionized water, and dried to constant weight. Biomass (dry weight of cells) was determined, and the cells were digested using the nitric acid-perchloric acid digestion method (referring to GB 5009.93-2017). Intracellular total selenium content was determined by inductively coupled plasma mass spectrometry. Three dominant strains were selected based on a biomass ≥5 g / L and an intracellular total selenium content ≥800 μg / g, and were designated ZKCM-1252, ZKCM-1251, and ZKCM-33074, respectively.

[0033] Step S5: Ion beam mutagenesis and screening of positive mutants. The three dominant strains selected in step S4 were prepared into strains with concentrations of approximately [missing information]. A bacterial suspension of 100 μL was spread onto a sterile empty petri dish and dried under sterile air to form a bacterial film. Ion implantation was then performed. Ion implantation, with an implantation energy of 10 keV, at which... , and Three doses were used for mutagenesis treatment. After mutagenesis, the bacterial films treated with each dose were washed off with 1 mL of sterile water and spread onto a substrate containing 200 μg / mL... On YEPD solid agar plates, the cultures were incubated at 30°C for 48 hours. The survival rate of each plate was calculated, and single colonies with rapid growth and large diameter were selected to obtain positive mutant strains. The results showed that... The highest positive mutation rate was observed at the specified dosage.

[0034] Step S6: Adaptive evolution. The positive mutant strain obtained in step S5 was inoculated with a solution containing 200 μg / mL After culturing in YEPD liquid medium at 30°C and 220 rpm for 24 h, the culture was transferred at a 5% inoculum to a medium containing the same concentration. In fresh YEPD medium, continue culturing for 24 hours. Thereafter, subculture every 24 hours, and repeat every 20 generations. The concentration was increased by 100 μg / mL, with a total of 90 consecutive transfers. A final concentration of 3200 μg / mL was obtained. Selenium-enriched Saccharomyces cerevisiae strains ZKCM-1252, ZKCM-1251, and ZKCM-33074 that can still grow normally at certain concentrations.

[0035] Tests showed that when the number of transfers was less than 80 (e.g., 60 or 70 times), the strain maintained a concentration of 3200 μg / mL. The growth capacity on the plate decreased significantly, and a stable, genetically highly selenium-tolerant strain could not be obtained. When the number of subcultures reached 80, the strain exhibited stable high selenium tolerance. Further increasing the number of subcultures to 90 resulted in optimal selenium tolerance and selenium accumulation. After exceeding 100 subcultures (e.g., 110 and 120), the strain's performance plateaued, with no further improvement observed. Therefore, 80–100 subcultures were determined to be the suitable range, with 90 subcultures being the optimal choice.

[0036] Strain preservation: The finally obtained selenium-enriched Saccharomyces cerevisiae strains ZKCM-1252, ZKCM-1251, and ZKCM-33074 were deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, with accession numbers CGMCC No. 34511, CGMCC No. 38811, and CGMCC No. 38812, respectively. The deposit date for ZKCM-1252 is May 12, 2025, and the deposit dates for ZKCM-1251 and ZKCM-33074 are May 21, 2026.

[0037] Performance test results: Selenium tolerance: the starting strain in At a concentration of 400 μg / mL, the growth of bacteria was completely inhibited, and no colonies grew on the plate. However, the ZKCM-1252 strain obtained by screening could still grow at a high concentration of 3200 μg / mL, and the colony diameter was about 65% of that of the selenium-free control plate.

[0038] Intracellular total selenium content and organic selenium percentage: ZKCM-1252 strain was subjected to a concentration of 200 μg / mL The cells were cultured in MEB medium at 30℃ and 220 r / min for 48 h. After centrifugation, the cells were collected, washed, dried, and their selenium content was determined. The results are shown in Table 1. Table 1. Intracellular total selenium content and organic selenium percentage of different strains , Note: Table 1 shows the screening results for samples containing 200 μg / mL. The results of culturing in MEB medium for 48 h were used to screen dominant strains; Example 2 shows the results of yeast selenium preparation after optimizing fermentation conditions. The two tables show the results in terms of medium formulation, ... The concentrations added and the fermentation conditions are different, so the data are not directly comparable.

[0039] Example 2: Application of selenium-enriched Saccharomyces cerevisiae strains in the preparation of yeast selenium Experimental materials: Strains: Selenium-enriched Saccharomyces cerevisiae strains ZKCM-1252, ZKCM-1251 and ZKCM-33074 obtained in Example 1.

[0040] Culture medium: 1LMEB liquid medium: 6g malt extract, 1.8g maltose, 6g glucose, 1.2g yeast extract, pH 4.7±0.2, adjusted with 1mol / L HCl or NaOH, sterilized at 121℃ for 15min.

[0041] Main reagents: analytical grade (Purity ≥ 99%), analytical grade 3,3-diaminobenzidine, 1000 μg / mL selenium standard solution, analytical grade nitric acid, analytical grade perchloric acid, analytical grade hydrochloric acid, EDTA-2Na, hydroxylamine hydrochloride, analytical grade toluene, and all other reagents are analytical grade.

[0042] Main instruments: UV-Vis spectrophotometer, constant temperature shaker, centrifuge, electric constant temperature drying oven, electronic balance, electric digestion apparatus, pH meter.

[0043] like Figure 2 The specific steps of the yeast selenium preparation method are shown below: Step L1: Strain activation. The selenium-enriched Saccharomyces cerevisiae strain ZKCM-1252 obtained in Example 1 was inoculated onto MEB solid slant medium and incubated statically at 30°C for 24-48 hours until single colonies grew. Single colonies were picked and inoculated into 100 mL of MEB liquid medium and cultured in a constant temperature shaker at 30°C and 220 rpm for 16-24 hours as seed culture.

[0044] Step L2: Fermentation culture. During fermentation culture, the activated seed culture was transferred to 500 mL of MEB liquid medium at an inoculation rate of 5% (v / v), with a liquid volume of 500 mL / 1000 mL Erlenmeyer flask, and cultured at 30 °C and 220 r / min with shaking.

[0045] Step L3: Induction. After 6 hours of incubation, sterilized medium filtered through a 0.22 μm membrane was added to the culture medium. The mother liquor was used to achieve a final selenium concentration of 25 μg / mL, and the culture was continued under the same conditions for 48 h.

[0046] Step L4: Cell Collection. Centrifuge the fermentation broth at 8000 rpm for 10 min and collect the cell precipitate. Resuspend the cells in deionized water, centrifuge and wash again, repeating 2-3 times. Place the washed cells in a 60℃ electric thermostatic drying oven and dry until constant weight to obtain the yeast selenium product. Weigh and calculate the dry weight of the cells to determine the biomass, which will be used for selenium content determination.

[0047] Selenium content determination: Step 1: Selenium standard curve preparation: The 1000 μg / mL selenium standard solution was serially diluted with deionized water to prepare selenium standard working solutions of 0.2 μg / mL, 0.4 μg / mL, 0.6 μg / mL, 0.8 μg / mL and 1.0 μg / mL; Color development: Take 10 mL of each concentration of standard working solution into a separatory funnel, add 15 mL of distilled water, adjust the pH to 2.5 with 1 mol / L hydrochloric acid solution, add 5 mL of 0.5% 3,3-diaminobenzidine solution, shake well and react in the dark for 30 min. Extraction: Adjust the pH to 6.5-7.0 with 1 mol / L NaOH solution, add 10 mL of toluene, shake and extract for 1 min, and allow to stand for separation; Colorimetric method: Take the toluene layer and measure the absorbance at a wavelength of 420 nm; A selenium standard curve was plotted with selenium content on the x-axis (in μg) and absorbance on the y-axis, as shown below. Figure 4 As shown. The regression equation for the standard curve is... Correlation coefficient This indicates that the selenium content has a good linear relationship with absorbance in the range of 0~10μg.

[0048] Step 2: Total Selenium Content Determination: Accurately weigh 0.1g of the dried yeast selenium sample (accurate to 0.0001g), place it in a digestion tube, add 10mL of a nitric acid-perchloric acid mixture (volume ratio 9:1), and place in an electric heating digester. Digest magnetically in a boiling water bath for 1 hour until the solution is colorless and transparent. After cooling, add 5mL of a 6mol / L hydrochloric acid solution and continue heating until the solution becomes clear and colorless with the appearance of white fumes. Continue concentrating until the remaining volume is approximately 2mL, until the white fumes cease. Remove and cool, add 5mL of a 100g / L hydroxylamine hydrochloride solution, rinse the digestion tube with approximately 20mL of a 5g / L EDTA-2Na solution, transfer to a 100mL volumetric flask, and dilute to the mark with deionized water. Shake well. Adjust the pH to 2.0–2.5 with a 1mol / L hydrochloric acid solution to prepare the test solution. Prepare a blank control solution using the same method.

[0049] Take 10 mL of the above test solution into a separatory funnel, and perform colorimetric analysis, extraction, and colorimetric determination according to the method in step 1. Measure the absorbance at a wavelength of 420 nm. Calculate the total selenium content in the sample based on the selenium standard curve.

[0050] Step 3: Determination of Inorganic Selenium Content: Accurately weigh 0.1 g (accurate to 0.0001 g) of the dried yeast selenium sample, add 10 mL of hydrochloric acid solution (6 mol / L), heat in a boiling water bath for 30 min, cool, and then bring the volume to 100 mL. Take 10 mL of the above solution and perform colorimetric determination, extraction, and colorimetric analysis according to the method in Step 1 to calculate the inorganic selenium content. Calculate the inorganic selenium content in the sample based on the selenium standard curve.

[0051] Organic selenium content calculation: Organic selenium content = Total selenium content − Inorganic selenium content.

[0052] Organic selenium percentage (%) = (organic selenium content / total selenium content) × 100%.

[0053] The experimental results are as follows: The selenium content of yeast prepared from three strains (ZKCM-1252, ZKCM-1251, and ZKCM-33074) was determined using the above method. It should be noted that the culture conditions in this example (Example 2) differ from those in Table 1 of Example 1. The data in Table 1 of Example 1 were obtained using a culture medium containing 200 μg / mL selenium during the screening stage. The results were measured after culturing in MEB medium for 48 h. The purpose was to compare the relative superiority of different strains under the same conditions in order to screen for dominant strains. In this example, optimized fermentation conditions were used. Yeast selenium was prepared by adding 25 μg / mL of yeast extract at an inoculum size of 5% and a culture time of 48 h, with the aim of obtaining the best selenium enrichment effect. Therefore, the two sets of data reflect the performance of the strain under different culture conditions and should not be directly compared, as they each serve different experimental purposes. The results are shown in Table 2.

[0054] Table 2. Selenium content determination results of different selenium-enriched Saccharomyces cerevisiae strains , The above results indicate that the selenium-enriched Saccharomyces cerevisiae strain obtained by screening in this invention is significantly superior to the starting strain in terms of biomass, total intracellular selenium content, and proportion of organic selenium.

[0055] As shown in Table 2: All three selenium-enriched Saccharomyces cerevisiae strains exhibited high total selenium content and a high proportion of organic selenium. Strain ZKCM-1252 had the highest total selenium content at 5266.59 μg / g, with an organic selenium content of 5030.16 μg / g and an organic selenium content of 95.51%. Strain ZKCM-33074 had the highest organic selenium content at 99.46%, with a total selenium content of 4334.16 μg / g. Strain ZKCM-1251 had a total selenium content of 1033.16 μg / g and an organic selenium content of 70.03%.

[0056] The proportion of organic selenium in all three strains was significantly higher than that in the starting strain, which had an organic selenium content of 62.3%. This indicates that the strains obtained by the gradient screening-ion beam mutagenesis-adaptive evolution combination strategy of Example 1 of this invention all have excellent inorganic selenium biotransformation capabilities, and can efficiently convert inorganic selenium in the culture medium into organic selenium forms, which are then enriched in the bacterial cells in the form of selenoproteins, selenopeptides, etc.

[0057] Results Analysis: The results of this embodiment show that the yeast selenium prepared using the selenium-enriched Saccharomyces cerevisiae strain obtained by screening in this invention has the following significant advantages: High selenium content: The total selenium content of the yeast prepared by the three strains all exceeded 1000 μg / g, of which ZKCM-1252 and ZKCM-33074 had a total selenium content of 5266.59 μg / g and 4334.16 μg / g, respectively, which is far higher than the level of selenium-enriched yeast products reported in the prior art.

[0058] High proportion of organic selenium: The proportion of organic selenium in all three strains reached over 70%, with ZKCM-1252 and ZKCM-33074 having proportions of 95.51% and 99.46%, respectively. This indicates that the majority of the inorganic selenium absorbed by the strains has been converted into a form of organic selenium that is highly safe and bioavailable.

[0059] Performance differences among strains reveal screening value: The three strains showed certain differences in total selenium content and organic selenium ratio. Among them, ZKCM-1252 had the best total selenium content, reaching 5266.59 μg / g, while ZKCM-33074 had the best organic selenium conversion rate, reaching 99.46%. This provides a technical basis for strain selection under different application scenarios - if high selenium content is required, ZKCM-1252 can be selected, and if the ultimate organic selenium purity is required, ZKCM-33074 can be selected.

[0060] The process is stable and reliable: The yeast selenium preparation method in this embodiment is simple to operate, has mild conditions, and good repeatability. It is suitable for large-scale industrial production and can be widely used in selenium-enriched foods, selenium-enriched feed additives, and selenium-enriched functional raw materials.

[0061] Example 3: Application of selenium-enriched Saccharomyces cerevisiae strain ZKCM-1252 in the preparation of selenium-enriched yeast selenopeptides Experimental materials: Strain: The selenium-enriched Saccharomyces cerevisiae strain ZKCM-1252 obtained in Example 1; Culture medium: 1LMEB liquid medium: 6g malt extract, 1.8g maltose, 6g glucose, 1.2g yeast extract, pH 4.7±0.2, sterilized at 121℃ for 15min.

[0062] Main reagents: analytical grade The reagents used included trypsin with an enzyme activity of not less than 250 U / mg, PBS buffer at a concentration of 0.01 M and a pH of 7.2, Tris-HCl, EDTA, hydrochloric acid, NaOH, and all other reagents of analytical grade.

[0063] Main instruments: high-pressure homogenizer, ultrafiltration device with a molecular weight cutoff of 3kDa, high-speed refrigerated centrifuge, ultraviolet-visible spectrophotometer, pH meter, constant temperature shaking incubator, electronic balance.

[0064] like Figure 3 As shown, the preparation method of selenium-enriched yeast selenopeptides specifically includes the following steps: Step M1: Strain activation. The selenium-enriched Saccharomyces cerevisiae strain ZKCM-1252 obtained in Example 1 was inoculated onto MEB solid slant medium and incubated statically at 30°C for 24-48 hours until single colonies grew. A single colony was picked and inoculated into 100 mL of MEB liquid medium and cultured in a constant temperature shaker at 30°C and 220 rpm for 16-24 hours as the seed culture.

[0065] Step M2: Fermentation culture. The activated seed culture was transferred to 500 mL of MEB liquid medium at an inoculation rate of 10% (v / v) in a 500 mL / 1000 mL Erlenmeyer flask and cultured at 30 °C with shaking at 220 rpm.

[0066] Step M3: Induction. When the bacterial culture... When the value reaches 0.6, add sterilized solution filtered through a 0.22μm filter membrane. The mother liquor was used to achieve a final selenium concentration of 25 μg / mL, and the culture was continued under the same conditions for 40 h.

[0067] Step M4: Cell Collection and Washing. Centrifuge the fermentation broth at 8000 rpm and 4°C for 10 min, and collect the cell pellet. Resuspend the cells in pre-cooled 0.01M, pH 7.2 PBS buffer, and centrifuge and wash again. Repeat 2-3 times to remove residual culture medium and external impurities. The washed cells are used for subsequent cell disruption.

[0068] Step M5: Cell resuspending. Resuspend the washed bacterial cells in pre-chilled cell disruption buffer A and mix thoroughly. The cell disruption buffer formula is: 50 mM Tris-HCl, 1 mM EDTA, pH 8.0.

[0069] Step M6: High-pressure homogenization for cell disruption. Place the cell suspension in a high-pressure homogenizer for cell disruption at a pressure of 1200 bar for 20 minutes. Repeat this process 2-3 times until the cell disruption rate is ≥95% as determined by microscopic examination.

[0070] Step M7: Pretreatment of cell wall disruption solution. Centrifuge the cell wall disruption solution at 10000 r / min and 4℃ for 20 min to remove unbroken cell debris and large particulate impurities. Collect the supernatant as the substrate for subsequent enzymatic digestion.

[0071] Step M8: Trypsin digestion. Adjust the pH of the collected supernatant to 8.0 with 1 mol / L NaOH solution. Add trypsin at a mass ratio of 1:50 to substrate, mix well, and incubate at 37℃ with gentle shaking at 100 rpm for 4-6 hours.

[0072] Step M9: Post-enzymatic hydrolysis treatment. After enzymatic hydrolysis, centrifuge the hydrolysate at 10000 r / min and 4℃ for 15 min to remove undissolved residue and collect the clarified liquid. Ultrafiltration is performed on the clarified liquid using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The permeate is collected; if its molecular weight is ≤3 kDa, it is the product rich in low molecular weight selenopeptides.

[0073] Selenopeptide content determination: The content of peptides was determined by the biuret method: Take an appropriate amount of sample solution, add biuret reagent, and measure the absorbance at 540 nm. Use bovine serum albumin as a standard to plot a standard curve and calculate the peptide content.

[0074] The total selenium content in the sample was determined using the method described in Example 2, and the selenium content in the selenopeptide was calculated.

[0075] The experimental results are as follows: Basic properties of the selenium peptide product: The selenium-enriched yeast selenium peptide prepared by the above method is a pale yellow to light brown transparent liquid with a characteristic yeast fermentation odor and a pH of approximately 6.5–7.0. After freeze-drying, a powdered selenium peptide product is obtained, which is pale yellow in color and readily soluble in water.

[0076] Molecular weight distribution of selenopeptides: The selenopeptide products obtained by ultrafiltration were low molecular weight selenopeptides with a molecular weight ≤3kDa. Peptides in this molecular weight range have good intestinal absorption performance and high bioavailability.

[0077] Selenium content of selenopeptides: The total selenium content in the dry powder of selenopeptide products is 432.58 μg / g, of which organic selenium accounts for ≥92%, indicating that selenium is effectively retained in the peptide components during the preparation process.

[0078] Peptide content: The peptide content of the selenium peptide product dry powder is 68.32%, indicating that the enzymatic hydrolysis efficiency is high and most of the yeast protein has been degraded into low molecular weight peptides.

[0079] Results analysis: The results of this embodiment demonstrate that using the selenium-enriched Saccharomyces cerevisiae strain ZKCM-1252 obtained by screening in this invention to prepare selenium-enriched yeast selenopeptides has the following significant advantages: Synergistic effect of selenium and peptides: The selenium peptides prepared in this invention organically combine selenium with low molecular weight peptides, possessing the dual biological activities of selenium and peptides. Selenium exists in the peptides in an organic form, namely selenoamino acids, and is characterized by safety, non-toxicity, and high bioavailability; the low molecular weight peptides are easily absorbed directly by the intestines and can serve as effective carriers of selenium.

[0080] Moderate molecular weight: Low molecular weight selenium peptides obtained by ultrafiltration membrane with a molecular weight of ≤3kDa conform to the intestinal absorption characteristics of small molecule peptides, have high biological activity, and are an ideal form of selenium nutritional supplement.

[0081] The preparation process is simple: This embodiment adopts a process route of high-pressure homogenization and cell disruption combined with trypsin enzymatic hydrolysis. The operating conditions are mild, the equipment is universal, and it is easy to scale up, making it suitable for industrial production. Trypsin, as a commonly used food-grade enzyme preparation, is safe and reliable. The enzymatic hydrolysis conditions are mild and controllable at pH 8.0, 37℃, and 4~6h, and have no significant impact on selenium content.

[0082] High product purity: Through centrifugation and ultrafiltration fractionation, impurities such as macromolecular proteins, nucleic acids and cell residues are effectively removed, resulting in high purity and uniform quality of selenium peptide products that can be directly applied in the fields of food, health food and feed additives.

[0083] This embodiment successfully established a complete process flow from the fermentation and cultivation of selenium-enriched Saccharomyces cerevisiae strain to the preparation of selenium-enriched yeast selenopeptides. Using the strain ZKCM-1252 of this invention, through selenium-enriched fermentation, high-pressure homogenization and cell wall disruption, trypsin hydrolysis, and ultrafiltration fractionation, a product rich in low molecular weight selenopeptides can be obtained. This selenopeptide product possesses the dual nutritional functions of selenium and peptides, and has advantages such as high safety, good absorption, and simple preparation process, showing broad application prospects in the fields of functional foods, health products, and feed additives.

[0084] This embodiment uses strain ZKCM-1252 as an example for detailed description. Those skilled in the art will understand that strains ZKCM-1251 or ZKCM-33074 can also obtain selenium-enriched yeast selenopeptide products of comparable quality under the same conditions, and all fall within the scope of protection of this invention. Although the three strains have certain differences in total selenium content and organic selenium ratio (see Table 2 of Example 2), they all have the ability to prepare selenium-enriched selenopeptides, and the preparation methods are exactly the same.

[0085] Comparative Example To verify the superior performance of the selenium-enriched Saccharomyces cerevisiae strains obtained by screening in this invention and the effectiveness of each key step in the screening method, the following comparative examples 1 to 5 were set up.

[0086] Comparative Example 1: Comparison of selenium enrichment performance of the starting strains Using the original starting strain without any screening or mutagenesis treatment, the brewing yeast isolated from the soy sauce-flavored koji sample was used as a control. Fermentation culture and yeast selenium preparation were carried out under the same culture conditions as in Example 2. The maximum selenium tolerance concentration, biomass, total selenium content and organic selenium ratio were determined. The results are shown in Table 3.

[0087] Comparative Example 2: Comparison of screening effects without ion beam mutagenesis To verify the necessity of the ion beam mutagenesis step (step S5 in Example 1) in the screening method, this comparative example was set up. Except for omitting the ion beam mutagenesis treatment in step S5, the remaining screening steps were exactly the same as in Example 1, including gradient plate initial screening, After resistance screening and 90 rounds of adaptive evolution, the final strain was named ZKCM-1252-CK2. It was cultured under the same fermentation conditions as in Example 2, and various performance indicators were measured. The results are shown in Table 3.

[0088] Comparative Example 3: Comparison of selection effects without adaptive evolution To verify the necessity of the adaptive evolution step (step S6 in Example 1) in the screening method, this comparative example was set up. Except for omitting the repeated transition operation in step S6, the remaining screening steps were exactly the same as in Example 1, including gradient plate initial screening, Resistance screening and ion beam mutagenesis were performed, and the resulting strain was named ZKCM-1252-CK3. It was cultured under the same fermentation conditions as in Example 2, and various performance indicators were measured. The results are shown in Table 3.

[0089] Comparative Example 4: Performance comparison between the strain of this invention and commercially available selenium-enriched yeast products A commercially available brand of selenium-enriched yeast product (purchased from the market, labeled with a selenium content ≥2000μg / g) was selected as a control and its performance was compared with that of yeast selenium prepared by the selenium-enriched brewer's yeast strain ZKCM-1252 of the present invention under the same fermentation conditions. The total selenium content and the proportion of organic selenium were determined according to the selenium content determination method in Example 2. The results are shown in Table 3.

[0090] Comparative Example 5: Effect of different trypsin hydrolysis times on the yield of selenopeptides To verify the optimality of the trypsin hydrolysis time (4-6h) in Example 3, four treatment groups were set up with hydrolysis times of 2h, 4h, 6h and 8h respectively. All other hydrolysis conditions were the same as in Example 3, namely pH 8.0, 37℃, and trypsin to substrate ratio of 1:50. After hydrolysis, the yield of selenopeptide and the selenium content in selenopeptide of each treatment group were determined according to the method of Example 3. The results are shown in Table 4.

[0091] The comparative results are summarized below: Table 3 Performance Comparison of Each Comparative Strain / Product , The commercially available selenium-enriched yeast product used in Comparative Example 4 was a pre-prepared selenium-enriched yeast product, not a live bacteria, so the maximum selenium tolerance concentration and biomass were not determined.

[0092] Table 4. Effects of different enzymatic hydrolysis times on the yield and selenium content of selenopeptides. , Note: The comprehensive score of selenium peptides = selenium peptide yield × selenium content in selenium peptides / 100. It is a relative evaluation index. The higher the value, the higher the comprehensive effectiveness.

[0093] To comprehensively evaluate the preparation effect of selenopeptides under different enzymatic hydrolysis times, this application defines a "selenopeptide comprehensive score" = selenopeptide yield × selenium content in selenopeptides / 100. This score can simultaneously reflect the yield and quality (selenium content) of selenopeptides; a higher score indicates a higher overall efficiency of the selenopeptide product obtained per unit fermentation broth. Table 4 shows that when the enzymatic hydrolysis time is extended from 2 h to 6 h, the selenopeptide yield continues to increase. Although the selenium content in the selenopeptides decreases slightly, the comprehensive score continues to rise. When the enzymatic hydrolysis time exceeds 6 h, the selenopeptide yield tends to plateau, while the selenium content in the selenopeptides continues to decrease, leading to a decrease in the comprehensive score. Therefore, 6 h is the optimal enzymatic hydrolysis time, and 4–6 h is the preferred range.

[0094] As shown in Table 3: Comparing Comparative Example 1 and Example 2: The highest selenium tolerance concentration of the starting strain was only 400 μg / mL, and the total selenium content was only 312.50 μg / g; while the highest selenium tolerance concentration of the ZKCM-1252 strain obtained by screening according to the present invention reached 3200 μg / mL, which is 8 times that of the starting strain, and the total selenium content reached 5266.59 μg / g, which is 16.9 times that of the starting strain. The proportion of organic selenium increased from 62.30% to 95.51%, an increase of 33.21 percentage points. This shows that the combined screening strategy of the present invention can achieve a leapfrog improvement in the selenium tolerance and selenium accumulation capacity of the strain.

[0095] Comparison Example 2 with Example 2: The highest selenium tolerance concentration of the unmutated strain ZKCM-1252-CK2 was only 1200 μg / mL, and the total selenium content was only 652.38 μg / g, which were 62.5% and 87.6% lower than those of the mutagenized ZKCM-1252, respectively. This indicates that ion beam mutagenesis is an indispensable key step in this screening method.

[0096] Comparison Example 3 with Example 2: The highest selenium tolerance concentration of strain ZKCM-1252-CK3 without adaptive evolution was only 1600 μg / mL, and the total selenium content was only 896.25 μg / g, which were 50.0% and 83.0% lower than those of ZKCM-1252 after 90 adaptive evolutions, respectively. This indicates that adaptive evolution is an indispensable key step in this screening method.

[0097] Comparison of Comparative Example 4 and Example 2: The total selenium content of commercially available selenium-enriched yeast products is 2134.50 μg / g, with an organic selenium content of 85.80%; while the total selenium content of yeast prepared by the ZKCM-1252 strain of this invention is 5266.59 μg / g, which is 2.47 times that of commercially available products, with an organic selenium content of 95.51%, an increase of 9.71 percentage points compared to commercially available products. This indicates that the strain of this invention is significantly superior to commercially available similar products in terms of selenium enrichment performance.

[0098] As shown in Table 4: Comparative Example 5: When the enzymatic hydrolysis time was extended from 2 h to 6 h, the yield of selenopeptides continuously increased from 8.2 g / L to 14.6 g / L, but the selenium content in the selenopeptides slightly decreased from 485.3 μg / g to 432.6 μg / g. After the enzymatic hydrolysis time exceeded 6 h, the increase in selenopeptide yield tended to level off (6 h: 14.6 g / L → 8 h: 14.9 g / L), while the selenium content in the selenopeptides continued to decrease to 401.2 μg / g, and the overall score began to decrease. Based on the overall score, 6 h of enzymatic hydrolysis reached the optimal value, i.e., 6316. This indicates that the selection of 4–6 h as the preferred enzymatic hydrolysis time in this invention is reasonable.

[0099] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A selenium-enriched brewer's yeast strain, characterized in that, The strain is Saccharomyces cerevisiae ZKCM-1252, with accession number CGMCC No.34511.

2. The application of the selenium-enriched Saccharomyces cerevisiae strain as described in claim 1 in the preparation of yeast selenium, characterized in that, Includes the following steps: Step L1: The selenium-enriched Saccharomyces cerevisiae strain is inoculated into MEB medium for activation and cultured to the logarithmic phase to obtain the activated bacterial solution; Step L2: Inoculate the activated bacterial solution at a volume ratio of 5% into MEB liquid medium and incubate at 30℃ and 220r / min with shaking. Step L3: After culturing for 6 hours, add [the following solution] to a final concentration of 25 μg / mL. The solution was cultured for another 48 hours to obtain a yeast culture medium. Step L4: Collect yeast cells by centrifugation from yeast culture medium, wash with deionized water by centrifugation, and dry to constant weight to obtain yeast selenium.

3. The application of the selenium-enriched Saccharomyces cerevisiae strain as described in claim 1 in the preparation of selenium-enriched yeast selenopeptides, characterized in that, Includes the following steps: Step M1: Inoculate the selenium-enriched Saccharomyces cerevisiae strain into MEB medium for activation culture; Step M2: Inoculate the activated bacterial solution into MEB liquid medium at a volume ratio of 10% and culture at 30℃ and 220r / min with shaking. Step M3: When the bacterial solution... Add to a final mass concentration of 25 μg / mL. The solution was incubated for another 40 hours. Step M4: Collect bacterial cells by centrifugation at 8000 r / min, 4℃, and 10 min, and wash with pre-cooled PBS buffer to remove residual culture medium and external impurities; Step M5: Resuspend the washed cells in pre-cooled cell disruption buffer and mix thoroughly; Step M6: Place the cell suspension in a high-pressure homogenizer for pressure cell disruption; Step M7: Centrifuge the cell wall disruption solution to remove unbroken cell debris and large particulate impurities, and collect the supernatant; Step M8: Adjust the pH of the supernatant to 8.0, add trypsin, mix well, and incubate at 37°C with shaking for enzymatic hydrolysis; Step M9: Centrifuge to remove undissolved residue, collect the clarified liquid, perform ultrafiltration using an ultrafiltration device, collect the permeate, and obtain the product rich in low molecular weight selenium peptides.

4. The application of the selenium-enriched Saccharomyces cerevisiae strain according to claim 3 in the preparation of selenium-enriched yeast selenopeptides, characterized in that: The cell disruption buffer in step M5 is 50mM Tris-HCl, 1mM EDTA, pH 8.0; the pressure of the high-pressure homogenizer in step M6 is 1200 bar, the cell disruption time is 20 min, and the cycle is 2-3 times.

5. The application of the selenium-enriched Saccharomyces cerevisiae strain according to claim 3 in the preparation of selenium-enriched yeast selenopeptides, characterized in that: In step M8, the mass ratio of trypsin to substrate is 1:50, and the enzymatic hydrolysis time is 4-6 hours; in step M9, the ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 3 kDa.

Images