Gastrodia elata products with high conversion rate of p-hydroxybenzyl alcohol and preparation method

Abstract

The present invention belongs to the field of application of microbial fermentation technology, and discloses a gastrodia elata product with high conversion rate of p-hydroxybenzyl alcohol and a preparation method thereof. The present invention adopts a combined method of complex enzymatic hydrolysis and co-fermentation with a mixed strain of yeast-lactic acid bacteria, and uses enzymatic hydrolysis and microbial metabolism to convert gastrodin into p-hydroxybenzyl alcohol, thereby increasing the content of p-hydroxybenzyl alcohol in the product. Finally, the content of p-hydroxybenzyl alcohol in the gastrodia elata fermented oral liquid reaches 0.744 mg / mL. At the same time, the fermentation time in the method of the present invention is short, and the production efficiency of the product is high.

Description

Technical Field

[0001] The present invention relates to the field of application of microbial fermentation technology, and particularly relates to a gastrodia elata product with a high conversion rate of p-hydroxybenzyl alcohol and a preparation method thereof. Background Art

[0002] Gastrodia elata is mainly divided into several variants such as Gastrodia elata Bl. f. glauca S. Chow, Gastrodia elata Bl. f. elata, Gastrodia elata Bl. f. flavida S. Chow, and Gastrodia elata Bl. f. viridis Makino according to variant differences. At present, it is considered that the active ingredients of gastrodia elata mainly include phenolic components such as gastrodin, p-hydroxybenzyl alcohol (gastrodigenin), and parishin glycosides, and have various pharmacological activities such as improving sleep, antidepressant, antiepileptic, and antioxidant.

[0003] Both gastrodin and p-hydroxybenzyl alcohol (gastrodigenin) are the main active ingredients of gastrodia elata, and their pharmacological effects are similar. Gastrodigenin, with the chemical name of p-hydroxybenzyl alcohol, is the aglycone of gastrodin and also the metabolite of gastrodin in vivo. From a structural perspective, gastrodigenin has a smaller relative molecular mass and is amphiphilic; in terms of function, the results of Caco-2 cell transport studies on gastrodin and gastrodigenin show that the oral absorption and utilization rate of gastrodin may not be high, which is related to its poor liposolubility, while the absorption of gastrodigenin is better. The results of MDCK-MDR1 and MDCK cell transport studies on gastrodin and gastrodigenin show that gastrodin is difficult to penetrate, while gastrodigenin is easy to penetrate. The results of the two cell transport experiments show that gastrodigenin has a stronger transmembrane ability than gastrodin and is more likely to pass through the blood-brain barrier. In addition, the results of the intestinal absorption characteristics of gastrodigenin show that p-hydroxybenzyl alcohol mainly enters the body through passive diffusion in the intestine, has good absorption in each intestinal segment, and there is no intestinal segment difference, which is more suitable for the development of oral preparations.

[0004] The research on the functional components of traditional Gastrodia elata products has mainly focused on gastrodin, with less research on p-hydroxybenzyl alcohol. In terms of medicinal value, there has been more research on gastrodin and p-hydroxybenzyl alcohol injection, and less research on oral liquid. In terms of edible value, most of the products are Gastrodia elata wine, and there is less Gastrodia elata fermentation broth. Application No. 201110211423.4 discloses a traditional Chinese medicine medicinal liquor with anti-fatigue and immune-enhancing effects. The raw materials are a combination of various traditional Chinese medicines, and it is soaked in 60-degree sorghum liquor by the traditional cold soaking method for 90 days. This process is the traditional cold soaking method of liquor, not a fermentation method. Application No. 202310667058.0 discloses a brewing method of Gastrodia elata wine. Through the fermentation of fresh Gastrodia elata, the liquid is distilled by heating the material at high temperature. This process solves the problem that the Gastrodia elata components in the Gastrodia elata health wine brewed by the traditional liquor soaking method cannot be completely integrated with the liquor, and the Gastrodia elata flavor is weak after soaking, but it does not verify its functional components. Application No. 201911125147.2 discloses a method for preparing Gastrodia elata wine using Gastrodia elata medicinal koji. Using glutinous rice and Gastrodia elata residue as raw materials, it is fermented with Gastrodia elata medicinal koji and distilled to obtain rice wine distilled liquor containing gastrodin, and the gastrodin in Gastrodia elata powder is extracted by reflux. The content of gastrodin in this Gastrodia elata wine is 1000 μg / g, and the research on active ingredients focuses on gastrodin, without reflecting p-hydroxybenzyl alcohol. Application No. 202110603564.4 discloses a Lactobacillus plantarum and its preparation method and application for fermenting Gastrodia elata oral liquid. The content of gastrodin in the fermented Gastrodia elata oral liquid prepared by this invention is 6.16 mg / ml, and it is clear that the product has health care functions such as improving sleep and promoting the balance of intestinal microecology, but it does not mention the component detection and efficacy of p-hydroxybenzyl alcohol. Application No. 201610635648.5 discloses an enzyme-treated Gastrodia elata health drink for improving sleep, its preparation process and application. This product uses fresh Gastrodia elata as the main raw material, and is fermented by the directional enzyme treatment process with fresh rose, coix seed, wheat, wild jujube seed, mulberry, and wolfberry as auxiliary materials. The detection of active ingredients in the product reflects gastrodin and gastrodigenin. The highest value of p-hydroxybenzyl alcohol in this enzyme-treated health drink is 384 μg / ml, but the content is relatively low, the conversion and utilization rate of gastrodin is not high, and the production process takes a long time, 3-5 months.

[0005] In view of this, the present invention is specifically proposed to solve the technical problem of the low conversion rate of p-hydroxybenzyl alcohol in Gastrodia elata in the prior art. Summary of the Invention

[0006] In order to overcome the defects of existing products, the present invention uses Gastrodia elata as raw material, and jointly ferments with cellulase and α-amylase composite enzymes, yeast and lactic acid bacteria composite strains to prepare a high-conversion rate p-hydroxybenzyl alcohol Gastrodia elata product, namely, Gastrodia elata fermented oral liquid. The p-hydroxybenzyl alcohol content in the Gastrodia elata product is 0.744 mg / mL, and gastrodin is almost completely converted into p-hydroxybenzyl alcohol. Because the product has a high conversion rate of p-hydroxybenzyl alcohol, which is increased by 94% compared with existing products, the product has stronger transmembrane ability, is easier to pass through the blood-brain barrier, and is easier to be absorbed by the intestine than gastrodin functional products.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for preparing a gastrodia elata product with a high conversion rate of p-hydroxybenzyl alcohol, comprising:

[0009] After the pre-treated Gastrodia elata is ultra-low temperature pre-frozen, vacuum freeze-drying is performed to obtain cooked Gastrodia elata;

[0010] The cooked Gastrodia elata is crushed into Gastrodia elata powder, water and white sugar are added and stirred to dissolve, a composite enzyme is added for enzymolysis to obtain a fermentation base liquid, and yeast and lactic acid bacteria are added for combined fermentation to obtain a fermented Gastrodia elata liquid;

[0011] The fermented gastrodia elata liquid is filtered and centrifuged to obtain the supernatant to obtain the gastrodia elata product.

[0012] Preferably, the pretreatment of Gastrodia elata is to wash the fresh Gastrodia elata, steam it until cooked through, and cool it.

[0013] Preferably, the ultra-low temperature pre-freezing temperature is -40 to -50°C, and the time is 10 to 12 hours; the vacuum freeze-drying temperature is 42 to 48°C, and the time is 8 to 12 hours.

[0014] Preferably, the complex enzyme is obtained by mixing α-amylase and cellulase in a mass ratio of 1:1 to 3:1.

[0015] Preferably, the added amount of the complex enzyme is 0.3% to 0.5% of the total mass of gastrodia powder, water and white sugar, the enzymatic hydrolysis temperature is 50 to 60° C., and the time is 20 to 30 min.

[0016] Preferably, the mass ratio of the yeast to the lactic acid bacteria is 1:1 to 5:1, and the total mass of the yeast and the lactic acid bacteria is 0.2% to 0.4% of the mass of the fermentation base liquid.

[0017] Preferably, the lactic acid bacteria include Lactobacillus bulgaricus Lactobacillus bulgaricus (LB57), Streptococcus thermophilus Streptococcus thermophilus (ST021), Lactobacillus plantarum Lactobacillus plantarum (LP121), Lactobacillus casei Lactobacillus casei (LC39), Lactobacillus salivarius Lactobacillus salivarius (LS17), Lactobacillus reuteri Lactobacillus reuteri (LR09), Lactobacillus reuteri Lactobacillus helveticus( (LH17), Bifidobacterium animalis Bifidobacterium animalis (BAA68), Leuconostoc mesenteroides, any one or more of them.

[0018] Preferably, the yeast includes any one or more of Saccharomyces cerevisiae 1305, Saccharomyces cerevisiae EC1118, and general wine yeast LA-C12.

[0019] Preferably, the temperature of the co-fermentation is 37 - 45 °C, the relative humidity is 85%, and the anaerobic fermentation is carried out for 14 - 21 days.

[0020] The present invention also discloses a Gastrodia elata product with a high conversion rate of p-hydroxybenzyl alcohol obtained by using any of the above preparation methods.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The present invention adopts a combined fermentation of complex enzymatic hydrolysis and yeast-lactic acid bacteria mixed strains. The conversion of gastrodin to p-hydroxybenzyl alcohol is achieved through enzymatic hydrolysis and microbial metabolism, thereby increasing the content of p-hydroxybenzyl alcohol in the product. Finally, the content of the target substance (p-hydroxybenzyl alcohol) in the Gastrodia elata fermentation oral liquid reaches 0.744 mg / mL, which is much higher than the content of 0.384 mg / mL of p-hydroxybenzyl alcohol in the Gastrodia elata fermented drink prepared in Patent CN201610635648.5. Based on the above invention, the content of p-hydroxybenzyl alcohol in the present invention is increased by 94%.

[0023] 2. The fermentation cycle of the present invention is shorter than that of other Gastrodia elata fermentation products. The fermentation time in Patent CN201610635648.5 is 3 - 5 months, while the fermentation process of the Gastrodia elata product of the present invention only takes up to 14 days at most, and the production efficiency of the product is high.

[0024] 3. The raw material of the present invention adopts vacuum freeze-drying, which can ensure the stability of the fermentation process and the content of functional ingredients in the product. Armillaria mellea is a symbiotic fungus of Gastrodia elata, which provides nutrients for the growth and development of Gastrodia elata. Different Armillaria mellea strains have different abilities to form rhizomorphs and the ability of rhizomorphs to invade the cortical tissue of Gastrodia elata. The cortex of fresh Gastrodia elata is prone to residual hyphae invaded by Armillaria mellea. Most existing Gastrodia elata fermentation products use fresh Gastrodia elata tubers as raw materials. If the raw materials are not properly treated or the fermentation substrate liquid is not thoroughly sterilized during the fermentation process, it is easy to cause contamination by miscellaneous bacteria, resulting in fermentation failure of the product, or a decrease in the content of target active ingredients in the product. Using freeze-dried Gastrodia elata as the raw material has low requirements for the sterilization process and can ensure the stability during the product fermentation process.

[0025] 4. The present invention adopts a vacuum freeze-drying process for gastrodia elata raw materials, which can ensure the quality stability of fermentation raw materials and is suitable for batch production in any period of the whole year in factories. Compared with fresh gastrodia elata, the vacuum freeze-dried gastrodia elata tablets prepared by vacuum-freezing gastrodia elata can solve the problem of product surplus during the concentrated listing period of gastrodia elata and insufficient raw material supply during the non-production period; compared with traditional dried gastrodia elata powder, freeze-dried gastrodia elata can retain the active ingredients of the product to the greatest extent, which is beneficial to the conversion of p-hydroxybenzyl alcohol during the subsequent fermentation process of the product.

[0026] 5. Since the present invention adopts a pretreatment method of steaming in water and then conducts vacuum freeze-drying, compared with directly freeze-drying raw gastrodia elata, the pretreatment of steaming gastrodia elata in water can improve the active ingredients of gastrodin and p-hydroxybenzyl alcohol in the raw materials, and further increase the content and conversion rate of p-hydroxybenzyl alcohol in the fermentation product. The content of p-hydroxybenzyl alcohol in raw gastrodia elata freeze-dried tablets is relatively high, while the content of gastrodin is relatively low, and vice versa for cooked gastrodia elata freeze-dried tablets. However, in terms of the total amount of gastrodin and p-hydroxybenzyl alcohol, the cooked gastrodia elata freeze-dried tablets are higher than the raw gastrodia elata freeze-dried tablets. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 图1 HPLC chromatogram of active ingredients of gastrodia elata fermentation oral liquid prepared in Example 1;

[0028] 图2 HPLC chromatogram of active ingredients of gastrodia elata fermentation oral liquid prepared in Example 2;

[0029] 图3 HPLC chromatogram of active ingredients of gastrodia elata fermentation oral liquid prepared in Example 3;

[0030] 图4 HPLC chromatogram of active ingredients of gastrodia elata fermentation oral liquid prepared in Example 4;

[0031] 图5 HPLC chromatogram of active ingredients of gastrodia elata fermentation oral liquid prepared in Example 5;

[0032] 图6 HPLC chromatogram of active ingredients of gastrodia elata fermentation oral liquid prepared in Comparative Example 2;

[0033] 图7 HPLC chromatogram of active ingredients of gastrodia elata fermentation oral liquid prepared in Comparative Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention will be specifically described below through examples. It is necessary to point out here that the following examples are only used to further illustrate the present invention and should not be construed as limiting the protection scope of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention according to the above content of the present invention.

[0035] The present invention provides a method for preparing a Gastrodia elata product with a high conversion rate of p-hydroxybenzyl alcohol, comprising:

[0036] After the pre-treated Gastrodia elata is ultra-low temperature pre-frozen, vacuum freeze-drying is performed to obtain cooked Gastrodia elata;

[0037] The cooked Gastrodia elata is crushed into Gastrodia elata powder, water and white sugar are added and stirred to dissolve, a composite enzyme is added for enzymolysis to obtain a fermentation base liquid, and yeast and lactic acid bacteria are added for combined fermentation to obtain a fermented Gastrodia elata liquid;

[0038] The fermented gastrodia elata liquid is filtered and centrifuged to obtain the supernatant to obtain the gastrodia elata product.

[0039] The embodiment of the present invention uses yeast and lactic acid bacteria to jointly ferment the fermented gastrodia elata base liquid after enzymatic hydrolysis, and can utilize microbial metabolism to convert gastrodin into p-hydroxybenzyl alcohol, thereby significantly increasing the content of p-hydroxybenzyl alcohol in the gastrodia elata product. The gastrodia elata product prepared in the embodiment of the present invention is also the fermented gastrodia elata oral liquid.

[0040] There is no special limitation on the selection of Gastrodia elata raw materials in the embodiment of the present invention. This embodiment is applicable to all varieties of Gastrodia elata, for example, Gastrodia elata from Dafang County, Hezhang County, Dejiang County and other Guizhou production areas, Gastrodia elata from Yunnan production areas, and Gastrodia elata from Shaanxi, Anhui and other production areas.

[0041] The pretreatment of Gastrodia elata is to wash the fresh Gastrodia elata, steam it until it is cooked, and cool it. In a preferred embodiment, steaming the Gastrodia elata until it is cooked is preferably done by steaming in a water bath, which can increase the active ingredients of gastrodin and p-hydroxybenzyl alcohol in the raw material.

[0042] Before the pre-treated Gastrodia elata is subjected to ultra-low temperature pre-freezing, the pre-treated Gastrodia elata can be sliced ​​to make the freezing more uniform; there is no need to specifically limit the thickness of the slices, which can be adjusted according to actual production needs, for example, the thickness of the Gastrodia elata slices can be 5-8 mm.

[0043] There is no need to specifically define the specific parameters of ultra-low temperature pre-freezing. The method of ultra-low temperature pre-freezing is a conventional technical means in the art. The specific parameters can be adaptively adjusted according to the actual production needs for the raw materials. For example, the temperature of ultra-low temperature pre-freezing is -40~-50°C, and the time is 10~12h. The parameters for vacuum freeze drying are also the parameters commonly used in the art for preparing freeze-dried products. There is no need to specifically define them. They can be adjusted according to actual production needs. For example, the temperature of vacuum freeze drying is 42°C~48°C, and the time is 8~12h. The moisture content in the cooked Gastrodia elata obtained after vacuum freeze drying is less than 5%. The Gastrodia elata slices treated with vacuum freeze drying are slightly yellow, uniform in color, and maintain their original shape. There are no obvious visible pores on the surface of Gastrodia elata, and the red spores are intact.

[0044] In a preferred embodiment, the water added to the gastrodia powder is sterile water. The water addition ratio is the mass ratio of gastrodia powder to water volume (g / mL) of 1:8 to 1:10, and the addition amount of white granulated sugar is 8-10% of the total mass of the gastrodia powder and water mixture.

[0045] In a preferred embodiment, the composite enzyme is obtained by mixing α-amylase and cellulase in a mass ratio of 1:1 to 3:1. The addition amount of the composite enzyme is 0.3% to 0.5% of the total mass of the gastrodia powder, water, and white granulated sugar. The temperature of enzymatic hydrolysis is 50 to 60 °C, and the time is 20 to 30 min.

[0046] In a preferred embodiment, the mass ratio of the yeast to the lactic acid bacteria is 1:1 to 5:1. In the co-fermentation process of the present invention, the yeast plays a leading role, and the conversion rate of p-hydroxybenzyl alcohol is increased by controlling the dosage relationship between the yeast and the lactic acid bacteria; the total mass of the yeast and the lactic acid bacteria is 0.2% to 0.4% of the mass of the fermentation substrate liquid.

[0047] It should be noted that the key to achieving a high conversion rate of p-hydroxybenzyl alcohol in the present invention lies in the co-fermentation of lactic acid bacteria and yeast. Therefore, for the specific strain selection of lactic acid bacteria and yeast, there is no special limitation. The fermentation mechanisms of strains belonging to the same genus are similar, and the final effects achieved are also similar or the same. Commonly used lactic acid bacteria and yeast can all achieve the present invention. Specifically, for example, the lactic acid bacteria include Lactobacillus bulgaricus Lactobacillus bulgaricus (LB57), Streptococcus thermophilus Streptococcus thermophilus (ST021), Lactobacillus plantarum Lactobacillus plantarum (LP121), Lactobacillus casei Lactobacillus casei (LC39), Lactobacillus salivarius Lactobacillus salivarius (LS17), Lactobacillus reuteri Lactobacillus reuteri (LR09), Lactobacillus reuteri Lactobacillus helveticus( LH17), Bifidobacterium animalis Bifidobacterium animalis (BAA68) Leuconostoc mesenteroides, any one or more of them. For example, by weight, the lactic acid bacteria can be composed of 20% Lactobacillus bulgaricus, 10% Lactobacillus plantarum, 10% Streptococcus thermophilus, and 10% Leuconostoc mesenteroides. At this time, the yeast is also 50%.

[0048] For example, the yeast includes any one or more of Saccharomyces cerevisiae 1305, Saccharomyces cerevisiae EC1118, and general wine yeast LA-C12.

[0049] In a preferred embodiment, the temperature of the co-fermentation is 37-45 °C, the relative humidity is 85%, and anaerobic fermentation is carried out for 14-21 days.

[0050] The Gastrodia elata product prepared by the embodiment of the present invention, namely the Gastrodia elata fermented oral liquid, has clear liquid, slightly yellow color, slightly sour and sweet taste, with a slight Gastrodia elata flavor and a unique aroma.

[0051] To make the technical solution of the present invention clearer, the preparation method and performance of the Gastrodia elata product will be described below through multiple examples.

[0052] Some of the product manufacturers or models used in the embodiments of the present invention are shown in Table 1.

[0053] Table 1 Some of the product manufacturers or models used in the examples

[0054] 。

[0055] Example 1

[0056] (1) Raw material pretreatment: Select fresh Gastrodia elata, wash it, steam it over water until half-cooked, cool it in a clean environment, and then slice it after cooling. The thickness of the Gastrodia elata slices is 6 mm.

[0057] (2) Ultra-low temperature precooling and vacuum freeze-drying: Place the Gastrodia elata slices in an ultra-low temperature freezer for precooling. The ultra-low temperature is -45 °C and the time is 12 h. Then transfer them to a vacuum cooler, with the parameters set at 45 °C and 12 h. After vacuum freeze-drying, cooked Gastrodia elata freeze-dried slices are obtained.

[0058] (3) Mixed fermentation: Crush the cooked Gastrodia elata freeze-dried slices to obtain Gastrodia elata freeze-dried powder. Add sterile water according to the mass-volume ratio (g / mL) of Gastrodia elata: sterile water of 1:8. Further add granulated sugar at 10% of the total mass of the Gastrodia elata powder-sterile water mixture, and stir well to dissolve to obtain a Gastrodia elata freeze-dried paste. Add a composite enzyme, α-amylase: cellulase = 2:1, and the addition amount is 0.3% of the Gastrodia elata freeze-dried paste. The enzymatic hydrolysis temperature is 55 °C and the enzymatic hydrolysis time is 20 min. After enzymatic hydrolysis, a Gastrodia elata fermentation base liquid is obtained. Further add a mixed bacteria of yeast and lactic acid bacteria for combined fermentation. The composition of the mixed bacteria is 50% Saccharomyces cerevisiae, 20% Lactobacillus bulgaricus, 10% Lactobacillus plantarum, 10% Streptococcus thermophilus, and 10% Leuconostoc mesenteroides. The addition amount is 0.2% of the mass of the fermentation base liquid. The fermentation temperature is 37 °C, the fermentation humidity is 85%, and anaerobic fermentation is carried out for 14 d.

[0059] (4) Filtration and centrifugation: After fermentation is completed, filter with sterile gauze. The filtered liquid is centrifuged at low temperature. The centrifugation speed is 5000 rpm and the centrifugation time is 5 min. Take the supernatant to obtain Gastrodia elata fermented oral liquid.

[0060] Example 2

[0061] (1) Raw material pretreatment: Select fresh Gastrodia elata, wash it, steam it over water until half-cooked, cool it in a clean environment, and then slice it after cooling. The thickness of the Gastrodia elata slices is 6 mm.

[0062] (2) Ultra-low temperature precooling and vacuum freeze-drying: Place the gastrodia elata slices in an ultra-low temperature freezer for precooling. The ultra-low temperature is -45°C and the time is 12 h. Then transfer them to a vacuum cooler with the parameters set at 45°C for 12 h. After vacuum freeze-drying, cooked gastrodia elata freeze-dried slices are obtained.

[0063] (3) Mixed fermentation: Crush the cooked gastrodia elata freeze-dried slices to obtain gastrodia elata freeze-dried powder. Add sterile water according to the mass-volume ratio of gastrodia elata to sterile water (g / mL) of 1:8. Further add granulated sugar at 10% of the total mass of the gastrodia elata powder-sterile water mixture and stir well to dissolve to obtain a gastrodia elata freeze-dried paste. Add a composite enzyme, α-amylase: cellulase = 2:1, and the addition amount is 0.3% of the gastrodia elata freeze-dried paste. The enzymatic hydrolysis temperature is 55°C and the enzymatic hydrolysis time is 20 min. After enzymatic hydrolysis, a gastrodia elata fermentation base liquid is obtained. Further add a mixed strain of yeast and lactic acid bacteria for combined fermentation. The composition of the mixed strain is 55% Saccharomyces cerevisiae, 25% Lactobacillus bulgaricus, and 20% Streptococcus thermophilus. The addition amount is 0.2% of the mass of the fermentation base liquid. The fermentation temperature is 37°C, the fermentation humidity is 85%, and anaerobic fermentation is carried out for 14 d.

[0064] (4) Filtration and centrifugation: After fermentation is completed, filter with a sterile gauze. The filtered liquid is centrifuged at a low temperature. The centrifugation speed is 5000 rpm and the centrifugation time is 5 min. Take the supernatant to obtain gastrodia elata fermentation oral liquid.

[0065] Example 3

[0066] (1) Raw material pretreatment: Select fresh gastrodia elata, wash it, steam it until half-cooked over water, cool it in a clean environment, and then slice it after cooling. The thickness of the gastrodia elata slices is 6 mm.

[0067] (2) Ultra-low temperature precooling and vacuum freeze-drying: Place the gastrodia elata slices in an ultra-low temperature freezer for precooling. The ultra-low temperature is -45°C and the time is 12 h. Then transfer them to a vacuum cooler with the parameters set at 45°C for 12 h. After vacuum freeze-drying, cooked gastrodia elata freeze-dried slices are obtained.

[0068] (3) Mixed fermentation: The cooked Gastrodia elata freeze-dried slices are crushed to obtain Gastrodia elata freeze-dried powder. Sterile water is added according to the mass-volume ratio of Gastrodia elata to sterile water (g / mL) of 1:8. Further, granulated sugar is added at 10% of the total mass of the Gastrodia elata powder-sterile water mixture, and it is fully stirred and dissolved to obtain a Gastrodia elata freeze-dried paste. A compound enzyme is added, α-amylase: cellulase = 2:1, and the addition amount is 0.5% of the Gastrodia elata freeze-dried paste. The enzymatic hydrolysis temperature is 55 °C, and the enzymatic hydrolysis time is 20 min. After enzymatic hydrolysis, a Gastrodia elata fermentation base liquid is obtained. Further, a mixed bacteria of yeast and lactic acid bacteria are added for combined fermentation. The composition of the mixed bacteria is 60% Saccharomyces cerevisiae, 20% Lactobacillus plantarum, and 20% Leuconostoc mesenteroides. The addition amount is 0.4% of the mass of the fermentation base liquid. The fermentation temperature is 37 °C, the fermentation humidity is 85%, and anaerobic fermentation is carried out for 14 days.

[0069] (4) Filtration and centrifugation: After fermentation is completed, it is filtered with sterile gauze. The filtered liquid is centrifuged at low temperature. The centrifugation speed is 3000 - 5000 rpm, and the centrifugation time is 5 min. The supernatant is taken to obtain Gastrodia elata fermented oral liquid.

[0070] Example 4

[0071] (1) Raw material pretreatment: Fresh Gastrodia elata is selected, washed, steamed until half-cooked in a water bath, cooled in a clean environment, and then sliced. The thickness of the Gastrodia elata slices is 6 mm.

[0072] (2) Ultra-low temperature precooling and vacuum freeze-drying: The Gastrodia elata slices are placed in an ultra-low temperature freezer for precooling. The ultra-low temperature freezing temperature is -45 °C, and the time is 12 h. Then it is transferred to a vacuum cooler, and the parameters are set at 45 °C for 12 h. After vacuum freeze-drying, cooked Gastrodia elata freeze-dried slices are obtained.

[0073] (3) Mixed fermentation: The cooked Gastrodia elata freeze-dried slices are crushed to obtain Gastrodia elata freeze-dried powder. Sterile water is added according to the mass-volume ratio of Gastrodia elata to sterile water (g / mL) of 1:8. Further, granulated sugar is added at 10% of the total mass of the Gastrodia elata powder-sterile water mixture, and it is fully stirred and dissolved to obtain a Gastrodia elata freeze-dried paste. A compound enzyme is added, α-amylase: cellulase = 3:1, and the addition amount is 0.3% of the Gastrodia elata freeze-dried paste. The enzymatic hydrolysis temperature is 55 °C, and the enzymatic hydrolysis time is 20 min. After enzymatic hydrolysis, a Gastrodia elata fermentation base liquid is obtained. Further, a mixed bacteria of yeast and lactic acid bacteria are added for combined fermentation. The composition of the mixed bacteria is 50% Saccharomyces cerevisiae and 50% Lactobacillus plantarum. The addition amount is 0.3% of the mass of the fermentation base liquid. The fermentation temperature is 37 °C, the fermentation humidity is 85%, and anaerobic fermentation is carried out for 14 days.

[0074] (4) Filtration and centrifugation: After fermentation is completed, it is filtered with sterile gauze. The filtered liquid is centrifuged at low temperature. The centrifugation speed is 5000 rpm, and the centrifugation time is 5 min. The supernatant is taken to obtain Gastrodia elata fermented oral liquid.

[0075] Example 5

[0076] (1)Raw material pretreatment: Select fresh gastrodia elata, wash it, steam it over water until half-cooked, cool it in a clean environment, and slice it after cooling. The thickness of the gastrodia elata slices is 6 mm.

[0077] (2)Ultra-low temperature precooling and vacuum freeze-drying: Place the gastrodia elata slices in an ultra-low temperature freezer for precooling. The ultra-low temperature is -45 °C and the time is 12 h. Then transfer them to a vacuum cooler with parameters set at 45 °C for 12 h. After vacuum freeze-drying, cooked gastrodia elata freeze-dried slices are obtained.

[0078] (3)Mixed fermentation: Crush the cooked gastrodia elata freeze-dried slices to obtain gastrodia elata freeze-dried powder. Add sterile water according to the mass-volume ratio (g / mL) of gastrodia elata: sterile water of 1:8. Further add granulated sugar at 10% of the total mass of the gastrodia elata powder-sterile water mixture, and stir well to dissolve to obtain a gastrodia elata freeze-dried paste. Add a composite enzyme, α-amylase: cellulase = 1:1, and the addition amount is 0.4% of the gastrodia elata freeze-dried paste. The enzymatic hydrolysis temperature is 55 °C and the enzymatic hydrolysis time is 20 min. After enzymatic hydrolysis, a gastrodia elata fermentation base liquid is obtained. Further add a mixed strain of yeast and lactic acid bacteria for combined fermentation. The composition of the mixed strain is 83% Saccharomyces cerevisiae, 10% Lactobacillus bulgaricus, and 7% Streptococcus thermophilus. The addition amount is 0.3% of the mass of the fermentation base liquid. The fermentation temperature is 37 °C, the fermentation humidity is 85%, and anaerobic fermentation is carried out for 14 d.

[0079] (4)Filtration and centrifugation: After fermentation, filter with a sterile gauze. The filtered liquid is centrifuged at low temperature. The centrifugation speed is 5000 rpm and the centrifugation time is 5 min. Take the supernatant to obtain gastrodia elata fermentation oral liquid.

[0080] Test Example 1: Example 1 - Enzyme-fermented health drink 1 in Chinese Patent CN201610635648.5.

[0081] Test Example 2: Example 2 - Enzyme-fermented health drink 2 in Chinese Patent CN201610635648.5.

[0082] Comparative Example 1

[0083] The difference compared with Example 2 is only that there is no enzymatic hydrolysis, and a mixed strain of yeast and lactic acid bacteria is directly added for combined fermentation. The other steps are the same as those in Example 2.

[0084] Comparative Example 2

[0085] The difference compared with Example 1 is only that during the combined fermentation process, only yeast is used for fermentation and lactic acid bacteria are not used. The other steps are the same as those in Example 1.

[0086] Comparative Example 3

[0087] The difference compared with Example 1 is only that in the co-fermentation process, only lactic acid bacteria fermentation is used, and yeast fermentation is not used, and the remaining steps are the same as those in Example 1.

[0088] Test Example

[0089] According to the quality evaluation requirements of gastrodia elata related products in the Chinese Pharmacopoeia 2020, the raw materials of Example 1 (processed gastrodia elata freeze-dried tablets), the gastrodia elata fermentation oral liquids prepared in Examples 1-5, Comparative Examples 1-3, and the enzyme-activated health care drinks in Test Examples 1-2 were detected, and two indexes of gastrodin and p-hydroxybenzyl alcohol were analyzed. The specific detection methods are as follows: Detection equipment: Agilent 1100 HPLC. Chromatographic conditions: Chromatographic column CAPCELLPAK C18; Mobile phase: acetonitrile-0.05% phosphoric acid solution (3:97); Detection wavelength: 220 nm; Flow rate: 0.8 mL / min; Column temperature: 30°C.

[0090] According to the HPLC analysis results, the detection results of the processed gastrodia elata freeze-dried tablet raw materials, Examples 1-5, and Comparative Examples 1-3 are shown in Table 2. Among them, in Comparative Example 1, because there was no enzymatic hydrolysis, the fermentation base liquid was in a paste state and extremely viscous, and it was impossible to obtain the gastrodia elata fermentation liquid, so there was no detection significance and no data. The detection results of Test Examples 1-2 are detailed in the measurement results in paragraph

[0079] of the specification of Chinese Patent CN201610635648.5. The detection results of Test Example 1 - enzyme-activated health care drink 1 and Test Example 2 - enzyme-activated drink 2 are summarized in Table 2.

[0091] Table 2 Detection Results

[0092] 。

[0093] The detection spectrum of Example 1 is as 图1 shown, and the content of p-hydroxybenzyl alcohol is 74.4%. The detection spectrum of Example 2 is as 图2 shown, and the content of p-hydroxybenzyl alcohol is 74.1%. The detection spectrum of Example 3 is as 图3 shown, and the content of p-hydroxybenzyl alcohol is 70.5%. The detection spectrum of Example 4 is as 图4 shown, and the content of p-hydroxybenzyl alcohol is 68.6%. The detection spectrum of Example 5 is as 图5As shown, the content of p-hydroxybenzyl alcohol is 67.0%. The raw material - the content of gastrodin in the freeze-dried slices of cooked gastrodia elata is relatively high (0.37%), but the gastrodin in Example 1 after fermentation is only 0.016 mg / mL (1.6%); the raw material - the content of p-hydroxybenzyl alcohol in the freeze-dried slices of cooked gastrodia elata is relatively low (0.04%), but the content of p-hydroxybenzyl alcohol in Example 1 after fermentation is as high as 0.744 mg / mL (74.4%). Combining the results of Examples 1 - 5, it can be seen that through the manufacturing process of the present invention, almost all of the gastrodin in the gastrodia elata raw material has been converted into p-hydroxybenzyl alcohol. P-hydroxybenzyl alcohol has a stronger transmembrane ability relative to gastrodin, is more likely to pass through the blood-brain barrier, and p-hydroxybenzyl alcohol is well absorbed in each section of the intestine, making it more suitable for the development of oral preparations. From the results of Comparative Example 1 - Comparative Example 3 and Examples 1 - 5, it can be seen that complex enzymatic hydrolysis and co-fermentation with mixed bacteria synergize with each other to jointly achieve a high conversion rate of p-hydroxybenzyl alcohol. Whether enzymatic hydrolysis is cancelled, or co-fermentation with mixed bacteria is cancelled, or only single-strain fermentation with yeast or lactic acid bacteria is carried out, the conversion rate of p-hydroxybenzyl alcohol in gastrodia elata products cannot be significantly improved.

[0094] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. 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. Preparation method of gastrodia elata product with high conversion rate of p-hydroxybenzyl alcohol, characterized in that, The method comprises: pre-freezing the pre-treated gastrodia elata at ultra-low temperature, and then performing vacuum freeze drying to obtain cooked gastrodia elata; crushing the cooked gastrodia elata into gastrodia elata powder, adding water and white sugar to fully stir and dissolve, adding a composite enzyme to hydrolyze to obtain a fermentation base liquid, and then adding yeast and lactic acid bacteria to perform combined fermentation to obtain a fermented gastrodia elata liquid; filtering the fermented gastrodia elata liquid, centrifuging and taking the supernatant to obtain the gastrodia elata product; The pretreatment of Gastrodia elata is as follows: washing the fresh Gastrodia elata, steaming it until it is cooked, and cooling it; The compound enzyme is obtained by mixing α-amylase and cellulase in a mass ratio of 1:1 to 3:1; the addition amount of the compound enzyme is 0.3% to 0.5% of the total mass of gastrodia powder, water and white sugar, the enzymolysis temperature is 50 to 60°C, and the time is 20 to 30 minutes; The total mass of the yeast and lactic acid bacteria is 0.2% to 0.4% of the mass of the fermentation base liquid; the mass ratio of the yeast to the lactic acid bacteria is 1:1 to 5:1; The temperature of the ultra-low temperature pre-freezing is -40~-50°C, and the time is 10~12h; the temperature of the vacuum freeze drying is 42°C~48°C, and the time is 8~12h.

2. The preparation method according to claim 1, wherein, The lactic acid bacteria include any one or more of Lactobacillus bulgaricus LB57, Streptococcus thermophilus ST021, Lactobacillus plantarum LP121, Lactobacillus casei LC39, Lactobacillus salivarius LS17, Lactobacillus reuteri LR09, Lactobacillus helveticus LH17, Bifidobacteriumanimalis BAA68, and Leuconostoc mesenteroides.

3. The preparation method according to claim 1, characterized in that, The yeast comprises any one or more of Saccharomyces cerevisiae 1305, wine yeast EC1118, and fruit wine universal yeast LA-C12.

4. The preparation method according to claim 1, characterized in that, The combined fermentation temperature is 37-45° C., the relative humidity is 85%, and the anaerobic fermentation is performed for 14-21 days.

5. A Gastrodia elata product with a high conversion rate of p-hydroxybenzyl alcohol obtained by the preparation method as described in any one of claims 1 to 4.

Images