Meyrowia guilliermondii strain with high yield of beta-glucan and application of Meyrowia guilliermondii strain

Abstract

The invention relates to the technical field of strain breeding, in particular to a high-yield beta-glucan Meyrowia guilliermondii strain and application thereof. According to the present invention, the high-yield beta-glucan mutant strain obtained through ARTP mutagenesis has the name of Meyerozyma guilliermondii NCUASD7, has been preserved in Guangdong Microbial Culture Collection Center on November 28, 2025, and has the preservation number of CDMCC NO: 67365; aRTP mutagenesis is high in mutation rate, good in hereditary stability and safer to operate, a large number of mutation libraries can be obtained within a short time, high-throughput primary screening and shake flask secondary screening are conducted through a 24-deep-hole plate, a yeast mutant strain with the high beta-glucan yield capacity is obtained, then subculture is conducted on the high-yield mutant strain, the hereditary stability of the strain is verified, and the high beta-glucan yield is obtained. The mutant strain not only has the characteristic of high yield of beta-glucan, but also can maintain good genetic stability.

Description

Technical Field

[0001] This invention relates to the field of strain selection and breeding technology, specifically to a high-β-glucan-producing *Saccharomyces giardi* strain and its applications. Background Technology

[0002] Yeast β-glucan, found in the inner lining of yeast cell walls, is a cell wall polysaccharide primarily linked by β-1,3-glycosidic bonds and secondarily by β-1,6-glycosidic bonds. It possesses a variety of unique physiological activities, including immunomodulatory activity, antitumor activity, anti-radiation effects, cholesterol and lipid-lowering effects, and promotion of beneficial gut bacteria growth. It has found wide application in medical, health food, food and beverage, cosmetic, and animal feed industries.

[0003] Currently, wild-type yeast strains have insufficient capacity to synthesize β-glucan, limiting its yield. Optimizing extraction methods such as chemical, physical, and enzymatic methods often leads to more complex processes, increased costs, and yield losses. Therefore, obtaining a high-yield β-glucan-producing yeast strain through mutagenesis using specific techniques is crucial.

[0004] Mutagenesis breeding is an important method for strain selection. Conventional mutagenesis techniques include ultraviolet mutagenesis, heavy particle mutagenesis, and chemical mutagenesis. However, traditional mutagenesis methods generally suffer from poor genetic stability, low mutagenesis efficiency, and environmental pollution. Atmospheric and Room Temperature Plasma (ARTP) mutagenesis breeding technology, developed in recent years, combines numerous possible biological mutagenesis factors such as electrons, ions, photons, excited-state neutral particles, free radicals, and ultraviolet radiation. Therefore, it has shown great application potential in biological breeding and is widely used in various fields of microbial research. Furthermore, its simple operation, high safety, environmental friendliness, and rapid mutation rate have made it an effective method for obtaining high-yield strains.

[0005] Fermented foods contain a rich variety of microbial resources, among which yeast is the main functional microorganism. This invention is the first to screen a strain of *Saccharomyces giardi* capable of producing β-glucan, and through ARTP mutagenesis, a high-β-glucan-producing yeast mutant strain was obtained, which can effectively increase the glucan content in its cell wall and achieve higher β-glucan yield. Currently, there are no publicly available reports on the fermentation production of β-glucan by this strain. At the same time, the high-yield yeast β-glucan-producing strain obtained through mutagenesis can be applied to the large-scale fermentation production of yeast β-glucan, and will also provide technical reference and experience accumulation for the development of other microbial fermentation products, promote the improvement of production efficiency and quality of various fermentation products, open up new possibilities for the application of biotechnology in the food, pharmaceutical, and cosmetic industries, and has broad application prospects. Summary of the Invention

[0006] To address the shortcomings of the existing technology, this invention aims to provide a high-β-glucan-producing *Saccharomyces giardi* strain and its application, offering a new approach for breeding high-β-glucan-producing *Saccharomyces giardi* strains.

[0007] To solve the above problems, the present invention adopts the following technical solution: In a first aspect, the present invention provides a strain of *Saccharomyces giardi* that produces high levels of β-glucan through ARTP mutagenesis, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 28, 2025, with accession number CDMCC NO:67365.

[0008] Secondly, the present invention provides a method for screening the aforementioned *Saccharomyces cerevisiae* strain, comprising: (1) Yeast strains were screened using naturally fermented foods from different sources as raw materials; (2) Screening for yeast strains capable of synthesizing β-glucan; (3) ARTP mutagenesis was performed on yeast strains capable of synthesizing β-glucan; (4) Screening for yeast mutants with high β-glucan production capacity; (5) Using yeast mutant strains with high β-glucan production capacity as starting strains, ARTP iterative mutagenesis was carried out to further improve the ability of the strains to synthesize β-glucan; (6) Primary and secondary screening of yeast mutant strains that produce high β-glucan were performed using 24-well plates; (7) The high β-glucan-producing yeast mutant strains after secondary screening were passaged and liquid fermentation and β-glucan extraction were performed every 2 generations to verify the genetic stability of the mutant strains.

[0009] Furthermore, including: (1) Using naturally fermented foods from different sources as samples, the yeast was serially diluted on WL agar medium for initial screening of morphological identification, and the ITS region sequence was used for species identification and secondary screening. (2) After activating the yeast in YPD solid medium, fermentation culture was carried out. The fermentation liquid cells were collected for yeast β-glucan extraction and polysaccharide content determination. Yeast strains with the ability to synthesize β-glucan were screened. (3) Growth curves of yeast strains capable of synthesizing β-glucan were determined to identify the logarithmic phase of the strains. The strains were then subjected to ARTP mutagenesis under the following conditions: power of 120 W, distance of 2 mm, gas flow rate of 10 SLM, and mutagenesis time of 0-150 s. (4) Calculate the lethality of the ARTP-mutated strains. The strains with a lethality of over 90% are positive mutants. The positive mutants are fermented and cultured to extract sugar, and then the mutants with high β-glucan production are screened. (5) The mutant yeast strains with high β-glucan production capacity after ARTP mutagenesis were subjected to ARTP iterative mutagenesis to obtain a mutant library; (6) A yeast mutant strain with high β-glucan production capacity was obtained by primary screening through 24-well plate fermentation and secondary screening through shake flask fermentation; (7) The mutant strains with high β-glucan production obtained by iterative mutagenesis and shake flask screening were subjected to genetic stability tests. Finally, a yeast mutant strain with high stability and high β-glucan production was obtained, which was the yeast strain Gynordimium Mayerii.

[0010] Furthermore, in step (2), the growth curve determination includes activating a yeast strain capable of synthesizing β-glucan in YPD solid medium, determining the logarithmic growth phase of the strain to be 16-20 h, transferring the overnight logarithmic phase strain to YPD liquid fermentation medium, fermenting at 30℃ and 200 rpm for 4 days, collecting the fermentation broth for yeast β-glucan extraction, and screening for yeast strains capable of synthesizing β-glucan.

[0011] Furthermore, in (3), the mutagenesis time is 0, 30, 60, 90, 100, 120, or 150 s.

[0012] Furthermore, in (5), the mutagenesis time is 100 s.

[0013] Furthermore, in step (6), through 24-well plate fermentation, using OD value and biomass as the initial screening criteria, strains with high growth potential are screened for shake-flask fermentation for secondary screening and β-glucan extraction, ultimately obtaining yeast mutant strains with high β-glucan production capacity.

[0014] Thirdly, this invention provides the application of the described *Saccharomyces cerevisiae* strain and / or its metabolites in food, beverages, cosmetics, and animal feed.

[0015] The beneficial effects of this invention are as follows: Compared with traditional mutagenesis techniques, ARTP mutagenesis has the characteristics of high mutation rate, good genetic stability, and safer operation. It can screen yeast mutants with high β-glucan production capacity in a short time, and obtain yeast mutants with high β-glucan production capacity through high-throughput primary screening in 24-well plates and secondary screening in shake flasks. Secondly, the high-yield mutants are passaged to verify the genetic stability of the strains. The mutants not only have the characteristic of high β-glucan production, but also maintain good genetic stability. Attached Figure Description

[0016] Figure 1 This is a gel electrophoresis image of PCR products after partial yeast gene amplification.

[0017] Figure 2 Phylogenetic tree of *Saccharomyces cerevisiae* D7.

[0018] Figure 3 The image shows the screening of ARTP-induced mutant strains; where A is the growth curve of *Saccharomyces giardi* D7, B is the ARTP-induced lethality curve of *Saccharomyces giardi* D7, and C is the β-glucan yield of the mutant strain.

[0019] Figure 4 The image shows the screening of mutant strains induced by ARTP iterative mutagenesis; A represents the OD value index of the mutant strain, B represents the biomass index of the mutant strain, C represents the correlation analysis between OD value and biomass, and D represents the β-glucan yield of the mutant strain.

[0020] Figure 5 The genetic stability assessment diagrams for five high-yielding strains are shown; A represents the β-glucan yield of the mutant strain after passage, B represents the OD value of the mutant strain after passage, and C represents the biomass after passage.

[0021] Figure 6 The graph shows the growth differences between the original strain and the mutant strain after ARTP mutagenesis; A is the growth curve of the original strain and the high-yielding mutant strain, and B is the growth of the high-yielding mutant strain of the original strain on a solid plate after being diluted by the same factor. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to specific embodiments.

[0023] It should be noted that these embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Simple improvements to the method under the premise of the present invention are all within the scope of protection claimed by the present invention.

[0024] Example 1 A strain of *Saccharomyces giardi* that produces high levels of β-glucan through ARTP mutagenesis was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 28, 2025, with accession number CDMCC NO:67365.

[0025] Example 2 Screening methods for β-glucan-producing yeast strains (1) Yeast screening: Naturally fermented foods from different sources (Guizhou fermented sour soup, pickled vegetables, etc.) were used as raw materials and serially diluted to 10. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 For later use, 100 μL of each gradient was spread onto WL agar medium and incubated in a 30℃ constant temperature biochemical incubator for 2-3 days. Based on the differences in colony morphology and growth characteristics in the culture dishes, the culture dishes were numbered and the bacteria were preserved. (2) A total of 20 yeast strains were screened in the initial plate screening. As shown in Table 1, most of the yeast strains were round and white in color on the plate, with large colonies that appeared oval under a microscope; Table 1. Morphological characteristics of some yeasts

[0026] (3) Extract yeast genomic DNA according to the operating instructions of the yeast genomic DNA kit; (4) Yeast genes were amplified by PCR using the universal fungal primers ITS1 / ITS4. The amplification system was 25 μL, and the reaction system is shown in Table 2. Table 2 PCR reaction system

[0027] The PCR reaction conditions were: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 30 cycles, and 72℃ extension for 5 min.

[0028] (5) Subsequently, 10 μL of the PCR product was subjected to 1% agarose gel electrophoresis for detection, and the results are as follows. Figure 1 As shown, the amplified bands were correct. The remaining PCR products were then subjected to molecular biological identification. In the figure, M represents the marker, and different numbers represent yeast cell numbers.

[0029] (6) Molecular biological identification: PCR products with correct electrophoretic bands were sent to Shanghai Sangon Biotech Co., Ltd. for ITS sequencing of the strain. Table 3 shows the spliced ​​sequence identified by sequencing NCUD7 (Saccharomyces cerevisiae). The phylogenetic tree of Saccharomyces cerevisiae D7 is shown in Table 3. Figure 2 .

[0030] Table 3. ITS gene sequence of NCUD7

[0031] (7) Screening of β-glucan-producing yeast: Yeast was activated in YPD solid medium (2% glucose, 2% peptone, 1% yeast extract) for 2-3 days until single colonies grew. Single colonies were selected and inoculated into YPD liquid medium for overnight culture, and then transferred to YPD fermentation medium for fermentation. The volume of the liquid was 50 mL out of 250 mL. After fermentation, the yeast cells in the fermentation broth were collected for yeast β-glucan extraction, and yeast strains with the ability to synthesize β-glucan were screened.

[0032] Example 3 ARTP selects high-yield β-glucan-producing *Saccharomyces gimmickii* strains. (1) The growth curve of *Saccharomyces cerevisiae* NCUD7 is as follows: Figure 3 As shown in Figure A, the strain is in the lag phase from 1 to 4 hours, during which the strain grows slowly. From 4 to 24 hours, it is in the logarithmic growth phase, during which the strain grows rapidly and is suitable for subculturing. After 24 hours, it enters the stationary phase. To ensure the growth viability and bacterial quantity of the strain, bacterial culture from 16 to 20 hours was selected for ARTP mutagenesis.

[0033] (2) For example Figure 3 As shown in Figure B, ARTP mutagenesis of a yeast strain capable of synthesizing β-glucan resulted in a lethality of 95.93% at a mutagenesis time of 100 s. Therefore, 100 s was selected as the optimal treatment time for screening mutants. (3) Screening of yeast mutant strains with high β-glucan production: The bacterial suspension induced by 100 s mutagenesis was spread on solid plates. After single colonies grew, 44 smooth, large, and round colonies were selected for fermentation. Figure 3 As shown in Figure C, strain NCUAFD7-8 had the highest yield, approximately 2.0 g / L, therefore ARFD7-8 was selected for further study. (4) In order to further improve the ability of the strain to synthesize β-glucan, a yeast mutant strain with high β-glucan production capacity was used as the starting strain for ARTP iterative mutagenesis. The mutagenesis time was set to 100 s, and a large number of mutant libraries were obtained through ARTP iterative mutagenesis. (5) After initial screening using a 24-well plate with a liquid volume of 3 mL, 149 mutant strains were obtained based on OD value and biomass. Figure 4 (A, B, C). Perform sterilization; (6) The 25 mutant strains with outstanding growth ability obtained from the initial screening using 24-well plates were subjected to shake-flask fermentation for secondary screening. For example... Figure 4 As shown in (D), the yeast β-glucan yield ranged from 2.239 to 2.878 g / L, which was 17.59%-51.14% higher than the initial ARTP-induced mutant strain NCUARFD7-8 and 103.54%-161.60% higher than the original strain NCUD7. (7) Select the five mutant strains with the highest yield for genetic stability testing, and monitor the growth, biomass, and polysaccharide yield of each generation. For example... Figure 5 As shown, after 10 generations of subculturing, the polysaccharide production of the five strains remained stable with no significant differences between generations, indicating good genetic stability. Combined with OD values ​​( Figure 5 (A) and biomass ( Figure 5 Through screening (B), a high-yielding and stable yeast mutant strain, NCUASD7, was identified, with a polysaccharide yield of 3.154 g / L.

[0034] (8) To confirm the effectiveness of the mutagenesis, we performed phenotypic analysis on the strain. For example... Figure 6 As shown in the growth curves, both NCUD7 and NCUASD7 were in the early stage of logarithmic growth after 8 hours of culture. They entered the stationary phase after 24 hours of culture. From the start of the logarithmic phase, NCUASD7 grew slightly faster than NCUD7. p <0.05), see Figure 6 In step A, NCUD7 and NCUASD7 were diluted to the same concentration and spotted onto agar plates. The results showed that NCUASD7 grew slightly faster than NCUD7. p <0.05), see Figure 6 B.

[0035] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described with reference to preferred embodiments, those skilled in the art should understand that various changes in form and detail can be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A strain of Guymardia quiuiomontana selected for high production of β-glucans by ARTP mutagenesis, characterized in that, It was deposited at Guangdong Microbial Culture Collection Center on November 28, 2025, and the deposit number is CDMCC NO: 67365.

2. The method for screening of the strain of the Yarrowia lipolytica according to claim 1, characterized in that, It comprises: (1) taking different sources of naturally fermented food as raw materials, screening yeast strains; (2) screening yeast strains with the ability to synthesize β-glucan; (3) ARTP mutagenesis of yeast strains with the ability to synthesize β-glucan; (4) screening of yeast mutant strains with high β-glucan production capacity; (5) taking yeast mutant strains with high β-glucan production capacity as starting strains for ARTP iterative mutagenesis to further improve the ability of the strains to synthesize β-glucan; (6) initial screening and rescreening of yeast mutant strains with high β-glucan production capacity by 24 deep well plates; (7) subculture of yeast mutant strains with high β-glucan production capacity after rescreening, and liquid fermentation and β-glucan extraction every 2 generations to verify the genetic stability of the mutant strains.

3. The screening method of a strain of Guymardia segbwela according to claim 2, characterized in that, It comprises: (1) taking different sources of naturally fermented food as samples, gradient dilution in WL agar medium for yeast morphological identification initial screening, ITS region sequence for species identification rescreening; (2) after activation of yeast in YPD solid medium, fermentation culture, collection of fermentation broth for yeast β-glucan extraction and polysaccharide content determination, screening of yeast strains with the ability to synthesize β-glucan; (3) growth curve determination of yeast strains with the ability to synthesize β-glucan, determination of logarithmic phase of the strains, ARTP mutagenesis of the strains, mutagenesis conditions are power 120 w, distance 2 mm, gas flow 10 SLM, mutagenesis time 0~150 s; (4) calculation of the mortality rate of ARTP mutant strains, strains with mortality rate above 90% are positive mutant strains, fermentation culture of positive mutant strains for sugar extraction, screening of mutant strains with high β-glucan production capacity; (5) ARTP iterative mutagenesis of yeast mutant strains with high β-glucan production capacity after ARTP mutagenesis to obtain a mutant library; (6) obtaining yeast mutant strains with high β-glucan production capacity through 24 deep well plate fermentation initial screening and shake flask fermentation rescreening; (7) genetic stability test of mutant strains with high β-glucan production capacity screened by iterative mutagenesis shake flask, finally screening yeast mutant strains with high stability and high β-glucan production capacity, which are Saccharomyces boulardii strains.

4. The screening method of a strain of Guymardia segbwela according to claim 3, characterized in that, In the (2), the growth curve determination includes activating β-glucan capacity yeast strains in YPD solid medium, determining the logarithmic phase growth period of the strains at 16-20 h, and transferring the overnight bacteria in the logarithmic phase to YPD liquid fermentation medium for fermentation at 30°C and 200 rpm for 4 days, collecting the fermentation broth for yeast β-glucan extraction, and screening yeast strains with the ability to synthesize β-glucan.

5. The screening method of a strain of Guymardia segbwela according to claim 3, characterized in that, In the (3), the mutagenesis time is 0, 30, 60, 90, 100, 120, 150 s.

6. The screening method of a strain of Guymardia segbwela according to claim 3, characterized in that, In the (5), the mutagenesis time is 100 s.

7. The screening method of a strain of Guymardia segbwela according to claim 3, characterized in that, In the (6), through 24 deep well plate fermentation, with OD value and biomass as the primary screening standard, the strain with high growth potential is screened for shake flask fermentation re-screening and β-glucan extraction, and finally the yeast mutant strain with high β-glucan production capacity is obtained.

8. Use of the strain of the Saccharomyces quilliermiodii according to claim 1 and / or its metabolites in food and beverages, cosmetics and animal feed.

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