Gastrodia elata fermentation liquor as well as preparation method and application thereof

Abstract

The invention relates to gastrodia elata fermentation liquor as well as a preparation method and application thereof, and belongs to the technical field of bioengineering. The invention provides a preparation method of gastrodia elata fermentation liquor, which comprises the following steps: (1) mixing gastrodia elata, mixed enzyme and water, and performing enzymolysis to obtain gastrodia elata enzymatic hydrolysate; and (2) inoculating zymophyte into the gastrodia elata enzymatic hydrolysate obtained in the step (1), fermenting to obtain fermentation liquor, centrifuging the fermentation liquor, taking supernate, and filtering the supernate to obtain gastrodia elata fermentation liquor. The gastrodia elata fermentation liquor obtained according to the scheme disclosed by the invention contains high concentration of p-hydroxybenzyl alcohol and parishin, so that the capacity of removing DPPH free radicals and OH-free radicals is improved, and the total antioxidant capacity of ABTS is also improved.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a gastrodia elata fermentation liquid and a preparation method and application thereof. Background Art

[0002] Plant lactic acid bacteria fermentation is an environmentally friendly and sustainable technology used in food processing and healthcare. It not only diversifies product flavors but also provides health benefits. Therefore, research in this area is crucial. During the fermentation process, microorganisms use polysaccharides, proteins, fibers and other substances as nutrient sources. This process enhances the release of active ingredients and converts large molecules into small molecules, thereby increasing their biological activity. In addition, the growth of microorganisms during fermentation leads to the production of various metabolites, such as amino acids and organic acids, which further enhance the flavor.

[0003] Traditional Chinese medicine fermentation is the use of microbial fermentation to produce traditional Chinese medicine. In traditional Chinese medicine production, natural microbial fermentation is used to improve the efficacy of traditional Chinese medicine, eliminate the toxic side effects of traditional Chinese medicine, and change the medicinal properties of traditional Chinese medicine. For example, traditional Chinese medicines such as Liushenqu, Dandouchi, and Banxiaqu are all fermented by natural microorganisms. However, in the prior art, there is little research on the use of fermentation to process traditional Chinese medicine.

[0004] Para-hydroxybenzyl alcohol and parison glycosides are the main active ingredients of Gastrodia elata. They have antioxidant effects and can be used for Alzheimer's disease and cardiovascular diseases. The existing technology does not provide a method for increasing the content of these active ingredients.

[0005] The present invention is proposed based on this. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing a fermented gastrodia elata liquid, so as to increase the concentration of active substances in the fermented gastrodia elata liquid and improve its antioxidant effect.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing a fermented gastrodia elata liquid, comprising the following steps:

[0009] (1) mixing Gastrodia elata, mixed enzyme and water, and performing enzymolysis for 1.5 to 2.5 hours to obtain Gastrodia elata enzymolysis solution;

[0010] (2) inoculating fermentation bacteria into the Gastrodia elata enzymatic hydrolysate obtained in step (1), fermenting for 18 to 26 hours to obtain a fermentation liquid, centrifuging the fermentation liquid to obtain a supernatant, and filtering the supernatant to obtain a Gastrodia elata fermentation liquid;

[0011] The mixed enzyme is cellulase and pectinase, with a mass ratio of 1-2:1-2;

[0012] The fermentation bacteria are Lactobacillus plantarum LZU-J-TSL6 and Lactobacillus plantarum LZU-LJY-1Q1, with a volume ratio of 1-2:1-2;

[0013] The Latin name of the Lactobacillus plantarum LZU-J-TSL6 is Lactobacillus plantarum, which is deposited in Guangdong Microbiological Culture Collection Center on October 23, 2020, at the 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 61242;

[0014] The Latin name of the plant lactobacillus LZU-LJY-1Q1 is Lactiplantibacillus plantarum, which is preserved in the Guangdong Microbial Culture Collection Center on July 1, 2024. The preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the preservation number is GDMCC No: 64797.

[0015] Preferably, the mass volume ratio of Gastrodia elata to water in step (1) is 1 g: 20-30 mL.

[0016] Preferably, the mass volume ratio of the mixed enzyme to water in step (1) is 4-7 g:1000 mL.

[0017] Preferably, the temperature of the enzymatic hydrolysis in step (1) is 65-75°C.

[0018] Preferably, the inoculation volume of the fermentation bacteria in step (2) is 2% to 4% of the volume of the Gastrodia elata enzymatic hydrolysate.

[0019] Preferably, the fermentation temperature in step (2) is 35-37°C.

[0020] Preferably, the centrifugal speed in step (2) is 5800-6200 rpm, and the centrifugal time is 3-7 min.

[0021] Preferably, the filtration in step (2) is filtration through a microporous membrane, and the pore size of the microporous membrane is 0.20 to 0.24 μm.

[0022] The invention also provides the Gastrodia elata fermentation liquid prepared by the preparation method.

[0023] The present invention also provides the use of the Gastrodia elata fermentation liquid in the preparation of antioxidant medicines, cosmetics, medical devices or foods.

[0024] The present invention has the following technical effects and advantages:

[0025] The present invention provides a method for preparing a fermented gastrodia elata liquid, comprising the following steps: (1) mixing gastrodia elata, a mixed enzyme and water, and performing enzymolysis to obtain a gastrodia elata liquid; (2) inoculating fermentation bacteria into the gastrodia elata liquid obtained in step (1), fermenting to obtain a fermented liquid, centrifuging the fermented liquid to obtain a supernatant, and filtering the supernatant to obtain a gastrodia elata liquid. The gastrodia elata liquid prepared according to the method of the present invention has a high concentration of p-hydroxybenzyl alcohol and balisong glycosides, and improves the scavenging of DPPH free radicals and OH free radicals. - The ability of free radicals also enhances the overall antioxidant capacity of ABTS. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Graphs of standard solutions of gastrodin and p-hydroxybenzyl alcohol, wherein Figure A is a graph of standard solution of gastrodin, and Figure B is a graph of standard solution of p-hydroxybenzyl alcohol;

[0027] Figure 2 is the total content of gastrodin and p-hydroxybenzyl alcohol in the Gastrodia elata hydrolysate at different enzymatic hydrolysis times;

[0028] Figure 3 is the total content of gastrodin and p-hydroxybenzyl alcohol in Gastrodia elata enzymatic hydrolysate with different material-liquid ratios;

[0029] Figure 4 is the total content of gastrodin and p-hydroxybenzyl alcohol in Gastrodia elata hydrolysate at different enzymatic hydrolysis temperatures;

[0030] Figure 5 is the total content of gastrodin and p-hydroxybenzyl alcohol in the Gastrodia elata hydrolysate with different enzyme addition amounts;

[0031] Figure 6 The pH and total acid content of the fermentation liquid of Gastrodia elata with different fermentation strains;

[0032] Figure 7 is the total content of total phenolic acid, gastrodin and p-hydroxybenzyl alcohol in the fermentation liquid of Gastrodia elata with different fermentation strains;

[0033] Figure 8 The total polysaccharide content and viable bacterial count in the fermentation liquid of Gastrodia elata of different fermentation strains;

[0034] Fig. 9 is the number of viable bacteria in the fermented liquid of Gastrodia elata with different inoculation amounts;

[0035] Fig.10 is the number of viable bacteria in the fermentation liquid of Gastrodia elata at different fermentation times;

[0036] Fig.11 is the number of viable bacteria in the fermentation liquid of Gastrodia elata at different fermentation temperatures;

[0037] Fig.12 is the pH value in groups F, G, and L at different fermentation times;

[0038] Fig.13 is the total acid content in groups F, G, and L at different fermentation times;

[0039] Fig.14 is the number of viable bacteria in groups F, G, and L at different fermentation times;

[0040] Fig.15 is the total polysaccharide content in groups F, G, and L at different fermentation times;

[0041] Fig.16 is the total phenol content in groups F, G, and L at different fermentation times;

[0042] Fig.17 It is a high performance liquid chromatography analysis chart of a mixed standard solution of gastrodin, barisonoside B, barisonoside C, barisonoside E and p-hydroxybenzyl alcohol;

[0043] Fig.18 This is a high performance liquid chromatography analysis diagram of Gastrodia elata fermentation liquid;

[0044] Fig.19 The content of gastrodin in groups F and G at different fermentation times;

[0045] Fig. 20 is the content of p-hydroxybenzyl alcohol in groups F and G at different fermentation times;

[0046] Fig.21 is the content of balisenoside E in groups F and G at different fermentation times;

[0047] Fig. 22 is the content of balisenoside A in groups F and G at different fermentation times;

[0048] Fig.23 is the content of balisenoside B in groups F and G at different fermentation times;

[0049] Fig.24 is the content of balisenoside C in groups F and G at different fermentation times;

[0050] Fig.25 is the ABTS free radical inhibition rate of groups F, G, and L at different fermentation times;

[0051] Fig.26 is the DPPH free radical inhibition rate of groups F, G, and L at different fermentation times;

[0052] Fig. 27 OH of F, G, and L groups at different fermentation times - Free radical inhibition rate.

[0053] Collection Instructions

[0054] The Latin name of Lactobacillus plantarum LZU-J-TSL6 is Lactobacillus plantarum, which is deposited in Guangdong Microbiological Culture Collection Center on October 23, 2020. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 61242;

[0055] The Latin name of Lactobacillus plantarum LZU-LJY-1Q1 is Lactiplantibacillus plantarum. It is deposited in Guangdong Microbiological Culture Collection Center. The preservation date is July 1, 2024. The preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City. The preservation number is GDMCC No: 64797. DETAILED DESCRIPTION

[0056] The present invention provides a method for preparing gastrodia elata fermented liquid, comprising the following steps:

[0057] (1) mixing Gastrodia elata, mixed enzyme and water, and performing enzymolysis for 1.5 to 2.5 hours to obtain Gastrodia elata enzymolysis solution;

[0058] (2) inoculating fermentation bacteria into the Gastrodia elata enzymatic hydrolysate obtained in step (1), fermenting for 18 to 26 hours to obtain a fermentation liquid, centrifuging the fermentation liquid to obtain a supernatant, and filtering the supernatant to obtain a Gastrodia elata fermentation liquid;

[0059] The mixed enzyme is cellulase and pectinase, with a mass ratio of 1-2:1-2, preferably 1:1;

[0060] The fermentation bacteria are Lactobacillus plantarum LZU-J-TSL6 and Lactobacillus plantarum LZU-LJY-1Q1, with a volume ratio of 1-2:1-2, preferably 1:1;

[0061] The Latin name of the Lactobacillus plantarum LZU-J-TSL6 is Lactobacillus plantarum, which is deposited in Guangdong Microbiological Culture Collection Center on October 23, 2020, at the 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 61242;

[0062] The Latin name of the plant lactobacillus LZU-LJY-1Q1 is Lactiplantibacillus plantarum, which is preserved in the Guangdong Microbial Culture Collection Center on July 1, 2024. The preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the preservation number is GDMCC No: 64797.

[0063] In the present invention, the particle size of the Gastrodia elata in step (1) is preferably 60 mesh.

[0064] In the present invention, the step (1) further includes a sterilization step after the enzymatic hydrolysis, specifically sterilization at 115° C. for 30 minutes.

[0065] In the present invention, the mass volume ratio of Gastrodia elata to water in step (1) is 1 g:20-30 mL, preferably 1 g:30 mL.

[0066] In the present invention, the mass volume ratio of the mixed enzyme to water in step (1) is 4-7 g:1000 mL, preferably 5 g:1000 mL.

[0067] In the present invention, the temperature of the enzymatic hydrolysis in step (1) is 65-75°C, preferably 70°C; the time of the enzymatic hydrolysis is preferably 2.0 h.

[0068] In the present invention, the inoculation volume of the fermentation bacteria in step (2) is 2% to 4% of the volume of the Gastrodia elata enzymatic hydrolysate, preferably 3%.

[0069] In the present invention, the fermentation temperature in step (2) is 35-37° C., preferably 37° C.; the fermentation time is preferably 18 hours.

[0070] In the present invention, the centrifugal speed in step (2) is 5800-6200 rpm, preferably 6000 rpm, and the centrifugal time is 3-7 min, preferably 5 min.

[0071] In the present invention, the filtration in step (2) is filtration through a microporous membrane, and the pore size of the microporous membrane is 0.20 to 0.24 μm, preferably 0.22 μm.

[0072] The invention also provides the Gastrodia elata fermentation liquid prepared by the preparation method.

[0073] The present invention also provides the use of the Gastrodia elata fermentation liquid in the preparation of antioxidant medicines, cosmetics, medical devices or foods.

[0074] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0075] Example 1: Parameter optimization of the preparation method of Gastrodia elata fermentation liquid

[0076] Optimization of Enzymatic Hydrolysis for Extraction of Active Components from Gastrodia elata Using Response Surface Method

[0077] Taking the total content of gastrodin and p-hydroxybenzyl alcohol, the active ingredients in Gastrodia elata, as indicators, high performance liquid chromatography was used to establish standard curves with gastrodin and p-hydroxybenzyl alcohol respectively, and the total content of gastrodin and p-hydroxybenzyl alcohol in Gastrodia elata was determined.

[0078] 1. Preparation of Gastrodia elata sample solution

[0079] The impurities of the Gastrodia elata sample (purchased from Boyu Tibetan Medicinal Materials Processing Co., Ltd., Zhouqu County, Gansu Province) were removed, and then crushed with a grinder, passed through a 60-mesh sieve to obtain a uniform sample powder, and stored in a desiccator for later use. Weigh 5g of Gastrodia elata powder, put it into a wall-breaking food processor, add 150ml of water to make a homogenate, and then add 0.75g of mixed enzyme (cellulase and pectinase mass ratio is 1:1), and enzymolysis at a certain temperature. Then heat the solution to 115℃ for sterilization for 30min, cool it, centrifuge it at 6000rpm for 5min, and take the supernatant and filter it through a 0.22μm microporous filter membrane to obtain it.

[0080] 2. Preparation of gastrodin and p-hydroxybenzyl alcohol standard solutions

[0081] Accurately weigh 10.14 mg of gastrodin and place it in a 50 mL volumetric flask, and accurately weigh 10.29 mg of p-hydroxybenzyl alcohol and place it in a 100 mL volumetric flask. Add acetonitrile-water (3:97) solution to make up to volume to prepare gastrodin and p-hydroxybenzyl alcohol standard solutions, 1 ml containing 101.40 μg of gastrodin and 51.54 μg of p-hydroxybenzyl alcohol. Filter through a 0.22 μm microporous filter membrane to obtain the product.

[0082] 3. Chromatographic conditions

[0083] The chromatographic column was a SVEAC18 chromatographic column (250 mm×4.6 mm, 5 μm); the mobile phase was acetonitrile-0.05% phosphoric acid aqueous solution (3:97); the volume flow rate was 0.8 mL / min; the detection wavelength was 220 nm; and the column temperature was 30° C.

[0084] 4. Methodological investigation

[0085] 4.1 Linear Relationship

[0086] The prepared gastrodin standard solution and p-hydroxybenzyl alcohol standard solution were diluted into solutions of different concentrations, injected into a high performance liquid chromatograph, and the peak area was measured. The chromatographic peak area (y) was linearly regressed against the concentration (x) to obtain a regression equation. The concentration of gastrodin and p-hydroxybenzyl alcohol solution was used as the abscissa (x), and the chromatographic peak area was used as the ordinate (Y). The regression equation of gastrodin was obtained as Y=5785246.40X-4861.75, R 2 =0.9999, the concentration showed a good linear relationship with the peak area in the range of 0.020-0.307 mg / mL, such as Figure 1A shows; the regression equation of p-hydroxybenzyl alcohol is Y=10889966.65X-4411.39, R 2 =0.9999, the concentration showed a good linear relationship with the peak area in the range of 0.010-0.051 mg / mL, such as Figure 1 B. Within this range, the concentration and the peak area showed a good linear relationship, so the gastrodin standard solution and p-hydroxybenzyl alcohol standard solution could be used for further experimental analysis.

[0087] 4.2 Precision test

[0088] Accurately draw the Gastrodia sample solution, inject according to the chromatographic conditions in "3. Chromatographic conditions", inject 5 μL each time, repeat the injection 6 times, and calculate the RSD of the retention time (tR) and peak area (S) of each target compound. The RSD of the peak area of ​​gastrodin is 0.76% (n=6), and the RSD of the retention time is 0.06% (n=6); the RSD of the peak area of ​​p-hydroxybenzyl alcohol solution is 0.13% (n=6), and the RSD of the retention time is 0.04% (n=6), indicating that the instrument has good precision.

[0089] 4.3 Stability test

[0090] Accurately draw the Gastrodia sample solution, and inject according to the chromatographic conditions in "3. Chromatographic conditions", inject 5 μL each time, and measure at 0, 3, 6, 9, 12, 18h, 24h, respectively, and calculate the RSD of the retention time (tR) and peak area (S) of each target compound. The RSD of the peak area of ​​gastrodin is 1.53% (n=6), and the RSD of the retention time is 0.12% (n=6); the RSD of the peak area of ​​p-hydroxybenzyl alcohol solution is 0.08% (n=6), and the RSD of the retention time is 0.47% (n=6), indicating that the instrument has good precision.

[0091] 4.4 Repeatability test

[0092] Six samples of the same batch of Gastrodia elata were accurately weighed to prepare the test solution, and 5 μL of each sample was injected to calculate the RSD of the retention time (tR) and peak area (S) of each target compound. The RSD of the peak area of ​​GAS was 0.09% (n=6), and the RSD of the retention time was 2.66% (n=6); the RSD of the peak area of ​​HBA was 0.37% (n=6), and the RSD of the retention time was 0.02% (n=6), indicating that the method had good repeatability.

[0093] 5. Single Factor Experiment

[0094] 5.1 Optimization of enzymatic hydrolysis time

[0095] Gastrodia elata was crushed, 5 g was weighed, 50 mL of water was added, 0.2 g of complex enzyme (cellulase: pectinase mass ratio was 1:1, cellulase and pectinase were purchased from Nanning Pangbo Bioengineering Co., Ltd.), and enzymolysis was performed in a 65°C water bath for 0.5 h, 1 h, 1.5 h, 2 h, and 2.5 h, respectively. The total content of gastrodin and p-hydroxybenzyl alcohol was measured, and 3 replicates were set for each gradient. The results are shown in the figure. Figure 2 As shown in the figure, the horizontal axis represents the enzymatic hydrolysis time (h), the vertical axis represents the total content of gastrodin and p-hydroxybenzyl alcohol in the enzymatic hydrolysate (mg / g), and different letters indicate significant differences (P<0.05).

[0096] according to Figure 2 It can be seen that when the enzymatic hydrolysis time is 2h, the total content of gastrodin and p-hydroxybenzyl alcohol in Gastrodia elata is high, and there is no significant difference in the total content after enzymatic hydrolysis for 2.5h.

[0097] 5.2 Optimization of material-liquid ratio

[0098] Gastrodia elata was crushed, 5 g was weighed, and 50 mL, 75 mL, 100 mL, 125 mL, 150 mL, and 175 mL of water were added respectively, and 0.2 g, 0.3 g, 0.4 g, 0.5 g, 0.6 g, and 0.7 g of complex enzyme (the mass ratio of cellulase: pectinase was 1:1) was added accordingly. Stirred in a 65°C water bath, enzymolysis was performed for 2 h, and the total content of gastrodin and p-hydroxybenzyl alcohol was measured. Three replicates were set for each gradient. The results are shown in the following table. Figure 3 As shown in the figure, the horizontal axis represents the liquid ratio of water to Gastrodia elata (mL:g), that is, the volume mass ratio of water to Gastrodia elata, and the vertical axis represents the total content of gastrodin and p-hydroxybenzyl alcohol in the enzymatic hydrolysate (mg / g). Different letters indicate significant differences (P<0.05).

[0099] according to Figure 3 It can be seen that when the volume mass ratio of water to Gastrodia elata is 30:1 (mL / g), the total content of gastrodin and p-hydroxybenzyl alcohol in Gastrodia elata is the highest.

[0100] 5.3 Optimization of enzymatic hydrolysis temperature

[0101] Gastrodia elata was crushed, 5 g was weighed, 150 mL of water was added, 0.6 g of complex enzyme (cellulase: pectinase mass ratio was 1:1) was added, and the mixture was heated and stirred at 50°C, 55°C, 60°C, 65°C, 70°C, and 75°C in a water bath, respectively, and enzymolysis was performed for 2 h. The total contents of gastrodin and p-hydroxybenzyl alcohol in the enzymolysis solutions obtained at different enzymolysis temperatures were measured, and 3 replicates were set for each gradient. The results are shown in the figure. Figure 4 As shown in the figure, the horizontal axis represents the enzymatic hydrolysis temperature (°C), the vertical axis represents the total content of gastrodin and p-hydroxybenzyl alcohol in the enzymatic hydrolysate (mg / g), and different letters indicate significant differences (P<0.05).

[0102] according to Figure 4 It can be seen that when the enzymatic hydrolysis temperature is 70℃, the total content of gastrodin and p-hydroxybenzyl alcohol in Gastrodia elata is the highest.

[0103] 5.4 Optimization of enzyme dosage

[0104] Gastrodia elata was crushed, 5 g was weighed, 150 mL of water was added, and 0.3 g, 0.45 g, 0.6 g, 0.75 g, 0.9 g, and 1.05 g of compound enzyme (cellulase: pectinase mass ratio was 1:1) were added respectively. The mixture was heated and stirred in a 70°C water bath and enzymolyzed for 2 h. The total contents of gastrodin and p-hydroxybenzyl alcohol in the enzymolyzed solutions obtained with different enzyme addition amounts were measured. Three replicates were set for each gradient. The results are shown in the figure. Figure 5 As shown in the figure, the horizontal axis represents the amount of enzyme added (%), that is, the mass volume percentage of the complex enzyme and water, and the vertical axis represents the total content of gastrodin and p-hydroxybenzyl alcohol in the enzymatic hydrolyzate (mg / g), and different letters indicate significant differences (P<0.05).

[0105] according to Figure 5 It can be seen that when the mass volume percentage of the complex enzyme and water is 0.5%, that is, when the added amount of the complex enzyme is 0.75g, the total content of gastrodin and p-hydroxybenzyl alcohol in Gastrodia elata is the highest.

[0106] 6. Box-Behnken response surface experiment

[0107] On the basis of the single factor experiment, a Box-Behnken design was adopted, with enzymatic hydrolysis time (A), water to Gastrodia elata volume mass ratio (B), enzymatic hydrolysis temperature (C) and mass volume percentage of complex enzyme to water (D) as variables, and total content of gastrodin and p-hydroxybenzyl alcohol (Y) as response values ​​to conduct a four-factor three-level experiment. The design of experimental factors and levels is shown in Table 1.

[0108] Table 1 Response surface design factors and levels

[0109]

[0110] The experiment was conducted according to the response surface design and the results are shown in Table 2.

[0111] Table 2 Response surface test results

[0112]

[0113]

[0114] Taking the total content of gastrodin and p-hydroxybenzyl alcohol in Gastrodia elata as the response value, the calculation results in Table 2 were subjected to multivariate linear regression analysis, and the quadratic equation was fitted. The regression mathematical model of the total content of gastrodin and p-hydroxybenzyl alcohol (Y) on the enzymatic hydrolysis time (A), the volume mass ratio of water to Gastrodia elata (B), the enzymatic hydrolysis temperature (C) and the mass volume percentage of the complex enzyme to water (D) was obtained as follows: Y=8.9340+0.3858A-0.0800B+0.1267C+0 .0342D+0.0350AB-0.0100AC+0.0275AD-0.02BC-0.22BD-0.02CD-0.0845A2-0.1382B2-0.2258C2-0.187D2, the regression model was subjected to variance analysis, and the results are shown in Table 3. In the table, *** indicates that the difference is highly significant, P < 0.0001, ** indicates that the difference is extremely significant, P < 0.01; * indicates that the difference is significant, P < 0.05. Table 3 shows that the P value of the model is < 0.0001, and the quadratic model used in the test is highly significant and statistically significant. The P value of the lack of fit term is 0.171> 0.05, and the difference is not significant. Therefore, the regression equation can be used to replace the actual test point to analyze the test results. In the first-order terms, the linear effect of A enzymatic hydrolysis time on gastrodin and p-hydroxybenzyl alcohol was highly significant (P < 0.0001), B the volume mass ratio of water to gastrodia elata had a significant effect on the content of gastrodin and p-hydroxybenzyl alcohol (P < 0.05), C the linear effect of enzymatic hydrolysis temperature on the content of gastrodin and p-hydroxybenzyl alcohol was extremely significant (P < 0.01), and D the mass volume percentage of complex enzyme and water had no significant linear effect on the content of gastrodin and p-hydroxybenzyl alcohol (P > 0.05). In the second-order terms, C 2 The effect was highly significant (P<0.001), B 2 , D 2 The effect was extremely significant (P < 0.01), and the effect of BD in the interaction term was extremely significant (P < 0.01). The correlation coefficient of this model correction model R 2 The value is 0.9361, and the coefficient of determination R 2 The Adj value is 0.8723, indicating that the selected model fits the actual situation well with small errors, and can better reflect the relationship between various factors and the content of gastrodin and p-hydroxybenzyl alcohol in Gastrodia elata. From the F value, it can be seen that within the test range, the influence of various factors on the content is A (enzyme hydrolysis time) > C (enzyme hydrolysis temperature) > B (water to gastrodia elata volume mass ratio) > D (mass volume percentage of complex enzyme to water).

[0115] Table 3 Analysis of variance of regression model

[0116]

[0117]

[0118] The regression equation was solved by using Design-Expert13 statistical software, and the optimal process conditions for enzymatic hydrolysis of Gastrodia elata were obtained: the solid-liquid ratio was 1:28.89 (g / mL), the enzymatic hydrolysis time was 2.19h, the enzymatic hydrolysis temperature was 66.79℃, the mass volume percentage of the complex enzyme and water was 0.47%, and the predicted content was 8.85mg / g. Considering the convenience in actual operation, the various factors were corrected to a solid-liquid ratio of 1:30 (g / mL), an enzymatic hydrolysis time of 2h, an enzymatic hydrolysis temperature of 68℃, and a mass volume percentage of the complex enzyme and water of 0.5%, that is, 5g:1000mL.

[0119] Under this condition, three parallel extractions of gastrodin and p-hydroxybenzyl alcohol were performed, and the average content was 8.88 mg / g, which was basically consistent with the predicted value. This shows that this model can well simulate the actual situation of gastrodin and p-hydroxybenzyl alcohol extraction, and the model is effective.

[0120] Preparation of strains for fermentation of Gastrodia elata enzymatic hydrolysate

[0121] 1. Fermentation strains

[0122] Lactobacillus plantarum LZU-J-TSL6 and Lactobacillus plantarum LZU-LJY-1Q1 are both laboratory-independent isolated strains. They have been identified as Lactobacillus plantarum and have been experimentally verified to have good probiotic properties. The Latin name of Lactobacillus plantarum LZU-J-TSL6 is Lactobacillus plantarum, which is deposited in the Guangdong Microbiological Culture Collection Center on October 23, 2020. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 61242. The Latin name of Lactobacillus plantarum LZU-LJY-1Q1 is Lactiplantibacillus plantarum, which is deposited in the Guangdong Microbiological Culture Collection Center on July 1, 2024. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 64797.

[0123] 2. Preparation of Lactobacillus plantarum

[0124] The preparation method of Lactobacillus plantarum LZU-J-TSL6 bacterial liquid is as follows: the preserved Lactobacillus plantarum LZU-J-TSL6 is inoculated and streaked on a solid culture medium, and activated at 37°C for 24 hours, and the activated single colony is inoculated into 8 mL of MRS liquid culture medium containing 52.24 g broth / L, and cultured at 37°C for 18 hours to obtain a first-generation bacterial liquid; 200 μL of the first-generation bacterial liquid is inoculated into 9.8 mL of MRS liquid culture medium containing 52.24 g broth / L, and cultured at 37°C for 18 hours to obtain a second-generation bacterial liquid. The second-generation bacterial liquid is diluted to obtain 1×108 CFU / mL, which is the bacterial liquid required for fermentation.

[0125] The preparation method of Lactobacillus plantarum LZU-LJY-1Q1 bacterial liquid is the same as above.

[0126] 3. Determine the composite ratio of fermentation strains

[0127] Lactobacillus plantarum LZU-J-TSL6 bacterial liquid (abbreviated as: TSL-6), Lactobacillus plantarum LZU-LJY-1Q1 bacterial liquid (abbreviated as: LJY-1Q1) and composite bacterial liquid with a volume ratio of LJY-1Q1:TSL-6 of 1:1, 1:3 and 3:1 were inoculated as fermentation bacteria into Gastrodia elata enzymatic hydrolysate, the volume of the inoculated bacterial liquid was 3% of the volume of the Gastrodia elata enzymatic hydrolysate, and the culture was carried out for 18 hours to obtain Gastrodia elata fermentation liquid.

[0128] 3.1 Comparison of pH and total acid content of Gastrodia elata fermentation broth from different fermentation strains

[0129] The pH and total acid content in the fermented broth of Gastrodia elata inoculated with different strains were measured using an acidometer and according to the national standard for the determination of total acid in food (GB12456-2021). The results are as follows Figure 6 As shown in the figure, the horizontal axis represents the Gastrodia elata fermentation liquid of different fermentation bacteria, the vertical axis on the left represents the total acid content (mg / mL), and the vertical axis on the right represents the pH value. The red broken line in the figure represents the total acid content, the black broken line represents the pH value, and different letters represent significant differences (P<0.05).

[0130] according to Figure 6 It can be seen that there are no significant differences in pH and total acid content in the Gastrodia elata fermentation broth of TSL-6, LJY-1Q1 and composite strains in different volume ratios.

[0131] 3.2 Comparison of the total contents of total phenolic acids, gastrodin and p-hydroxybenzyl alcohol in the fermentation broth of Gastrodia elata from different fermentation strains

[0132] The total contents of total phenolic acids, gastrodin, p-hydroxybenzyl alcohol and total polysaccharides in the fermented broth of Gastrodia elata inoculated with different strains were determined. Figure 7As shown in the figure, the horizontal axis represents the Gastrodia elata fermentation broth of different fermentation bacteria, the left vertical axis represents the total content of gastrodin and p-hydroxybenzyl alcohol (mg / mL), the right vertical axis represents the total phenolic acid content (mg / mL), the red broken line in the figure represents the total content of gastrodin and p-hydroxybenzyl alcohol, the black broken line represents the total phenolic acid content, and different letters indicate significant differences (P<0.05).

[0133] according to Figure 7 It can be seen that the total content of gastrodin and p-hydroxybenzyl alcohol and the total phenolic acid content in the Gastrodia elata fermentation broth of the composite strain LJY-1Q1:TSL-6 with a volume ratio of 1:1 were significantly higher than those of other strains.

[0134] 3.3 Comparison of total polysaccharide content and viable bacterial count in Gastrodia elata fermentation broth from different fermentation strains

[0135] The total polysaccharide content and viable bacterial count in the fermented broth of Gastrodia elata inoculated with different strains were determined. Figure 8 As shown in the figure, the horizontal axis represents the Gastrodia elata fermentation liquid of different fermentation bacteria, the vertical axis on the left represents the number of viable bacteria (lgCFU / mL), and the vertical axis on the right represents the total polysaccharide content (mg / mL). The red broken line in the figure represents the number of viable bacteria, and the black broken line represents the total polysaccharide content. Different letters indicate significant differences (P<0.05).

[0136] according to Figure 8 It can be seen that there is no significant difference in the number of live bacteria in the Gastrodia elata fermentation broth of the composite strain LJY-1Q1:TSL-6 with a volume ratio of 1:1 and TSL-6, and both are higher than other strains; there is a significant difference in the total polysaccharide content in the Gastrodia elata fermentation broth of the composite strain LJY-1Q1:TSL-6 with a volume ratio of 1:1 and 1:3, and both are significantly higher than other strains.

[0137] By comparing the physicochemical indices in the fermentation broth of different strains of Gastrodia elata, the composite strain with a volume ratio of LJY-1Q1:TSL-6 of 1:1 was determined to be the fermentation strain.

[0138] Optimization of the process of fermentation of Gastrodia elata by Lactobacillus plantarum

[0139] The screening results showed that the composite strain of L. plantarum LJY-1Q1:TSL-6 with a volume ratio of 1:1 was used as the fermentation bacteria to optimize the fermentation conditions.

[0140] 1. Single Factor Exploration

[0141] Taking the number of live bacteria in the fermented liquid of Gastrodia elata as an indicator, the optimal fermentation temperature, fermentation time and bacterial inoculum amount were explored.

[0142] 1.1 Optimization of inoculation amount

[0143] A certain amount of Gastrodia elata hydrolysate was added to a sterile conical flask, and fermentation bacteria were inoculated at 1%, 2%, 3%, 4%, and 5% of the volume of Gastrodia elata hydrolysate, respectively. The fermentation was carried out at 37°C for 18 hours, and the number of live bacteria in the bacterial solution was calculated by plating on the plates. Three replicates were set for each ratio. The results were as follows: Fig. 9 As shown in the figure, the horizontal axis represents the inoculation volume of the fermentation bacteria (%), the vertical axis on the left represents the number of viable bacteria (lgCFU / mL), and different letters in the figure represent significant differences (P<0.05).

[0144] pass Fig. 9 It was found that when the volume of the fermentation bacteria was 3%, the number of live bacteria in the fermentation liquid was significantly higher than that of other inoculation amounts, so the inoculation amount was determined to be 3%.

[0145] 1.2 Optimization of fermentation time

[0146] A certain amount of Gastrodia elata hydrolysate was added to a sterile conical flask, and fermentation bacteria was inoculated at 3% of the volume of Gastrodia elata hydrolysate. Fermentation was performed at 37°C for 14 h, 18 h, 22 h, 26 h, and 30 h. The number of live bacteria in the bacterial solution was calculated by plating on the plates. Three replicates were set for each ratio. The results were as follows: Fig.10 As shown in the figure, the horizontal axis represents different fermentation times (h), the vertical axis on the left represents the number of viable bacteria (lgCFU / mL), and different letters in the figure indicate significant differences (P<0.05).

[0147] pass Fig.10 It was found that when the fermentation time was 18 hours, the number of live bacteria in the fermentation liquid was significantly higher than that at other fermentation times, so the fermentation time was determined to be 18 hours.

[0148] 1.3 Optimization of fermentation temperature

[0149] A certain amount of Gastrodia elata hydrolysate was added to a sterile conical flask, and 3% of the volume of Gastrodia elata hydrolysate was inoculated with bacterial solution. The solution was fermented at 33°C, 35°C, 37°C, 39°C, and 41°C for 18 h, and the number of live bacteria in the bacterial solution was calculated by plating on the plate. Three replicates were set for each ratio. The results were as follows: Fig.11 As shown in the figure, the horizontal axis represents different fermentation temperatures (℃), the vertical axis represents the number of viable bacteria (lgCFU / mL), and different letters in the figure represent significant differences (P<0.05).

[0150] pass Fig.11 It was found that when the fermentation temperature was 37°C, the number of live bacteria in the fermentation liquid was significantly higher than that at other temperatures, so the fermentation temperature was determined to be 37°C.

[0151] 1.4 Orthogonal test

[0152] Based on the single-factor experiment, SPSSAU was used to select the volume of inoculated fermentation bacteria, fermentation time and fermentation temperature as scalar factors, and the number of viable bacteria was used as an indicator for orthogonal experiment. Three levels were set for each factor, and the blank group was used as the control. The orthogonal experimental factors are shown in Table 4.

[0153] Table 4 Orthogonal test factor levels

[0154]

[0155] Orthogonal experiments were carried out at different factor levels, and the results are shown in Table 5.

[0156] Table 5 Analysis of orthogonal test results

[0157]

[0158]

[0159] The orthogonal test was subjected to variance analysis, and the results are shown in Table 6, in which ** indicates extremely significant difference, P < 0.01; * indicates significant difference, P < 0.05.

[0160] Table 6 Analysis of variance of orthogonal test

[0161] factor Sum of Squared Deviations Degrees of Freedom Mean Square F Significance Inoculated fermentation bacteria volume (A) 0.173 2 0.086 6.646 0.131 Fermentation time (B) 0.257 2 0.129 9.89 0.092 Fermentation temperature (C) 0.643 2 0.322 24.713 * error 0.026 2 0.013

[0162] The results showed that factors A and B had no significant effect on the number of viable bacteria in the fermentation broth, while factor C had a significant effect, and the primary and secondary relationships of the factors on the number of viable bacteria in the fermentation broth were C>A>B. Considering the production cost, the best combination of optimization levels under experimental conditions was A2B1C3, that is, the volume of inoculated fermentation bacteria was 3%, the fermentation time was 18h, and the fermentation temperature was 37℃.

[0163] Verification test: The Gastrodia elata hydrolysate was fermented according to the above optimal conditions, and the number of viable bacteria was obtained by plating on the plate, and the parallel test was repeated 3 times. The results showed that the average number of viable bacteria in the fermentation liquid was 10.38 (lgCFU / ml), which was higher than the maximum value of the orthogonal test.

[0164] The optimal conditions for fermenting Gastrodia elata hydrolysate were as follows: the volume of the inoculated fermentation bacteria was 3% of the volume of the hydrolysate, the fermentation time was 18 h, and the fermentation temperature was 37°C.

[0165] It is generally known that the preparation process of Gastrodia elata fermentation liquid is as follows: crush Gastrodia elata through a 60-mesh sieve, mix Gastrodia elata powder, a mixed enzyme with a mass ratio of 1:1 of cellulase and pectinase, and water to obtain a mixed liquid, the mass volume ratio of Gastrodia elata to water is 1g:30mL, and the mass volume ratio of the mixed enzyme to water is 5g:1000mL, and the mixed liquid is enzymolyzed at 70°C for 2h to obtain an enzymolyzate, the enzymolyzate is heated to 115°C and sterilized for 30min to obtain Gastrodia elata enzymolyzate, the fermentation bacteria are Lactobacillus plantarum LZU-J-TSL6 and Lactobacillus plantarum LZU-LJY-1Q1 in a volume ratio of 1:1, the fermentation bacteria are inoculated into the enzymolyzate, the inoculation amount is 3% of the volume of the Gastrodia elata enzymolyzate, the inoculated enzymolyzate is fermented at 37°C for 18h to obtain a fermentation liquid, the fermentation liquid is centrifuged at 6000rpm for 5min, the supernatant is taken, and the supernatant is filtered using a 0.22μm microporous filter membrane.

[0166] Example 2: Changes in in vitro activity of Gastrodia elata fermented liquid before and after fermentation

[0167] Microbial fermentation can change the original properties of substances, enhance or produce new effects. During the microbial fermentation process, polysaccharides, fibers, proteins and other components in traditional Chinese medicine can be used to obtain nutrients. This not only promotes the release of active ingredients, but also converts macromolecules into small molecules, thereby enhancing their biological activity. In addition, microorganisms will produce various secondary metabolites during their growth, including amino acids, organic acids, vitamins, phenols, and aroma components such as alcohols, aldehydes, and esters, which can also affect the antioxidant activity of the original substances.

[0168] Crush Gastrodia elata, weigh 5 g, add 150 mL of water, add 0.75 g of complex enzyme (cellulase: pectinase mass ratio is 1:1), heat and stir in a 70°C water bath, enzymolysis for 2 hours, then heat the solution to 115°C for sterilization for 30 minutes, centrifuge at 6000 rpm for 5 minutes after cooling, take the supernatant and filter it through a 0.22 μm microporous filter membrane to obtain Gastrodia elata enzymolysis solution for use.

[0169] In order to determine the changes in in vitro activity of Gastrodia elata fermented liquid before and after fermentation, Gastrodia elata was treated by three groups. F was the Gastrodia elata fermentation group, and 100 mL of Gastrodia elata hydrolysate was taken and inoculated with 3 mL of fermentation bacteria of Lactobacillus plantarum LZU-J-TSL6 and Lactobacillus plantarum LZU-LJY-1Q1 in a volume ratio of 1:1; G was the Gastrodia elata hydrolysate group, and 100 mL of Gastrodia elata hydrolysate was taken and inoculated with 3 mL of water; L was the fermentation bacteria group, Gastrodia elata was crushed, 5 g was weighed, and 150 mL of water was added to obtain a Gastrodia elata mixed liquid. 100 mL of the Gastrodia elata mixed liquid was taken and inoculated with 3 mL of fermentation bacteria of Lactobacillus plantarum LZU-J-TSL6 and Lactobacillus plantarum LZU-LJY-1Q1 in a volume ratio of 1:1. The solutions of groups F, G, and L were placed at 37°C for fermentation for 18 h, respectively.

[0170] 1. Changes in pH, titratable acid and viable count during fermentation

[0171] The number of live Lactobacillus plantarum cells increases significantly during the fermentation process. The growth of the bacteria will produce certain organic acids, such as lactic acid, which will reduce the pH of the fermentation liquid and increase the total acid content of the fermentation. These are two important indicators for evaluating the degree of fermentation. The pH values ​​of groups F, G, and L at different fermentation times were measured. The results are as follows: Fig.12 As shown in the figure, the horizontal axis represents the fermentation time (h), and the vertical axis represents the pH value; the titratable acid content is determined, and the results are as follows Fig.13 As shown in the figure, the horizontal axis represents the fermentation time (h), and the vertical axis represents the titratable acid content (g / 100mL); the number of viable bacteria was determined, and the results are as follows Fig.14 As shown in the figure, the horizontal axis represents the fermentation time (h), and the vertical axis represents the number of viable bacteria (lgCFU / mL). Figures 12-14 The blue line represents the F group, the yellow line represents the G group, and the red line represents the L group. Different letters indicate significant differences (P<0.05); *** indicates that P<0.001 is significantly different from the G group; ### indicates that P<0.001 is significantly different from the L group.

[0172] according to Figures 12-14 It can be seen that compared with group G, the number of live bacteria in groups F and L gradually increased with time, the pH gradually decreased, and the total acid content gradually increased, indicating that Lactobacillus plantarum TSL-6 and LJY-1Q1 can grow in Gastrodia elata enzymatic hydrolysate and produce metabolites.

[0173] 2. Changes of total polysaccharides and total phenols during fermentation

[0174] Polysaccharides and total phenolic acids are the main active substances in Gastrodia elata, but some macromolecular substances have low oral availability. Microbial fermentation can convert macromolecular substances into small molecules, improve bioavailability and thus improve effectiveness. The total polysaccharide content in groups F, G, and L at different fermentation times was determined. Fig.15 As shown in the figure, the horizontal axis represents the fermentation time (h), and the vertical axis represents the total polysaccharide content (mg / mL); the total phenol content was determined, and the results are as follows Fig.16 As shown in the figure, the horizontal axis represents the fermentation time (h), the vertical axis represents the total phenol content (μg / L), the blue broken line represents the F group, the yellow broken line represents the G group, and the red broken line represents the L group. Different letters indicate significant differences (P<0.05); *** indicates that P<0.001 is significantly different from the G group; ### indicates that P<0.001 is significantly different from the L group.

[0175] according to Fig.15 It was found that compared with group G, the total polysaccharide content in group F did not change significantly, but there was an upward trend; Fig.16 It was found that compared with group G, the total polyphenol content of group F increased significantly during the fermentation process, and the total polyphenols increased significantly.

[0176] 3. Changes of monomer compounds such as gastrodin and p-hydroxybenzyl alcohol during fermentation

[0177] 3.1 Chromatographic conditions

[0178] SVEAC18 chromatographic column (250mm×4.6mm, 5μm); mobile phase: acetonitrile (A)-water (containing 0.1% phosphoric acid) (B), gradient elution (0-8.5min, 3-10% A; 8.5-17min, 10-12% A; 15-25min, 12-18% A; 25-35min, 18% A; 35-42min, 18-25% A); volume flow rate 0.8mL / min; column temperature 30°C; detection wavelength 220nm, injection volume 5μL.

[0179] 3.2 Preparation of Gastrodia elata fermentation broth

[0180] The Gastrodia elata fermented liquid was prepared by referring to the preferred Gastrodia elata fermented liquid preparation process in Example 1.

[0181] 3.3 Preparation of mixed standard solution of gastrodin, barleysonoside B, barleysonoside C, barleysonoside E and p-hydroxybenzyl alcohol

[0182] Gastrodin (mass fraction 96.7%), barisonoside B (mass fraction 98%), barisonoside C (mass fraction 98%), barisonoside E (mass fraction 98%) and p-hydroxybenzyl alcohol (mass fraction 97.4%) were weighed respectively and added with 50% methanol to prepare a mixed standard solution. In the mixed standard solution, the concentration of gastrodin (GAS) was 0.01137 mg / mL, the concentration of p-hydroxybenzyl alcohol (HBA) solution was 0.03661 mg / mL, the concentration of barisonoside A (PA) solution was 0.01297 mg / mL, the concentration of barisonoside B (PB) solution was 0.01286 mg / mL, the concentration of barisonoside C (PC) solution was 0.01262 mg / mL, and the concentration of barisonoside E (PE) solution was 0.01326 mg / mL.

[0183] 3.4 Methodological Review

[0184] 3.4.1 Results of linear relationship investigation

[0185] The prepared mixed standard solution was diluted into solutions of different concentrations, injected into a high performance liquid chromatograph, and the peak area was measured. A linear regression was performed between the chromatographic peak area (y) and the concentration (x) to obtain a regression equation. The results are shown in Table 7.

[0186] Table 7 Linear regression equations for 6 standard reference substances

[0187]

[0188]

[0189] 3.4.2 Results of precision test, stability test and repeatability test

[0190] Accurately aspirate the Gastrodia elata fermentation broth, and inject it according to "3.1 Chromatographic Conditions". Repeat the injection 6 times, and calculate the RSD of the retention time (tR) and peak area (S) of each target compound. Accurately aspirate the Gastrodia elata fermentation broth, and inject it according to "3.1 Chromatographic Conditions". Determine at 0, 3, 6, 9, 12, 18h, and 24h, respectively, and calculate the RSD of the retention time (tR) and peak area (S) of each target compound. Accurately weigh 6 portions of the same batch of Gastrodia elata samples to prepare Gastrodia elata fermentation broth, inject 5μL of each portion, and calculate the RSD of the retention time (tR) and peak area (S) of each target compound. The results are shown in Table 8. The mixed standard solution and Gastrodia elata fermentation broth sample solution were subjected to HPLC analysis. The HPLC analysis of the mixed standard solution is shown in Table 8. Fig.17 As shown in Figure 2, the HPLC analysis of Gastrodia elata fermentation broth was as follows: Fig.18 shown.

[0191] Table 8 Results of precision test, stability test and repeatability test of different Gastrodia elata fermentation broths

[0192]

[0193] 3.5 Changes in the contents of gastrodin, p-hydroxybenzyl alcohol, barisonoside E, barisonoside A, barisonoside B, and barisonoside C before and after fermentation

[0194] The data of the contents of gastrodin, p-hydroxybenzyl alcohol, barisonoside E, barisonoside A, barisonoside B and barisonoside C in groups F and G at different fermentation times were determined. The results are as follows Figures 19 to 24 As shown, Fig.19 The middle abscissa is the fermentation time (h), and the ordinate is the gastrodin content (μg / mL); Fig. 20 The middle abscissa is the fermentation time (h), and the ordinate is the p-hydroxybenzyl alcohol content (μg / mL); Fig.21 The middle abscissa is the fermentation time (h), and the ordinate is the barisin E content (μg / mL); Fig. 22 The middle abscissa is the fermentation time (h), and the ordinate is the balisin A content (μg / mL); Fig.23 The middle abscissa is the fermentation time (h), and the ordinate is the barisin B content (μg / mL); Fig.24 The horizontal axis is the fermentation time (h), and the vertical axis is the content of balisenoside C (μg / mL). Figures 19 to 24In the figure, the blue line represents the F group, the red line represents the G group, different letters represent significant differences (P<0.05), and *** represents P<0.001 compared with the G group, and the difference is extremely significant.

[0195] according to Fig.19 It can be seen that the content of gastrodin in group F decreased significantly during the fermentation process. Compared with group G, the content of gastrodin decreased significantly. Fig. 20 It can be seen that the content of p-hydroxybenzyl alcohol in group F increased significantly during the fermentation process. Compared with group G, the content of p-hydroxybenzyl alcohol increased significantly. Figures 21 to 24 It can be seen that PE in group F gradually decreased during the fermentation process, and its content increased significantly compared with that in group G; PA, PB and PC in group F gradually increased during the fermentation process, and their contents increased significantly compared with that in group G.

[0196] In summary, fermenting Gastrodia elata with Lactobacillus plantarum LZU-LJY-1Q1 and Lactobacillus plantarum LZU-J-TSL6 in a volume ratio of 1:1 can significantly increase the content of p-hydroxybenzyl alcohol, total phenols, barisonoside A, barisonoside B and barisonoside C in Gastrodia elata. Gastrodin belongs to a small molecule phenolic glycoside compound. Part of gastrodin is decomposed to produce p-hydroxybenzyl alcohol (gastrodin aglycone) after the action of microorganisms. In addition, barisonoside is transformed under the action of microorganisms.

[0197] 4. Changes in the total antioxidant capacity of ABTS and the scavenging capacity of DPPH and OH- free radicals during fermentation

[0198] 4.1 ABTS total antioxidant capacity

[0199] The ABTS free radical stock solution is a mixture of 50 mL of 7 mM ABTS solution and 880 μL of 140 mM potassium persulfate solution, which is stored in the dark at room temperature for 18 h and diluted with distilled water until the absorbance at 734 nm reaches 0.70±0.02 to obtain the ABTS free radical working solution.

[0200] The total antioxidant capacity of ABTS is expressed as ABTS free radical inhibition rate. The determination method of ABTS free radical inhibition rate is as follows: sample: 100 μL of the sample to be tested (after centrifugation and removal of the supernatant) is mixed with 100 μL ABTS working solution; control: 100 μL of the sample to be tested is mixed with 100 μL distilled water, blank: 100 μL distilled water is mixed with 100 μL ABTS free radical working solution (3 portions each), react at room temperature in a dark place away from light for 30 minutes, use an enzyme marker to measure the absorbance values ​​of the sample, control and blank at 734 nm and record them as A1, A2, and A0, and calculate the ABTS free radical inhibition rates of F (Gastrodia elata fermentation group), G (Gastrodia elata enzymatic hydrolysate group), and L (fermentation bacteria group), respectively. The results are shown in Fig.25As shown in the figure, the horizontal axis represents the fermentation time (h), the vertical axis represents the ABTS free radical inhibition rate (%), the blue broken line in the figure represents the F group, the yellow broken line represents the G group, and the red broken line represents the L group. Different letters indicate significant differences (P<0.05); *** indicates that P<0.001 compared with the G group, and the difference is extremely significant; ### indicates that P<0.001 compared with the L group, and the difference is extremely significant.

[0201] The calculation formula of ABTS free radical inhibition rate is as follows:

[0202]

[0203] 4.2DPPH free radical scavenging rate

[0204] The DPPH free radical scavenging ability detection method is as follows: sample: 1mL of the sample to be tested (after centrifugation and removal of the supernatant) is mixed with 1mL of 0.2mM DPPH (dissolved in anhydrous ethanol); control: 1mL of the sample to be tested is mixed with 1mL of distilled water; blank: 1mL of distilled water is mixed with 1mL of DPPH solution, shaken thoroughly, centrifuged at 4000rpm for 5min, and kept in the dark for 30min. The reaction was carried out in a dark place at room temperature for 30min, and the absorbance values ​​of the sample, control and blank were measured at 517nm using an ELISA instrument, which were recorded as A1, A2, and A0, respectively. The DPPH free radical scavenging rates of F (Gastrodia elata fermentation group), G (Gastrodia elata enzymatic hydrolysate group), and L (fermentation bacteria group) were calculated, and the results are shown in Figure 2. Fig.26 As shown in the figure, the horizontal axis represents the fermentation time (h), the vertical axis represents the DPPH free radical inhibition rate (%), the blue broken line in the figure represents the F group, the yellow broken line represents the G group, and the red broken line represents the L group. Different letters indicate significant differences (P<0.05); *** indicates that P<0.001 compared with the G group, and the difference is extremely significant; ### indicates that P<0.001 compared with the L group, and the difference is extremely significant.

[0205] The calculation formula of DPPH free radical inhibition rate is as follows:

[0206]

[0207] 4.3OH - Free radical scavenging rate

[0208] OH - The free radical scavenging ability was determined using a hydroxyl radical assay kit (Nanjing Jiancheng Bioengineering Institute). The hydroxyl radical inhibition rate of all samples was determined using the assay kit. The blank group was 0.4 mL of double distilled water, recorded as A blank The control group consisted of 0.2 mL double distilled water and 0.2 mL substrate application solution, recorded as A contrast; The sample group is 0.2mL of the sample to be tested and 0.2mL of the substrate application solution, recorded as A sample , calculate OH - Free radical inhibition rate, the results are as follows Fig. 27 As shown in the figure, the horizontal axis represents the fermentation time (h), and the vertical axis represents OH - Free radical scavenging rate (%), the blue line represents group F, the yellow line represents group G, and the red line represents group L. Different letters indicate significant differences (P<0.05); *** indicates that P<0.001 is significantly different from group G; ### indicates that P<0.001 is significantly different from group L.

[0209] The calculation formula of OH- free radical inhibition rate is as follows:

[0210]

[0211] Depend on Figures 25-27 It can be seen that with the change of time, the three antioxidant capacities of group L increased slightly, indicating that the probiotics themselves would produce antioxidant active substances during the fermentation process; with the change of time, the three antioxidant capacities of group G remained unchanged; the antioxidant activity of group F increased with time, and was significantly higher than that of groups G and L, indicating that the antioxidant activity of Gastrodia elata can be significantly improved after enzymatic fermentation.

[0212] As can be seen from the above embodiments, the present invention provides a method for preparing a fermented gastrodia elata liquid, comprising the following steps: (1) mixing gastrodia elata, a mixed enzyme and water, and performing enzymolysis to obtain a gastrodia elata liquid; (2) inoculating fermentation bacteria into the gastrodia elata liquid obtained in step (1), fermenting to obtain a fermented liquid, centrifuging the fermented liquid to obtain a supernatant, and filtering the supernatant to obtain a gastrodia elata liquid. The fermented gastrodia elata liquid obtained according to the scheme of the present invention contains high concentrations of p-hydroxybenzyl alcohol and balisonglucosides, which improves the ability to remove DPPH free radicals and OH radicals. - The ability of free radicals also enhances the overall antioxidant capacity of ABTS.

[0213] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing Gastrodia elata fermented liquid, characterized in that: The steps include: (1) mixing Gastrodia elata, mixed enzyme and water, and performing enzymolysis for 1.5 to 2.5 hours to obtain Gastrodia elata enzymolysis solution; (2) inoculating fermentation bacteria into the Gastrodia elata enzymatic hydrolysate obtained in step (1), fermenting for 18 to 26 hours to obtain a fermentation liquid, centrifuging the fermentation liquid to obtain a supernatant, and filtering the supernatant to obtain a Gastrodia elata fermentation liquid; The mixed enzyme is cellulase and pectinase, with a mass ratio of 1-2:1-2; The fermentation bacteria are Lactobacillus plantarum LZU-J-TSL6 and Lactobacillus plantarum LZU-LJY-1Q1, with a volume ratio of 1-2:1-2; The Latin name of the Lactobacillus plantarum LZU-J-TSL6 is Lactobacillus plantarum, which is deposited in Guangdong Microbiological Culture Collection Center on October 23, 2020, at the 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 61242; The Latin name of the plant lactobacillus LZU-LJY-1Q1 is Lactiplantibacillus plantarum, which is preserved in the Guangdong Microbial Culture Collection Center on July 1, 2024. The preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the preservation number is GDMCC No: 64797.

2. The preparation method according to claim 1, characterized in that: The mass volume ratio of Gastrodia elata to water in step (1) is 1 g: 20-30 mL.

3. The preparation method according to claim 2, characterized in that: The mass volume ratio of the mixed enzyme to water in step (1) is 4-7 g:1000 mL.

4. The preparation method according to claim 3, characterized in that: The temperature of the enzymatic hydrolysis in step (1) is 65-75°C.

5. The preparation method according to claim 4, characterized in that: The inoculation volume of the fermentation bacteria in step (2) is 2% to 4% of the volume of the Gastrodia elata enzymatic hydrolysate.

6. The preparation method according to claim 5, characterized in that: The fermentation temperature in step (2) is 35-37°C.

7. The preparation method according to claim 6, characterized in that: The centrifugal speed in step (2) is 5800-6200 rpm, and the centrifugal time is 3-7 min.

8. The preparation method according to claim 7, characterized in that: The filtration in step (2) is filtration through a microporous membrane, and the pore size of the microporous membrane is 0.20 to 0.24 μm.

9. The Gastrodia elata fermentation liquid prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the Gastrodia elata fermented liquid according to claim 9 in the preparation of antioxidant drugs, cosmetics, medical devices or foods.

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