Preparation method for improving quality of gastrodia elata, gastrodia elata fermentation liquor and gastrodia elata powder

Abstract

The invention relates to the technical field of natural medicine fermentation, in particular to a preparation method for improving the quality of gastrodia elata, gastrodia elata fermentation liquor and gastrodia elata powder. The preparation method comprises the step of fermenting gastrodia elata by using a sugar tea water fermentation product. According to the preparation method, the contents of total acids, crude polysaccharides and alcohol of the gastrodia elata are increased, meanwhile, the production of gastrodin, p-hydroxybenzyl alcohol and other active substances can be promoted, and the contents of total polyphenols and total flavonoids of the gastrodia elata as well as the antioxidant activity and the hypoglycemic activity of the gastrodia elata are remarkably improved. And microbial fermentation obviously improves the quality of the gastrodia elata and endows the gastrodia elata with mellow and sweet smells.

Description

Technical Field

[0001] The present invention relates to the technical field of natural medicine fermentation, and more particularly, to a preparation method for improving the quality of Gastrodia elata, a fermentation broth of Gastrodia elata, and Gastrodia elata powder. Background Art

[0002] Gastrodia elata is the dried tuber of Gastrodia elata Blume of the Orchidaceae family. It is a precious traditional Chinese medicinal material in China and has now been included in the list of substances that are both food and traditional Chinese medicine according to tradition. When searching for the active ingredients of Gastrodia elata, a series of small molecule compounds were discovered, including gastrodin, p-hydroxybenzyl alcohol, β-sitosterol, etc. Modern research shows that Gastrodia elata has the effects of improving cognitive memory, improving sleep, regulating blood lipids, blood sugar and blood pressure, and neuroprotection. Therefore, consuming Gastrodia elata is of great benefit to human health. At present, the edible methods of Gastrodia elata mostly follow the tradition and are relatively single, with fresh Gastrodia elata being mostly processed by traditional Chinese medicine processing methods. In contrast, using microbial fermentation technology to process Gastrodia elata can produce fermentation products rich in active ingredients and functional enzymes. In this process, microorganisms cause complex reactions among the components in the raw materials through their metabolic activities, not only producing new active substances and functional enzymes, but also retaining the original nutritional components to a certain extent.

[0003] However, even the quality of Gastrodia elata obtained by single-strain fermentation is still not high. In view of this, the present invention is specifically proposed. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method for improving the quality of Gastrodia elata, a fermentation broth of Gastrodia elata, and Gastrodia elata powder. The embodiments of the present invention provide a new preparation method, which can greatly improve the quality of Gastrodia elata.

[0005] The present invention is implemented as follows:

[0006] In a first aspect, the present invention provides a preparation method for improving the quality of Gastrodia elata, including: fermenting Gastrodia elata with a sugar tea fermentation product.

[0007] In an alternative embodiment, the sugar tea fermentation product is formed by fermenting sugar tea with a compound microbial inoculum, and the compound microbial inoculum is composed of lactic acid bacteria, acetic acid bacteria, and yeast.

[0008] In an alternative embodiment, the ratio of the colony counts of the lactic acid bacteria, the acetic acid bacteria, and the yeast is (0.5 - 5):(0.1 - 1):1.

[0009] In an alternative embodiment, the preparation steps of the sugar tea include: mixing tea leaves, sugar, and water, boiling, and then filtering, wherein the concentration of the tea leaves is 8 - 12 g / L, and the concentration of the sugar is 120 - 220 g / L.

[0010] In an alternative embodiment, the compound microbial inoculum is added to the sugar tea water for fermentation at a volume fraction of 3-7%;

[0011] Preferably, the conditions for fermenting the sugar tea water include: the temperature is 25-32 °C, and the fermentation time is 5-7 days.

[0012] In an alternative embodiment, it further includes forming a gastrodia elata culture medium;

[0013] Preferably, the preparation steps of the gastrodia elata culture medium include: mixing gastrodia elata with sugar water, then adding an enzyme for enzymatic hydrolysis, and sterilizing after enzymatic hydrolysis.

[0014] In an alternative embodiment, the material-liquid ratio of the gastrodia elata to the sugar water is 1:(8-12); the mass concentration of the sugar water is 70-90 g / L;

[0015] Preferably, the enzyme includes any two of cellulase, amylase, and pectinase;

[0016] The concentrations of the cellulase, the amylase, and the pectinase are respectively 0.2-0.4 g / L;

[0017] Preferably, the process of enzymatic hydrolysis includes enzymatic hydrolysis with cellulase or pectinase at 40-60 °C for 45-75 minutes, and enzymatic hydrolysis with amylase at 70-90 °C for 45-75 minutes.

[0018] In an alternative embodiment, the fermentation product of the sugar tea water is inoculated into the gastrodia elata culture medium for cultivation at a volume fraction of 3-7%;

[0019] Preferably, the conditions for cultivation include: the temperature is 28-32 °C, and the time is 4-5 days.

[0020] In a second aspect, the present invention provides a gastrodia elata fermentation broth, which is prepared by the preparation method for improving the quality of gastrodia elata according to any one of the foregoing embodiments.

[0021] In a third aspect, the present invention provides a gastrodia elata powder, which is formed by freeze-drying the gastrodia elata fermentation broth described in the foregoing embodiments.

[0022] The present invention has the following beneficial effects: In the embodiments of the present invention, by fermenting gastrodia elata with the fermentation product of sugar tea water, the contents of total acid, crude polysaccharide, and alcohol are increased. At the same time, it can promote the production of gastrodin, p-hydroxybenzyl alcohol, and other active substances, significantly improving the contents of total polyphenols and total flavonoids in gastrodia elata, as well as its antioxidant activity and hypoglycemic activity. And microbial fermentation significantly improves the quality of gastrodia elata and imparts it with mellow and sweet scents. Description of the Drawings

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0024] Figure 1 Schematic diagram of the contents of active compounds in different Gastrodia elata treatment groups provided by the present invention;

[0025] Figure 2 Chromatogram of compound peak results in different Gastrodia elata treatment groups provided by the present invention;

[0026] Figure 3 Schematic diagram of the contents of total polyphenols and total flavonoids in different Gastrodia elata treatment groups provided by the present invention;

[0027] Figure 4 Schematic diagram of the ferric ion reducing ability of different Gastrodia elata treatment groups provided by the present invention;

[0028] Figure 5 Schematic diagram of the DPPH radical scavenging ability of different Gastrodia elata treatment groups provided by the present invention;

[0029] Figure 6 Schematic diagram of the ABTS radical scavenging ability of different Gastrodia elata treatment groups provided by the present invention;

[0030] Figure 7 Schematic diagram of the hydroxyl radical scavenging ability of different Gastrodia elata treatment groups provided by the present invention;

[0031] Figure 8 Schematic diagram of the α-glucosidase inhibitory ability of different Gastrodia elata treatment groups provided by the present invention;

[0032] Figure 9 Sensory evaluation radar chart provided by the present invention;

[0033] Figure 10 Result chart of the correlation analysis between the contents of TPC and TFC and antioxidant and hypoglycemic abilities provided by the present invention. Detailed implementation manners

[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0035] In a first aspect, the present invention provides a preparation method for improving the quality of Gastrodia elata, comprising:

[0036] S1. Preparation of the sugar tea fermentation product:

[0037] Mix tea leaves (such as, including but not limited to, black tea), sugar (such as, including but not limited to, white sugar or brown sugar) and water and boil them. After boiling, filter to obtain the sugar tea.

[0038] Among them, the concentration of the tea leaves is 8 - 12 g / L, such as 8 g / L, 9 g / L, 10 g / L, 11 g / L, and 12 g / L, etc., any value between 8 - 12 g / L. The concentration of the sugar is 120 - 220 g / L, such as 120 g / L, 150 g / L, 170 g / L, 200 g / L, and 220 g / L, etc., any value between 120 - 220 g / L.

[0039] Then, add a compound microbial inoculum with a water volume fraction of 3 - 7% to the sugar tea for fermentation. Specifically, the added amount of the compound microbial inoculum is any value between 3% and 7%, such as 3%, 4%, 5%, 6%, and 7%.

[0040] Among them, the compound microbial inoculum is composed of lactic acid bacteria, acetic acid bacteria, and yeast. The ratio of the colony counts of the lactic acid bacteria, the acetic acid bacteria, and the yeast is (0.5 - 5):(0.1 - 1):1. For example, the ratio of the colony counts of the lactic acid bacteria, the acetic acid bacteria, and the yeast is 1:1:1, 5:2:1, 0.5:0.1:1, 0.5:0.2:1, and 3:0.5:1, etc., any value between (0.5 - 5):(0.1 - 1):1.

[0041] Acetic acid bacteria, yeast, and lactic acid bacteria are common fermentation inoculants. However, when using a compound microbial inoculum formed by mixing the three for fermentation, under the synergistic action of multiple microorganisms, the product metabolism and synthesis are further improved; during the fermentation process, the mutual transformation of various substances such as alcohols, aldehydes, acids, and esters may occur, generating rich nutritional functional components and flavor substances with a sweet aroma. The main metabolic products include acetic acid, ethanol, gluconic acid, glucuronic acid, amino acids, and vitamins, etc. In the embodiments of the present invention, lactic acid bacteria, acetic acid bacteria, and yeast are used to ferment the sugar tea, and then Gastrodia elata is fermented, which can increase the content of active substances in the Gastrodia elata fermentation product, improve its functional activity, and improve its original unpleasant odor.

[0042] Furthermore, the fermentation conditions include a temperature of 25 - 32 °C, a time of 5 - 7 d, and aerobic fermentation.

[0043] S2. Form a Gastrodia elata culture medium;

[0044] Mix Gastrodia elata with sugar water (such as including but not limited to brown sugar water or white sugar water), wherein the concentration of the sugar water is 70 - 90 g / L, such as any value between 70 - 90 g / L like 70 g / L, 75 g / L, 80 g / L, 85 g / L, and 90 g / L. The material - liquid ratio of Gastrodia elata to the sugar water is 1:(8 - 12), such as any value between 1:(8 - 12) like 1:8, 1:9, 1:10, 1:11, and 1:12.

[0045] Then add enzymes for enzymatic hydrolysis. Among them, the enzymes used are any two of cellulase, amylase, and pectinase; the concentrations of the cellulase, the amylase, and the pectinase are respectively 0.2 - 0.4 g / L; such as any value between 0.2 - 0.4 g / L like 0.2 g / L, 0.25 g / L, 0.3 g / L, 0.35 g / L, and 0.4 g / L.

[0046] The process of enzymatic hydrolysis includes enzymatic hydrolysis with cellulase or pectinase at 40 - 60 °C for 45 - 75 minutes, and enzymatic hydrolysis with amylase at 70 - 90 °C for 45 - 75 minutes.

[0047] The embodiments of the present invention adopt the above - mentioned enzymatic hydrolysis process and enzymes, which are beneficial to the enzymatic hydrolysis of Gastrodia elata by the enzymes, and then beneficial to the fermentation of Gastrodia elata by the fermentation products of sugar - tea water, improving the quality of Gastrodia elata.

[0048] After enzymatic hydrolysis, sterilization treatment is carried out. The conditions of sterilization treatment adopt existing conditions, such as sterilization at 121 °C for 20 min.

[0049] S3. Inoculate and ferment;

[0050] Inoculate the fermentation products of sugar - tea water into the Gastrodia elata culture medium for cultivation; specifically, the inoculation amount of the fermentation products of sugar - tea water is 3 - 7% by volume fraction, such as any value between 3 - 7% like 3%, 4%, 5%, 6%, and 7%.

[0051] The conditions of cultivation include: the temperature is 28 - 32 °C, and the time is 4 - 5 d.

[0052] In the second aspect, the present invention provides a Gastrodia elata fermentation broth, which is prepared by the preparation method for improving the quality of Gastrodia elata according to any one of the foregoing embodiments.

[0053] In the third aspect, the present invention provides a Gastrodia elata powder, which is formed by freeze - drying the Gastrodia elata fermentation broth described in the foregoing embodiments.

[0054] The features and properties of the present invention will be further described in detail below in conjunction with embodiments.

[0055] Example 1

[0056] An embodiment of the present invention provides a preparation method for improving the quality of Gastrodia elata, including:

[0057] Preparation of sugar tea water fermentation product: 10 g / L of black tea leaves are added to 100 g / L of sugar water and boiled, and then filtered to obtain sugar tea water; a compound microbial inoculant with a volume fraction of 5% (lactic acid bacteria: acetic acid bacteria: yeast = 1:1:1) is added to the sugar tea water, and it is placed in an incubator at 28 °C for 7 days to obtain a sugar tea water fermentation product.

[0058] Gastrodia elata culture medium: Gastrodia elata powder is added to brown sugar water (80 g / L) according to a material-liquid ratio of 1:10 by mass, and cellulase (0.3 g / L) and amylase (0.3 g / L) are added and enzymolyzed at 55 °C and 80 °C for 1 h respectively. After the enzymolysis is completed, it is sterilized at 121 °C for 20 min.

[0059] Inoculation and fermentation: The sugar tea water fermentation product is inoculated into the Gastrodia elata culture medium according to a volume fraction of 5%, placed in an incubator at 30 °C, and statically fermented for 5 days to complete the fermentation.

[0060] The fermentation product is the Gastrodia elata fermentation broth.

[0061] Then, the Gastrodia elata fermentation broth is freeze-dried, and the conditions for freeze-drying include: the sublimation temperature is 35 °C, the pre-freezing temperature is -80 °C, the vacuum degree is 1 Pa, the shelf temperature is 20 °C, and the drying time is 48 h.

[0062] Examples 2 - 4

[0063] Examples 2 - 4 prepare Gastrodia elata powder with reference to the preparation method for improving the quality of Gastrodia elata provided in Example 1. Only some conditions are different, and the rest of the operations are the same as those in Example 1. Specifically as follows:

[0064] Example 2: The ratio of the colony numbers of lactic acid bacteria, the acetic acid bacteria, and the yeast is 0.5:0.1:1; the concentration of cellulase is 0.2 g / L, and the concentration of amylase is 0.4 g / L; the inoculation amount of the sugar tea water fermentation product is 3%.

[0065] Example 3: The ratio of the colony numbers of lactic acid bacteria, the acetic acid bacteria, and the yeast is 3:0.5:1; the concentration of cellulase is 0.3 g / L, and the concentration of the amylase is 0.3 g / L; the inoculation amount of the sugar tea water fermentation product is 5%.

[0066] Example 4: The ratio of the colony numbers of lactic acid bacteria, the acetic acid bacteria, and the yeast is 5:1:1; the concentration of cellulase is 0.4 g / L, and the concentration of pectinase is 0.2 g / L; the inoculation amount of the sugar tea water fermentation product is 7%.

[0067] Comparative Example 1

[0068] This comparative example prepared gastrodia powder according to the preparation method for improving the quality of gastrodia provided in Example 1, with the only difference being that the sugar tea fermentation product was replaced with an equal amount of sterile water, and other operations were the same as those in Example 1. (Recorded as the MJ group below).

[0069] Comparative Example 2

[0070] This comparative example prepared gastrodia powder according to the preparation method for improving the quality of gastrodia provided in Example 1, with the only difference being that enzymatic hydrolysis was not carried out, that is, cellulase (0.3 g / L) and amylase (0.3 g / L) were not added. ； At the same time, the sugar tea fermentation product was not inoculated either. (Recorded as the CK group below).

[0071] Comparative Example 3

[0072] This comparative example prepared gastrodia powder according to the preparation method for improving the quality of gastrodia provided in Example 1, with the only difference being that lactic acid bacteria, acetic acid bacteria, and yeast were directly inoculated into the gastrodia culture medium, and the inoculation amounts were the same as those in Example 1. The three did not undergo sugar tea fermentation. (Recorded as the QJ group below).

[0073] Pretreatment before detection

[0074] The gastrodia powder of Example 1 (recorded as the FJ group below) and Comparative Examples 1 - 3 was processed. Specifically, 1 g of gastrodia samples from different groups (accurate to 0.01 g) was weighed, 10 mL of methanol with a volume fraction of 80% was added, and it was fully dissolved. It was ultrasonically treated at 40 °C for 30 min, then centrifuged at 6000 r / min for 15 min, and the supernatant was collected. The extraction was repeated 2 times in the same way and the supernatants were combined, and it was made up to 50 mL with methanol with a volume fraction of 80%. An appropriate amount of the sample solution was filtered through a 0.22 μm filter membrane for testing.

[0075] Detection method:

[0076] 1. Determination of physical and chemical indexes

[0077] (a) pH determination

[0078] The pH value of the sample solution was measured using a pH meter.

[0079] (b) Total acid determination

[0080] The total acid of the sample solution was determined with reference to GB 12456 - 2021 "National Food Safety Standard - Determination of Total Acid in Foods".

[0081] (c) Crude polysaccharide determination

[0082] Method for ultrasonic extraction of polysaccharides. Pretreatment: Take an appropriate amount of Gastrodia elata powder obtained in Example 1 and Comparative Examples 1-3, add an ethanol solution with a volume fraction of 80%, ultrasonicate for 45 min (material-liquid mass ratio 1:20, to remove fat-soluble impurities), centrifuge at 8000 r / min for 5 min, and dry to constant weight. Extraction: Weigh 1 g of the pretreated sample, use pure water for ultrasonic extraction for 45 min (material-liquid ratio mass ratio 1:40), collect the supernatant after centrifuging at 8000 r / min for 5 min, repeat the extraction 2 times, and mix the combined supernatants uniformly. Alcohol precipitation for sample preparation: Concentrate the supernatant to about 1 / 4 of the original volume, add 100 mL of ethanol (95%). Precipitate overnight at low temperature, centrifuge at 8000 r / min for 5 min, dry the precipitate, dissolve in water and make up the volume to 50 mL, and place at 4°C for measurement.

[0083] The content of crude polysaccharides was determined by the anthrone-sulfuric acid method. Adjust the sugar concentration of the sample solution to the measurement range, accurately pipette 2 mL and place it in a dry and clean test tube, add 6 mL of anthrone reagent, shake well, heat in a boiling water bath for 15 min, and quickly immerse it in an ice-water bath for cooling for 15 min after taking it out. Measure the absorbance at a wavelength of 625 nm. Using the mass concentration (mg / mL) of the glucose solution as the abscissa and the absorbance as the ordinate, the standard curve was made as y = 1.2911x + 0.0408, R 2 = 0.9992; The calculation formula for the content of crude polysaccharides is shown in Equation (1).

[0084]

[0085] In the formula: ρ1 is the mass concentration of crude polysaccharides in the sample solution, μg / mL; N1 is the dilution factor; V1 is the total volume of the sample solution, mL; m1 is the mass of the sample taken, g.

[0086] (d) Determination of alcohol content

[0087] The alcohol content of the fermentation broth refers to the alcohol meter method in the national standard GB 5009.225 2016 "National Food Safety Standard - Determination of Ethanol Concentration in Alcohol".

[0088] 2. Determination of active compounds in Gastrodia elata samples

[0089] 2.1. Preparation of standard solution

[0090] Accurately weigh 20 mg of each of the standard products of gastrodin, p-hydroxybenzyl alcohol, p-hydroxybenzaldehyde, citric acid ester E, citric acid ester B, and baloside A, dissolve them with methanol and make up the volume to 10 mL as the standard stock solution, and prepare a series of standard solutions with different mass concentration gradients (μg / mL) respectively for standby.

[0091] 2.2. Chromatographic conditions

[0092] Chromatographic column, Poroshell 120 PFP (4.6 mm × 100 mm, 2.7 μm); DAD detector, detection wavelength 220 nm; column temperature 30 °C; injection volume 1 μL; flow rate 0.8 mL / min; mobile phase: phase A is phosphoric acid with a volume fraction of 0.1%, and phase B is acetonitrile. Gradient elution program: 0 - 5 min, 2% - 5% B; 5 - 10 min, 5% - 10% B; 10 - 20 min, 10% - 25% B; 20 - 25 min, 25% - 80% B.

[0093] 3. Determination of total polyphenols (TPC) and total flavonoids (TFC) in Gastrodia elata samples

[0094] 3.1. Determination of total polyphenols (TPC)

[0095] Take 20 μL of the extract and 20 μL of Folin-Ciocalteu reagent (2 mol / L) and mix them for reaction for 5 min. Then add 160 μL of 5% (mass fraction) Na 2 CO 3 , shake well, react in the dark at room temperature for 60 min, and measure the absorbance at a wavelength of 765 nm. Using the gallic acid mass concentration (μg / mL) as the abscissa and the absorbance as the ordinate, draw a standard curve as y = 0.0034x + 0.4205, R 2 = 0.9992. The total polyphenol content in the sample is expressed as gallic acid equivalents (mg GAE / g). The calculation formula for the total polyphenol content is shown in Equation (2).

[0096]

[0097] In the formula: ρ 2 is the mass concentration of total polyphenols in the sample to be measured, μg / mL; N 2 is the dilution factor; V 2 is the total volume of the extract, mL; m 2 is the mass of the sample weighed, g.

[0098] 3.2. Determination of total flavonoids (TFC)

[0099] Take 20 μL of the extract and 15 μL of 5% (mass fraction) NaNO 2 and mix them. React in the dark at room temperature for 6 min. Then add 15 μL of 10% (mass fraction) AlCl 3 ·6H 2 O, react at room temperature for 5 min. Finally, add 100 μL of NaOH (1 mol / L) and measure the absorbance at a wavelength of 510 nm. Using the rutin mass concentration (μg / mL) as the abscissa and the absorbance as the ordinate, draw a standard curve as y = 0.0005x + 0.046, R 2= 0.9995. The total flavonoid content is expressed as rutin equivalent (mg RE / g). The calculation formula for the total flavonoid content is shown in Equation (3).

[0100]

[0101] In the formula: ρ 3 is the mass concentration of total flavonoids in the sample solution to be measured, μg / mL; N 3 is the dilution factor; V 3 is the total volume of the extract, mL; m3 is the mass of the sample taken, g.

[0102] 4. Determination of the antioxidant activity of the Gastrodia elata Blume sample extract

[0103] 4.1. Determination of ferric reducing antioxidant power (FRAP)

[0104] Preparation of FRAP working solution: Mix 0.3 mol / L (pH = 3.6) sodium acetate solution, 10 mmol / L tripyridyltriazine (TPTZ) and 20 mmol / L FeCl 3 in a volume ratio of 10:1:1 to obtain the FRAP working solution. Take 30 μL of the extract and 265 μL of the FRAP working solution, mix well, react at a constant temperature of 37 °C for 30 min, and measure the absorbance at a wavelength of 593 nm. Using the mass concentration (μg / mL) of the Trolox solution as the abscissa and the absorbance as the ordinate, the standard curve is y = 0.0154x + 0.0953, R 2 = 0.9992.

[0105] The ferric reducing ability is expressed as water-soluble vitamin E (Trolox) equivalent (mg TE / g).

[0106]

[0107] In the formula: ρ 4 is the mass concentration of Trolox equivalent in the sample solution to be measured, μg / mL; N 4 is the dilution factor; V 4 is the total volume of the extract, mL; m 4 is the mass of the sample taken, g.

[0108] 4.2. Determination of DPPH radical scavenging ability

[0109] Prepare a DPPH solution with a mass concentration of 128.50 μg / mL using a methanol solution with a volume fraction of 80%. Dilute the extract to sample test solutions with different mass concentrations (μg / mL) for standby. Take 100 μL of the test solution, add 100 μL of the above DPPH solution (128.50 μg / mL), react at room temperature for 30 min, and then measure the absorbance at a wavelength of 517 nm. Use the above methanol solution as a blank control to measure the absorbance A 0 , and use Trolox as a positive control. The calculation formula for the DPPH radical scavenging rate is shown in Equation (5).

[0110]

[0111] In the formula: A 0 is the absorbance of the blank group; A 1 is the absorbance of the sample group or the positive group.

[0112] 4.3 Determination of ABTS radical scavenging ability

[0113] The ABTS stock solution is prepared by mixing a 7 mmol / L ABTS solution and a 2.45 mmol / L potassium persulfate solution in a volume ratio of 1:1, and used after reacting for 16 h at room temperature in the dark; ABTS working solution: Dilute the ABTS stock solution with a methanol solution with a volume fraction of 80% so that the absorbance of this solution is 0.70 ± 0.02 at a wavelength of 734 nm. Dilute the extract to sample test solutions with different mass concentrations (μg / mL) for standby. Take 40 μL of the test solution, add 160 μL of the ABTS working solution, react at room temperature for 6 min, and then measure the absorbance at a wavelength of 734 nm. Use methanol with a volume fraction of 80% to replace the sample solution as the blank group to measure the absorbance A 2 , and use Trolox as a positive reference. The calculation formula for the ABTS radical scavenging rate is shown in Equation (6).

[0114]

[0115] In the formula: A 2 is the absorbance of the blank group; A 3 is the absorbance of the sample group or the positive group.

[0116] 4.4 Determination of hydroxyl radical scavenging ability

[0117] Prepare o-phenanthroline as an anhydrous ethanol solution with a concentration of 0.75 mmol / L. Take 1.0 mL of the o-phenanthroline solution and add 2.0 mL of phosphate buffer solution (0.2 mol / L, pH 7.40) and 1.0 mL of distilled water respectively. After mixing well on a vortex mixer, add a ferrous sulfate solution with a concentration of 0.75 mmol / L (FeSO 4) 1.0 mL, mix well, and finally add 1.0 mL of 0.01% hydrogen peroxide (H 2 O 2 ), react in a water bath at 37 °C for 60 min, and measure the absorbance A 4 at a wavelength of 536 nm. Use vitamin C as the positive control. The calculation formula for the hydroxyl radical scavenging rate is shown in Equation (7).

[0118]

[0119] In the formula: A 4 is the absorbance of the sample solution; A 5 is the absorbance of the sample blank group (using absolute ethanol instead of the sample solution); A 6 is the absorbance of the sample control group (using distilled water instead of H 2 O 2 ).

[0120] 5. Determination of α-glucosidase inhibitory activity of Gastrodia elata Blume sample extract

[0121] Mix 40 μL of Gastrodia elata Blume sample extract (0.1 - 10 mg / g) and 30 μL of α-glucosidase solution (0.2 U / mL) in a 96-well plate, and react at 37 °C for 10 min. Add 30 μL of 5 mmol / L p-nitrophenyl-β-D-galactoside (pNPG, dissolved in 0.1 mol / L phosphate buffer solution with pH 6.8) to each well, mix evenly, and after reacting at 37 °C for 30 min, add 100 μL of 1 mol / L Na 2 CO 3 solution, and measure the absorbance A a at a wavelength of 405 nm. Use acarbose as the positive control. The calculation of the α-glucosidase activity inhibition rate is shown in Equation (8).

[0122]

[0123] In the formula: A a is the absorbance of the sample and the enzyme solution; A b is the absorbance with phosphate buffer solution replacing the sample; A c is the absorbance of the enzyme solution and the phosphate buffer solution; A d is the absorbance with phosphate buffer solution replacing the enzyme.

[0124] 6. Determination of volatile flavor substances of Gastrodia elata Blume

[0125] The volatile components of the fermentation broth were analyzed by solid phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS). 5 mL of the sample was placed in a 15 mL headspace vial, and 2-octanol (10 μL, 300 mg / L) as the internal standard and 1 g of sodium chloride were added. The headspace vial was kept at a constant temperature of 50 °C in a water bath for 30 min. After 30 min of headspace extraction at 50 °C using a manual injector with a 2 cm-50 / 30 μm DVB / CAR / PDMS StableFlex extraction head, the extraction head was removed and immediately inserted into the injection port of the GC instrument (temperature 250 °C) for thermal desorption for 5 min and then injection.

[0126] GC conditions: HP-5MS (60 m × 0.25 mm, 0.25 μm); maintained at 40 °C for 3 min, heated to 100 °C at a rate of 10 °C / min, maintained for 2 min, then heated to 160 °C at a rate of 3 °C / min, maintained for 5 min, and finally heated to 250 °C at a rate of 10 °C / min; splitless injection; high-purity N 2 The flow rate was 1 mL / min.

[0127] 7. Sensory evaluation

[0128] Using the CK group as the control, sensory evaluations were performed on the FJ, MJ, and QJ groups by quantitative descriptive analysis. The scores for color, odor, taste, texture, and appearance were based on a total score of 100 points. The scoring results of 12 professional sensory evaluation personnel were recorded, and the average value of each sensory item was taken. The sensory evaluation criteria are shown in Table 1.

[0129] Table 1 Sensory evaluation criteria for the fermentation broth

[0130]

[0131]

[0132] Results and analysis

[0133] 1. Physicochemical test results

[0134] The results are shown in Table 2 for reference.

[0135] Table 2 Physicochemical properties of different Gastrodia elata

[0136]

[0137] Note: Different lowercase letters in the same row indicate significant differences (P < 0.05), and the same applies hereinafter; " / " indicates not detected.

[0138] According to Table 2, after fermentation, the pH value of the fermentation broth in the FJ group decreased to 3.37 ± 0.04, and the total acid content increased significantly (P < 0.05), indicating that a large amount of acidic substances were produced under the action of microorganisms during the fermentation of Gastrodia elata. It is speculated that the fermentation products of the inoculated sugar tea can metabolize to produce lactic acid, acetic acid and other short-chain fatty acids, which play an important role in human intestinal health and immune function. Among them, compared with the QJ group, the FJ group showed better performance in various physical and chemical indexes, indicating that the use of the fermentation products of sugar tea to further ferment Gastrodia elata has a better effect on improving the quality of Gastrodia elata. At the same time, during the growth and metabolism of microorganisms, various biochemical reactions are catalyzed by the intracellular enzyme system, extracellular polysaccharides are secreted and ethanol is metabolized. Compared with the MJ group, the crude polysaccharide content in the FJ group increased by 70.21%, and the alcohol content increased to 0.54% vol. It can be seen that the fermentation with the compound microbial inoculant makes the FJ group metabolize more functional components and produce an alcohol fragrance.

[0139] 2. Analysis of Active Compounds in Gastrodia elata Samples

[0140] After HPLC analysis, with the mass concentration X (μg / mL) of the standard product as the abscissa and the average peak area Y as the ordinate, a standard curve was drawn, and regression analysis was performed based on the standard curve. The results showed that the linear relationships of the 6 standard products were good, and the regression equations and correlation coefficients are shown in Table 3.

[0141] Based on HPLC analysis of the active compounds in Gastrodia elata samples of different groups, the results are as Figure 1 shown. The peak results are as Figure 2 shown.

[0142] Table 3 Linear Relationships of Active Compounds (n = 6)

[0143]

[0144] According to Figure 1It can be seen that after enzymatic hydrolysis and fermentation, the active compounds in Gastrodia elata all showed differential changes. Among them, the contents of gastrodin, p-hydroxybenzyl alcohol, and baliside A changed most significantly (p<0.05). Relevant research shows that under the action of enzymes, balisoside A can be degraded into various products, including gastrodin, p-hydroxybenzyl alcohol, and balisoside B, etc. In the CK group of the embodiments of the present invention after enzymatic hydrolysis, a large amount of balisoside A was decomposed, and the contents of gastrodin and p-hydroxybenzyl alcohol increased significantly (p<0.05). Compared with the CK group, the contents of gastrodin and p-hydroxybenzyl alcohol in the MJ group increased by 66.17% and 25.76% respectively, and those in the FJ group increased by 80.14% and 35.65% respectively. Through the fermentation effect, the contents of gastrodin and p-hydroxybenzyl alcohol increased by 8.41% and 7.87% respectively compared with the MJ group, indicating that microbial metabolism can further degrade balisoside A and other substances that may contain gastrodin and p-hydroxybenzyl alcohol aglycones in Gastrodia elata. Secondly, after enzymatic hydrolysis and fermentation, the contents of citric acid ester E, citric acid ester B, and p-hydroxybenzaldehyde in Gastrodia elata also changed to a certain extent, but the changes were relatively small. And the content of active substances in the QJ group was slightly lower than that in the FJ group.

[0145] In addition, from the peak emergence results Figure 2 analysis shows that compared with the CK and MJ groups, more substances were detected in the FJ and QJ groups. For example, the substance with an emergence time of 1.527 min, and the peak area of such substances is relatively large and was not detected in the CK and MJ groups, indicating that this substance is a metabolite produced during the fermentation process. The overall situation shows that through compound microbial fermentation, it is beneficial for Gastrodia elata to metabolize and produce more active substances during the fermentation process, improving the added value of the product.

[0146] 3. Analysis of the contents of total polyphenols (TPC) and total flavonoids (TFC) in Gastrodia elata samples

[0147] The results are shown in Figure 3 , in the FJ group, the contents of TPC and TFC reached 39.87±1.55 mg GAE / g and 17.52±0.21 mg RE / g respectively, both significantly greater than those in the QJ group (35.86±1.25 mg GAE / g, 15.52±0.31 mg RE / g), the MJ group (30.80±0.89 mg GAE / g, 12.16±0.57 mg RE / g), and the CK group (24.95±0.97 mg GAE / g, 9.08±0.10 mg RE / g) (p<0.05), indicating that microbial fermentation can increase the contents of polyphenols and flavonoids in Gastrodia elata, and using sugar tea culture to ferment Gastrodia elata (i.e., the FJ group), the content of its metabolites is higher than that in the QJ group; overall, the contents of TPC and TFC showed FJ>QJ>MJ>CK, and this situation basically conforms to the HPLC determination results.

[0148] 4. Determination of the antioxidant activity of Gastrodia elata samples

[0149] The results are shown in Figures 4 - 7 . According to Figure 4 , by comparing the differences in the ferric ion reducing ability of Gastrodia elata samples in the CK group, MJ group, FJ group, and QJ group, it was found that there were significant differences in the ferric ion reducing ability among the four groups of samples (P<0.05). The order of the ferric ion reducing ability was FJ group > QJ group > MJ group > CK group. At the same time, the differences in the scavenging abilities of three groups of Gastrodia elata samples against DPPH radicals, ABTS radicals, and hydroxyl radicals were compared.

[0150] As Figures 5 - 7 , the smaller the IC 50 value, the stronger the radical scavenging ability. It was found that there were significant differences in the radical scavenging abilities of the four groups of samples, and they all showed the same trend: FJ group > QJ group > MJ group > CK group, but their antioxidant activities were all lower than that of the positive control group. The overall situation indicated that the in vitro antioxidant ability of fermented Gastrodia elata samples (FJ and QJ groups) was enhanced, but the antioxidant ability of the QJ group was slightly lower than that of the FJ group. Microbial fermentation could change the structure and content of active ingredients in Gastrodia elata. This result also showed that there was a positive correlation between antioxidant ability and the contents of total polyphenols and total flavonoids, which was in line with the relevant research results. Secondly, the antioxidant ability of enzymatically hydrolyzed Gastrodia elata samples (MJ group) was greater than that of the CK group. Active compounds such as total polyphenols and total flavonoids were decomposed during the enzymatic hydrolysis process, improving the antioxidant activity of Gastrodia elata samples.

[0151] 5. Analysis of α-glucosidase inhibitory ability

[0152] The results are shown in Figure 8 . There were significant differences in the α-glucosidase inhibitory abilities of different groups of Gastrodia elata. Among them, the FJ group showed the best performance in the four test groups (IC 50 = 0.69 ± 0.12 mg / mL), followed by the QJ group (IC 50 = 0.88 ± 1.14 mg / mL) and the MJ group (IC 50 = 1.17 ± 1.14 mg / mL), but they were lower than the α-glucosidase inhibitory ability of the positive control group. Through microbial fermentation, the inhibitory ability of Gastrodia elata against α-glucosidase activity was significantly improved (P<0.05), which was of great significance for the research on reducing postprandial blood glucose in the deep processing field of the Gastrodia elata industry.

[0153] 6. Sensory evaluation

[0154] The samples of each group were subjected to sensory evaluation, and their color, body state, odor, taste, and texture were quantitatively described and scored. The results are shown in Figure 9As shown. Through microbial fermentation, not only the active ingredients and functional activities of Gastrodia elata were changed, but also the Gastrodia elata samples in the fermentation groups (FJ, QJ) were better improved in terms of color, body, odor, taste, and texture. The sensory scores of the two groups were similar. The odor showed obvious mellow and sweet scents, and there was almost no unpleasant odor of Gastrodia elata. The color was reddish-brown, the body was relatively clear, and the taste was moderately sour and sweet; while the color of the MJ group was dull, and there was still an obvious unpleasant odor of Gastrodia elata. Overall, the sensory characteristics of Gastrodia elata products were significantly improved by microbial fermentation.

[0155] 7. Correlation analysis

[0156] Based on the above results, the correlation analysis of total polyphenols and total flavonoids with antioxidant capacity and hypoglycemic capacity in Gastrodia elata samples of each group was carried out as Figure 10 Analysis (*p <= 0.05, **p <= 0.01, ***p <= 0.001) showed that the total polyphenol content was significantly positively correlated with the antioxidant activity (DPPH radical scavenging ability, ABTS radical scavenging ability, hydroxyl radical scavenging ability, ferric ion reducing ability) and hypoglycemic activity (α-glucosidase inhibitory ability) of the samples; at the same time, the total flavonoid content was also significantly positively correlated with the antioxidant activity and hypoglycemic activity of the samples. This result indicates that the total polyphenol and total flavonoid contents have a positive effect on antioxidant activity and hypoglycemic activity. At the same time, relevant studies have also shown that flavonoid compounds and coumarin compounds have antioxidant pharmacological activities, and even they may jointly scavenge free radicals and reduce the level of oxidative stress through synergistic effects.

[0157] In summary, the basic physical and chemical properties, active substance components, in vitro antioxidant activity, and in vitro hypoglycemic activity of the fermented Gastrodia elata samples have all changed. The specific results show that compared with the CK group, the other treatment groups have improved in various physical and chemical indexes and active abilities. Among them, the gastrodin and p-hydroxybenzyl alcohol in the MJ group increased by 66.17% and 25.76% respectively, and the gastrodin and p-hydroxybenzyl alcohol in the FJ group increased by 80.14% and 35.65% respectively. Compared with the MJ group, the total acid content of the FJ group's Gastrodia elata fermentation broth increased by 2.24 times, the pH value decreased, and the crude polysaccharide and alcohol contents also increased significantly; the contents of gastrodin and p-hydroxybenzyl alcohol in Gastrodia elata after microbial fermentation increased significantly, and at the same time, some substances with higher contents were additionally metabolized, but they were not identified in this study; at the same time, the TPC and TFC contents of the FJ group were the highest, reaching 39.87±1.55mg GAE / g and 17.52±0.21mg RE / g respectively, increasing by 29.45% and 44.08% respectively compared with the control group CK. At the same time, the antioxidant ability and hypoglycemic ability were significantly improved, all showing: FJ group > QJ group > MJ group > CK group, indicating that microbial fermentation has a significant effect on enhancing antioxidant activity and hypoglycemic activity. Through sensory evaluation analysis, the FJ group has better quality, which confirms the role of microbial fermentation in improving product flavor and helps to further develop new Gastrodia elata functional foods.

[0158] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method for improving the quality of Gastrodia elata, characterized in that: include: Ferment Gastrodia elata using the fermentation products of sugar tea.

2. The method for preparing gastrodia elata for improving its quality according to claim 1, characterized in that: The sugar tea fermentation product is formed by fermenting the sugar tea with a composite microbial agent, wherein the composite microbial agent consists of lactic acid bacteria, acetic acid bacteria and yeast.

3. The method for preparing gastrodia elata with improved quality according to claim 2, characterized in that: The ratio of the colony counts of the lactic acid bacteria, the acetic acid bacteria and the yeast is (0.5-5):(0.1-1):

1.

4. The method for preparing gastrodia elata for improving its quality according to claim 2, characterized in that: The steps of preparing the sugar tea include: mixing tea leaves, sugar and water, boiling them, and then filtering them, wherein the concentration of tea leaves is 8-12 g / L, and the concentration of sugar is 120-220 g / L.

5. The method for preparing gastrodia elata with improved quality according to any one of claims 2 to 4, characterized in that: The composite microbial agent is added into the sugar tea water at a volume fraction of 3-7% for fermentation; Preferably, the conditions for fermenting the sugar tea include: a temperature of 25-32° C. and a fermentation time of 5-7 days.

6. The method for preparing gastrodia elata with improved quality according to claim 1, characterized in that: Also included is forming a Gastrodia elata culture medium; Preferably, the preparation step of the Gastrodia elata culture medium comprises: mixing Gastrodia elata with sugar water, then adding enzyme for enzymolysis, and sterilizing after enzymolysis.

7. The method for preparing gastrodia elata for improving its quality according to claim 6, characterized in that: The solid-liquid ratio of the gastrodia elata to the sugar water is 1:(8-12); the mass concentration of the sugar water is 70-90g / L; Preferably, the enzyme comprises any two of cellulase, amylase and pectinase; The concentrations of the cellulase, the amylase and the pectinase are 0.2-0.4 g / L respectively; Preferably, the enzymatic hydrolysis process comprises enzymatic hydrolysis with cellulase or pectinase at 40-60°C for 45-75 minutes, and enzymatic hydrolysis with amylase at 70-90°C for 45-75 minutes.

8. The method for preparing gastrodia elata for improving its quality according to claim 6, characterized in that: The sugar tea fermentation product is inoculated into the Gastrodia elata culture medium at a volume fraction of 3-7% for cultivation; Preferably, the culture conditions include: a temperature of 28-32°C and a culture time of 4-5 days.

9. A fermented gastrodia elata liquid, characterized in that: The gastrodia elata is prepared by the preparation method for improving the quality of gastrodia elata as described in any one of claims 1 to 8.

10. A gastrodia powder, characterized in that: The method is formed by freeze-drying the Gastrodia elata fermentation liquid according to claim 9.

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