SOD-producing saccharomyces cerevisiae strain isolated from deep-sea hydrothermal vent and its application

Abstract

The application discloses a deep-sea hot spring separated SOD yeast strain and application thereof, and belongs to the technical field of biology. The application provides a Saccharomyces cerevisiae SC-SH18, the strain has good temperature tolerance, the strain has good growth state in the range of 28 DEG C to 45 DEG C, has the best growth effect at 42 DEG C, and can meet the production demand of fermentation under high-temperature conditions. The application provides a high-temperature-resistant SOD enzyme, the SOD enzyme is prepared by fermentation of the Saccharomyces cerevisiae SC-SH18, the SOD enzyme has good heat resistance, has almost no enzyme activity loss after 90 DEG C heat treatment for 120 min, retains more than 80% of enzyme activity after 100 DEG C heat treatment for 30 min, and retains more than 57% of enzyme activity after 100 DEG C heat treatment for 60 min.

Description

Technical Field

[0001] This invention relates to a strain of SOD-producing Saccharomyces cerevisiae isolated from deep-sea hydrothermal vents and its application, belonging to the field of biotechnology. Background Technology

[0002] With the improvement of living standards, people are becoming more aware of their own health and are paying more and more attention to the demand for antioxidants and anti-inflammatories. Superoxide dismutase (SOD) is an important antioxidant enzyme in living organisms, widely distributed in animals, plants, and microorganisms. Because of its ability to scavenge superoxide anion free radicals in living organisms, it can effectively resist the toxicity of free radicals and other oxidative free radicals to cell membranes, maintain normal cellular metabolism, and play an important role in protecting the body. This has attracted widespread attention and is therefore widely used in the fields of medicine, food, and cosmetics as an additive in foods, medicines, and cosmetics for anti-oxidation, anti-aging, anti-inflammation, and treatment of autoimmune diseases. Experts call it the most promising medicinal enzyme of the 21st century.

[0003] Traditionally, SOD is derived from animal tissues or plant extracts. However, due to bottlenecks in cost and activity retention, microbial fermentation has become the mainstream approach, with yeast or engineered strains (such as Escherichia coli and Bacillus subtilis) as representatives. Regarding activity retention, heat-resistant SOD is currently a research hotspot. SOD production involves fermentation and purification, but the purification process typically involves high-temperature treatment. Furthermore, as a protease, it is frequently affected by environmental factors during storage and transportation, making it prone to denaturation and loss of activity. This significantly limits its application in food, pharmaceuticals, and cosmetics. Summary of the Invention

[0004] To address the shortcomings of the existing technologies, this invention provides a SOD-producing Saccharomyces cerevisiae strain isolated from deep-sea hydrothermal vents and its applications. The aim is to solve the technical problems in the existing technologies where the purification process of SOD enzymes usually involves high-temperature treatment, and the storage and transportation processes are often affected by the environment, making them prone to denaturation and loss of activity. This greatly limits their application in the fields of food, medicine, and cosmetics.

[0005] The first technical solution provided by the present invention is a thermoresistant SOD enzyme, the amino acid sequence of which is shown in SEQ ID NO.2.

[0006] The second technical solution provided by the present invention is to encode the gene for the heat-resistant SOD enzyme described in the first technical solution.

[0007] In some embodiments, the nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0008] The third technical solution provided by the present invention is a recombinant vector carrying the gene described in the second technical solution.

[0009] The fourth technical solution provided by the present invention is to express the heat-resistant SOD enzyme described in the first technical solution, or to contain the gene described in the second technical solution, or to transform a host cell with the recombinant vector described in the third technical solution.

[0010] The fifth technical solution provided by this invention is a brewing yeast strain that produces the heat-resistant SOD enzyme described in the first technical solution. Saccharomyces cerevisiae SC-SH18, the brewing yeast SC-SH18, was deposited at the China General Microbiological Culture Collection Center on June 18, 2026, with accession number CGMCC No. 39426.

[0011] The sixth technical solution provided by the present invention is a microbial preparation, wherein the microbial preparation contains the brewing yeast SC-SH18 or its fermentation broth as described in the fifth technical solution, or contains brewing yeast SC-SH18 dried cells obtained by freeze-drying, or contains brewing yeast SC-SH18 cells obtained by solidification technology.

[0012] The seventh technical solution provided by the present invention is a product containing the heat-resistant SOD enzyme described in the first technical solution, the brewing yeast SC-SH18 described in the fifth technical solution, or the microbial preparation described in the sixth technical solution.

[0013] In some embodiments, the product is a chemical.

[0014] The eighth technical solution provided by the present invention is the application of the brewing yeast SC-SH18 described in the fifth technical solution or the microbial preparation described in the sixth technical solution in the preparation of fermented products.

[0015] In some embodiments, the fermentation product includes a fermentation product containing the thermostable SOD enzyme described in the first technical solution.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a brewing yeast ( Saccharomyces cerevisiae SC-SH18 is a strain with good temperature tolerance. It exhibits good growth in the range of 28℃ to 45℃, with the best growth effect at 42℃, which can meet the production needs of fermentation under high temperature conditions.

[0017] This invention provides a heat-resistant SOD enzyme, wherein the SOD enzyme is *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomyces cerevisiaeThe SOD enzyme prepared by fermentation of SC-SH18 has good heat resistance. There is almost no loss of enzyme activity after heat treatment at 90℃ for 120 min, more than 80% of the enzyme activity is retained after heat treatment at 100℃ for 30 min, and more than 57% of the enzyme activity is retained after heat treatment at 100℃ for 60 min.

[0018] Preservation of biological materials A strain of brewer's yeast ( Saccharomyces cerevisiae SC-SH18, taxonomically named *Saccharomyces cerevisiae*. Saccharomyces cerevisiae It was deposited on June 18, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39426. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Attached Figure Description

[0019] Figure 1 For brewing yeast ( Saccharomyces cerevisiae The flat plate form of SC-SH18.

[0020] Figure 2 For brewing yeast ( Saccharomyces cerevisiae Determination of the optimal growth temperature of SC-SH18.

[0021] Figure 3 For brewing yeast ( Saccharomyces cerevisiae Growth curve of SC-SH18 at 42℃.

[0022] Figure 4 For brewing yeast ( Saccharomyces cerevisiae ) SC-SH18 self-SOD enzyme activity assay.

[0023] Figure 5 For brewing yeast ( Saccharomyces cerevisiae The thermal stability of SOD enzyme prepared by SC-SH18 at 90℃.

[0024] Figure 6 This study aims to determine the thermal stability of SOD enzyme at 100℃. Detailed Implementation

[0025] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0026] The detection methods involved in the following embodiments are as follows: SOD enzyme activity assay method: Using the method in GB / T5009.171-2003, SOD enzyme activity is defined as the amount of SOD required to inhibit the auto-oxidation rate of pyrogallol by 50% at 25℃, which is 1 activity unit.

[0027] The culture media involved in the following examples are as follows: YPD medium: 1L of medium contains: 10g yeast extract, 20g peptone, 20g glucose. If preparing solid medium, add 1.5% agar powder (filter out and add glucose before inoculation).

[0028] Example 1: Screening and identification of Saccharomyces cerevisiae SC-SH18 strain The specific steps are as follows: 1. Isolation of brewing yeast (1) Sample processing Take 0.1g of silt collected from the deep sea and dissolve it in an Erlenmeyer flask containing 50mL of YPD medium. Incubate at 42℃ and 200rpm for 24h. After incubation, take 1mL of fermentation broth and add it to the YPD medium. Subculture for 24h. Repeat this subculture 5 times. Then take 1mL and spread it for dilution.

[0029] (2) Diluting and coating the isolated strains Take 1 mL of the fermentation broth after 5 subcultures and dilute it 1000, 10000, and 100000 times in YPD solid medium, respectively. After incubating in a 42℃ incubator for 48 h, randomly select single colonies with typical yeast colony characteristics into 5 mL YPD medium test tubes and incubate at 42℃ and 200 rpm for 12 h to obtain candidate yeast strains.

[0030] (3) Initial screening Using TTC (2,3,5-triphenyltetrazolium chloride) as a chromogenic agent, a double-layer culture medium was used for chromogenic culture. The specific method is as follows: First, 10 mL of YPD medium containing 2% agarose was poured into a petri dish. After solidification and cooling, the candidate yeast strain obtained above was diluted 10,000 times and inoculated onto the medium. The dish was then incubated at 42℃ for 36 h. Then, 10 mL of YPD medium mixed with TTC chromogenic agent and 2% agarose was placed on top of the previously cultured petri dish. The dish was incubated at 42℃ in the dark for 4 h. After removal, the colony color was observed immediately. Single red colonies were transferred to test tubes containing 5 mL of YPD medium and incubated at 42℃ and 200 rpm for 24 h to obtain the initial screening yeast strain.

[0031] (4) Identification For the yeast strains obtained from the initial screening, preliminary morphological identification was performed by observing the morphological indicators of the cells using a microscope and a stereomicroscope, according to the standard methods for yeast identification in "Research on Yeast Taxonomy". The selection criteria for strains were: smooth and moist surface, viscous texture when tapped with a pipette tip, uniform texture and color of the entire cell, and milky white colony characteristics.

[0032] Simultaneously, sequencing primers were designed to target the ITS of Saccharomyces cerevisiae for PCR. The primers are as follows: F: 5'-AAAGAAATTTAATAATTTTGAAATGGATTTTTTTGTTT-3'; R: 5'-ACTTTAAGAACATTGTTGCCTAGAC-3'.

[0033] The obtained product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. After gene sequence alignment analysis, it was sequenced using ITS (sequence shown in SEQ ID NO.1). Analysis of the similarity between the strain and *Saccharomyces cerevisiae* in the NCBI database showed a homology of 99.86%, thus identifying it as *Saccharomyces cerevisiae*. Saccharomyces cerevisiae ), named brewer's yeast ( Saccharomyces cerevisiae SC-SH18. The final streaking result yielded the following yeast strain morphology: Figure 1 As shown.

[0034] Example 2: Saccharomyces cerevisiae ( Saccharomyces cerevisiae SC-SH18 temperature tolerance brewing yeast ( Saccharomyces cerevisiae SC-SH18 was inoculated into YPD medium and cultured at 38℃, 42℃, and 45℃ for 30 h at 200 rpm. Temperature tolerance was assessed every 2 h. Results are shown below. Figure 2 As shown.

[0035] Growth curves (600 nm) of strain SC-SH18 at 38℃, 42℃, and 45℃ were plotted. The results showed that Saccharomyces cerevisiae SC-SH18 could grow in the range of 38℃ to 45℃, but 42℃ was the optimal growth temperature.

[0036] Example 3: Saccharomyces cerevisiae ( Saccharomyces cerevisiae Growth curve of strain SC-SH18 The specific steps are as follows: (1) Add brewing yeast ( Saccharomyces cerevisiae The SC-SH18 strain was inoculated into a 5 mL YPD medium test tube and cultured at 42 °C and 200 rpm for 24 h to prepare the seed culture.

[0037] (2) The seed culture prepared in step (1) was inoculated into an Erlenmeyer flask containing 100 mL of YPD medium at an inoculation rate of 1% (v / v). The flask was incubated at 42°C and 200 rpm in a constant temperature shaker. The growth of the strain was observed over 72 hours. Samples were taken every 2 hours in the early stage of inoculation and at 4, 6, and 12 hours in the later stage. The absorbance at 600 nm was measured using a spectrophotometer. The experiment was repeated three times, and the growth curve of the strain was plotted (see [link to relevant documentation]). Figure 3).

[0038] Example 4: Saccharomyces cerevisiae ( Saccharomyces cerevisiae SC-SH18 fermentation produces SOD gene The specific steps are as follows: 1. Fermentation for enzyme production (1) Add brewing yeast ( Saccharomyces cerevisiae The SC-SH18 strain was inoculated into 5 mL YPD medium test tubes and cultured at 42℃ and 200 rpm for 12 h, 24 h, 36 h and 48 h respectively to prepare fermentation broth.

[0039] (2) The fermentation broth prepared in step (1) was centrifuged at 5000 rpm for 10 min to collect the cells. The supernatant was discarded, and 1 mL of pH 7.0, 50 mM PBS buffer was added to the cells to resuspend them. The cells were then broken up using an ultrasonic disruptor with the following parameters: 400 W, 7 s sonication, 7 s interval, and 10 min sonication time. After disruption, the cells were centrifuged at 10000 rpm for 10 min to obtain the supernatant, which is the crude SOD enzyme solution.

[0040] (3) The enzyme activity of the above crude enzyme solution was tested respectively, and the results are as follows: Figure 4 As shown.

[0041] The results showed that the SOD activity of Saccharomyces cerevisiae SC-SH18 reached its highest level of 679.67±5.03 U / mL after 36 hours of fermentation.

[0042] (4) Determination of SOD gene sequence After whole-genome sequencing of Saccharomyces cerevisiae SC-SH18, the SOD sequence of Saccharomyces cerevisiae (number: NP_011872.1) in the NCBI public database was used as the starting sequence. The sequencing results were compared and analyzed to obtain the amino acid sequence of SOD enzyme in Saccharomyces cerevisiae SC-SH18 as shown in SEQ ID NO.2. The amino acid coverage of SOD enzyme in Saccharomyces cerevisiae SC-SH18 was 99% and the similarity was 61.14% compared with NP_011872.1.

[0043] Example 5: Detection of the enzymatic properties of SOD enzyme The specific steps are as follows: 1. Thermal stability determination (1) Inoculate the Saccharomyces cerevisiae SC-SH18 strain into a 5mL YPD medium test tube, and culture at 42℃ and 200rpm for 36h to prepare the fermentation broth. Centrifuge the fermentation broth at 5000rpm for 10min to collect the cells and discard the supernatant.

[0044] (2) Add 1 mL of pH 7.0, 50 mM PBS buffer to the bacterial cells obtained in step (1) to resuspend the bacterial cells, and use an ultrasonic disruptor to disrupt them (under the same conditions as described in Example 4). After centrifuging at 10000 rpm for 10 min, the bacterial cells are disrupted and the supernatant is obtained as crude SOD enzyme solution. (3) The obtained crude SOD enzyme solution was divided into a control group and an experimental group. The control group was left untreated and placed at room temperature; the experimental group was placed in boiling water at 90℃ and boiled for 10 min, 30 min, 60 min, and 120 min, and the SOD enzyme activity was measured. The results are as follows: Figure 5 As shown.

[0045] The results showed that the SOD enzyme activities of the samples placed at room temperature at 10 min, 30 min, 60 min, and 120 min were 676.33±6.02 U / mL, 676±5.29 U / mL, 674.67±4.51 U / mL, and 670±5.68 U / mL, respectively.

[0046] After heat treatment at 90℃ for 10 min, 30 min, 60 min, and 120 min, the SOD enzyme activities of the experimental groups were 673.67±5.51 U / mL, 673.33±4.04 U / mL, 679.67±8.02 U / mL, and 675.33±6.11 U / mL, respectively.

[0047] The experimental results show that the SOD enzyme activity in the experimental group was basically the same as that in the control group, indicating that the SOD enzyme prepared by fermentation of Saccharomyces cerevisiae SC-SH18 exhibits extremely high heat resistance.

[0048] (4) The specific implementation method is the same as steps (1) to (3), except that in step (3), the experimental group is placed in boiling water at 100℃ and boiled for 0 min, 30 min, 60 min, 90 min, and 120 min, and then the SOD enzyme activity is measured. The results are as follows. Figure 6 As shown.

[0049] The results showed that the SOD enzyme activities after heat treatment at 100℃ for 0 min, 30 min, 60 min, 90 min, and 120 min were 676.33±6.02 U / mL, 551.33±11.01 U / mL, 389.67±9.01 U / mL, 211±11.13 U / mL, and 185.6±10.78 U / mL, respectively.

[0050] It can be seen that when heat-treated at 100℃ for 30 min, the SOD enzyme activity is retained by more than 80%, and when heat-treated at 100℃ for 60 min, the SOD enzyme activity is retained by more than 57%.

[0051] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A thermostable SOD enzyme, characterized in that, The amino acid sequence of the thermoresistant SOD enzyme is shown in SEQ ID NO.

2.

2. The gene encoding the thermostable SOD enzyme of claim 1.

3. A recombinant vector containing the gene of claim 2.

4. A yeast host cell expressing the thermostable SOD enzyme of claim 1, or containing the gene of claim 2, or transformed with the recombinant vector of claim 3.

5. A strain of *Saccharomyces cerevisiae* producing the thermostable SOD enzyme as described in claim 1 (… Saccharomyces cerevisiae SC-SH18, characterized in that, The brewing yeast SC-SH18 was deposited at the China General Microbiological Culture Collection Center on June 18, 2026, with accession number CGMCC No. 39426.

6. A microbial preparation, characterized in that, The microbial preparation contains the brewer's yeast SC-SH18 as described in claim 5.

7. The microbial preparation according to claim 6, characterized in that, The microbial preparation contains freeze-dried Saccharomyces cerevisiae SC-SH18.

8. The microbial preparation according to claim 6, characterized in that, The microbial preparation contains Saccharomyces cerevisiae SC-SH18 cells obtained through solidification technology.

9. A microbial preparation, characterized in that, The microbial preparation contains the thermostable SOD enzyme as described in claim 1.

10. The use of the brewing yeast SC-SH18 according to claim 5 or the microbial preparation according to any one of claims 6 to 8 in the preparation of fermented products.

11. The application according to claim 10, characterized in that, The fermented product includes a fermentation product containing the thermostable SOD enzyme as described in claim 1.

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