Yeast strain for efficiently transforming saponin and polysaccharide and application thereof

Abstract

The application discloses a strain of yeast for efficiently transforming ginsenosides and polysaccharides and application thereof, and belongs to the technical field of functional component transformation. According to the total ginsenoside and polysaccharide content in ginseng, a strain of yeast for efficiently transforming ginsenosides and polysaccharides is screened by taking a comprehensive score as an index, and is identified as Zygosaccharomyces bisporus by morphology, physiology and biochemistry and molecular biology, and is preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: M 2020912. After transformation, the total ginsenoside content in American ginseng and ginseng is respectively increased by 67.81% and 107.03%, and the polysaccharide content is respectively increased by 238.61% and 205.87%. The DPPH antioxidant capacity, ABTS antioxidant capacity, blood sugar lowering capacity and blood lipid lowering capacity of the total ginsenoside extract and polysaccharide extract of the American ginseng and ginseng after transformation are all improved. The yeast screened by the application has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of functional ingredient conversion technology, and more specifically to a yeast strain that efficiently converts saponins and polysaccharides and its applications. Background Technology

[0002] Ginseng and American ginseng have a long history in China. They are not only abundant in quantity but also possess rich medicinal potential, including antioxidant and blood sugar-regulating bioactivities. Given their outstanding nutritional and medicinal value, ginseng was officially included in the list of foods and medicines with the same origin in 2012, and American ginseng in 2023. This move has opened a new chapter in the development and utilization of ginseng and American ginseng, promoting their further development in the health industry.

[0003] Ginseng and American ginseng mainly consist of dietary fiber, starch, protein, polysaccharides, and saponins, among which polysaccharides and saponins are considered key active ingredients. However, studies have shown that the content of active ingredients such as saponins and polysaccharides in ginseng and American ginseng is relatively low, and some rare saponins are even absent in natural raw materials. This situation seriously restricts the development potential and application scope of ginseng and American ginseng. Therefore, how to improve the content of active ingredients in ginseng and American ginseng has become the focus of current research and a key problem that urgently needs to be solved in the industrialization process.

[0004] Biotransformation strategies, with their rich variety, high targeting, and high conversion rates, have become the dominant technology for converting active ingredients. Microbial transformation technology, particularly the process of generating rare saponins by producing glycoside hydrolases to break the glycosidic bonds within saponins, demonstrates its unique value. Furthermore, ginseng and American ginseng can convert carbohydrates such as starch and dietary fiber into biopolysaccharides through the respiratory metabolism of microorganisms, thereby enhancing their efficacy. Among numerous microorganisms, yeast, lactic acid bacteria, molds, Bacillus, and Bifidobacterium all possess advantages such as high safety and good conversion performance.

[0005] Therefore, how to screen microbial strains with high conversion potential and apply them to the conversion process of saponins and polysaccharides, with the aim of effectively increasing the content of active ingredients in ginseng or American ginseng and thus optimizing its overall quality, is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a yeast strain that efficiently converts saponins and polysaccharides and its applications.

[0007] The purpose of this invention is to screen for superior microbial strains capable of converting total saponins and polysaccharides, and to apply them to the conversion of active ingredients in ginseng, American ginseng, etc. Based on economic considerations, this invention first selects ginseng as raw material to screen for microbial strains with high conversion efficiency, then uses these strains to convert ginseng and American ginseng, analyzes the components of the conversion products, and evaluates the efficacy of the conversion products.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0009] A yeast strain, named Lan-S5, is classified as *Diplosporon zygosaccharomyces* (Bizarreta zygosaccharomyces). Zygosaccharomyces bisporus It was deposited at the China Center for Type Culture Collection on December 16, 2020, with accession number CCTCC NO: M 2020912, at Wuhan University, Wuhan, China.

[0010] The above-mentioned yeast is used in the fermentation and transformation of ginsenosides and polysaccharides.

[0011] Furthermore, the ginseng mentioned belongs to the ginseng family, specifically ginseng and American ginseng.

[0012] Furthermore, it is used to increase the content of saponins and polysaccharides.

[0013] Furthermore, it can be used to improve antioxidant capacity, blood sugar lowering capacity, and blood lipid lowering capacity.

[0014] A microbial agent for converting saponins and polysaccharides includes the aforementioned yeast.

[0015] A method for increasing saponins and polysaccharides in ginseng / American ginseng, using the aforementioned yeast.

[0016] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: This invention screened a yeast strain highly efficient at converting saponins and polysaccharides based on the total saponin and polysaccharide content of ginseng, using a comprehensive score as the indicator. Morphological, physiological, biochemical, and molecular biological identification confirmed it to be a disporin conjugated yeast, and it is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2020912. After conversion, the total saponin content in American ginseng and ginseng increased by 67.81% and 107.03%, respectively, and the polysaccharide content increased by 238.61% and 205.87%, respectively. The DPPH antioxidant capacity, ABTS antioxidant capacity, hypoglycemic capacity, and hypolipidemic capacity of the total saponin and polysaccharide extracts of American ginseng and ginseng were all improved after conversion. The yeast strain screened in this invention has broad application prospects. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0018] Figure 1 The image shows the colony morphology of yeast M 2020912 in Example 2 of the present invention, where (a) shows the morphology in solid culture medium and (b) shows the morphology under a microscope.

[0019] Figure 2 The results of PCR electrophoresis of yeast M 2020912 in Example 2 of this invention are shown.

[0020] Figure 3 This is the phylogenetic tree of yeast M 2020912 in Example 2 of the present invention.

[0021] Figure 4 This is a liquid phase diagram of ginseng before and after conversion in Example 3 of the present invention.

[0022] Figure 5 This is a liquid phase diagram of American ginseng before and after transformation in Example 3 of the present invention.

[0023] Figure 6 The image shows the liquid chromatography-mass spectra of ginseng before and after conversion in positive ion mode in Example 4 of this invention.

[0024] Figure 7 The images show the liquid chromatography-mass spectra of American ginseng before and after conversion in the positive ion mode in Example 4 of this invention.

[0025] Figure 8 The image shows the liquid chromatography-mass spectra of ginseng before and after conversion in the negative ion mode in Example 4 of this invention.

[0026] Figure 9 The images show the liquid chromatography-mass spectra of American ginseng before and after conversion in the negative ion mode in Example 4 of this invention. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1 Screening of superior transforming strains 1. Microbial strain activation and amplification (1) Lactic acid bacteria Lactic acid bacteria strains (isolated from kimchi, cheese, yogurt, etc., numbered JL-RS1, JL-RS2, ..., JL-RS7) screened and preserved by the Agricultural Product Processing and Storage Engineering Laboratory of the College of Food Science and Engineering, Jilin Agricultural University, were inoculated into MRS medium at a 1% inoculum (1 mL of frozen solution inoculated into 100 mL of medium) and cultured in a shaker at 37℃ and 180 r / min for 24 h to obtain the activated bacterial solution (OD). 600 Activation is complete when the concentration reaches 1.8. The activated bacterial solution is inoculated into MRS medium at a 2% inoculum volume (2 mL of activated bacterial solution inoculated into 100 mL of medium), and cultured at 37°C and 180 r / min in a shaker for 24 h to obtain the expanded bacterial culture (OD). 600 The expansion is considered complete when the 1.8 ohm diameter is reached.

[0029] (2) Yeast: Yeasts collected and preserved by the Laboratory of Agricultural Product Processing and Storage Engineering, College of Food Science and Engineering, Jilin Agricultural University, with serial numbers M 2020912 and CICC 32636; commercially available yeasts with serial numbers QB / T4577, CH0599, CH0465, and CF10928. A 1% inoculum (1 g of yeast powder inoculated into 100 mL of culture medium) was added to YPD medium and cultured at 30 ℃ and 160 r / min for 24 h to obtain the activated bacterial solution (OD). 600 Activation is complete when the concentration reaches 1.8. Inoculate the activated bacterial solution into YPD medium at a 2% inoculum (2 mL of activated bacterial solution into 100 mL of medium) and incubate at 30℃ and 160 rpm for 24 hours to obtain the expanded bacterial culture (OD). 600 The expansion is considered complete when the 1.8 ohm diameter is reached.

[0030] (3) Molds: *Russula versicolor*, *Trichoderma reesei*, and *Aspergillus niger* 1, collected and preserved by the Agricultural Product Processing and Storage Engineering Laboratory of the College of Food Science and Engineering, Jilin Agricultural University, were numbered CICC 2625, CICC 13052, and CICC 40048, respectively; and commercially available *Aspergillus niger* 2 and *Aspergillus niger* 3, numbered BNCC 186380 and SHBCC D11667, respectively. A 1% inoculum (1 mL of frozen solution inoculated into 100 mL of culture medium) was added to potato dextrose medium and cultured at 30 ℃ and 160 r / min for 24 h to obtain the activated bacterial solution (OD). 600 Activation is complete when the OD reaches 1.8. Inoculate the activated bacterial solution into potato dextrose medium at a 2% inoculation rate (2 mL of activated bacterial solution into 100 mL of medium), and incubate at 30 ℃ and 160 r / min for 24 h to obtain the expanded bacterial culture (OD). 600 The expansion is considered complete when the 1.8 ohm diameter is reached.

[0031] (4) Bacillus subtilis: Bacillus subtilis 1, collected and preserved by the Laboratory of Agricultural Product Processing and Storage Engineering, College of Food Science and Engineering, Jilin Agricultural University (CICC 22384); and commercially available Bacillus subtilis 2 and 3, (BNCC 109047 and SHBCC D10486), were inoculated into LB medium at a 1% inoculation rate (1 mL of frozen solution inoculated into 100 mL of medium) and cultured at 37℃ and 180 r / min for 24 h to obtain activated bacterial solution (OD). 600 Activation is complete when the OD reaches 1.8. Inoculate the activated bacterial solution into LB medium at a 2% inoculum volume (2 mL of activated bacterial solution into 100 mL of medium) and incubate at 37°C and 180 r / min for 24 h to obtain the expanded bacterial culture (OD). 600 The expansion is considered complete when the 1.8 ohm diameter is reached.

[0032] (5) Bifidobacteria: Bifidobacterium 1, collected and preserved by the Laboratory of Agricultural Product Processing and Storage Engineering, College of Food Science and Engineering, Jilin Agricultural University, was designated CICC 6069; commercially available Bifidobacterium 2 and Bifidobacterium 3, designated BNCC341605 and CICC 6068, were inoculated into MRS medium at an inoculation rate of 1% (1 mL of frozen solution inoculated into 100 mL of medium) and cultured at 37℃ and 180 r / min for 24 h to obtain activated bacterial solution (OD). 600 Activation is complete when the concentration reaches 1.8. The activated bacterial solution is inoculated into MRS medium at a 2% inoculum volume (2 mL of activated bacterial solution inoculated into 100 mL of medium), and cultured at 37°C and 180 r / min in a shaker for 24 h to obtain the expanded bacterial culture (OD). 600 The expansion is considered complete when the 1.8 ohm diameter is reached.

[0033] 2. Raw material pretreatment Commercially available ginseng is coarsely ground in a food processor to obtain coarse powder with a diameter of about 0.5 to 1 cm. The coarse powder is then transferred to an ultrafine grinder for further grinding and sieved to obtain ginseng powder with a diameter of 150 to 200 mesh.

[0034] 3. Preparation of fermentation broth Ginseng powder obtained by sieving was diluted with purified water at a ratio of 1:10 to obtain ginseng fermentation broth. The broth was then sterilized at 121°C for 15-25 minutes to prevent the influence of contaminating bacteria on the sieving process.

[0035] 4. Microbial transformation treatment (1) Lactic acid bacteria: The expanded lactic acid bacteria culture was inoculated into the ginseng fermentation broth at a 10% inoculation rate (accounting for 10% of the total liquid volume to be fermented after inoculation), and placed in a shaker at 180 r / min and cultured at 37 ℃ for 7 days. The transformed ginseng transformation broth was then removed, sterilized at 121 ℃ for 20 min, and freeze-dried. The ginseng transformation product was obtained.

[0036] (2) Yeast: The expanded yeast culture was inoculated into the ginseng fermentation broth at a 10% inoculation rate (the percentage of the total liquid to be fermented after inoculation), and placed in a shaker at 160 r / min and cultured at 30 ℃ for 7 days. The transformed ginseng transformation broth was then removed, sterilized at 121 ℃ for 20 min, and freeze-dried. The ginseng transformation product was obtained.

[0037] (3) Mold: The expanded mold culture was inoculated into the ginseng fermentation broth at a 10% inoculation rate (the inoculation rate after inoculation accounts for the total volume of the broth to be fermented), and placed in a shaker at 160 r / min and cultured at 30℃ for 7 days. The transformed ginseng transformation broth was taken out, sterilized at 121℃ for 20 min, and freeze-dried. The ginseng transformation product was obtained.

[0038] (4) Bacillus subtilis: The expanded Bacillus subtilis culture was inoculated into the ginseng fermentation broth at a 10% inoculation rate (the percentage of the total liquid to be fermented after inoculation), and placed in a shaker at 180 r / min and cultured at 37 ℃ for 7 days. The transformed ginseng transformation broth was then removed, sterilized at 121 ℃ for 20 min, and freeze-dried. The ginseng transformation product was obtained.

[0039] (5) Bifidobacterium: The prepared Bifidobacterium culture was inoculated into the ginseng fermentation broth at a 10% inoculation rate (which accounts for 10% of the total volume of the broth to be fermented after inoculation), and placed in a shaker at 180 r / min and cultured at 37 ℃ for 7 days. The transformed ginseng transformation broth was then removed, sterilized at 121 ℃ for 20 min, and freeze-dried. The ginseng transformation product was obtained.

[0040] 5. Determine the optimal transformation strain (1) Characteristic strain screening method By comparing the total saponin and polysaccharide contents in the ginseng transformation products of different strains, the weights of total saponins and polysaccharides were determined by the entropy method. The comprehensive score of the transformation effect of each strain was calculated, and the superior strains with better transformation effect were screened using the comprehensive score as an indicator.

[0041] (2) Comprehensive scoring method The weights of each indicator are obtained according to the entropy method weight calculation formula. After the weights are allocated using the entropy method, the membership values ​​of the two indicators in each experiment are obtained according to the indicator normalization (membership value), and the comprehensive score of each experiment is obtained.

[0042] Normalization of each indicator ( For the nth indicator Membership value of the subsample For n indicators, the first The value of the subsample index. This represents the maximum value of the nth index. The formula for calculating the minimum value of the nth index is as follows:

[0043] Non-negative translation ( For the nth indicator The formula for calculating the non-negative translation result of the subsample is as follows:

[0044] Proportion( For the first The formula for calculating the proportion of a sample on the nth indicator (where m is the number of samples) is:

[0045] Entropy ( The formula for calculating the entropy value of the nth index is as follows:

[0046] Weights ( For the first n The weight of each indicator, j The formula for calculating the number of indicators is as follows:

[0047] Combined weighted comprehensive score ( For the nth indicator , The formula for calculating the overall score is as follows:

[0048] Results of screening for superior strains:

[0049] The above microorganisms were screened, and the transformation effects of the obtained strains are shown in Table 1. Among them, yeast M 2020912 showed the strongest transformation ability in total saponins and polysaccharides (the total saponin content increased by 76.09% after transformation, from 2.7441 mg / g to 4.8320 mg / g; the polysaccharide content increased by 168.77%, from 8.2429 mg / g to 22.1541 mg / g), and had the highest overall score (0.9826).

[0050] Example 2 Identification of yeast strain M 2020912 1. Morphological identification of strains Yeast strain M2020912 was placed in a yeast solid culture medium and cultured at 30 ℃ for 5 days before morphological observation. Visual observation revealed that yeast colonies of M2020912 were milky white, spherical, smooth, uniformly viscous, slightly convex in the center, and with neat edges. Figure 1 (a)). Under a biological microscope, the cells appear oval, and the asci contain 1-4 smooth, oval ascospores. Figure 1 (b) in the middle.

[0051] 2. Physiological and biochemical identification of the strain Physiological and biochemical identification of yeast M 2020912 was performed with reference to the "Handbook of Characteristics and Identification of Yeasts".

[0052] As shown in Table 2, yeast M 2020912 can utilize glucose for fermentation, but cannot utilize other sugar sources such as maltose / sucrose; yeast M 2020912 can assimilate glycerol as a carbon source, but cannot assimilate cellobiose, inulin, or other carbon sources; yeast M 2020912 can assimilate ammonium sulfate as a nitrogen source, but cannot assimilate nitrate as a nitrogen source.

[0053]

[0054] 3. Molecular biological identification Yeast cells M 2020912 were dissolved in 40 μL TaKaRa Lysis Buffer for Microorganism to Direct PCR (Code No. D304) for denaturation. After centrifugation, the supernatant was used as template DNA. The reaction conditions were 70 °C for 13 min.

[0055] The target fragment was amplified by PCR using a 2×TransTaq® High Fidelity (HiFi) PCR SuperMix I (TransGenBiotech, Code No: AS131). The negative control used 2 μL of 26S-free H2O to replace the template DNA; the positive control used 3 μL of 26S rDNA from a known bacterial strain to replace the template DNA.

[0056] The PCR reaction system is: Template DNA 3 μL, Forward primer (10 pmol / μL) 1 μL, Reverse primer (10 pmol / μL) 1 μL, 2×TransTaq® HiFi PCR SuperMix I 25 μL, 16S-free H2O 20 μL, Total 50 μL.

[0057] The PCR amplification conditions were as follows: 94 ℃ pre-denaturation for 5 min; 95 ℃ for 10 s, 55 ℃ for 20 s, 72 ℃ for 1.5 min, for 35 cycles; 72 ℃ extension for 10 min.

[0058] 26S rDNA sequencing was performed using NL1 and NL4 primers. Upstream primer NL1: 5'-GCATATCAATAAGCGGAGGAAAAG-3', SEQ ID No. 1; Downstream primer NL4: 5'-GGTCCGTGTTTCAAGACGG-3', SEQ ID No. 2.

[0059] PCR electrophoresis results as follows Figure 2 As shown.

[0060] The gene sequence of yeast M 2020912 is as follows: , SEQ ID No.3.

[0061] The sequencing results were input into Genebank, and the BLAST function was used to compare the homology of the obtained gene sequences with the 26S rDNA sequences in the Genebank database. The 26S rDNA sequences of bacteria with high scores in the BLAST results were selected, and phylogenetic analysis was performed using MEGA 5.2.1 software based on Neighbor-Joining. The results are as follows. Figure 3 As shown.

[0062] In summary, based on morphological, physiological, biochemical, and molecular biological identification, yeast M 2020912 was determined to be a disporum conjugated yeast. Zygosaccharomyces bisporus ).

[0063] 4. Preservation of bacterial strains Yeast strain M 2020912, named Lan-S5, is taxonomically named *Diplosporon zygosacchari*, with the Latin name... Zygosaccharomyces bisporusIt was deposited at the China Center for Type Culture Collection on December 16, 2020, with accession number CCTCC NO: M 2020912, and the deposit address is Wuhan University, Wuhan, China.

[0064] Example 3 Comparative experiment on the content of components of ginseng and American ginseng before and after transformation 1. Activation and expansion of Lan-S5 disporin yeast The dispora zygosacchariformis Lan-S5 was inoculated into YPD medium at a 1% inoculum (1 mL of frozen solution inoculated into 100 mL of medium) and cultured in a shaker at 30 ℃ and 160 r / min for 24 h to obtain the activated bacterial solution (OD). 600 Activation is complete when the OD reaches 1.8. Inoculate the activated bacterial solution into YPD medium at a 2% inoculum (2 mL of activated bacterial solution into 100 mL of medium) and incubate at 30 ℃ and 160 r / min for 24 h to obtain the expanded bacterial culture (OD). 600 The expansion is considered complete when the 1.8 ohm diameter is reached.

[0065] 2. Raw material pretreatment Commercially available ginseng and American ginseng are coarsely ground in a food processor to obtain coarse powder with a diameter of about 0.5 to 1 cm. The coarse powder is then transferred to an ultrafine grinder for further grinding and sieving to obtain ginseng powder and American ginseng powder with a diameter of 150 to 200 mesh.

[0066] 3. Preparation of fermentation broth Using sieved ginseng powder and American ginseng powder, the powder was diluted with purified water at a ratio of 1:10 to obtain the ginseng and American ginseng fermentation liquid. The liquid was then sterilized in an autoclave at 121°C for 20 minutes to prevent the influence of other microorganisms in the fermentation liquid.

[0067] 4. Transformation of American ginseng and ginseng by the two-spore conjugated yeast Lan-S5 The expanded yeast culture was inoculated into the ginseng and American ginseng fermentation broth at an inoculation rate of 10%, a culture temperature of 30℃, and a culture time of 7 days. After the transformation was completed, the transformed ginseng and American ginseng transformation broth was taken out, sterilized at 121℃ for 20 minutes, and freeze-dried to obtain the ginseng and American ginseng transformation products.

[0068] 5. Determination of dietary fiber content The contents of total dietary fiber, soluble dietary fiber, and insoluble dietary fiber were determined using the enzymatic hydrolysis method in GB 5009.88-2023.

[0069] 6. Starch content determination Starch content was determined using the enzymatic hydrolysis method in GB 5009.9-2023.

[0070] 7. Protein content determination Protein content was determined using the Kjeldahl method as specified in GB 5009.5-2025.

[0071] 8. Determination of saponin content (1) Determination of total saponin content The total saponin content was determined by spectrophotometry according to DB 22 / T 1668-2012.

[0072] (2) Determination of monomeric saponin content Weigh 1 g (accurate to 0.001 g) of ginseng powder, American ginseng powder, and ginseng and American ginseng conversion products (ground through a 60-mesh sieve). Wrap the powder in neutral filter paper and place it in a Soxhlet extractor. Add 50 mL of ether and reflux at 80 °C for 2 h. Discard the ether solution, evaporate the ether solvent from the sample packet, tear the packet into pieces, pour the contents into a 250 mL Erlenmeyer flask, add 50 mL of water-saturated n-butanol, and soak overnight. The next day, place the Erlenmeyer flask in an ultrasonic cleaner at 50 / 60 Hz and 1500 W for 30 min, then centrifuge at 4000 r / min for 10 min. Transfer the supernatant to an evaporating dish. Repeat the above extraction process twice, 10 min each time. Combine the n-butanol solutions and evaporate to dryness at 90 °C. Dissolve the evaporated substances in methanol several times and bring the volume to 5 mL. This is the monomeric saponin test solution, for later use.

[0073] Chromatographic conditions for monomeric saponin determination: column: C18 column (4.6×250 mm, 5 µm), mobile phase: acetonitrile-water, column temperature: 35 ℃, flow rate: 1 mL / min, detection wavelength: 203 nm, injection volume: 20 µm, gradient elution program is shown in Table 3.

[0074]

[0075] 9. Polysaccharide content determination The polysaccharide content was determined by spectrophotometry according to DB 22 / T1685-2012.

[0076] 10. Ash content determination Ash content was determined using the combustion method in GB 5009.4-2016.

[0077] 11. Determination of fat content Fat content was determined using the acid hydrolysis method in GB5009.6-2016.

[0078] 12. Determination of flavonoid content The flavonoid content was determined by spectrophotometry according to SN / T 4592-2016.

[0079] The comparison results of the main component contents of ginseng and American ginseng before and after yeast transformation are shown in Tables 4 and 5. Figures 4-5 As shown.

[0080]

[0081] Analysis of the main components in Table 4 revealed that ginseng exhibited increased levels of polysaccharides, saponins, ash, flavonoids, protein, and soluble dietary fiber, while decreasing levels of dietary fiber, insoluble dietary fiber, starch, and fat. The polysaccharide content saw the largest increase, rising by 205.87% (from 72.43 mg / g to 221.54 mg / g). Total saponin content increased by 107.03% (from 23.34 mg / g to 48.32 mg / g). Ash content increased by 57.28% (from 33.36 mg / g to 52.47 mg / g). Flavonoid content increased by 35.43% (from 1.27 mg / g to 1.72 mg / g). Soluble dietary fiber content increased by 14.52% (from 126.92 mg / g to 145.35 mg / g). Protein content increased by 17.38% (from 110.95 mg / g to 130.23 mg / g). Dietary fiber content decreased by 33.46% (from 373.64 mg / g to 248.63 mg / g). Insoluble dietary fiber decreased by 58.14% (from 246.72 mg / g to 103.28 mg / g). Starch content decreased by 68.33% (from 232.57 mg / g to 73.65 mg / g). Fat content decreased by 5.92% (from 26.5 mg / g to 24.93 mg / g).

[0082] After ginseng transformation, the levels of saponins Rg1, Re, Rf, Rc, Rb2, Rb3, and Rd all increased to varying degrees, while saponin Rb1 decreased slightly. Saponin Rd, which was virtually absent in ginseng, increased from 0.1 mg / g to 0.38 mg / g after transformation.

[0083]

[0084] Analysis of the main components in Table 5 revealed that the contents of polysaccharides, soluble dietary fiber, saponins, ash, flavonoids, and protein in American ginseng increased, while the contents of dietary fiber, insoluble dietary fiber, starch, and fat decreased. The polysaccharide content saw the largest increase, rising by 238.61% (from 70.18 mg / g to 237.64 mg / g). The total saponin content increased by 67.81% (from 34.05 mg / g to 57.14 mg / g). The soluble dietary fiber content increased by 16.11% (from 81.4 mg / g to 94.51 mg / g). The protein content increased by 13.92% (from 118.67 mg / g to 135.19 mg / g). The ash content increased by 12.32% (from 37.26 mg / g to 41.85 mg / g). Flavonoid content increased by 28.47% (from 1.37 mg / g to 1.76 mg / g). Dietary fiber content decreased by 9.73% (from 329.77 mg / g to 297.68 mg / g). Insoluble dietary fiber decreased by 18.2% (from 248.37 mg / g to 203.17 mg / g), and starch content decreased by 73.99% (from 201.32 mg / g to 52.37 mg / g). Fat content decreased by 15.5% (from 15.94 mg / g to 13.47 mg / g).

[0085] After conversion, the levels of saponins Rg1, Rf, Re, Rb2, Rb3, and Rd in American ginseng increased to varying degrees, while the levels of saponins Rb1 and Rc decreased slightly. Saponin Rf, which was virtually absent in American ginseng, increased from 0.18 mg / g to 1.14 mg / g after conversion.

[0086] Example 4 Comparative experiment on the conversion of non-target saponins before and after transformation in ginseng and American ginseng 1. Activation and expansion of Lan-S5 disporin yeast The dispora zygosacchariformis Lan-S5 was inoculated into YPD medium at a 1% inoculum (1 mL of frozen solution inoculated into 100 mL of medium) and cultured in a shaker at 30 ℃ and 160 r / min for 24 h to obtain the activated bacterial solution (OD). 600 Activation is complete when the OD reaches 1.8. Inoculate the activated bacterial solution into YPD medium at a 2% inoculum (2 mL of activated bacterial solution into 100 mL of medium) and incubate at 30 ℃ and 160 r / min for 24 h to obtain the expanded bacterial culture (OD). 600 The expansion is considered complete when the 1.8 ohm diameter is reached.

[0087] 2. Raw material pretreatment Commercially available ginseng and American ginseng are coarsely ground in a food processor to obtain coarse powder with a diameter of about 0.5 to 1 cm. The coarse powder is then transferred to an ultrafine grinder for further grinding and sieving to obtain ginseng powder and American ginseng powder with a diameter of 150 to 200 mesh.

[0088] 3. Preparation of fermentation broth Using sieved ginseng powder and American ginseng powder, the powder was diluted with purified water at a ratio of 1:10 to obtain the ginseng and American ginseng fermentation liquid. The liquid was then sterilized in an autoclave at 121°C for 20 minutes to prevent the influence of other microorganisms in the fermentation liquid.

[0089] 4. Transformation of American ginseng and ginseng by the two-spore conjugated yeast Lan-S5 The expanded yeast culture was inoculated into the ginseng and American ginseng fermentation broth at an inoculation rate of 10%, a culture temperature of 30℃, and a culture time of 7 days. After the transformation was completed, the transformed ginseng and American ginseng transformation broth was taken out, sterilized at 121℃ for 20 minutes, and freeze-dried to obtain the ginseng and American ginseng transformation products.

[0090] 5. Non-target saponin conversion analysis Weigh 1 g (accurate to 0.001 g) of ginseng powder, American ginseng powder, and ginseng and American ginseng conversion products (ground through a 60-mesh sieve). Wrap the powder in neutral filter paper, place it in a Soxhlet extractor, add 50 mL of diethyl ether, and reflux at 80 °C for 2 h. Discard the ether solution, evaporate the ether solvent from the sample packet, tear the packet into pieces, pour the contents into a 250 mL Erlenmeyer flask, add 50 mL of water-saturated n-butanol, and soak overnight. The next day, place the Erlenmeyer flask in an ultrasonic cleaner at 50 / 60 Hz and 1500 W for 30 min, centrifuge at 4000 r / min for 10 min, and transfer the supernatant to an evaporating dish. Repeat the above extraction process twice, 10 min each time. Combine the n-butanol solutions and evaporate to dryness at 90 °C. Dissolve the evaporated substances in methanol several times, and bring the volume to 5 mL. This is the monomeric saponin test solution, for later use.

[0091] Ginsenosides were identified using ultra-high performance liquid chromatography-time-of-flight mass spectrometry (UHPLC-TOF-MS). Chromatographic column: Phenomenex C18 (100 mm × 2.1 mm, 1.7 μm); column temperature: 35℃; injection volume: 5 μL; mobile phase: 0.1% formic acid-water (phase A)-acetonitrile (phase B). Samples were analyzed in both positive and negative ion modes; ion source: ESI; nebulization voltage: 5500 V (-4500 V); nebulization temperature: 500℃; de-clustering voltage: 80 V (-80 V); collision energy: 10 V (-10 V); spray gas, auxiliary gas, and curtain gas: 55, 55, and 35 psi, respectively. Information-dependent acquisition (IDA) mode was enabled. In both positive and negative ion modes, the TOF-MS acquisition range was m / z 103–1500, and the TOF-MS acquisition range was m / z 50–1500. The gradient elution program for the mobile phase monomer saponins is shown in Table 6.

[0092]

[0093] Comparison of saponin conversion before and after transformation in ginseng and American ginseng under positive ion mode is as follows: Figure 6 , Figure 7 As shown.

[0094] The liquid chromatography-mass spectra of ginseng before and after conversion in positive ion mode show that ginsenosides 20(S)-Rh2, 20(R)-Rh2, and CK, which were present before conversion, completely disappeared after conversion, indicating that these components had been degraded or transformed. A series of saponins and their homologues, represented by pseudo-ginsenoside F11, ginsenosides Rg1, Rf, bamboo ginsenoside V, and pearl ginsenosides F1-F5, were significantly enriched after conversion, with their retention time stabilizing at about 6.48 minutes. The total peak area increased significantly from about 1.99 million to 4.53 million, indicating that these components underwent directional enrichment or structural modification.

[0095] The liquid chromatography-mass spectra of American ginseng before and after conversion in positive ion mode show that ginsenosides 20(S)-Rh2, 20(R)-Rh2, and CK, which were present before conversion, completely disappeared after conversion, indicating that these components had been degraded or transformed. A series of saponins and their homologues, represented by pseudo-ginsenoside F11, ginsenosides Rg1, Rf, bamboo ginsenoside V, and pearl ginsenosides F1-F5, were significantly enriched after conversion, with their retention time stabilizing at about 6.48 minutes. The total peak area changed from about 5.34 million to about 4.71 million, indicating that these components underwent directional enrichment or structural modification.

[0096] Comparison of saponin conversion before and after transformation in ginseng and American ginseng under negative ion mode is as follows: Figure 8 , Figure 9 As shown.

[0097] The liquid chromatography-mass spectra of ginseng before and after the negative ion mode show that ginsenoside Rh8, which was present before the transformation, completely disappeared after the transformation, indicating that this component had been degraded or transformed. A series of saponins, represented by ginsenosides Rb3, Rb2, and Rc, and their homologues were significantly enriched after the transformation, with their total peak area increasing dramatically from about 2.57 million to about 13.23 million. In particular, the component group represented by ginsenosides Rg2, Rg3, and rare saponins F2-F10 showed explosive growth, with the peak area surging from about 120,000 to about 17.37 million, an increase of more than 140 times, and the retention time remained stable at about 12.42 minutes. This series of components underwent directional enrichment or structural modification.

[0098] The liquid chromatography-mass spectra of American ginseng before and after conversion in negative ion mode show that ginsenoside Rh8, which was present before conversion, completely disappeared after conversion, indicating that this component had been degraded or transformed. A series of saponins, represented by ginsenosides Rb3, Rb2, and Rc, were significantly enriched after conversion, with the total peak area of ​​the corresponding components increasing dramatically from about 5.92 million to about 12.57 million. The peak areas of ginsenosides Rg2, Rg3 series, and rare saponins F2-F10 surged from extremely low levels to about 17.33 million after conversion, and the retention time remained stable at about 12.44 minutes, indicating that this series of components underwent explosive directional enrichment or structural modification.

[0099] Example 5 Comparative Experiment on the Efficacy of Ginseng and American Ginseng Before and After Yeast Transformation 1. Activation and expansion of Lan-S5 disporin yeast The dispora zygosacchariformis Lan-S5 was inoculated into YPD medium at a 1% inoculum (1 mL of frozen solution inoculated into 100 mL of medium) and activated by incubation at 30 ℃ and 160 r / min for 24 h to obtain the activated bacterial solution (OD). 600 Activation is complete when the OD reaches 1.8. Inoculate the activated bacterial solution into YPD medium at a 2% inoculum (2 mL of activated bacterial solution into 100 mL of medium) and incubate at 30 ℃ and 160 r / min for 24 h to obtain the expanded bacterial culture (OD). 600 The expansion is considered complete when the 1.8 ohm diameter is reached.

[0100] 2. Raw material pretreatment Commercially available ginseng and American ginseng are coarsely ground in a food processor to obtain coarse powder with a diameter of about 0.5 to 1 cm. The coarse powder is then transferred to an ultrafine grinder for further grinding and sieving to obtain ginseng powder and American ginseng powder with a diameter of 150 to 300 mesh.

[0101] 3. Preparation of fermentation broth Using sieved ginseng powder and American ginseng powder, the powder was diluted with purified water at a ratio of 1:10 to obtain the ginseng and American ginseng fermentation liquid. The liquid was then sterilized in an autoclave at 121°C for 20 minutes to prevent the influence of other microorganisms in the fermentation liquid.

[0102] 4. Transformation of ginseng and American ginseng by the two-spore conjugated yeast Lan-S5 The expanded yeast culture was inoculated into the ginseng and American ginseng fermentation broth at a 10% inoculation rate (representing the total volume of the fermentation liquid after inoculation). The broth was then placed in a shaker at 160 r / min and incubated at 30 ℃ for 7 days. After transformation, the transformed ginseng and American ginseng broth was removed, sterilized at 121 ℃ for 20 min, and then freeze-dried. The transformed ginseng and American ginseng products were obtained.

[0103] 5. Preparation of test solutions for ginseng and American ginseng (1) Preparation of test solution for total saponins of ginseng and American ginseng Weigh 1 g (accurate to 0.001 g) of ginseng powder, American ginseng powder, and ginseng and American ginseng conversion products (ground through a 60-mesh sieve). Wrap the powder in neutral filter paper, place it in a Soxhlet extractor, add 50 mL of ether, and reflux at 80 °C for 2 h. Discard the ether solution, evaporate the ether solvent from the sample packet, tear the packet into pieces, pour the contents into a 250 mL Erlenmeyer flask, add 50 mL of water-saturated n-butanol, and soak overnight. The next day, place the Erlenmeyer flask in an ultrasonic cleaner at 50 / 60 Hz and 1500 W for 30 min, centrifuge at 4000 r / min for 10 min, and transfer the supernatant to an evaporating dish. Repeat the above extraction process twice, 10 min each time. Combine the n-butanol solutions and evaporate to dryness at 90 °C. Dissolve the evaporated substances in methanol several times and bring the volume to 10 mL. The following test solutions were obtained: unconverted ginseng total saponins test solution, ginseng conversion product total saponins test solution, unconverted American ginseng total saponins test solution, and American ginseng conversion product total saponins test solution, for later use.

[0104] (2) Preparation of test solutions for ginseng and American ginseng polysaccharides Weigh 1 g (accurate to 0.001 g) of ginseng powder, American ginseng powder, and ginseng and American ginseng conversion products (ground through a 60-mesh sieve) into a 30 mL test tube, add 10 mL of water, and extract in an ultrasonic bath for 30 min. Place the extract in a 100 ℃ water bath for 4 h, cool to room temperature, add 15 mL of 80% ethanol solution to the extract and mix well. Centrifuge at 10000 r / min for 10 min on a high-speed centrifuge and discard the supernatant. Mix well with 15 mL of 80% ethanol solution and centrifuge twice, discarding the supernatant each time. Dissolve the residue in water in a 25 mL volumetric flask to obtain unconverted ginseng polysaccharide test solution, ginseng conversion product polysaccharide test solution, unconverted American ginseng polysaccharide test solution, and American ginseng conversion product polysaccharide test solution for later use.

[0105] The test solutions include: unconverted ginseng total saponins test solution, ginseng conversion product total saponins test solution, unconverted American ginseng total saponins test solution, American ginseng conversion product total saponins test solution, unconverted ginseng polysaccharide test solution, ginseng conversion product polysaccharide test solution, unconverted American ginseng polysaccharide test solution, and American ginseng conversion product polysaccharide test solution.

[0106] 6. DPPH free radical scavenging rate determination Add the nitrogen free radical standard solution, test solution, anhydrous ethanol, and DPPH solution sequentially to a 1 mL centrifuge tube according to the order in Table 7, mix well, and incubate at room temperature in the dark for 30 min. Take a 96-well plate and add 0.3 mL of each solution sequentially to the 96-well plate. Measure the absorbance at 517 nm using a microplate reader. Calculate the DPPH free radical scavenging rate according to the formula.

[0107] E1 = [1 - (A1 - A2) / A0] × 100% In the formula: the absorbance of the blank group is recorded as A0; the absorbance of the test solution group is recorded as A1; and the absorbance of the test solution control group is recorded as A2.

[0108]

[0109] 7. ABTS + Free radical scavenging rate determination ABTS + Working solution, test solution, PBS buffer, and purified water were added to the 96-well plate in the order shown in Table 8. After reacting for 6 minutes, the absorbance was measured at 734 nm using a microplate reader. ABTS was calculated according to the formula. + Free radical scavenging rate.

[0110] E = [1 - (A1 - A2) / A0] × 100% In the formula: the absorbance of the blank group is A0; the absorbance of the test solution group is A1; and the absorbance of the test solution control group is A2.

[0111]

[0112] 8. Measurement of blood glucose lowering capacity (α-glucosidase inhibition rate) The PNPG method was used to determine the α-glucosidase inhibition rate of the samples. Test solution assay group, test solution blank group, control group, and control blank group were set up. PBS solution, test solution, and α-glucosidase solution were added sequentially according to the reaction system in Table 9. After incubating at 37 ℃ for 15 min, PNPG solution was added, and the reaction was continued for another 15 min. The reaction was terminated by adding Na2CO3 solution. The absorbance was measured, and the inhibition rate was calculated. The α-glucosidase inhibition rate was calculated according to the formula.

[0113] E1 = [1 - (A2 - A3) / (A0 - A1)] × 100% In the formula: the absorbance of the control group is A0; the absorbance of the blank control group is A1; the absorbance of the test sample group is A2; and the absorbance of the blank test sample group is A3.

[0114]

[0115] 9. Measurement of lipid-lowering ability (pancreatic lipase inhibition rate) The inhibition rate of pancreatic lipase in the samples was determined using the PNPB method. Test sample group, test sample blank group, control group, and control blank group were set up. The test solution and pancreatic lipase solution were added sequentially according to the reaction system in Table 10, and the mixture was incubated at 37 ℃ for 10 min. Then, PNPB solution and Tris-HCl solution were added, mixed, and incubated at 37 ℃ for 15 min. The absorbance was measured at 405 nm using a microplate reader, and the pancreatic lipase inhibition rate was calculated according to the formula. E1 = [1 - (A4 - A3) / (A2 - A1)] × 100% In the formula: the absorbance of the control group is A2; the absorbance of the blank control group is A1; the absorbance of the test sample measurement group is A4; and the absorbance of the blank test sample group is A3.

[0116]

[0117] The results of the DPPH free radical scavenging rate test before and after the transformation of ginseng and American ginseng are as follows.

[0118]

[0119] Table 11 shows that the DPPH free radical scavenging rate of American ginseng and ginseng transformed by *Diplostomum diplostomum* Lan-S5 was significantly improved compared with that before transformation. The DPPH free radical scavenging rate of total ginsenosides increased from 47.63% to 69.79%, that of total American ginseng saponins increased from 64.35% to 85.64%, that of ginseng polysaccharides increased from 36.81% to 60.72%, and that of American ginseng polysaccharides increased from 31.27% to 68.95%.

[0120] ABTS before and after conversion of ginseng and American ginseng + The results of the free radical scavenging rate test are as follows.

[0121]

[0122] Table 12 shows that the ginseng and American ginseng transformed by Lan-S5 of Diplostomum bifidum are significantly better than the ABTS products before transformation. + Free radical scavenging rate is significantly improved. Ginsenosides ABTS+ The free radical scavenging rate increased from 42.73% to 63.75%, and the total saponins ABTS of American ginseng were also increased. + The free radical scavenging rate increased from 58.69% to 84.07%, and ginseng polysaccharide ABTS... + The free radical scavenging rate increased from 33.58% to 58.84%, and the ginseng polysaccharide ABTS was also improved. + The free radical scavenging rate increased from 28.63% to 63.71%.

[0123] The results of the blood glucose lowering ability (α-glucosidase inhibition rate) test of ginseng and American ginseng before and after conversion are as follows.

[0124]

[0125] Table 13 shows that the α-glucosidase inhibition rate of ginseng and American ginseng transformed by *Diplostomum diplostomum* Lan-S5 was significantly increased compared with that before transformation. The α-glucosidase inhibition rate of total ginsenosides increased from 28.93% to 43.34%, that of total American ginseng saponins increased from 40.79% to 57.64%, that of ginseng polysaccharides increased from 24.87% to 43.68%, and that of American ginseng polysaccharides increased from 23.74% to 48.57%.

[0126] The results of the lipid-lowering ability (pancreatic lipase inhibition rate) of ginseng and American ginseng before and after transformation are as follows.

[0127]

[0128] Table 14 shows that the pancreatic lipase inhibition rates of ginseng and American ginseng transformed by *Diplostomum diplostomum* Lan-S5 were significantly increased compared to the untransformed products. The pancreatic lipase inhibition rate of total ginseng saponins increased from 37.63% to 42.58%, that of total American ginseng saponins increased from 20.17% to 49.27%, that of ginseng polysaccharides increased from 23.58% to 38.95%, and that of American ginseng polysaccharides increased from 20.26% to 40.34%.

[0129] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0130] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of yeast in the fermentation and transformation of ginseng or American ginseng, characterized in that, The application increases the content of saponins and polysaccharides in fermented ginseng or American ginseng; The saponins are total saponins, Rg1, Re, Rf, Rb2, Rb3 or Rd; The preservation number of the yeast is CCTCC NO: M 2020912.

2. The application as described in claim 1, characterized in that, The application enhances the in vitro DPPH free radical scavenging ability, ABTS free radical scavenging ability, α-glucosidase inhibition rate, and pancreatic lipase inhibition rate of the transformed American ginseng or ginseng total saponin extract or polysaccharide extract.

3. A method for increasing the saponins and polysaccharides in fermented ginseng or American ginseng, characterized in that, Fermentation of ginseng or American ginseng raw materials using yeast; The saponins are total saponins, Rg1, Re, Rf, Rb2, Rb3 or Rd; The preservation number of the yeast is CCTCC NO: M 2020912.

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