Saccharomyces cerevisiae C16I12 strain and application thereof

Abstract

The invention relates to a Saccharomyces cerevisiae C16I12 strain which is preserved in the China Center for Type Culture Collection on May 21, 2025, and the preservation number of the Saccharomyces cerevisiae C16I12 strain is CCTCC (China Center for Type Culture Collection) No. M20251151. The strain disclosed by the invention has good oxidation resistance and can be applied to the fields of skin care products, health care products, anti-aging foods and the like. Especially, extracellular fermentation broth of the strain, namely a fermentation product or a fermentation extract of the strain, has excellent oxidation resistance, can scavenge free radicals and relieve oxidative damage, has the effects of protecting skin, resisting inflammation, delaying senescence and the like, and can be applied to the fields of skin care products, health care products, anti-aging foods and the like as a natural anti-aging agent.

Description

Technical Field

[0001] This invention relates to the field of microbial biotechnology, and in particular to a strain of Saccharomyces cerevisiae C16I12 and its applications. Background Technology

[0002] Oxidative stress refers to the abnormal accumulation of reactive oxygen species (ROS) within cells, exceeding the cells' own clearance capacity, and subsequently triggering a series of damages to biomolecules, including DNA, proteins, and lipids. ROS include superoxide anions, hydrogen peroxide, and hydroxyl radicals, which are unavoidable byproducts of cellular metabolism. Under normal physiological conditions, cells possess antioxidant defense mechanisms, such as superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH), to maintain redox homeostasis. However, under high-intensity or sustained oxidative stress, these defense mechanisms often become imbalanced, leading to impaired cell function and even cell death.

[0003] In industrial fermentation processes, microorganisms are often exposed to strong oxidative stress due to factors such as high-density cultivation, dissolved oxygen fluctuations, organic acid accumulation, and the buildup of metabolic byproducts. Oxidative stress not only causes lipid peroxidation of the cell membrane, mitochondrial damage, and energy metabolism disorders, but also directly inhibits enzyme activity and anabolism, resulting in slowed cell growth, decreased product synthesis efficiency, and prolonged fermentation cycles. Therefore, constructing or screening strains with strong antioxidant capacity is an important means to improve the stability and fermentation yield of industrial microorganisms. For example, in fields such as alcoholic fermentation, organic acid production, and enzyme expression, enhancing the antioxidant capacity of strains can significantly prolong the time for efficient product expression, increase the product yield per unit volume of fermentation broth, and reduce production costs.

[0004] Meanwhile, antioxidant microorganisms and their metabolites are increasingly being used in the development of cosmetics and functional skincare products. Skin is frequently exposed to ultraviolet radiation, pollutants, and other environmental stimuli, leading to the excessive generation of reactive oxygen species (ROS) and causing problems such as skin cell aging, pigmentation, and collagen degradation. Microbial extracts with antioxidant activity have been proven to scavenge free radicals and reduce oxidative damage, offering protective, anti-inflammatory, and anti-aging benefits to the skin, and are widely used in high-end cosmetics such as serums, masks, and toners. Therefore, screening for microbial strains with high antioxidant properties not only has promising industrial applications but also provides microbial resource support for the development of natural and safe functional anti-aging ingredients for the skin.

[0005] Currently, screening methods for antioxidant microbial strains mainly include in vitro antioxidant enzyme activity assays (such as SOD and CAT activity), free radical scavenging rate determinations (such as DPPH and ABTS methods), flow cytometry, and high-throughput imaging screening. While these methods are precise, they typically rely on expensive instruments or are complex to operate, making large-scale initial screening difficult. In contrast, screening methods using hydrogen peroxide (H2O2) as the selection pressure have become an effective preliminary screening tool due to their simplicity, low cost, and strong physiological relevance. H2O2 can penetrate cell membranes and cause oxidative damage, mimicking natural oxidative stress. Continuous passage in a culture environment with progressively increasing H2O2 concentrations can screen for microbial variants with stronger ROS stress tolerance. These strains often exhibit phenotypic characteristics such as upregulated expression of antioxidant systems and enhanced ROS scavenging ability, making them excellent candidates for subsequent antioxidant performance verification and functional studies.

[0006] Prior to this invention application, two patents disclosed methods for enhancing the antioxidant capacity of yeast. Chinese invention patent application "A brewer's yeast strain with high antioxidant capacity" (publication number: CN108559714A) uses a chemical mutagenesis method, stimulating brewer's yeast with 10mM H2O2, followed by screening for mutant strains with enhanced antioxidant capacity using 96-well microplate liquid culture. After 10-20 generations of directed evolution, strain P-12-7 (CMCC No. 15504) with strong antioxidant capacity was obtained. This strain showed improvements in intracellular ROS content, ATP level, and cell viability compared to the original strain, with a maximum tolerance to hydrogen peroxide concentration of 8mM. Chinese invention patent application "A method for enhancing the antioxidant capacity of yeast" (publication number: CN109401986A) uses 10mM H2O2 for a short time (1... h) Multiple yeast strains (such as brewer's yeast, saccharitomyces, and Rhodotorula roux) were subjected to shock treatment. Single colonies were isolated by culturing in 96-well plates. Strains with high antioxidant capacity were screened by intracellular ROS and ATP content. The treated strains showed significant tolerance in 6mM H2O2 plate culture, with an average decrease of about 30% in intracellular ROS content and an average increase of more than 50% in ATP content. Summary of the Invention

[0007] This invention provides a strain of brewing yeast. Saccharomyces cerevisiae Strain C16I12 was deposited at the China Center for Type Culture Collection on May 21, 2025, with accession number CCTCC No. M20251151.

[0008] The strain of this invention was obtained from the original starting strain Y017 through space mutagenesis, screening, and domestication. The original starting strain Y017 was deposited at the China General Microbiological Culture Collection Center on April 26, 2020, with accession number CGMCC No. 19732. The function and application of this strain are described in Chinese invention patent CN114081862B, which has been granted patent rights, and its full disclosure is cited in this application. Based on this, the present invention, through space mutagenesis, microbial antioxidant screening, and domestication, utilizes a gradient of increasing hydrogen peroxide concentration for stepwise domestication and screening, ultimately obtaining a new strain C16I12 with excellent antioxidant properties. Experimental testing shows that it can tolerate hydrogen peroxide concentrations of not less than 0.4%; furthermore, the extracellular fermentation broth of this strain exhibits antioxidant properties and the ability to scavenge free radicals, with the ABTS free radical scavenging rate of its original fermentation broth being 168% higher than that of the original starting strain Y017. The stable strain C16I12 obtained after screening and domestication exhibits stable traits and fermentation performance after multiple generations of inheritance, and can be used to construct high-efficiency industrial fermentation strains.

[0009] Furthermore, the screening process specifically involves: stepwise increasing concentration of hydrogen peroxide in liquid culture for screening, with a concentration increase gradient of 0.05% to 0.15% by mass-volume ratio for each step, in 12-well plates, and by progressively increasing the hydrogen peroxide concentration, strains with strong tolerance to oxidative stress are screened out.

[0010] The acclimatization process is as follows: In a 12-well plate, liquid culture medium with progressively increasing hydrogen peroxide concentration is added to each well. The mass-to-volume ratio of hydrogen peroxide used ranges from 0.10% to 0.50%, and each concentration increase is set to be 10% to 20% higher than the hydrogen peroxide concentration in the previous round of culture medium, so as to achieve a gradually increasing oxidative stress condition. The selected Saccharomyces cerevisiae strain is pre-cultured to obtain a bacterial solution, which is thoroughly mixed and then inoculated into liquid culture medium with a further increased hydrogen peroxide concentration. Each strain is set up with 3 to 5 replicate wells and incubated statically at 30°C for 48 to 72 hours. After the strain grows stably under these conditions, the above inoculation and concentration increase steps are repeated for 10 to 20 generations, so that the target Saccharomyces cerevisiae strain gradually adapts to and tolerates the oxidative stress environment of 0.4% to 0.6% hydrogen peroxide, and finally the successfully acclimatized target strain is obtained.

[0011] The strain of this invention possesses excellent antioxidant properties and can be applied in skincare products, health supplements, and anti-aging foods. In particular, the extracellular fermentation broth of this strain, i.e., its fermentation product or fermentation extract, exhibits outstanding antioxidant properties, capable of scavenging free radicals and reducing oxidative damage. It has protective, anti-inflammatory, and anti-aging effects on the skin and can be used as a natural anti-aging agent in skincare products, health supplements, and anti-aging foods. Microbial Preservation Instructions

[0012] brewing yeast Saccharomyces cerevisiae C16I12 was deposited on May 21, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Bayi Road, Wuchang District, Wuhan, Hubei Province, China, with accession number CCTCC No. M20251151. The deposit acceptance notice (receipt) was received on May 28, 2025, and is submitted as an attachment. Attached Figure Description

[0013] Figure 1 This describes the colony morphology of the yeast strain of the present invention on YPD solid culture medium; Figure 2 This is an optical microscope image of the morphology of the yeast strain of the present invention; Figure 3 This is a growth diagram of the strain of the present invention in medium with different concentrations of hydrogen peroxide; Figure 4 This is a growth diagram of the strain of the present invention in a medium with a high concentration of hydrogen peroxide; Figure 5 The free radical scavenging rate of the strains of this invention was detected by the ABTS method. Detailed Implementation

[0014] The present invention will be further described below with reference to embodiments, but this is not intended to limit the invention in any way. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection of the present invention.

[0015] The brewing yeast provided by this invention S. cerevisiae The C16I12 culture method involves inoculating *Saccharomyces cerevisiae* into a self-made non-oxidizing G40 medium. The medium consists of: glucose: 20-50 g / L, (NH4)2SO4: 5-10 g / L, KH2PO4: 0.5-2 g / L, MgSO4·7H2O: 0.5-2 g / L, natural pH, prepared using standard laboratory water, and sterilized at 115℃ for 15 min.

[0016] The *Saccharomyces cerevisiae* C16I12 provided by this invention exhibits a 60% increase in semi-lethal hydrogen peroxide tolerance compared to the original strain *Saccharomyces cerevisiae* Y017. At the original strain's semi-lethal hydrogen peroxide concentration, the biomass of the *Saccharomyces cerevisiae* C16I12 provided by this invention is increased by 45%. In a hydrogen peroxide-free culture medium, the ABTS scavenging rate of the yeast C16I12 provided by this invention is increased by more than 150% per unit biomass, reaching 168%.

[0017] Example 1: Screening of antioxidant yeast On May 30, 2023, the original strain Y017 was launched into space aboard the Shenzhou-16 manned spacecraft from the Jiuquan Satellite Launch Center by a Long March 2F carrier rocket. It underwent mutation in the space experimental module environment and was recovered on October 31, 2023, by the return capsule of the Shenzhou-16 manned spacecraft in the designated area of ​​the Dongfeng Landing Site. A small amount of dried bacterial powder sample (frozen at -80℃) was taken from the spacecraft and revived on YPD medium under suitable conditions. 50 µL of this sample and 50 µL of the original starting strain Y017 (as a control) were added to separate 15 mL centrifuge tubes containing 5 mL of YPD medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, natural pH, prepared with distilled water, sterilized at 115℃ for 15 min). The mixture was incubated at 150-200 rpm with shaking for 24-36 h. The culture was then inoculated at a 1:50-100 (v / v) ratio into 250 mL shake flasks containing 50 mL of YPD medium and incubated at 30℃ with shaking for 24-36 h. The fermentation broth was centrifuged at 8000 rpm, and the bacterial cells were collected. The cells were washed 2-3 times with sterile water and then inoculated into 15 mL centrifuge tubes containing 5 mL of G40 medium. In mL centrifuge tubes, the initial inoculation OD600nm is in the range of 0.05-0.2, and the cells are cultured at 30℃ and 150-200 rpm for 24-36 h with shaking.

[0018] Take 50-100µL of the cultured bacterial solution and inoculate it into 12-well plates containing 3 mL of G40 culture medium with a concentration of 0.05%-0.15% (w / v, all percentages of hydrogen peroxide concentration below are w / v). Incubate at 30℃ for 48-72 h.

[0019] Prepare YPD solid culture plates containing 0.05%-0.15% hydrogen peroxide. Autoclave using standard methods, cool to a suitable temperature, add pre-prepared hydrogen peroxide working solution, mix well, and quickly pour onto the plates to avoid H2O2 inactivation. Take 30-100 µL of the fermentation broth from 12-well plates cultured for 48-72 h, mix well, and add to the prepared YPD solid plates. Spread evenly and incubate at 30°C inverted for 24-36 h. Transfer 5-10 single colonies to 15 mL centrifuge tubes containing 5 mL of YPD liquid culture medium. Incubate at 30°C and 150-200 rpm with shaking for 24-36 h, then store at -80°C with 30% glycerol for later use.

[0020] When a strain does not show visible growth in a 12-well plate in G40 liquid medium containing 0.05%~0.15% hydrogen peroxide, it indicates that its antioxidant capacity has not reached the minimum threshold set for screening. Therefore, the strain is judged to have no further screening potential and is removed.

[0021] Conversely, when a strain exhibits superior growth characteristics compared to other strains in G40 medium within this concentration range, and continues to form stable colonies in YPD solid medium containing 0.10%–0.20% hydrogen peroxide, it is considered to have strong tolerance to oxidative stress. In this case, the screening concentration of hydrogen peroxide should be appropriately increased, and the above-mentioned stepwise screening and culture experiments should be continued to obtain candidate strains with even stronger tolerance.

[0022] Example 2: Domestication of antioxidant yeast The hydrogen peroxide-resistant strain obtained in Example 1 was acclimatized in 12-well plates.

[0023] First, the hydrogen peroxide-resistant strain was inoculated into a 15 mL centrifuge tube containing 5 mL of YPD liquid medium and cultured at 30℃ and 200 rpm for 24-36 h with shaking to obtain a bacterial suspension. The bacterial cells were washed 2-3 times with sterile water, and the resuspended cells were inoculated into a 15 mL centrifuge tube containing 5 mL of G40 liquid medium, ensuring the initial absorbance (OD600nm) was within the range of 0.05-0.2. The suspension was then cultured at 30℃ and 200 rpm with shaking for 24-36 h. In a 12-well plate, 3 mL of G40 medium with a hydrogen peroxide concentration of 0.10%-0.20% was prepared, and 50-100 µL of the cultured bacterial suspension was added to each well, with 3-5 replicates for each strain. The plates were then incubated statically at 30℃ for 48-72 h to obtain a *Saccharomyces cerevisiae* suspension tolerant to this concentration of hydrogen peroxide.

[0024] In a 12-well plate, 3 mL of G40 liquid medium with progressively increasing hydrogen peroxide concentration was added to each well. The mass-volume fraction of hydrogen peroxide used ranged from 0.10% to 0.50%. Each concentration increase was set to be 10% to 20% higher than the hydrogen peroxide concentration in the previous round of medium, in order to achieve a gradually increasing oxidative stress condition, thereby screening for yeast strains with strong tolerance to hydrogen peroxide.

[0025] Thoroughly mix the pre-cultured *Saccharomyces cerevisiae* culture in a 15 mL centrifuge tube, and inoculate 50–150 µL of the culture into G40 liquid medium with a further increased hydrogen peroxide concentration. Set up 3–5 replicate wells for each strain and incubate statically at 30°C for 48–72 h. After the strain has stabilized under these conditions, repeat the inoculation and concentration increase steps, subculturing for 10–20 generations to allow the target *Saccharomyces cerevisiae* strain to gradually adapt to and tolerate the oxidative stress environment of 0.4%–0.6% hydrogen peroxide. Prepare YPD solid culture plates with a hydrogen peroxide concentration of 0.40%-0.6%. Take *Saccharomyces cerevisiae* cultured for 48-72 h in a 12-well plate, mix the fermentation broth, and add 30-100 µL of the bacterial culture to a YPD solid culture plate with a hydrogen peroxide concentration of 0.40%-0.6%. Spread the mixture evenly and incubate at 30℃ inverted for 36-48 h, observing colony growth. Collect single colonies, culture them in YPD liquid medium, and store them at -80℃ to complete the acclimatization process of the antioxidant *Saccharomyces cerevisiae* strain.

[0026] To evaluate the phenotypic stability and antioxidant capacity retention of the high-antioxidant-capacity Saccharomyces cerevisiae strain C16I12 after cryopreservation, the highly tolerant strain, cryopreserved at -80℃, was retrieved and inoculated onto YPD solid plates without hydrogen peroxide. The plates were then incubated at 30℃ for 24–36 h with the plates inverted. Single colonies were collected and inoculated into 15 mL centrifuge tubes containing 5 mL of YPD liquid medium. The tubes were then incubated at 30℃ with shaking at 200 rpm for 24–36 h. After incubation, colony morphology and cell morphology under an optical microscope (e.g., [missing information]) were observed. Figure 1 and Figure 2 As shown in the figure, the strain was compared with the original control strain that had not undergone mutagenesis treatment to assess whether there were significant differences in morphology. The results showed no significant differences.

[0027] The bacterial cells obtained from the above culture were washed three times with sterile water to remove residual culture medium components and then resuspended. 50-100 µL of the resuspended cells were inoculated into a 15 mL centrifuge tube containing 5 mL of G40 liquid medium with a hydrogen peroxide concentration of 0.40%-0.60%. The tubes were then cultured at 30°C and 200 rpm with shaking for 24-36 h to assess whether the cells maintained their tolerance to high concentrations of hydrogen peroxide.

[0028] Simultaneously, optical microscopic images of the strain were acquired in G40 high hydrogen peroxide concentration medium and compared with optical microscopic images of the same strain in YPD liquid medium to determine the cell morphological stability of the strain under oxidative stress after preservation. The results showed good stability.

[0029] Example 3: Antioxidant index test of antioxidant yeast The antioxidant capacity of the Saccharomyces cerevisiae strain C16I12 (i.e., CCTCC No. M20251151) domesticated in Example 2 and the original ground strain Y017 (i.e., CGMCC No. 19732) were tested for hydrogen peroxide tolerance gradient in 250 mL shake flasks containing 50 mL of G40 liquid medium at 30°C and 200 rpm.

[0030] A hydrogen peroxide concentration gradient of 0%, 0.1%, 0.2%, 0.3%, and 0.4% (w / v) was used in G40 liquid medium to assess the antioxidant capacity of each bacterial strain. Figure 3 As shown, the domesticated Saccharomyces cerevisiae antioxidant strain C16I12 can still survive and maintain considerable growth activity under 0.2% and 0.4% hydrogen peroxide conditions, showing superior resistance to oxidative stress compared to the original strain C16I12 and the model strain S288C.

[0031] The growth rate of C16I12 in 0.2% hydrogen peroxide G40 medium is approximately 80% of that of Y017 in G40 medium without hydrogen peroxide (0%); under 0.4% hydrogen peroxide conditions, its growth rate can still reach 122% of that of Y017 under 0.3% hydrogen peroxide conditions (e.g., Figure 4 (As shown in the figure). These results indicate that C16I12 has significant physiological adaptability and antioxidant potential under oxidative stress, and is a potential candidate strain of highly antioxidant industrial yeast.

[0032] Example 4: Free radical scavenging rate test of antioxidant brewer's yeast The antioxidant capacity of the Saccharomyces cerevisiae strain C16I12 selected in Example 2 was compared with that of the original starting strain Y017 by ABTS testing.

[0033] Two yeast strains were cultured in 250 mL shake flasks containing 50 mL of G40 liquid medium at 30°C and 200 rpm for 36-48 h. 3-5 mL of the fermentation broth was centrifuged at 6000-8000 rpm for 5-10 min, and the supernatant was used for analysis. 100-200 µL of the supernatant was transferred to a 2 mL centrifuge tube and heated in boiling water at 100°C for 3-5 min, then immediately transferred to room temperature water to cool, yielding an inactivated fermentation broth. 10 mL of 7 mM ABTS aqueous solution and 179 µL of 140 mM potassium persulfate aqueous solution were incubated at room temperature in the dark for 12-15 h to obtain the ABTS stock solution. 300 µL of the ABTS stock solution was diluted with distilled water to 10 mL to obtain the ABTS working solution. Mix 170-200 µL of ABTS working solution with 20 µL of supernatant in an ELISA plate, incubate at 37 °C for 10-15 min, and measure the absorbance (A1) at 734 nm. Use deionized water as a blank control (A0) and ascorbic acid as a positive control. Calculate the ABTS free radical scavenging rate using the formula:

[0034] Test results as follows Figure 5As shown, the ABTS scavenging rate of the original fermentation broth of the antioxidant Saccharomyces cerevisiae strain C16I12 was 168% higher than that of the original starting strain Y017 without mutagenesis, and the ABTS scavenging rate of the inactivated fermentation broth of the antioxidant Saccharomyces cerevisiae strain C16I12 was 136% higher than that of the original starting strain Y017 without mutagenesis.

[0035] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of brewer's yeast Saccharomyces cerevisiae strain C16I12, characterized in that, The strain was deposited at the China Center for Type Culture Collection on May 21, 2025, with accession number CCTCC No. M20251151.

2. The strain according to claim 1, characterized in that, The strain was obtained from the original starting strain Y017 through space mutagenesis, screening, and domestication.

3. The strain according to claim 2, characterized in that, The original strain Y017 was deposited at the China General Microbiological Culture Collection Center on April 26, 2020, with accession number CGMCC No. 19732.

4. The strain according to claim 2, characterized in that, The screening process is as follows: liquid culture screening with hydrogen peroxide at progressively increasing concentrations, with each step increasing the concentration by 0.05% to 0.15% by mass-volume ratio, is carried out in 12-well plates. By gradually increasing the hydrogen peroxide concentration, strains with strong tolerance to oxidative stress are screened out.

5. The strain according to claim 2, characterized in that, The acclimatization process is as follows: In a 12-well plate, liquid culture medium with progressively increasing hydrogen peroxide concentration is added to each well. The mass-to-volume ratio of hydrogen peroxide used ranges from 0.10% to 0.50%, and each concentration increase is set to be 10% to 20% higher than the hydrogen peroxide concentration in the previous round of culture medium, so as to achieve a gradually increasing oxidative stress condition. The selected Saccharomyces cerevisiae strain is pre-cultured to obtain a bacterial solution, which is thoroughly mixed and then inoculated into liquid culture medium with a further increased hydrogen peroxide concentration. Each strain is set with 3 to 5 replicate wells and incubated statically at 30°C for 48 to 72 hours. After the strain grows stably under these conditions, the above inoculation and concentration increase steps are repeated for 10 to 20 generations, so that the target Saccharomyces cerevisiae strain gradually adapts to and tolerates the oxidative stress environment of 0.4% to 0.6% hydrogen peroxide, and finally the successfully acclimatized target strain is obtained.

6. The strain according to claim 1, characterized in that, The strain has antioxidant properties and can tolerate hydrogen peroxide concentrations of not less than 0.4%.

7. The strain according to claim 1, characterized in that, The extracellular fermentation broth of the strain has antioxidant properties and the ability to scavenge free radicals. The ABTS free radical scavenging rate of its original fermentation broth is 168% higher than that of the original starting strain Y017.

8. The application of the strain described in claim 1 in the fields of skin care products, health products, and anti-aging foods.

9. The fermentation product or fermentation extract of the strain described in claim 1.

10. The application of the fermentation product or fermentation extract as a natural anti-aging agent in the fields of skin care products, health products, and anti-aging foods, according to claim 9.

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