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

Abstract

The invention relates to saccharomyces cerevisiae capable of producing soluble beta-glucan at high yield and application of the saccharomyces cerevisiae. According to the invention, the saccharomyces cerevisiae with high yield of soluble beta-glucan is obtained by screening in a manner of separating and screening from soil. The saccharomyces cerevisiae MF-001 disclosed by the invention 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 obtained through screening and separation can reach the logarithmic phase more quickly, the fermentation period is short, the fermentation efficiency is improved, meanwhile, the saccharomyces cerevisiae MF-001 has the high beta-glucan yield, a liquid product which is high in purity, colorless, transparent, low in viscosity and high in flowability can be obtained through the fermentation liquor obtained through fermentation of the saccharomyces cerevisiae MF-001 through a simple purification step, and the yield of beta-glucan is improved. The subsequent purification production efficiency can be improved. And the liquid product is proved to have good biological activity and wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, and specifically, to a Saccharomyces cerevisiae strain with high yield of soluble β-glucan and its application. Background Art

[0002] β-glucan is a natural polysaccharide widely present in bacteria, fungi, yeasts, 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 according to different sources. For example, β-glucan in yeasts and fungi is mainly a mixed polysaccharide of β-(1,3)-glucan with a small amount of β-(1,6)-glucan.

[0003] β-glucan has various biological activities, such as immunomodulation, anti-tumor, antioxidant, lipid-lowering and anti-inflammatory effects. And studies have shown that β-glucan has a large number of hydrophilic groups, can produce strong hydrophilicity, and thus has a strong water-locking and moisturizing effect; and it also has anti-allergic and anti-inflammatory, scar fading, anti-aging and wrinkle-removing effects. Therefore, it has a wide range of applications in the fields of medicine, food, cosmetics and feed.

[0004] In the trend of advocating green and natural cosmetics, yeast β-glucan is highly favored because it is a natural polysaccharide and has various effects, and has a good market prospect. However, at present, the domestic production capacity of yeast β-glucan cannot meet the market requirements, and there are mainly disadvantages such as many impurities in the fermentation products. Therefore, obtaining high-purity β-glucan usually requires multiple steps of complex purification processes, which not only increases the production cost, but also significantly reduces the product yield in multiple steps of purification.

[0005] Therefore, screening and obtaining Saccharomyces cerevisiae with high yield of soluble β-glucan is very important for industrial application. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a Saccharomyces cerevisiae strain with high yield of soluble β-glucan. The Saccharomyces cerevisiae with high yield of β-glucan produces few impurities during the fermentation and metabolism process. Therefore, β-glucan products with high purity, colorless, transparent, low viscosity and strong fluidity can be obtained through simple purification steps.

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

[0008] In a first aspect, the present invention provides a strain of Saccharomyces cerevisiae with high yield of soluble β-glucan, named Saccharomyces cerevisiae MF-001, with a Latin scientific name of Saccharomyces cerevisiae. The Saccharomyces cerevisiae MF-001 was deposited at the Guangdong Provincial Microbial Culture Collection Center on January 3, 2025, at the address of the 5th floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, China, with a deposit number of GDMCC No: 65715.

[0009] In a second aspect, the present invention provides a microbial inoculum, which comprises the above-mentioned Saccharomyces cerevisiae.

[0010] In a third aspect, the present invention provides the use of the Saccharomyces cerevisiae described in the first aspect in the preparation of soluble β-glucan.

[0011] In a fourth aspect, the present invention provides the use of the Saccharomyces cerevisiae described in the first aspect in the preparation of cosmetics containing soluble β-glucan.

[0012] In a fifth aspect, the present invention provides the use of the Saccharomyces cerevisiae 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, which comprises fermenting and producing by using the Saccharomyces cerevisiae 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 by using the Saccharomyces cerevisiae described in the first aspect, which is characterized in that the method comprises steps such as strain activation, seed liquid culture, fermentation culture and purification.

[0017] Advantages of the present invention:

[0018] 1. Through a large number of screenings in the early stage by the inventors of the present invention, the finally screened and isolated Saccharomyces cerevisiae MF-001 can reach the logarithmic growth phase faster than other strains, shortening the fermentation cycle and improving the efficiency of production fermentation. At the same time, Saccharomyces cerevisiae MF-001 has a higher β-glucan yield than other strains. The fermentation broth can obtain a liquid product with high purity, colorless, transparent, low viscosity and strong fluidity through simple purification steps, which is of great significance for improving the subsequent production efficiency.

[0019] 2. The fermentation supernatant of Saccharomyces cerevisiae MF-001 screened by the present invention has excellent antioxidant activity. Compared with the fermentation supernatants of other strains, it has better antioxidant performance, indicating that the β-glucan obtained from Saccharomyces cerevisiae MF-001 maintains good biological activity and has broad industrial application prospects. Brief Description of the Drawings

[0020] To further understand the present invention, the present invention will be described in detail below in conjunction with the drawings:

[0021] Figure 1 It is the growth curve diagrams of No. 72, No. 37, No. 55 and the strains of the prior art. Detailed Embodiments

[0022] Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.

[0023] The products and methods of the present invention have been described through preferred embodiments. It is obvious that relevant personnel can make changes or appropriate alterations and combinations to the products and methods described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0024] For the experimental methods without specific conditions indicated in the following examples, they are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. The materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels. Unless otherwise stated, the substances described in the present invention are calculated by mass percentage and mass fraction.

[0025] In the present invention:

[0026] Rose Bengal medium: Rose Bengal 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: 2% agar is added to the YEPD liquid medium;

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

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

[0031] The above media are sterilized at 115°C for 25 minutes.

[0032] Prior art strain: Saccharomyces cerevisiae producing soluble β-glucan with the preservation number of CCTCC NO: M 20211406 disclosed in CN114292763A.

[0033] Isolation and screening of the strain in Example 1

[0034] 1. Strain discovery and isolation

[0035] Collect 100 soil samples near Guangzhou Miaofucheng Biotechnology Co., Ltd. as the isolation source of yeast. Add the collected soil samples to sterile normal saline to make a bacterial solution and perform gradient dilution. Pipette 200 μl of the bacterial solution diluted 1000 times onto a plate of Rose Bengal solid medium, spread it evenly with a spreader, and culture it at 28 °C for 2 d.

[0036] Select 100 single colonies with good growth and typical yeast colony characteristics, purify them by streaking for 3 generations, and inoculate the isolated and purified yeast into YEPD slant medium as the original strains for preservation at 4 °C, and mark the corresponding numbers as 1 - 100.

[0037] 2. Strain screening

[0038] Optimize and screen the yeast strains obtained by the above isolation and purification: Inoculate each strain preserved on the slant into a flask containing 50 ml of YEPD liquid medium, culture it with shaking at 28 °C, transfer and inoculate it into 100 ml of YEPD liquid medium after 12 h, and culture it with shaking at 28 °C for 3 d. Select 5 strains with higher biomass and β-glucan yield, namely the strains numbered 13, 37, 55, 72, and 86.

[0039] Evaluate the fermentation performance of the above 5 selected strains and select the optimal strain among them.

[0040] Fermentation performance evaluation in Example 2

[0041] 1 Activation and seed culture

[0042] 1.1 Seed activation

[0043] Respectively select the single colonies numbered 13, 37, 55, 72, and 86 with good growth from the YEPD solid plate, inoculate them into 50 mL of YEPD liquid medium, and culture them in a constant temperature shaker at 28 °C and 200 r / min for 20 h.

[0044] 1.2 Seed culture

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

[0046] 2 Fermentation culture

[0047] 2.1 Inoculation

[0048] The obtained seed solution was inoculated into a 10 L fermenter containing fermentation medium that had been verified to be free of contaminants by microscopy at an inoculation amount of 10 v / v%, and the total volume of the fermentation broth was 5 L.

[0049] 2.2 Initial culture condition setting

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

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

[0052] A dissolved oxygen probe was used to monitor the dissolved oxygen level in the tank to ensure that the dissolved oxygen content was controlled at 20 - 22%, and the pH was maintained at about 4.5.

[0053] 2.4 Obtaining the fermentation broth:

[0054] The fermentation broth was obtained after 48 h of fermentation.

[0055] 3 Growth curve detection

[0056] To determine the time when the strain reached the stationary phase, the growth curve of the yeast was measured, and samples were taken every 2 h to measure the OD. 600 , and when the sample was too concentrated, it needed to be appropriately diluted before measurement. Three parallel controls were set, and the results were averaged. The growth situation of the strain could be analyzed through the optical density value (OD value). Generally speaking, the larger the OD value, the higher the corresponding bacterial solution concentration. The growth curve diagrams of some strains are shown in Figure 1 (The strains selected to display the results were No. 37, 55, 72 and the prior art strains).

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

[0058] 4.1 Instruments used

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

[0060] 4.2 Detection methods

[0061] Determination of the biomass of the fermentation broth: Filter 100 ml of the fermentation broth, centrifuge and wash it with deionized water at 4 °C until the supernatant is titrated to colorless with the phenol-sulfuric acid method. The washed yeast cells are dried to a constant weight at 80 °C, and their weight is measured with a balance. Repeat the process 3 times in parallel and take the average value.

[0062] β-glucan content of the fermentation broth: Accurately weigh 100 ml of the fermentation broth, and then detect it according to the detection method of QB / T 4572-2021 "Yeast β-glucan". Repeat the process 3 times in parallel and take the average value of the results.

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

[0064] Take the strains of the prior art and perform activation, seed culture, fermentation culture, growth curve detection, and detection of the biomass and β-glucan content of the 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 of the strains numbered 37, 55, and 72 and the strains of the prior art are shown in Figure 1 .

[0068] 6.2 β-glucan content and biomass results

[0069] Table 1 Detection results of β-glucan content and biomass

[0070]

[0071]

[0072] The results show that:

[0073] Figure 1 The growth curve detection results show that: The strain numbered 72 reaches the exponential growth phase at about 8 h, and the other strains reach the exponential growth phase at about 10 h, indicating that the strain numbered 72 can reach the logarithmic growth phase faster, which is helpful to improve the fermentation efficiency in production.

[0074] The detection results of β-glucan content and biomass in Table 1 show that: In the fermentation broth of the strain numbered 72, not only is the biomass the largest, but also the soluble β-glucan content in its fermentation broth is the highest, reaching 437.21 mg / 100 ml. Its yield is significantly better than that of the other screened strains and the existing soluble β-glucan-producing strains in the prior art. In summary, it is determined that the strain numbered 72 screened and isolated in the present invention is the optimal strain for high-yield soluble β-glucan.

[0075] Example 3 Molecular biological identification of the strain

[0076] 3.1 Strain Morphological Identification

[0077] Colony morphology: The colony is circular, shiny, with a neat edge, and milky white in color.

[0078] Morphology under the microscope: It is oval or spherical in shape, and the reproduction method is budding.

[0079] 3.2 Gene Identification

[0080] The strain numbered 72 obtained by screening was identified using molecular biology techniques. The 26S rDNA sequence of this strain is shown in SEQ ID NO.1. The sequencing results were subjected to BLAST alignment in the NCBI database. The sequence alignment results showed that: the sequence of this strain was highly homologous to the Saccharomyces cerevisiae gene sequence, and it was identified as Saccharomyces cerevisiae.

[0081] 4. Strain Preservation

[0082] Based on the colony morphological characteristics and molecular identification results of the strain numbered 72 described above, this strain was named Saccharomyces cerevisiae MF-001, with the Latin name Saccharomyces cerevisiae. And this strain was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on January 3, 2025. The address is the 5th floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, China, with the postal code 510070 and the deposit 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 supernatants of the strains numbered 13, 37, 55, 72, 86 in Example 2 and the strain of the prior art, sterilize them through instantaneous sterilization technology, and then dilute them to a concentration of 10 wt% with deionized water.

[0086] Experimental protocol: DPPH was prepared into a 2×10 -4 mol / L DPPH solution with absolute ethanol. Take several portions of 2 mL each of the test sample solution, DPPH solution, and absolute ethanol. Mix 2 mL of the test sample solution and 2 mL of the DPPH solution, and let it stand 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 and 2 mL of absolute ethanol, and measure the absorbance according to the above method as Aj;

[0088] Mix 2 mL of DPPH solution and 2 mL of absolute ethanol, and measure the absorbance according to the aforementioned method as Ac.

[0089] Each sample was measured in parallel 3 times, and the average value was taken.

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

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

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

[0093] Table 2 DPPH scavenging rate

[0094] Group DPPH scavenging rate 13 74.41％ 37 83.35％ 55 85.25％ 72 91.33％ 86 82.08％ Prior art strain 76.13％

[0095] Conclusion analysis:

[0096] As can be seen from Table 2, the antioxidant activity of the yeast fermentation product is positively correlated with the content of β-glucan in the fermentation broth. The Saccharomyces cerevisiae MF-001 (i.e., No. 72) screened in the present invention has a higher β-glucan yield compared to other strains, and its fermentation supernatant also has excellent antioxidant activity, and has better antioxidant performance compared to the fermentation product supernatants of other screened strains and strains in the prior art.

[0097] Combined with the above experiments, it can be known that the Saccharomyces cerevisiae MF-001 screened in the present invention grows fast and has a short fermentation cycle, which can improve production efficiency; and this strain has a high β-glucan yield, and it can be reasonably expected that a liquid product with high purity, colorless, transparent, low viscosity and strong fluidity can be obtained after simple purification steps of the fermentation of this strain, which is of great significance for improving the production efficiency of β-glucan; and the liquid product also maintains good biological activity and has broad application prospects.

[0098] Although the specific embodiments of the present invention have been described above, those skilled in the art should understand that this is only an example, and the protection scope of the present invention is defined by the appended claims. Without departing from the principles and essence of the present invention, those skilled in the art can make various changes or modifications to these embodiments, but these changes and modifications all fall within the protection scope of the present invention.

Claims

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

2. A microbial agent, characterized in that: The microbial agent comprises the brewer's yeast according to claim 1.

3. Use of the brewer's yeast according to claim 1 in the preparation of soluble β-glucan.

4. Use of the brewer's yeast according to claim 1 in preparing cosmetics containing soluble β-glucan.

5. Use of the brewer's yeast according to claim 1 in preparing a health product containing soluble β-glucan.

6. A method for preparing soluble β-glucan, characterized in that: The preparation method comprises fermenting the brewer's yeast according to claim 1.

7. The preparation method according to claim 6, characterized in that: The fermentation temperature is 25-30°C.

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

9. The preparation method according to claim 6, characterized in that: The fermentation time is 48h.

10. A method for producing soluble β-glucan using the brewer's yeast as claimed in claim 1, characterized in that: The method comprises the steps of bacterial strain activation, seed liquid culture, fermentation culture and purification.

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