Saccharomyces cerevisiae with high yield of soluble beta-glucan and application thereof

Abstract

The present application relates to a high-yield soluble beta-glucan Saccharomyces cerevisiae and its application. The present application is screened by the way of isolation and screening from soil, and a high-yield soluble beta-glucan Saccharomyces cerevisiae is screened. The Saccharomyces cerevisiae MF-001 of the present application is preserved in Guangdong Microbial Culture Collection Center on January 3, 2025, and the preservation number is GDMCC No: 65715. The Saccharomyces cerevisiae MF-001 screened and separated by the present application can reach the logarithmic growth phase faster, the fermentation period is short, and the fermentation efficiency is improved. At the same time, the Saccharomyces cerevisiae MF-001 has a high beta-glucan yield, and the fermentation liquid obtained by fermentation can obtain a high-purity, colorless, transparent, low-viscosity, high-fluidity liquid product after a simple purification step, which can improve the subsequent purification production efficiency. And the liquid product has been verified to have good biological activity, and has broad application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a strain of Saccharomyces cerevisiae that produces high levels of soluble β-glucan and its applications. Background Technology

[0002] β-glucan is a natural polysaccharide widely found in bacteria, fungi, yeast, algae, and plants. It is composed of glucose molecules linked by β-(1,3), β-(1,4), or β-(1,6) glycosidic bonds; and its structure varies depending on its source. For example, β-glucan in yeast and fungi is mostly a mixed polysaccharide with β-(1,3) glucan as the main component and a small amount of β-(1,6) glucan.

[0003] β-glucan possesses a variety of biological activities, including immunomodulation, anti-tumor activity, antioxidant activity, lipid-lowering activity, and anti-inflammatory activity. Studies have shown that β-glucan has numerous hydrophilic groups, resulting in strong hydrophilicity and thus powerful water-locking and moisturizing effects; it also exhibits anti-allergic, anti-inflammatory, scar-fading, anti-aging, and wrinkle-reducing effects. Therefore, it has wide applications in medicine, food, cosmetics, and animal feed.

[0004] In the trend of advocating green and natural cosmetics, yeast β-glucan is highly favored due to its natural polysaccharide and multiple effects, and has a good market prospect. However, the current domestic production capacity of yeast β-glucan cannot meet market demand. The main drawback is that the fermentation product has many impurities. Therefore, obtaining high-purity β-glucan usually requires a complex purification process with multiple steps, which not only increases production costs, but also leads to a significant reduction in product yield.

[0005] Therefore, screening for brewing yeast that produces high levels of soluble β-glucan is crucial for industrial applications. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a high-yield soluble β-glucan-producing Saccharomyces cerevisiae strain. This high-yield β-glucan-producing Saccharomyces cerevisiae produces fewer impurities during fermentation and metabolism, thus allowing for the acquisition of high-purity, colorless, transparent, low-viscosity, and highly fluid β-glucan products through simple purification steps.

[0007] To achieve the above objectives, the present invention discloses the following technical solutions:

[0008] In a first aspect, the present invention provides a strain of Saccharomyces cerevisiae that produces high levels of soluble β-glucan. The Saccharomyces cerevisiae is named Saccharomyces cerevisiae MF-001. The Saccharomyces cerevisiae MF-001 was deposited on January 3, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, China, with accession number GDMCC No: 65715.

[0009] Secondly, the present invention provides a microbial inoculant comprising the aforementioned brewing yeast.

[0010] Thirdly, the present invention provides the application of the brewing yeast described in the first aspect in the preparation of soluble β-glucan.

[0011] Fourthly, the present invention provides the application of the brewing yeast described in the first aspect in the preparation of cosmetics containing soluble β-glucan.

[0012] Fifthly, the present invention provides the application of the brewing yeast described in the first aspect in the preparation of health products containing soluble β-glucan.

[0013] In a sixth aspect, the present invention provides a method for preparing soluble β-glucan, the method comprising fermentation production using the brewing yeast described in the first aspect.

[0014] Preferably, the fermentation temperature is 25-30°C, and more preferably, the fermentation temperature is 28°C.

[0015] Preferably, the fermentation time is 40-50 hours, and more preferably, the fermentation time is 48 hours.

[0016] In a seventh aspect, the present invention provides a method for producing soluble β-glucan using the brewing yeast described in the first aspect, characterized in that the method includes steps such as: strain activation, seed culture, fermentation culture, and purification.

[0017] The beneficial effects of this invention are:

[0018] 1. Through extensive screening, the inventors ultimately isolated the brewing yeast MF-001, which reaches the logarithmic growth phase faster than other strains, shortening the fermentation cycle and improving the efficiency of production fermentation. At the same time, brewing yeast MF-001 has a higher β-glucan yield than other strains. The fermentation broth can be purified into a high-purity, colorless, transparent, low-viscosity, and highly fluid liquid product, which is of great significance for improving subsequent production efficiency.

[0019] 2. The fermentation supernatant of the Saccharomyces cerevisiae MF-001 screened in this invention has excellent antioxidant activity. Compared with the fermentation supernatant of other strains, it has better antioxidant performance, indicating that the β-glucan prepared from Saccharomyces cerevisiae MF-001 maintains good biological activity and has broad industrial application prospects. Attached Figure Description

[0020] To further understand the present invention, the following detailed description is provided in conjunction with the accompanying drawings:

[0021] Figure 1 Growth curves for strains numbered 72, 37, 55, and those from the prior art. Detailed Implementation

[0022] Those skilled in the art can refer to the content of this document to appropriately improve the process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention.

[0023] The products and methods of the present invention have been described through preferred embodiments. Those skilled in the art will be able to make modifications or appropriate alterations and combinations to the products and methods described herein without departing from the content, spirit and scope of the present invention, so as to realize and apply the technology of the present invention.

[0024] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Materials, reagents, etc., used are commercially available unless otherwise specified. Unless otherwise stated, substances described in this invention are calculated as percentages and parts by mass.

[0025] In this invention:

[0026] Bengal Red Medium: Bengal Red 0.0033%, Chloramphenicol 0.01%, MgSO4 0.05%, KH2PO4 0.1%, Peptone 0.5%, Glucose 1%, Agar 1.5%;

[0027] YEPD liquid medium: 1% yeast extract, 2% glucose and 2% peptone;

[0028] YEPD solid medium: YEPD liquid medium with 2% agar added;

[0029] Seed culture medium: 1% yeast extract, 2% glucose and 2% peptone;

[0030] Fermentation medium: 5% glucose, 1% yeast extract and 2% peptone;

[0031] All the above culture media were sterilized at 115℃ for 25 min.

[0032] The existing strain is the soluble β-glucan-producing Saccharomyces cerevisiae disclosed in CN114292763A with accession number CCTCC NO:M 20211406.

[0033] Example 1: Isolation and Screening of Strains

[0034] 1. Strain discovery and isolation

[0035] One hundred soil samples were collected from the vicinity of Guangzhou Miaofucheng Biotechnology Co., Ltd. as the source for yeast isolation. The collected soil samples were added to sterile physiological saline to prepare bacterial suspensions and serially diluted. 200 μl of the 1000-fold diluted bacterial suspension was taken and spread evenly on Bengal Red solid medium plates with a spreader and incubated at 28°C for 2 days.

[0036] One hundred well-grown single colonies with typical yeast colony characteristics were selected, streaked to purify for three generations, and the isolated and purified yeasts were inoculated into YEPD slant medium as original strains and stored at 4℃. The corresponding labels were numbered 1 to 100.

[0037] 2. Strain screening

[0038] The yeast strains obtained from the above isolation and purification were optimized and screened: each strain preserved on the slant was inoculated into a shake flask containing 50 ml of YEPD liquid medium and cultured at 28°C with shaking. After 12 h, the strains were transferred to 100 ml of YEPD liquid medium and cultured at 28°C with shaking for 3 days. Five strains with high biomass and β-glucan production were selected, namely strains numbered 13, 37, 55, 72 and 86.

[0039] The fermentation performance of the five strains obtained from the above screening was evaluated, and the best strain was selected.

[0040] Example 2: Fermentation Performance Evaluation

[0041] 1. Activation and Seed Culture

[0042] 1.1 Seed Activation

[0043] Single colonies with good growth (numbered 13, 37, 55, 72, and 86) were selected from YEPD solid plates and inoculated into 50 mL of YEPD liquid medium. The colonies were then cultured for 20 h in a constant temperature shaker at 28 °C and 200 r / min.

[0044] 1.2 Seed Culture

[0045] The activated bacterial solution was inoculated into a 500 mL shake flask containing 200 mL of seed culture medium at an inoculation rate of 1 v / v%, and cultured at 28 °C and 200 r / min for about 20 h to further increase the number of fungi and obtain the seed solution.

[0046] 2. Fermentation culture

[0047] 2.1 Vaccination

[0048] The prepared seed culture was inoculated into a 10L fermenter containing fermentation medium that had been verified to be free of contaminants by microscopic examination at an inoculation rate of 10v / v%, with a total fermentation broth volume of 5L.

[0049] 2.2 Initial culture conditions settings

[0050] The initial rotation speed of the fermenter was set at 150 r / min, and then gradually increased to 200 r / min and maintained as the bacteria grew. The fermentation temperature was set at 28℃, and the initial aeration rate was 1.5 vvm. The aeration rate was then gradually increased according to the dissolved oxygen value to ensure a sufficient oxygen supply.

[0051] 2.3 Monitoring and control of dissolved oxygen and pH:

[0052] Use a dissolved oxygen probe to monitor the dissolved oxygen level in the tank, ensuring that the dissolved oxygen level is controlled at 20-22% and the pH is maintained at around 4.5.

[0053] 2.4 Obtaining the fermentation broth:

[0054] Fermentation broth was obtained after 48 hours of fermentation.

[0055] 3. Growth curve detection

[0056] To determine when the strain reached its plateau phase, the yeast growth curve was measured, with OD values ​​taken every 2 hours. 600 If the sample is too concentrated, it needs to be appropriately diluted before measurement. Three parallel controls should be set up, and the average result should be taken. The growth of the strain can be analyzed using the optical density value (OD value). Generally speaking, the higher the OD value, the higher the corresponding bacterial concentration. Growth curves for some strains are shown below. Figure 1 (The strains selected for display are numbered 37, 55, 72 and existing technology strains).

[0057] 4. Detection of biomass and β-glucan content in fermentation broth

[0058] 4.1 Instruments used

[0059] Incubator, analytical balance (0.001g).

[0060] 4.2 Detection Method

[0061] Fermentation broth biomass determination: Take 100ml of fermentation broth, filter it, centrifuge and wash it with deionized water at 4℃, and titrate the supernatant with phenol and sulfuric acid until it is colorless. Dry the washed yeast cells at 80℃ to constant weight, weigh them with a balance, repeat 3 times in parallel, and take the average value.

[0062] Fermentation broth β-glucan content: Accurately weigh 100ml of fermentation broth, and then test it according to the detection method of QB / T 4572-2021 "Yeast β-glucan". Repeat the test three times in parallel and take the average value of the results.

[0063] 5. Evaluation of fermentation performance in the control group

[0064] Take existing strains and perform activation and seed culture, fermentation culture, growth curve detection, and detection of biomass and β-glucan content in fermentation broth according to the aforementioned steps 1-4.

[0065] 6. Fermentation performance results

[0066] 6.1 Growth Curve Results

[0067] The growth curve detection results for strains numbered 37, 55, and 72 and existing technology strains are shown below. Figure 1 .

[0068] 6.2 Results of β-glucan content and biomass

[0069] Table 1. Results of β-glucan content and biomass detection

[0070]

[0071]

[0072] The results show that:

[0073] Figure 1 Growth curve analysis results showed that strain number 72 reached the exponential growth phase in about 8 hours, while the other strains took about 10 hours to reach the exponential growth phase. This indicates that strain number 72 can reach the logarithmic growth phase more quickly, which helps to improve fermentation efficiency in production.

[0074] The results of the β-glucan content and biomass analysis in Table 1 show that strain 72 not only had the highest biomass in its fermentation broth, but also the highest soluble β-glucan content, reaching 437.21 mg / 100 ml. Its yield was significantly superior to the other screened strains and existing soluble β-glucan-producing strains. In conclusion, strain 72, isolated and screened in this invention, is determined to be the optimal strain for high soluble β-glucan production.

[0075] Example 3: Molecular biological identification of the strain

[0076] 3.1 Identification of strain morphology

[0077] Colony morphology: Colonies are round, glossy, with neat edges, and milky white in color.

[0078] Microscopic morphology: Oval or spherical, reproduces by budding.

[0079] 3.2 Gene Identification

[0080] Strain number 72, obtained through screening, was identified using molecular biology techniques. Its 26S rDNA sequence is shown in SEQ ID NO.1. BLAST alignment of the sequencing results with the NCBI database showed that the strain sequence was highly homologous to the *Saccharomyces cerevisiae* gene sequence, thus identifying it as *Saccharomyces cerevisiae*.

[0081] 4. Strain preservation

[0082] Based on the colony morphology and molecular identification results of strain number 72, this strain was named Saccharomyces cerevisiae MF-001. It was deposited on January 3, 2025, at the Guangdong Provincial Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, China, postal code 510070, with accession number GDMCC No: 65715.

[0083] Example 4 Antioxidant activity of yeast fermentation supernatant

[0084] Determination of DPPH free radical scavenging ability:

[0085] Test sample solution: Take the fermentation supernatant of strains numbered 13, 37, 55, 72, 86 in Example 2 and the prior art strains, sterilize them by instantaneous sterilization technology, and then dilute them with deionized water to a concentration of 10 wt%.

[0086] Experimental protocol: DPPH was prepared with anhydrous ethanol to a concentration of 2×10⁻⁶. -4 Take several portions of 2 mL each of the sample solution to be tested, DPPH solution, and anhydrous ethanol. Mix 2 mL of the sample solution to be tested and 2 mL of DPPH solution and place at room temperature for 30 minutes. Measure the absorbance at a wavelength of 517 nm to obtain Ai.

[0087] Mix 2 mL of the test sample with 2 mL of anhydrous ethanol and measure the absorbance as Aj according to the aforementioned method.

[0088] The absorbance was measured as Ac after mixing 2 mL of DPPH solution and 2 mL of anhydrous ethanol according to the aforementioned method.

[0089] Each sample was measured in triplicate, and the average value was taken.

[0090] Calculate the scavenging rate of DPPH free radicals for each test sample according to the following formula (1) and record it in Table 2 below.

[0091] Clearance rate (%) = (1 - (Ai - Aj) / Ac) × 100% ... (1)

[0092] In the formula: Ai is the absorbance of the mixture of 2 mL test sample + 2 mL DPPH solution; Aj is the absorbance of the mixture of 2 mL test sample + 2 mL anhydrous ethanol; Ac is the absorbance of the mixture of 2 mL DPPH solution + 2 mL anhydrous ethanol.

[0093] Table 2 DPPH removal rate

[0094] Group DPPH removal rate 13 74.41％ 37 83.35％ 55 85.25％ 72 91.33％ 86 82.08％ Existing technology strains 76.13％

[0095] Conclusion Analysis:

[0096] As shown in Table 2, the antioxidant activity of yeast fermentation products is positively correlated with the β-glucan content in the fermentation broth. The Saccharomyces cerevisiae MF-001 (i.e., No. 72) screened in this invention has a higher β-glucan yield than other strains, and its fermentation supernatant also has excellent antioxidant activity. Compared with other screened strains and strains in the prior art, the supernatant of its fermentation products has better antioxidant performance.

[0097] Based on the above experiments, it can be concluded that the *Saccharomyces cerevisiae* MF-001 strain screened by this invention grows rapidly, has a short fermentation cycle, and can improve production efficiency. Furthermore, this strain has a high β-glucan yield, suggesting that a simple purification process after fermentation can yield a high-purity, colorless, transparent, low-viscosity, and highly fluid liquid product, which is of great significance for improving β-glucan production efficiency. Moreover, the liquid product maintains good biological activity and has broad application prospects.

[0098] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and the scope of protection of the present invention is defined by the appended claims. Those skilled in the art can make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, but all such changes and modifications fall within the scope of protection of the present invention.

Claims

1. A brewer's yeast strain that produces high levels of soluble β-glucan, characterized in that, The brewing yeast is named brewing yeast (Saccharomyces cerevisiae). Saccharomyces cerevisiae) MF-001, the brewing yeast MF-001, was deposited on January 3, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, China, with accession number GDMCC No: 65715.

2. A microbial inoculant, characterized in that, The microbial agent comprises the brewing yeast as described in claim 1.

3. The application of the brewing yeast of claim 1 in the preparation of soluble β-glucan.

4. The use of the brewing yeast of claim 1 in the preparation of cosmetics containing soluble β-glucan.

5. The use of the brewing yeast of claim 1 in the preparation of health products containing soluble β-glucan.

6. A method for preparing soluble β-glucan, characterized in that, The preparation method includes fermentation using the brewing yeast of claim 1; The fermentation temperature is 25-30℃.

7. The preparation method according to claim 6, characterized in that, The fermentation temperature is 28°C.

8. The preparation method according to claim 6, characterized in that, The fermentation time is 48 hours.

9. A method for producing soluble β-glucan using the brewing yeast as described in claim 1, characterized in that, The method includes the following steps: strain activation, seed culture, fermentation culture, and purification.

Images