Aspergillus oryzae-ginseng solid fermentation method and application

Abstract

The invention provides an Aspergillus oryzae-ginseng solid fermentation method and application, and belongs to the technical field of ferment.The Aspergillus oryzae-ginseng solid fermentation method includes activating Aspergillus oryzae strains, preparing Aspergillus oryzae spore suspension and preparing and fermenting Aspergillus oryzae-ginseng solid fermentation substrates. Technical parameters of solid fermentation of aspergillus oryzae and ginseng are optimized by using a single-factor test method. The best fermentation effect is determined under the conditions that the fermentation time is 8 days, the inoculum size is 2.5% and the fermentation temperature is 28 DEG C. The total sugar content and the total saponin content obtained by fermentation are respectively 3.853 + / -0.025 mg.mL <-1 > and 1.687 + / -0.002 mg.mL <-1 >. Compared with a control group, the total sugar content is increased by 2.048 times, and the total saponin content is increased by 1.448 times.

Description

Technical Field

[0001] The present invention relates to the field of fermentation technology, and in particular to an Aspergillus oryzae-ginseng solid fermentation method and application. Background Art

[0002] Aspergillus oryzae (AO) belongs to the phylum Deuteromycotina, subphylum Deuteromycotina, class Hypomycetes, orders Hypomycetes, family Pseudomonas, and genus Aspergillus. A. oryzae reproduces by spores, which germinate into hyphae. The hyphae are white in the early stages of growth, yellow-green in the later stages, and gradually turn brown with aging. As a filamentous fungus, A. oryzae plays an important role in the production of traditional fermented foods and is often used in the production of alcoholic beverages, sauces, and soy sauces. After years of practical testing, its safety has been widely recognized by relevant domestic and international organizations, and it has been designated as a generally recognized safe strain by the US FDA. Research reports indicate that A. oryzae primarily degrades macromolecules such as proteins, polysaccharides, and cellulose in the fermentation matrix by secreting enzymes such as proteases, amylases, and cellulases.

[0003] Ginseng (Panax ginseng C.A. Mey) is a perennial herbaceous plant of the genus Panax, in the Araliaceae family. It was first mentioned in the Shennong's Herbal Classic: "It nourishes the five internal organs, calms the nerves, calms the soul, relieves palpitations, eliminates evil spirits, improves eyesight and intelligence, and, with long-term consumption, lightens the body and prolongs life." Numerous modern studies have shown that ginseng contains multiple bioactive components, including saponins, polysaccharides, volatile oils, and alkaloids, with ginsenosides being the core active ingredient. Ginsenosides have medicinal properties such as anti-tumor, anti-aging, anti-fatigue, immune enhancement, and cardiovascular protection. However, common ginsenosides are difficult to absorb and utilize by the human body, while rare ginsenosides are more easily absorbed and utilized by the body due to the hydrolysis of their glycosidic bonds. Rare ginsenosides are present in extremely low concentrations in natural ginseng, making their mass production through artificial chemical methods difficult and yielding low yields. Therefore, there is an urgent need to find efficient and feasible methods for converting ginsenosides to address the challenges posed by the ginseng industry and expand its development potential. Currently, the three mainstream methods for converting common ginsenosides into rare ginsenosides are physical transformation, chemical transformation, and biological transformation. The physical transformation method has disadvantages such as being time-consuming, low-yield, and low-precision; the chemical method for synthesizing ginsenosides is inefficient, highly destructive to the environment, and difficult to achieve mass production. The biological transformation method mainly uses ginsenosides as substrates and utilizes enzymes or one or more enzymes produced during microbial metabolism to hydrolyze the glycosidic bonds of ginsenosides, thereby converting common ginsenosides into rare ginsenosides. At the same time, during the reaction, macromolecules such as proteins and sugars in ginseng are consumed by enzymes produced by microbial metabolism, thereby increasing the concentration of ginsenosides. Current research shows that microbial transformation technology is relatively mature and has advantages that physical and chemical transformation methods do not have, such as being green and environmentally friendly, safe experimental conditions, few side reactions, and a single reaction product. Summary of the Invention

[0004] The present invention aims to solve the above technical problems and provides an Aspergillus oryzae-ginseng solid fermentation method and application.

[0005] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0006] An Aspergillus oryzae-ginseng solid fermentation method comprises the following steps:

[0007] S1. Activation of Aspergillus oryzae:

[0008] Clean the clean bench, place the top of the freeze-dried tube on the outer flame of an alcohol lamp and burn it evenly. Continue heating for 3-5 minutes. Use a sterile dropper to draw 2-3 drops of sterile water on the heated part to break the freeze-dried tube wall. Use tweezers to tap the broken part, open the freeze-dried tube, use a sterile dropper to draw a small amount of sterilized PDA liquid culture medium, inject it into the freeze-dried tube, and fully dissolve the freeze-dried powder. Use a sterile syringe to draw the dissolved bacterial suspension and place it in two 4-5 mL liquid culture media respectively. Mix well and place in a shaking incubator at 28°C, 140 rpm. -1 Culture in the dark under the same conditions, and then subculture once using the same method to ensure that the activity of the strain is fully restored;

[0009] S2. Preparation of Aspergillus oryzae spore suspension:

[0010] Take the activated bacterial suspension in a clean bench, mix it well, and then dip the inoculating loop into the bacterial suspension and spread it on the PDA slant solid culture medium. Dip the inoculating loop into the bacterial suspension and turn it upside down for 5-10 minutes before incubating it in a dark incubator. Only when the slant is covered with white hyphae can the spore suspension be prepared.

[0011] In a clean bench, inject 3-5 mL of sterile water into each slant solid culture medium covered with white mycelium. Use an inoculation loop to scrape the white mycelium, filter the mycelium with gauze to obtain the Aspergillus oryzae spore suspension, and then use a sterile syringe to draw a small amount of spore suspension into the center of the hemocytometer. Cover it with a slide and place it under an optical microscope to calculate the concentration of the spore suspension. Use sterile water to adjust the concentration to 5.7 × 10 6 CFU·mL -1 ;

[0012] S3. Preparation of Aspergillus oryzae-ginseng solid fermentation matrix:

[0013] Grind white ginseng to obtain uniform ginseng powder. Weigh the ginseng powder into a fermentation tank, add 40% pure water by volume to the ginseng powder, stir evenly and sterilize. After sterilization, transfer the ginseng matrix to a clean bench and let it cool naturally to room temperature. Gently shake the matrix to loosen it and turn on the ultraviolet light for 30 minutes for sterilization.

[0014] S4. Fermentation:

[0015] The fermentation time is 7.5-8.5 days, the fermentation temperature is 26-29°C, and the inoculation amount is 2.4-2.6%.

[0016] Preferably, in step S1, the freeze-dried tube is wiped with a 75% alcohol cotton ball before use and is used after the alcohol evaporates naturally.

[0017] Preferably, the dark-proof culturing time in step S1 is 36 hours.

[0018] Preferably, after mixing in step S2, the bacterial solution is dipped into an inoculating loop and spread in a "Z" shape on the PDA slant solid culture medium.

[0019] Preferably, the method for sterilizing by stirring uniformly in step S3 is high temperature and high pressure sterilization at 121° C. for 30 minutes.

[0020] Preferably, the white ginseng is crushed in step S3 and then passed through a 15-20 mesh sieve.

[0021] A method for solid fermentation of Aspergillus oryzae and ginseng to obtain ginsenosides is used in the preparation of medicines, foods or health products for preventing and treating gastric mucosal damage.

[0022] After adopting the above method, the present invention has the following advantages:

[0023] This study optimized the process parameters for solid-state fermentation of Aspergillus oryzae and ginseng using a single-factor experiment, using total sugar and total saponin content as dual evaluation indicators. The optimal fermentation conditions were determined to be 8 days, a 2.5% inoculum size, and a fermentation temperature of 28°C. The total sugar and total saponin contents obtained from fermentation were 3.853 ± 0.025 mg mL, respectively. -1 and 1.687±0.002mg·mL -1 Compared with the control group, the total sugar content increased by 2.048 times, and the total saponin content increased by 1.448 times.

[0024] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or technical descriptions. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 It is the glucose standard curve diagram of the present invention;

[0027] Figure 2 is a standard curve diagram of ginsenoside Re of the present invention;

[0028] Figure 3 This is a verification diagram of the Aspergillus oryzae-ginseng solid fermentation single-factor experimental results of the present invention;

[0029] Figure 4 This is a graph showing the effects of fermentation time and fermentation temperature on the total sugar and total saponin contents in Aspergillus oryzae-ginseng fermentation;

[0030] Figure 5 is a graph showing the effect of the inoculum amount of the present invention on the total sugar and total saponin contents in Aspergillus oryzae-ginseng fermentation;

[0031] Figure 6 It is a verification diagram of the orthogonal experiment results of Aspergillus oryzae-ginseng solid fermentation of the present invention (A is a plan view of the orthogonal experiment result verification; B is a top view of the orthogonal experiment result verification; C is a bottom view of the orthogonal experiment result verification).

[0032] Figure 7 This is a graph showing the total ginsenoside content obtained using the three extraction methods of the present invention;

[0033] Figure 8 It is the total ion current chromatogram of AO and AOC of the present invention;

[0034] Figure 9 It is a graph of the AO and AOC saponin contents of the present invention. DETAILED DESCRIPTION

[0035] Specific embodiments of the present invention will now be mentioned in detail. Although the present invention is described in conjunction with these specific embodiments, it should be appreciated that the present invention is not intended to be limited to these specific embodiments. On the contrary, these embodiments are intended to cover substitutions, changes or equivalent embodiments that may be included in the spirit and scope of the invention defined by the claims. In the following description, a large amount of specific details are set forth to provide a comprehensive understanding of the present invention. The present invention can be implemented without some or all of these specific details. In other cases, in order not to make the present invention unnecessarily obscure, well-known process operations are not described in detail.

[0036] When used in conjunction with "including," "methods comprising," or similar language in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0037] The present invention will be described in further detail below in conjunction with the full text.

[0038] Combined with attachment Figure 1-Figure 2 , an Aspergillus oryzae-ginseng solid fermentation method, comprising the following steps:

[0039] S1. Activation of Aspergillus oryzae:

[0040] Clean the clean bench, place the top of the freeze-dried tube on the outer flame of an alcohol lamp and burn it evenly. Continue heating for 3-5 minutes. Use a sterile dropper to draw 2-3 drops of sterile water on the heated part to break the freeze-dried tube wall. Use tweezers to tap the broken part, open the freeze-dried tube, use a sterile dropper to draw a small amount of sterilized PDA liquid culture medium, inject it into the freeze-dried tube, and fully dissolve the freeze-dried powder. Use a sterile syringe to draw the dissolved bacterial suspension and place it in two 4-5 mL liquid culture media respectively. Mix well and place in a shaking incubator at 28°C, 140 rpm. -1 Culture in the dark under the same conditions, and then subculture once using the same method to ensure that the activity of the strain is fully restored;

[0041] S2. Preparation of Aspergillus oryzae spore suspension:

[0042] Take the activated bacterial suspension in a clean bench, mix it well, and then dip the inoculating loop into the bacterial suspension and spread it on the PDA slant solid culture medium. Dip the inoculating loop into the bacterial suspension and turn it upside down for 5-10 minutes before incubating it in a dark incubator. Only when the slant is covered with white hyphae can the spore suspension be prepared.

[0043] In a clean bench, inject 3-5 mL of sterile water into each slant solid culture medium covered with white mycelium. Use an inoculation loop to scrape the white mycelium, filter the mycelium with gauze to obtain the Aspergillus oryzae spore suspension, and then use a sterile syringe to draw a small amount of spore suspension into the center of the hemocytometer. Cover it with a slide and place it under an optical microscope to calculate the concentration of the spore suspension. Use sterile water to adjust the concentration to 5.7 × 10 6 CFU·mL -1 ;

[0044] S3. Preparation of Aspergillus oryzae-ginseng solid fermentation matrix:

[0045] Grind white ginseng to obtain uniform ginseng powder. Weigh the ginseng powder into a fermentation tank, add 40% pure water by volume to the ginseng powder, stir evenly and sterilize. After sterilization, transfer the ginseng matrix to a clean bench and let it cool naturally to room temperature. Gently shake the matrix to loosen it and turn on the ultraviolet light for 30 minutes for sterilization.

[0046] S4. Fermentation:

[0047] The fermentation time is 7.5-8.5 days, the fermentation temperature is 26-29°C, and the inoculation amount is 2.4-2.6%.

[0048] In step S1, the freeze-dried tube was wiped with a 75% alcohol cotton ball before use and used after the alcohol evaporated naturally.

[0049] The dark-protected culture time in step S1 is 36 hours.

[0050] After mixing in step S2, use an inoculating loop to dip the bacterial solution and spread it in a "Z" shape on the PDA slant solid culture medium.

[0051] The method for sterilizing by stirring evenly in step S3 is high temperature and high pressure sterilization at 121° C. for 30 minutes.

[0052] The white ginseng in step S3 is crushed and passed through a 15-20 mesh sieve.

[0053] Aspergillus oryzae (AO) was purchased from China Center for Type Culture Collection with the strain number CCTCC AF 2018017. Five-year-old white ginseng was purchased from Northeast Specialty Products Co., Ltd., Fusong County, Baishan City, Jilin Province.

[0054] The experimental reagents are shown in Table 1 below:

[0055] Table 1 Experimental reagents and manufacturers

[0056] Experimental reagents Reagent manufacturers glucose Tianjin Damao Chemical Reagent Factory yeast powder Xi'an Jinyuan Biotechnology Co., Ltd. agar powder Shanghai Yuanye Biotechnology Co., Ltd. Potato extract powder Sinopharm Chemical Reagent Co., Ltd. sulfuric acid Sinopharm Chemical Reagent Co., Ltd. phenol Sinopharm Chemical Reagent Co., Ltd. Vanillin Sinopharm Chemical Reagent Co., Ltd. perchloric acid Sinopharm Chemical Reagent Co., Ltd. Ginsenoside Re Standard Shanghai Yuanye Biotechnology Co., Ltd. Methanol Thermo Fisher Scientific

[0057] The experimental instruments are shown in Table 2 below:

[0058] Table 2 Experimental instruments and manufacturers

[0059]

[0060]

[0061] Activation of Aspergillus oryzae:

[0062] In the clean bench, first use a 75% alcohol cotton ball to wipe the surface of the freeze-dried tube. After the alcohol evaporates naturally, place the top of the freeze-dried tube on the outer flame of the alcohol lamp for even burning, and continue heating for 3-5 minutes. Then, use a sterile dropper to draw 2-3 drops of sterile water on the heated area to break the wall of the freeze-dried tube. Then, use tweezers to tap the broken part to open the freeze-dried tube. After that, use a sterile dropper to draw a small amount of sterilized PDA liquid culture medium and inject it into the freeze-dried tube to fully dissolve the freeze-dried powder. Use a sterile syringe to draw the dissolved bacterial suspension and place it in 2 4-5mL liquid culture media, mix them, and place them in a shaking incubator at 28°C, 140r·min -1 Culture in the dark for 36 hours under normal conditions. Then subculture once using the same method to ensure complete recovery of bacterial activity.

[0063] Preparation of Aspergillus oryzae spore suspension:

[0064] Take the activated bacterial suspension in a laminar flow hood, mix thoroughly, and then use an inoculating loop to apply the suspension in a "Z" pattern to a PDA slant. Place the slant upside down for 5-10 minutes, then incubate in a dark incubator. Only prepare the spore suspension after the slant is covered with white hyphae.

[0065] In a clean bench, inject 3-5 mL of sterile water into each slant of solid culture medium covered with white mycelium. Use an inoculating loop to scrape the white mycelium and filter it through gauze to obtain an Aspergillus oryzae spore suspension. Then, use a sterile syringe to draw a small amount of spore suspension into the center of a hemocytometer. Cover it with a slide and place it under an optical microscope to calculate the concentration of the spore suspension. Adjust the concentration to 5.7 × 10 6 CFU·mL -1 .

[0066] Preparation of Aspergillus oryzae-ginseng solid fermentation matrix:

[0067] Method: Aspergillus oryzae-ginseng fermentation matrix was prepared. An appropriate amount of white ginseng was crushed and passed through a 16-mesh sieve according to the pharmacopoeia to obtain ginseng powder with uniform particles. An appropriate amount of ginseng powder was weighed into a fermentation tank, and pure water with a volume ratio of 40% of the ginseng powder was added. After stirring evenly, the mixture was sterilized at high temperature and high pressure at 121°C for 30 minutes. After sterilization, the ginseng matrix was transferred to a clean bench and allowed to cool naturally to room temperature. The matrix was gently shaken to loosen it, and then sterilized under ultraviolet light for 30 minutes. In the design of the present invention, an appropriate amount of Aspergillus oryzae spore suspension was inoculated into a ginseng solid culture matrix as the experimental group, while a ginseng solid culture matrix not inoculated with the Aspergillus oryzae spore suspension was used as the control group.

[0068] Single-factor experimental design of Aspergillus oryzae-ginseng solid fermentation:

[0069] In the solid-state fermentation experiment of ginseng by Aspergillus oryzae, it is generally believed that fermentation time, inoculation size, and fermentation temperature have a relatively large impact on the fermentation results. Therefore, the present invention selects three key factors, fermentation time (A), inoculation size (B), and fermentation temperature (C), to conduct a single-factor experiment, and uses the total saponin content and total sugar content of the fermented substrate as evaluation indicators to determine the optimal fermentation process.

[0070] (1) Fermentation time: Under the conditions of fermentation temperature of 28°C and inoculum size of 2.5%, the effects of fermentation time (2, 4, 6, 8, 10 days) on the total sugar content and total saponin content of the Aspergillus oryzae-ginseng solid fermentation product were examined.

[0071] (2) Inoculation amount: Under the conditions of fermentation temperature of 28°C and fermentation time of 8 days, the effects of inoculation amount (1%, 2.5%, 5%, 7.5%, 10%) on the total sugar content and total saponin content of the Aspergillus oryzae-ginseng solid fermentation product were examined.

[0072] (3) Fermentation Temperature: The effects of fermentation temperature (25, 28, 31, 34, and 37°C) on the total sugar and total saponin contents of the Aspergillus oryzae-ginseng solid fermentation product were investigated under conditions of a 10-day fermentation time and a 2.5% inoculum size. The single-factor experimental design is shown in Table 3. Each group had five replicates, and the results are expressed as mean ± standard deviation.

[0073] Table 3 Single factor experimental design table

[0074] Factor levels 1 2 3 4 5 A(d) 2 4 6 8 10 B(％) 1 2.5 5 7.5 10 C(℃) 25 28 31 34 37

[0075] Orthogonal experimental design of Aspergillus oryzae-ginseng solid fermentation:

[0076] Based on the results of the single-factor experiment, an orthogonal experiment was designed using the three-factor four-level L16(3 4 An orthogonal array was used to optimize fermentation time (A), inoculum size (B), and fermentation temperature (C). The optimal fermentation conditions were determined by testing and comparing the effects of different fermentation conditions on the total sugar and saponin content of the fermentation product. The orthogonal experimental design is shown in Table 4. Five replicates were set up in each group, and the results are expressed as mean ± standard deviation.

[0077] Table 4 Orthogonal experiment level factors

[0078]

[0079] Determination of total saponin content

[0080] The total saponin content in ginseng matrix before and after fermentation was determined.

[0081] Preparation of standard: Accurately weigh ginsenoside Re standard and add appropriate amount of methanol to make 1 mg·mL -1 of the standard solution.

[0082] Preparation of a standard curve: Use a pipette to precisely pipette 20, 40, 80, 120, 160, and 200 μL of the reference solution into a test tube. Dose to 200 μL with purified water. Evaporate the solvent at low temperature and add 0.5 mL of 1% vanillin-perchloric acid solution. Place the test tube in a 60°C water bath for 15 minutes. Immediately remove and cool in ice water for 2 minutes. After cooling, add 5 mL of 77% sulfuric acid solution, shake thoroughly, and defoam. Measure the absorbance at 540 nm in a UV / Vis spectrophotometer. Plot the standard curve using absorbance as the y-axis and concentration as the abscissa.

[0083] Determination of total saponin content in samples: Accurately weigh 100 mg of sample and reference substance, dilute to 5 mL with methanol, shake well, and pipette 50 μL of the solution. Measure the absorbance at 540 nm using the same method as the calibration curve. Read the ginsenoside Re content in the sample and reference substance from the calibration curve and multiply by 0.84 to obtain the total saponin content.

[0084] Total sugar content determination method

[0085] The total sugar content in samples and controls was determined using the phenol-sulfuric acid method with minor modifications.

[0086] Prepare glucose standard solution: Accurately weigh 10 mg of glucose to a constant weight and dilute to 100 mL with pure water. Use a pipette to pipette 0.2, 0.4, 0.6, 0.8, and 1.0 mL of glucose solution into a 10 mL test tube, dilute to 10 mL with pure water, and shake to obtain the dilution.

[0087] To prepare a standard curve: Use a pipette to precisely pipette 0.2, 0.4, 0.6, 0.8, and 1.0 mL of glucose dilution, dilute to 1.0 mL with purified water, add 1 mL of 5% phenol solution, shake well, then add 5 mL of concentrated sulfuric acid and shake well. Cool, place in a boiling water bath for 20 minutes, and measure the absorbance at 490 nm in a UV / Vis spectrophotometer. Prepare a standard curve with absorbance as the y-axis and concentration as the x-axis.

[0088] Determination of total sugar content in samples: Accurately weigh 5 mg of sample and reference substance, dilute to 50 mL with pure water, centrifuge and obtain the supernatant. Accurately aspirate 100 μL with a pipette, then add 100 μL of 5% phenol solution and 500 μL of concentrated sulfuric acid in sequence. Measure the absorbance at a wavelength of 490 nm in a UV-visible spectrophotometer according to the method for preparing a standard curve. Read the total sugar content of the sample and reference substance on the standard curve based on the absorbance.

[0089] Statistical analysis

[0090] The data in this paper were organized using Excel worksheets and statistically analyzed using SPSS 22.0 software. The data were expressed as mean ± standard deviation. The two groups were compared using a two-sample t-test, and the multiple sample means were compared using one-way analysis of variance (One-Way ANOVA). *, #P < 0.05 was considered statistically significant. Statistical graphs were drawn using Graphpad Prism 9.5 and Origin 2021 software.

[0091] Determination of glucose standard curve

[0092] Glucose standard curve Figure 1 As shown, the concentration of glucose dilution and OD 490The values ​​showed a good linear relationship, with the concentration of glucose dilution as the horizontal axis x, OD 490 The value is the ordinate y, and the standard curve is drawn. The regression equation is: y = 3.0648x + 0.0944, R 2 =0.9996.

[0093] Determination of the standard curve of ginsenoside Re

[0094] The standard curve of ginsenoside Re is as follows Figure 2 As shown, the concentration of ginsenoside Re standard dilution solution and OD 540 The values ​​showed a good linear relationship, with the concentration of glucose dilution as the horizontal axis x, OD 540 The value is the ordinate y, and the standard curve is drawn. The regression equation is: y = 0.0006x + 0.001, R 2 =0.9997.

[0095] Fermentation time screening

[0096] Effects of fermentation time on the accumulation of total sugar and total saponins in solid fermentation of Aspergillus oryzae-ginseng Figure 3 AC and Figure 4 As shown in A and B, the total sugar content of both the experimental and control groups increased with time. However, starting from the 6th day of fermentation, the total sugar content of the experimental group was significantly higher than that of the control group (P < 0.05), and reached the highest level of 2.482 ± 0.002 mg mL on the 10th day of fermentation. -1 However, in terms of the accumulation of total saponin content, the difference between the experimental and control groups was not significant during the first four days of fermentation, with the control group slightly higher than the experimental group. Starting from the sixth day of fermentation, the saponin content of the experimental group was higher than that of the control group, reaching its highest value on the eighth day of fermentation and showing a significant difference from the control group (P<0.05). Thereafter, the total saponin content of both the experimental and control groups showed a downward trend. Therefore, considering the changes in total sugar content and total saponin content, 8 days was selected as the optimal fermentation time for subsequent experiments.

[0097] Fermentation temperature screening

[0098] Effects of fermentation temperature on the accumulation of total sugar and total saponins in solid fermentation of Aspergillus oryzae-ginseng Figure 4 As shown in C and D, the total sugar content and total saponin content of the experimental group reached their maximum values ​​at 28°C, which were 2.958±0.067 mg·mL -1 and 1.125 ± 0.004 mg mL -1, the total saponin content showed a significant difference compared to the control group (P<0.05). Thereafter, the total sugar content and total saponin content decreased with increasing fermentation temperature. Under different temperature conditions, the total sugar content of the control group was higher than that of the experimental group, and the total saponin content was lower than that of the experimental group. This is believed to be due to the fact that the addition of Aspergillus oryzae as a fermentation bacteria in the experimental group promoted the production and conversion of ginsenosides by utilizing the sugars produced during the fermentation process as energy. Therefore, 28°C was selected as the optimal fermentation temperature for subsequent experiments.

[0099] Screening of inoculum size

[0100] Effect of inoculum size on the accumulation of total sugar and total saponins in solid fermentation of Aspergillus oryzae-ginseng Figure 5 As shown in A and B. The total sugar content and total saponin content of the experimental group reached their maximum values ​​at an inoculum concentration of 2.5%, which were 3.119±0.008 mg·mL -1 and 1.117 ± 0.006 mg mL -1 , among which the total saponin content of the experimental group showed significant difference compared with that of the control group (P<0.05). The total sugar content of the experimental group was lower than that of the control group, while the experimental group was relatively better than the control group in terms of total saponin content. This is because during the fermentation process, the amount of inoculation directly affects the growth and reproduction rate of Aspergillus oryzae. When the inoculation amount is too high, it is easy to cause insufficient oxygen supply, which in turn hinders product synthesis and only promotes the accumulation of sugars; when the inoculation amount is too low, Aspergillus oryzae cannot fully interact with the ginseng matrix, resulting in a decrease in fermentation efficiency as the fermentation time increases, resulting in a reverse effect. In summary, an inoculation amount of 2.5% was selected as the optimal fermentation inoculation amount for subsequent experiments.

[0101] Results of orthogonal experiment of solid fermentation of Aspergillus oryzae and ginseng

[0102] The results of the orthogonal experiment are shown in Table 5

[0103] Table 5 Orthogonal experiment results

[0104]

[0105] Table 5 Orthogonal experiment results

[0106]

[0107]

[0108]

[0109] Table 5 Orthogonal experiment results

[0110]

[0111] From the orthogonal analysis range R value in Table 5, it can be seen that the factors affecting the total sugar content and total saponin content of Aspergillus oryzae-ginseng solid fermentation are ranked from high to low as: A>B>C, indicating that during the fermentation process, the total sugar content and total saponin content are most significantly affected by the fermentation time, followed by the inoculation amount, while the influence of the fermentation temperature is relatively small. Taking into account various factors, total sugar content and total saponin content and k value, the optimal combination of Aspergillus oryzae-ginseng solid fermentation is A2B2C3, that is, the fermentation effect is best under the conditions of 8 days of fermentation time, 2.5% inoculation amount and 28°C fermentation temperature. A verification experiment was carried out under this optimal condition, with 3 parallels set up for each group, and the total sugar content and total saponin content were measured 3 times each. The results are expressed as mean ± standard deviation. The total sugar content and total saponin content obtained by fermentation were 3.853±0.025 mg·mL -1 and 1.687±0.002mg·mL -1 Compared with the control group, the total sugar content increased by 2.048 times, and the total saponin content increased by 1.448 times, both showing significant differences (P<0.05). The results showed that the fermentation process conditions optimized by orthogonal experiments were reliable and reproducible.

[0112] The present invention experiments optimized the Aspergillus oryzae-ginseng solid fermentation process by single-factor experiments, and under the optimized process, detected the total sugar content and total saponin content produced by fermentation, and carried out orthogonal experiments on the basis of the single-factor experimental results to optimize the optimal Aspergillus oryzae-ginseng solid fermentation conditions. The specific results are as follows:

[0113] In this study, we optimized the process parameters for solid-state fermentation of Aspergillus oryzae and ginseng using a single-factor experimental approach, using total sugar and total saponin content as dual evaluation indicators. The study focused on the impact of three key factors, fermentation time, fermentation temperature, and inoculum size, on the quality of the fermentation product. The final optimized fermentation process conditions were: fermentation time of 8 days, fermentation temperature of 28°C, and inoculum size of 2.5%.

[0114] Based on this, a three-factor, four-level orthogonal experiment was designed. The results showed that the most influential factor in the solid-state fermentation of Aspergillus oryzae with ginseng was the fermentation time. The optimal fermentation conditions were 8 days, a 2.5% inoculum, and a temperature of 28°C. A validation experiment under these conditions showed that the total sugar content in the experimental group increased by 2.048 times and the total saponin content increased by 1.448 times compared to the control group without Aspergillus oryzae. These results provide data and theoretical support for related research on Aspergillus oryzae-ginseng solid-state fermentation and also provide a foundation for subsequent research on transformation mechanisms and bioactivity.

[0115] Screening of extraction methods

[0116] Take an appropriate amount of fermentation sample, crush it with a grinder, and then pass it through a 60-mesh sieve for later use. Based on the three commonly used saponin extraction methods summarized, use the saponin content detection method to calculate the extraction rate, and then select the best extraction method for subsequent experiments.

[0117] Method 1: Accurately weigh 1.0 g of fermentation sample and use a volume ratio of 1:50 g mL -1 Add 50 mL of 70% ethanol aqueous solution and extract by ultrasonication at 480W and 60℃ for 30 min. -1 The supernatant was collected after centrifugation for 15 min and made up to 50 mL with 70% ethanol. The same extraction operation was repeated three times.

[0118] Method 2: Accurately weigh 1.0 g of fermentation sample, place in a 100 mL conical flask, add 50 mL of 70% LC-MS methanol aqueous solution (v / v), and sonicate at 250W, 40kHz, 30°C water bath temperature for 30 min. After cooling to room temperature, add 70% LC-MS methanol aqueous solution to 50 mL. Repeat the same extraction procedure three times and then extract at 15,000 r / min. -1 Centrifuge for 15 min and collect the supernatant.

[0119] Method 3: 1.0 g of fermentation sample was accurately weighed and placed in a 100 mL conical flask. 50 mL of 70% LC-MS methanol aqueous solution (v / v) was added. Ultrasonication was performed at 300 W and 40 kHz for 2 h at room temperature. After cooling to room temperature, the volume was filled to 50 mL with 70% LC-MS methanol aqueous solution. The extraction solution was stirred at 13000 r·min. -1 Centrifuge for 15 min and collect the supernatant.

[0120] Each extraction method was performed in triplicate, and the saponin content in each replicate was determined 5 times. The results were expressed as mean ± standard deviation.

[0121] Preparation of standards

[0122] 1.0 mg of each of the 21 ginsenoside standards purchased was accurately weighed and dissolved in 70% LC-MS methanol aqueous solution (v / v) to a final concentration of 1 mg mL -1 The 21 standard stock solutions were diluted to 40, 30, 20, 10, and 1 μg·mL -1 Working solution. -1 For example, 21 ginsenoside standards were mixed in equal volume ratios to form 1.5 mL of mixed standard solution, filtered through a 0.22 μm organic filter membrane, and stored at -20°C for later use. 30, 20, 10, 1 μg·mL -1 The preparation method of the concentration mixed standard solution is the same as that of 40 μg·mL -1 .

[0123] In addition, to ensure the stability and consistency of mass spectrometry, 10 μg mL -1 The mixed standard solution was used as the quality control (QC) sample, and 5 QC injections were randomly performed during the sample collection period.

[0124] Sample preparation

[0125] Based on the optimal extraction method from the experimental results, sample (Aspergillus oryzae, AO) and control (Aspergillus oryzae control, AOC) solutions were obtained. Freeze-dried samples were obtained by vacuum freeze-drying. The freeze-dried samples were dissolved in 70% LC-MS methanol-water solution (v / v) and prepared to a concentration of 20 mg mL. -1 The test samples were filtered through a 0.22 μm organic filter membrane and then refrigerated at -20°C for later use.

[0126] Chromatographic analysis conditions

[0127] Chromatographic column: ACQUITY UPLC BEH C18 (2.1x100mm, 1.7μm); mobile phase: 0.1% formic acid in water (phase A), 0.1% formic acid in acetonitrile (phase B); flow rate: 0.3mL·min -1 ; Injection volume: 3 μL; Column temperature: 35°C; Sample plate temperature: 4°C; Mobile phase elution gradient table is shown in Table 6.

[0128] Table 6 Mobile phase elution gradient

[0129]

[0130]

[0131] Mass spectrometry analysis conditions

[0132] ESI - Source, MSE acquisition mode, negative ion mode m / z 554.2620 [MH] - ; The atomizing and cone gas is nitrogen, and the cone gas flow rate is 50L·h -1 , the desolvation gas flow rate is 800 L·h -1 , desolvation temperature 450°C, ion source temperature 120°C; cone voltage 40V, capillary voltage 3V; scan time 0.3s, scan interval 0.02s, scan range m / z 100-1500Da; 200pg·mL -1 Leucine enkephalin was used as an external reference, and the flow rate was 10 μL·min -1 Make corrections.

[0133] Statistical analysis

[0134] The experimental results of the extraction method were organized using Excel worksheets and statistically analyzed using SPSS 22.0 software. Data are expressed as mean ± standard deviation. MassLynx V4.2.Ink software was used for data acquisition, processing, and analysis of the LC-MS / MS data.

[0135] Screening of extraction methods

[0136] Three ultrasonic ginsenoside extraction methods with better extraction effects were summarized and compared. The extraction yields of ginsenosides by the three extraction methods are shown in Table 7. The results showed that the content of ginsenosides extracted by method 2 was the highest, at 1.668±0.002 mg·mL -1 , method three times, method one is the lowest. Figure 7 As shown in the results, the content of total ginsenosides extracted by method 2 was significantly higher than that by method 1 and method 3 (P<0.01), so method 2 was selected as the extraction method for total ginsenosides from the solid fermentation product of Aspergillus oryzae and ginseng.

[0137] Table 7 Comparison of three methods for extracting total ginsenosides

[0138]

[0139]

[0140] Identification of Ginsenoside Conversion Products by Aspergillus oryzae-Ginseng Solid-State Fermentation

[0141] The linear regression equation of ginsenoside standard was prepared with the concentration of ginsenoside standard as x and the peak area as y. The 18 ginsenosides were prepared at a concentration of 0.010 mg·mL -1 -0.500mg·mL -1 The linear relationship is good within the range, and the linear regression equation is shown in Table 8.

[0142] Table 8 Linear regression equation of ginsenoside standards

[0143]

[0144]

[0145] The total ion current chromatogram and mass spectrum of the Aspergillus oryzae-ginseng solid fermentation AO ginsenoside extract and AOC ginsenoside extract in negative ion mode are shown. Since there are many types of ginsenoside components, with similar polarity and close retention times, it is difficult to distinguish them. Therefore, the present invention uses Waters ion mobility Q-TOF high-resolution liquid chromatography-mass spectrometry to distinguish the ginsenoside components contained in the extract. Using the negative ion current diagram and mass spectrum, 24 chromatographic peaks can be identified with a retention time of 10-50min. Using the summary analysis of the negative ion current, accurate molecular weight, secondary mass spectrometry fragmentation and ginsenoside standards, a total of 18 ginsenosides were identified.

[0146] According to the total ion current Figure 8 The changes in ginsenosides before and after solid fermentation of Aspergillus oryzae-ginseng were analyzed. It was found that compared with AOC, the contents of ginsenosides Re4, Rg1, Rb2 / Rb3, IVA, and Rh1 in AO were significantly reduced (P<0.01), among which Rg1, Rb2 / Rb3, Rh1, and IVA decreased most significantly. In addition, it was found that the contents of rare ginsenosides Rf, Re, and PPT in AO increased significantly after fermentation by Aspergillus oryzae (P<0.01). In order to clarify the transformation effect of Aspergillus oryzae on ginsenosides, mass spectrometry analysis was performed on AO and AOC, as shown in Tables 9 and 10. Figure 9 As shown in the figure, the peak time of PPT in AO is later than that of AOC. Combined with the results of total ion current, it is believed that the transformation of ginsenosides by Aspergillus oryzae is mainly manifested in triol ginsenosides, and its metabolites and transformation pathways are determined as follows: ①Re→Rg1→Rh1→PPT; ②Rg1→Rh1→PPT; ③Rf→Rh1→PPT.

[0147] Table 9 Ginsenoside information of AO extract

[0148] Serial number name Molecular formula m / z Retention time (min) Content (μg·mL-1) 1 Re4 C47H80O18 977.5774 13.83 41.307±25.043 2 Rg1 C42H72O14 845.5308 14.31 72.744±71.378 3 Rf C42H72O14 845.5308 20.64 119.671±26.169 4 F3 C41H70O13 815.5116 23.81 61.770±21.090 5 Rb1 C54H92O23 1153.6542 24.19 254.141±4.089 6 Rb2 / Rb3 C53H90O22 1123.6329 26.76 88.431±33.106 7 PPD C56H94O24 1195.6570 28.15 231.769±28.309 8 Re C48H82O18 991.5839 30.70 268.193±43.227 9 Rk2 C42H70O12 811.5128 33.51 35.177±16.341 10 Rk3 C36H60O8 665.4519 34.14 36.171±13.371 11 IVA C42H66O14 793.4733 34.73 51.005±13.219 12 Rh1 C36H62O9 636.4377 34.81 31.743±6.197

[0149] Table 9: Ginsenoside information of AO extract

[0150]

[0151]

[0152] Table 10 Ginsenoside information of AOC extract

[0153]

[0154]

[0155] The optimal extraction method was screened by calculating the extraction rate and then used for subsequent experiments. The optimal extraction method was selected to obtain the test sample. The ginsenoside components contained in AO and AOC were resolved using a Waters ion mobility Q-TOF high-resolution liquid chromatography-mass spectrometry instrument. The transformation pathways of ginsenosides were also analyzed. The specific results are as follows:

[0156] Three extraction methods were screened based on saponin content, and method 2 was found to be the best extraction method. The ginsenoside content extracted using method 2 was 1.668±0.002mg·mL -1 , which was significantly higher than the other two methods (P<0.01). The total ion current and mass spectrometry information obtained by Waters ion mobility Q-TOF high-resolution liquid chromatography-mass spectrometry were used to analyze the transformation of ginsenosides in AO and AOC. It was found that the main type of saponin transformed was triol ginsenosides, and the specific transformation pathway was: ①Re→Rg1→Rh1→PPT; ②Rg1→Rh1→PPT; ③Rf→Rh1→PPT. Among them, the contents of ginsenosides Re4, Rg1, Rb2 / Rb3, Rh1, and IVA were significantly reduced, while the contents of ginsenosides Rf, Re, and PPT were significantly increased (P<0.01). Among them, Rg1, as an allelopathic substance affecting the ginseng emergence rate, had a content reduced by about 62% compared with before fermentation; ginsenosides Rf, Re, and PPT have biological activities such as analgesia, antioxidant, anti-tumor, and protection of the nervous system. After conversion, their contents increased by 1.12 times, 2.74 times, and 4.03 times, respectively.

[0157] This study, published in the journal Nature Communications, utilizes Aspergillus oryzae for the first time to transform ginsenosides via solid-state fermentation. The enzymes secreted by Aspergillus oryzae during fermentation also break down macromolecules such as sugars and proteins in the ginseng matrix, providing energy for ginsenoside conversion, increasing the concentration of total ginsenosides and boosting conversion efficiency. It is speculated that enzymes, primarily β-glucosidase, produced during fermentation by Aspergillus oryzae effectively act on the glycosyl moieties of ginsenosides, converting common ginsenosides into rarer ginsenosides. This biotransformation process not only significantly reduces the content of allelochemicals but also increases the concentration of bioactive components in ginseng. In summary, the Aspergillus oryzae-mediated ginsenoside biotransformation method exhibits high substrate utilization and conversion efficiency, while significantly reducing byproduct formation. This study demonstrates the feasibility and efficiency of Aspergillus oryzae in ginsenoside biotransformation, providing important data and theoretical support for the study of the biotransformation mechanism of ginsenosides. Furthermore, these results lay the foundation for further investigation into the specific molecular mechanisms of ginsenoside conversion by Aspergillus oryzae, and have significant scientific significance and application value for promoting further research in related fields. The protective effect of saponin extract on ethanol-induced GES-1 cell damage and ginsenosides provide effective research for the treatment of gastric inflammatory diseases such as gastric mucosal injury.

[0158] Analysis of the transformation pathways of ginsenosides revealed that triol-type ginsenosides were primarily transformed by Aspergillus oryzae, with the specific transformation pathways being: ①Re→Rg1→Rh1→PPT; ②Rg1→Rh1→PPT; and ③Rf→Rh1→PPT. The contents of ginsenosides Re4, Rg1, Rb2 / Rb3, Rh1, and IVA were significantly decreased, while the contents of ginsenosides Rf, Re, and PPT were significantly increased (P<0.01). Rg1, an allelopathic substance that affects ginseng seedling emergence, decreased by approximately 62% compared to pre-fermentation levels. Ginsenosides Rf, Re, and PPT, which possess analgesic, antioxidant, antitumor, and neuroprotective activities, increased by 1.12-fold, 2.74-fold, and 4.03-fold, respectively, after transformation. This experiment was the first to use Aspergillus oryzae to transform ginsenosides through solid fermentation. It was found that the enzymes produced by Aspergillus oryzae during the fermentation process can successfully act on the glycosyl part of ginsenosides, converting common ginsenosides into rare ginsenosides, and the conversion effect of triol ginsenosides is more obvious. In addition, the enzymes secreted by Aspergillus oryzae during the fermentation process can also enzymatically hydrolyze macromolecules such as sugars and proteins in the ginseng matrix, providing energy for the conversion of ginsenosides, increasing the content of total ginsenosides in ginseng, and increasing conversion efficiency.

[0159] The present invention and its embodiments are described above. This description is not restrictive. What is shown in the full text is only one embodiment of the present invention, and the actual structure is not limited thereto. In short, if a person skilled in the art is inspired by this and, without departing from the purpose of the present invention, designs a structure and embodiment similar to this technical solution without creatively designing, they shall all fall within the scope of protection of the present invention.

Claims

1. A solid fermentation method of Aspergillus oryzae and ginseng, characterized in that: The following steps are involved: S1. Activation of Aspergillus oryzae: Clean the clean bench, place the top of the freeze-dried tube on the outer flame of an alcohol lamp and burn it evenly. Continue heating for 3-5 minutes. Use a sterile dropper to draw 2-3 drops of sterile water on the heated part to break the freeze-dried tube wall. Use tweezers to tap the broken part, open the freeze-dried tube, use a sterile dropper to draw a small amount of sterilized PDA liquid culture medium, inject it into the freeze-dried tube, and fully dissolve the freeze-dried powder. Use a sterile syringe to draw the dissolved bacterial suspension and place it in two 4-5 mL liquid culture media respectively. Mix well and place in a shaking incubator at 28°C, 140 rpm. -1 Culture in the dark under the same conditions, and then subculture once using the same method to ensure that the activity of the strain is fully restored; S2. Preparation of Aspergillus oryzae spore suspension: Take the activated bacterial suspension in a clean bench, mix it well, and then dip the inoculating loop into the bacterial suspension and spread it on the PDA slant solid culture medium. Dip the inoculating loop into the bacterial suspension and turn it upside down for 5-10 minutes before incubating it in a dark incubator. Only when the slant is covered with white hyphae can the spore suspension be prepared. In a clean bench, inject 3-5 mL of sterile water into each slant solid culture medium covered with white mycelium. Use an inoculation loop to scrape the white mycelium, filter the mycelium with gauze to obtain the Aspergillus oryzae spore suspension, and then use a sterile syringe to draw a small amount of spore suspension into the center of the hemocytometer. Cover it with a slide and place it under an optical microscope to calculate the concentration of the spore suspension. Use sterile water to adjust the concentration to 5.7 × 10 6 CFU·mL -1 ; S3. Preparation of Aspergillus oryzae-ginseng solid fermentation matrix: Grind white ginseng to obtain uniform ginseng powder. Weigh the ginseng powder into a fermentation tank, add 40% pure water by volume to the ginseng powder, stir evenly and sterilize. After sterilization, transfer the ginseng matrix to a clean bench and let it cool naturally to room temperature. Gently shake the matrix to loosen it and turn on the ultraviolet light for 30 minutes for sterilization. S4. Fermentation: The fermentation time is 7.5-8.5 days, the fermentation temperature is 26-29 DEG C, the inoculation amount is 2.4-2.6%, and a fermentation product is obtained. Polysaccharide and ginsenoside are extracted from the fermentation product.

2. A Aspergillus oryzae-ginseng solid fermentation method according to claim 1, characterized in that: In step S1, the freeze-dried tube was wiped with a 75% alcohol cotton ball before use and used after the alcohol evaporated naturally.

3. A Aspergillus oryzae-ginseng solid fermentation method according to claim 1, characterized in that: The dark-protected culture time in step S1 is 36 hours.

4. A Aspergillus oryzae-ginseng solid fermentation method according to claim 1, characterized in that: After mixing in step S2, use an inoculating loop to dip the bacterial solution and spread it in a "Z" shape on the PDA slant solid culture medium.

5. The Aspergillus oryzae-ginseng solid fermentation method according to claim 1, wherein: The method for sterilizing by stirring evenly in step S3 is high temperature and high pressure sterilization at 121° C. for 30 minutes.

6. The Aspergillus oryzae-ginseng solid fermentation method according to claim 1, characterized in that: The white ginseng in step S3 is crushed and passed through a 15-20 mesh sieve.

7. Use of ginsenosides obtained by the Aspergillus oryzae-ginseng solid fermentation method according to any one of claims 1 to 6 in the preparation of medicines, foods or health products for preventing and treating gastric mucosal damage.

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