A gastrodia enzyme, its preparation method and application

Abstract

This invention provides a gastrodia enzyme, its preparation method, and its application. The preparation method of the gastrodia enzyme includes the following steps: washing fresh gastrodia, pulping, enzyme inactivation, cooling, ultrasonic extraction, enzymatic hydrolysis, filtration, homogenization, primary sterilization, inoculation fermentation, degassing, and secondary sterilization. The inoculation fermentation step involves adding an enzyme fermentation agent and baker's yeast for symbiotic fermentation. The preparation method of this invention has a stable process, can ensure a high gastrodia enzyme content, and inhibits the ethanol fermentation pathway, thus preserving the flavor of the gastrodia enzyme to the greatest extent.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, and in particular to a gastrodia elata enzyme, its preparation method, and its application. Background Technology

[0002] The genus *Gastrodia* belongs to the subfamily Epidyloides, tribe Gastrodieae, and subtribe Gastrodinae within the family Orchidaceae. Currently, *Gastrodia elata* is widely cultivated and distributed in my country. Within this species, many variations have emerged, often observed in flower color, flower stalk color, tuber shape, and tuber water content.

[0003] The processing technology of Gastrodia elata mostly adopts the traditional drying technology, which involves preheating, constant temperature drying, cooling and heat dissipation, and rehydration of fresh Gastrodia elata. The Gastrodia elata processed by this method is hard and inconvenient to eat. The gastrodin content will be greatly lost during storage, transportation, reprocessing or consumption. At the same time, most of the Gastrodia elata enzyme drinks on the market have problems such as long fermentation cycle, unstable fermentation, and poor flavor. Some products even have high alcohol content and do not meet the relevant national food standards. Therefore, there is an urgent need to develop a Gastrodia elata enzyme with a simple preparation process, stable fermentation, good flavor during fermentation, qualified alcohol concentration and high gastrodin content. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention proposes a gastrodia enzyme, its preparation method, and its application.

[0005] This invention provides a method for preparing Gastrodia elata enzyme, comprising the following steps: washing fresh Gastrodia elata, pulping, inactivating enzyme, cooling, ultrasonic extraction, enzymatic hydrolysis, filtration, homogenization, primary sterilization, inoculation fermentation, degassing, and secondary sterilization to obtain the enzyme;

[0006] The inoculation and fermentation step involves adding an enzyme starter and baker's yeast for symbiotic fermentation. During the first 10 hours of fermentation, the enzyme starter and baker's yeast promote each other: the baker's yeast rapidly activates with glucose and quickly enters the pyruvate pathway for fermentation, rapidly consuming oxygen and producing a large amount of carbon dioxide, quickly introducing an anaerobic environment into the fermentation system. During this period, the enzyme starter rapidly utilizes glycogen to complete activation, proliferation, and expansion, entering the lactic acid fermentation stage, causing the pH of the fermentation system to drop rapidly and producing acid and aroma. This is the period when the main flavor of the gastrodia elata enzyme is generated. After 10 hours of fermentation, the enzyme starter and baker's yeast inhibit each other, leading to a balanced fermentation stage. At this point, the pH of the system has dropped below 3.97, and the activity and fermentation efficiency of the baker's yeast are greatly inhibited. Simultaneously, the glycogen concentration in the system is very low, and the enzyme starter and baker's yeast begin to compete for limited glycogen to maintain their own fermentation. At this time, the acid production efficiency of the enzyme starter is also greatly reduced, so the pH drop at this stage is relatively slow. As the ester production capacity increases, the lipogenic aroma of the product is also enhanced. Therefore, the symbiotic fermentation method using baker's yeast and enzyme starter (Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus acidophilus) can shorten the fermentation cycle, maintain the stability of the fermentation process, and inhibit the ethanol fermentation pathway. The resulting Gastrodia elata enzyme product has an ethanol content that meets the relevant national or industry food safety standards for edible enzymes (requiring an ethanol content of ≤0.5g / 100g).

[0007] Immediately after pulping Gastrodia elata, enzyme inactivation can be performed to avoid the rapid decomposition of gastrodin by β-glycosidic bond enzymes that are rapidly activated during pulping, thus ensuring a high extraction rate of gastrodin in the next step.

[0008] Ultrasonic extraction of Gastrodia elata pulp can ensure that gastrodin in Gastrodia elata is preserved to a greater extent. Compared with similar products on the market that are only extracted by pulping, ultrasonic extraction can increase the gastrodin content from about 0.24% (on a dry basis) to about 0.29% (on a dry basis). It can be seen that the extraction efficiency of gastrodin is increased by nearly 20%, which provides great convenience for the research and development and production of subsequent functional products of Gastrodia elata.

[0009] Using pre-enzyme inactivation and ultrasonic extraction processes can control the high extraction efficiency of gastrodin at the source.

[0010] Enzymatic hydrolysis of Gastrodia elata extract can effectively dissolve high-quality starch (approximately 35% of the dried Gastrodia elata) and polysaccharides (approximately 20% of the dried Gastrodia elata) into the Gastrodia elata pulp. This prevents the pectin and starch in the Gastrodia elata peel from rapidly increasing the concentration of the pulp during heating, which would otherwise affect fermentation. After enzymatic hydrolysis, the yeast starter can more effectively utilize these monosaccharides or polysaccharides for fermentation, shortening the fermentation time, improving fermentation efficiency, and further reducing the production cost of Gastrodia elata enzyme.

[0011] Furthermore, in the pulping step, the material-to-water ratio is 1:3-8, such as 1:3, 1:4, 1:5, 1:6, 1:7, or 1:8, and the water temperature is 90-100℃, such as 90℃, 92℃, 94℃, 96℃, 98℃, or 100℃. The pulper is circulated twice using a colloid mill and pulped for 5 minutes using a high-shear mill at a speed of 2800-3800 rpm.

[0012] Furthermore, in the enzyme inactivation step, the enzyme inactivation temperature is 85-98℃ and the time is 5-12 minutes, such as 5 minutes, 6 minutes, 7 minutes, 8 minutes, 10 minutes, or 12 minutes.

[0013] Furthermore, the ultrasonic extraction step involves ultrasonic extraction at 20 kHz for 10-40 min to obtain Gastrodia elata extract, such as 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, or 40 min, preferably 20-40 min, with the extract temperature maintained below 50℃ (if the temperature exceeds this, it can be cooled by external circulation of cold water).

[0014] Further, the enzymatic hydrolysis step involves heating the Gastrodia elata extract to 88-98℃, then adding 5-20 U / g (based on the weight of fresh Gastrodia elata) of thermostable α-amylase, such as 5 U / g, 6 U / g, 7 U / g, 8 U / g, 9 U / g, 10 U / g, 12 U / g, 14 U / g, 16 U / g, 18 U / g, or 20 U / g. After incubating for 50-70 minutes, the temperature is lowered to 70-80℃, and then 10-25 U / g (based on the weight of fresh Gastrodia elata) of saccharifying enzyme, such as 10 U / g, 12 U / g, 14 U / g, 16 U / g, 18 U / g, 20 U / g, 22 U / g, or 24 U / g, is added. After incubating at 25 U / g for 50-70 minutes, cool to 50-55℃ and add 2-10 U / g (based on the weight of fresh Gastrodia elata) of pectinase, such as 2 U / g, 3 U / g, 4 U / g, 5 U / g, 6 U / g, 8 U / g, or 10 U / g. Incubate for 20-40 minutes to obtain Gastrodia elata enzymatic hydrolysate. The addition of α-amylase can rapidly degrade the starch in Gastrodia elata into oligosaccharides, and the saccharifying enzyme can rapidly decompose oligosaccharides, some polysaccharides, and oligosaccharides into monosaccharides. Pectinase decomposes the pectin in Gastrodia elata, reducing the viscosity of the Gastrodia elata extract, which is conducive to the dissolution of sugars and gastrodin, facilitates the fermentation of gastrodin enzyme, shortens the fermentation time, and reduces the fermentation cost.

[0015] Further, the filtration step involves filtering the Gastrodia elata enzymatic hydrolysate through a 100-300 mesh; the homogenization step involves adding 1-4% glucose by weight of the Gastrodia elata enzymatic hydrolysate to the filtrate and homogenizing it under two-stage high pressure at 20 MPa; and the primary sterilization step involves high-temperature sterilization at 90-95℃ for 1-1.1 hours to obtain sterilized enzymatic hydrolysate.

[0016] Further, the inoculation and fermentation step involves cooling the sterilized enzymatic hydrolysate to 32-38°C and adding 10-300 U / T of enzyme fermentation agent (based on the weight of the sterilized Gastrodia elata enzymatic hydrolysate), such as 10 U / T, 20 U / T, 40 U / T, 50 U / T, 80 U / T, 100 U / T, 120 U / T, 150 U / T, 180 U / T, 200 U / T, 220 U / T, 240 U / T, 250 U / T, 280 U / T, 300 U / T. U / T, preferably 120U / T-300U / T, and 0.01-0.02% (by weight) of sterilized enzymatic hydrolysate bread yeast, such as 0.01%, 0.011%, 0.012%, 0.013%, 0.015%, or 0.02%. After stirring for 15-20 minutes, fermentation begins. Fermentation is carried out at a temperature of 35-38℃ for 12-72 hours to obtain Gastrodia elata fermentation liquid. Fermentation time includes, but is not limited to, 12h, 24h, 36h, 48h, and 72h.

[0017] Furthermore, the degassing step involves degassing the Gastrodia elata fermentation broth under a vacuum of 0.04 MPa and a temperature of 50-60°C. After degassing, the Gastrodin content in the fermentation broth is 68-113 μg / mL, the pH value is 3.0-3.9, and the ethanol content is ≤0.5 (w / w). The secondary sterilization step involves sterilizing the degassed Gastrodia elata fermentation broth at 121°C for 5 seconds to terminate fermentation.

[0018] The present invention also provides gastrodia enzyme prepared by the preparation method described above.

[0019] The present invention also provides the application of the aforementioned Gastrodia elata enzyme in functional foods, which include any one of the following: oral liquids, health products, beverages, milk and dairy products, jelly, candy, biscuits, prepared dishes, and seasonings. When Gastrodia elata enzyme is used directly or in the form of food additives, its addition amount can be 1%-99%.

[0020] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0021] (1) The method for preparing Gastrodia elata enzyme provided by the present invention can effectively inhibit the fermentation pathway of ethanol, and the ethanol content of the finished Gastrodia elata enzyme product is ≤0.5g / 100g, which meets the relevant national or industry food safety standards for edible enzymes.

[0022] (2) The enzyme fermentation agent used in the preparation method of Gastrodia elata enzyme provided by the present invention is a highly active fermentation strain, so the fermentation cycle is short. At the same time, it is a multi-strain fermentation, with balanced acid and aroma production, and the fermented enzyme liquid has a better flavor.

[0023] (3) The method for preparing gastrodin enzyme provided by the present invention can effectively avoid the loss of gastrodin, retain gastrodin to the maximum extent, and the gastrodin enzyme obtained has a high gastrodin content.

[0024] (4) The fermentation path of the gastrodia enzyme preparation method provided by the present invention is stable. By redesigning the fermentation path, the edible enzyme produced fully meets the food safety requirements, and has good water solubility and stability, which effectively improves the palatability of the product.

[0025] (5) The gastrodia enzyme and preparation method provided by the present invention can be applied to the processing of a variety of foods. Attached Figure Description

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 This is a standard curve diagram of gastrodin content;

[0028] Figure 2 This is a graph showing the gastrodin content of the embodiments and comparative examples of the present invention;

[0029] Figure 3 This is a pH change curve of Gastrodia elata enzyme before fermentation in the embodiments and comparative examples of the present invention;

[0030] Figure 4 This is a pH change curve of the fermentation process of Gastrodia elata enzyme in the embodiments and comparative examples of this application;

[0031] Figure 5 The graph shows the ethanol content of the gastrodia enzyme in the embodiments and comparative examples of this application;

[0032] Figure 6 The graph shows the total acid value (calculated as lactic acid) of the gastrodia enzymes in the embodiments and comparative examples of this application;

[0033] Figure 7 The images show the sensory evaluation of Gastrodia elata enzymes in the embodiments and comparative examples of the present invention. Detailed Implementation

[0034] To enable those skilled in the art to better understand the present invention, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0035] I. Raw material sources for the examples and comparative examples

[0036] The substances used in the embodiments and comparative examples of this invention are all commercially available. Among them, the Gastrodia elata (Dejiang Wu Gastrodia elata variety) was purchased from Dejiang County, Guizhou Province and stored at 0-4℃; the enzyme fermentation agents (Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus acidophilus) were purchased from Shanghai Haoyue Biotechnology Co., Ltd. and Shanenkang Biotechnology (Suzhou) Co., Ltd.; and the baker's yeast was purchased from Meishan Mali Yeast Co., Ltd. and Angel Yeast Co., Ltd.

[0037] II. Performance Tests

[0038] (1) Sensory evaluation criteria for Gastrodia elata enzyme liquid

[0039]

[0040]

[0041] (2) Determination of gastrodin content

[0042] According to the Chinese Pharmacopoeia 2020, the content of gastrodin in the sample is determined by high performance liquid chromatography (HPLC).

[0043] ①Chromatographic column conditions: Hypersil Gold aQ, Size: 250mm x 4.6mm; Mobile phase: Acetonitrile: 0.05% phosphoric acid = 3:97 (V:V); Injection volume: 20μL; Injection mode: constant flow; Flow rate: 1mL / min; Wavelength: 220nm; Column temperature: room temperature;

[0044] ② Preparation of the standard curve: Accurately weigh 2 mg of Gastrodia elata reference standard and place it in a 100 mL volumetric flask. Dilute to volume with the mobile phase, shake well, and prepare a 20 μg / mL gastrodin standard solution. Measure 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0 mL of the gastrodin standard solution and place them in separate 10 mL volumetric flasks. Dilute to 6.0 mL with the mobile phase, shake well, filter through a 0.22 mm filter membrane, and inject 20 μL of each solution. Plot a standard curve with gastrodin concentration on the x-axis and peak area on the y-axis. The standard curve is shown below. Figure 1 As shown;

[0045] ③ Determination of gastrodin content in samples: Accurately transfer 6 mL of fermentation broth into a 20 mL centrifuge tube, add 4 mL of anhydrous ethanol, seal, and extract ultrasonically at 60℃ (40 kHz) for 30 min. Centrifuge the extract at 3500 r / min for 5 min, take 6 mL of the supernatant, concentrate to dryness at 70℃, wash into a 10 mL volumetric flask with the mobile phase (acetonitrile: 0.05% phosphoric acid = 3:97), filter the diluted liquid through a 0.22 μm filter membrane, and inject 20 μL for analysis. After analysis, calculate the gastrodin content in the sample according to the standard curve, as shown in Table 2.

[0046] (3) pH value measurement

[0047] The pH value of the sample was determined using the potentiometric method. The gastrodia enzyme solution and pH standard buffer solution were placed in a 25℃ environment for 1 hour. The pH meter was preheated and zeroed as required, and the "temperature" potentiometer was adjusted to match the temperature of the solution to be tested. Calibration was performed using pH buffer solutions (pH 4.0, 6.88, and 3.57). The electrode was rinsed several times with the gastrodia enzyme sample to be tested, then inserted into the sample for 1 minute. The "reading" switch was pressed, and the pH value indicated by the pointer was the pH value of the gastrodia enzyme sample to be tested. The pH value changes and curves before and after gastrodia enzyme fermentation are shown in Table 4. Figure 3 and Figure 4 .

[0048] (4) Determination of ethanol content in gastrodia enzyme

[0049] The ethanol content of Gastrodia elata enzyme was determined according to the standard GB / T 12143-2008, using the reference titration method.

[0050] ① Weigh 20g of Gastrodia elata enzyme sample into a small beaker, accurate to 0.001g, and transfer the sample to a 1000mL distillation flask, with the total volume not exceeding 1 / 2 of the flask's capacity;

[0051] ② Add 0.2 mL of 1% bromothymol blue indicator solution, titrate with 1 mol / L sodium hydroxide solution until a distinct blue color is obtained, add a few ceramic pieces or glass beads, and if there is a lot of foam, add 0.5 mL of silicone oil;

[0052] ③ Immediately connect the flask to the other parts of the still. Use a 100mL volumetric flask pre-filled with 10mL of water as the receiving container. The lower end of the receiving tube should be immersed in the water but not in contact with the bottom of the flask. Cool the volumetric flask with ice water. After connecting, immediately heat the flask with an adjustable electric furnace for distillation. The temperature can be increased quickly at the beginning. When it approaches boiling, reduce the temperature of the electric furnace to prevent foam from overflowing. After the foam dissipates, increase the temperature of the electric furnace again until the liquid in the volumetric flask is about 80mL. Disconnect the condenser from the connecting bend and remove it. Stop heating. Rinse the bend, condenser and receiving tube with water. Add the rinsing liquid to the volumetric flask. After the temperature rises to room temperature, dilute to the mark with water and mix well.

[0053] ④ Take 10 mL of the prepared test solution into a 250 mL iodine flask, add 10 mL to 15 mL (V3) potassium dichromate standard solution, stopper the flask tightly, shake gently, quickly measure 20 mL of sulfuric acid solution with a graduated cylinder, slightly open the stopper, pour it into the flask along the mouth of the flask, stopper the flask tightly and shake gently (be careful not to let the stopper pop out), place it in a 40℃ constant temperature water bath for oxidization for 1 hour, shaking it slightly during the process, take it out, if the solution in the flask is green, it means that the ethanol content in the solution is too high, and the sample amount should be reduced;

[0054] ⑤ Open the bottle stopper, rinse the bottle mouth with water, and then titrate with ferrous ammonium sulfate standard titration solution until it turns yellow-green. Add 0.2 mL of o-phenanthroline ferric indicator solution and continue titrating. It gradually turns blue-green until it suddenly turns brown-red, which is the endpoint. Record the volume of ferrous ammonium sulfate standard titration solution consumed (V2).

[0055] ⑥ Replace the gastrodia enzyme sample with water, and follow the above procedure for the rest. Record the volume (V1) of ferrous ammonium sulfate standard titration solution consumed.

[0056] ⑦ The ethanol content in the sample is calculated using the following formula:

[0057]

[0058] In the formula:

[0059] x — the ethanol content in the sample, expressed in grams per kilogram (g / kg);

[0060] V1—Volume of ferrous ammonium sulfate standard titration solution consumed in the blank test, in milliliters (mL); V2—Volume of ferrous ammonium sulfate standard titration solution consumed in the sample test, in milliliters (mL); V3—Volume of potassium dichromate standard solution added during ethanol oxidation, in milliliters (mL).

[0061] c s—1 mL of potassium dichromate standard solution is equivalent to the mass of ethanol, 0.0002349 g / mL; K—the dilution factor of the sample, in this invention, Examples 1-4 and Comparative Examples 1-3 are all 2 times, and Comparative Example 4 is 4 times.

[0062] m s —Sample mass, in grams (g).

[0063] The ethanol content in the gastrodia enzyme samples of each embodiment and comparative example is shown in Table 3.

[0064] (5) Determination of total acidity in Gastrodia elata enzyme

[0065] The test was conducted according to GB / T 12456-2021, using the acid-base indicator titration method:

[0066] ① Pipette 25.0 mL of the finished Gastrodia elata enzyme solution into a 250 mL volumetric flask, dilute to the mark with carbon dioxide-free water, shake well, filter with rapid filter paper, collect the filtrate, and use it for determination;

[0067] ② Add 2-4 drops (10g / L) of tyrosine indicator solution, and titrate with 0.1mol / L sodium hydroxide standard titration solution until the faint red color does not fade for 30 seconds. Record the volume of 0.1mol / L sodium hydroxide standard titration solution consumed.

[0068] ③ Use the same volume of carbon dioxide-free water to replace the sample for a blank test, and record the volume of sodium hydroxide standard titration solution consumed;

[0069] ④ The total acid content is calculated using the following formula:

[0070]

[0071] In the formula:

[0072] x — the total acid content in the sample, expressed in grams per kilogram (g / kg) or grams per liter (g / L);

[0073] c—Concentration of the standard titration solution, in moles per liter (mol / L);

[0074] V1—The volume of sodium hydroxide standard titration solution consumed during the titration of the test solution, in milliliters (mL);

[0075] V2—The volume of sodium hydroxide standard titration solution consumed in the blank test, in milliliters (mL);

[0076] k—Conversion factor for acids: 0.090 for lactic acid;

[0077] F—Dilution factor of the test solution, 5 times;

[0078] m—mass of the sample, in grams (g) or volume of the sample taken, in milliliters (mL); 1000—conversion factor.

[0079] The total acid (calculated as lactic acid) in each example and comparative example of Gastrodia elata enzyme samples is shown in Table 2.

[0080] Example 1

[0081] The method for preparing gastrodia enzyme in this embodiment includes the following steps: fresh gastrodia is washed, pulped, enzyme-inactivated, cooled, ultrasonically extracted, enzymatically hydrolyzed, filtered, homogenized, sterilized once, inoculated for fermentation, degassed, and sterilized a second time to obtain gastrodia enzyme liquid.

[0082] In the pulping step, the material-to-water ratio is 1:4, the water temperature is 100℃, the pulper is circulated twice using a colloid mill, and pulped for 5 minutes using a high-shear mill at a speed of 3800 rpm.

[0083] The enzyme inactivation step was performed at a temperature of 98℃ for 12 minutes.

[0084] In the ultrasonic extraction step, ultrasonic extraction was performed at 20 kHz for 40 min, and the temperature of the extract was kept below 50 ℃.

[0085] In the enzymatic hydrolysis step, 10 U / g (based on the weight of fresh Gastrodia elata) of thermoresistant α-amylase was added at 98℃, and the temperature was maintained for 60 min. After cooling to 70-80℃, 12 U / g (based on the weight of fresh Gastrodia elata) of saccharifying enzyme was added, and the temperature was maintained for 60 min. After cooling to 50-55℃, 4 U / g (based on the weight of fresh Gastrodia elata) of pectinase was added, and the temperature was maintained for 30 min to obtain Gastrodia elata enzymatic hydrolysate.

[0086] In the steps of filtration, homogenization, and primary sterilization, the gastrodia hydrolysate is filtered through a 300-mesh filter and then 4% glucose (based on the weight of the gastrodia hydrolysate) is added. After two-stage high-pressure homogenization at 20MPa, it is sterilized at a high temperature of 90-95℃ for 1 hour.

[0087] In the inoculation and fermentation step, the sterilized Gastrodia elata hydrolysate is cooled to 37℃, and 200U / T of enzyme fermentation agent (Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus acidophilus) (based on the weight of the sterilized hydrolysate) and 0.012% of baker's yeast (based on the weight of the sterilized Gastrodia elata hydrolysate) are added. Fermentation begins after stirring for 15-20 minutes, with the temperature controlled at 35-38℃ and the fermentation time being 48 hours.

[0088] In the degassing and secondary sterilization steps, the gastrodia fermentation broth with a gastrodin content of 113 μg / mL, a pH value of 3.60, and an ethanol content of 0.26% (w / w) was degassed under a vacuum of 0.04 MPa and a temperature of 50°C. After degassing, the fermentation was terminated by sterilization at 121°C for 5 seconds to produce gastrodia enzyme liquid. This gastrodia enzyme liquid has a unique flavor and no unpleasant odor.

[0089] Example 2

[0090] The method for preparing gastrodia enzyme in this embodiment includes the following steps: fresh gastrodia is washed, pulped, enzyme-inactivated, cooled, ultrasonically extracted, enzymatically hydrolyzed, filtered, homogenized, sterilized once, inoculated for fermentation, degassed, and sterilized a second time to obtain gastrodia enzyme liquid.

[0091] In the pulping step, the material-to-water ratio is 1:3, the water temperature is 100℃, the pulper is circulated twice using a colloid mill, and pulped for 5 minutes using a high-shear mill at a speed of 2800 rpm.

[0092] The enzyme inactivation step was performed at a temperature of 98℃ for 12 minutes.

[0093] In the ultrasonic extraction step, ultrasonic extraction was performed at 20 kHz for 10 min, and the temperature of the extract was kept below 50 ℃.

[0094] In the enzymatic hydrolysis step, 20 U / g (based on the weight of fresh Gastrodia elata) of thermoresistant α-amylase was added at 98℃, and the temperature was maintained for 60 min. The temperature was then lowered to 80-70℃, 25 U / g (based on the weight of fresh Gastrodia elata) of saccharifying enzyme was added, and the temperature was maintained for 60 min. The temperature was then lowered to 55-50℃, 10 U / g (based on the weight of fresh Gastrodia elata) of pectinase was added, and the temperature was maintained for 30 min to obtain the Gastrodia elata enzymatic hydrolysate.

[0095] In the steps of filtration, homogenization, and primary sterilization, the gastrodia hydrolysate is filtered through a 100-mesh filter and then 3% glucose (based on the weight of the gastrodia hydrolysate) is added. After two-stage high-pressure homogenization at 20MPa, it is sterilized at a high temperature of 90-95℃ for 1 hour.

[0096] In the inoculation and fermentation step, the sterilized Gastrodia elata hydrolysate is cooled to 37℃, and 300U / T of enzyme fermentation agent (Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus acidophilus) (based on the weight of the sterilized hydrolysate) and 0.01% of baker's yeast (based on the weight of the sterilized Gastrodia elata hydrolysate) are added. Fermentation begins after stirring for 15-20 minutes, with the temperature controlled at 35-38℃ and the fermentation time being 72 hours.

[0097] In the degassing and secondary sterilization steps, the gastrodia fermentation broth with a gastrodin content of 108 μg / mL, a pH value of 3.00, and an ethanol content of 0.32% (w / w) was degassed under a vacuum of 0.04 MPa and a temperature of 50°C. After degassing, the fermentation was terminated by sterilization at 121°C for 5 seconds to produce gastrodia enzyme liquid. This gastrodia enzyme liquid has a unique flavor and no unpleasant odor.

[0098] Example 3

[0099] The method for preparing gastrodia enzyme in this embodiment includes the following steps: fresh gastrodia is washed, pulped, enzyme-inactivated, cooled, ultrasonically extracted, enzymatically hydrolyzed, filtered, homogenized, sterilized once, inoculated for fermentation, degassed, and sterilized a second time to obtain gastrodia enzyme liquid.

[0100] In the pulping step, the material-to-water ratio is 1:6, the water temperature is 95℃, the pulper is circulated twice using a colloid mill, and pulped for 5 minutes using a high-shear mill at a speed of 3600 rpm.

[0101] The enzyme inactivation step was performed at a temperature of 92℃ for 8 minutes.

[0102] In the ultrasonic extraction step, ultrasonic extraction was performed at 20 kHz for 20 min, and the temperature of the extract was kept below 50 ℃.

[0103] In the enzymatic hydrolysis step, 12 U / g (based on the weight of fresh Gastrodia elata) of thermoresistant α-amylase was added at 98℃, and the temperature was maintained for 60 min. The temperature was then lowered to 80-70℃, 16 U / g (based on the weight of fresh Gastrodia elata) of saccharifying enzyme was added, and the temperature was maintained for 60 min. The temperature was then lowered to 55-50℃, 8 U / g (based on the weight of fresh Gastrodia elata) of pectinase was added, and the temperature was maintained for 30 min to obtain the Gastrodia elata enzymatic hydrolysate.

[0104] In the steps of filtration, homogenization and primary sterilization, the gastrodia hydrolysate is filtered through a 300-mesh filter and then 2% glucose (based on the weight of the gastrodia hydrolysate) is added. After two-stage high-pressure homogenization at 20MPa, it is sterilized at a high temperature of 90-95℃ for 1 hour.

[0105] In the inoculation and fermentation step, the sterilized Gastrodia elata hydrolysate is cooled to 37℃, and 160U / T of enzyme fermentation agent (Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus acidophilus) (based on the weight of the sterilized hydrolysate) and 0.016% of baker's yeast (based on the weight of the sterilized Gastrodia elata hydrolysate) are added. Fermentation begins after stirring for 15-20 minutes, with the temperature controlled at 35-38℃ and the fermentation time being 60 hours.

[0106] In the degassing and secondary sterilization steps, the fermentation broth of Gastrodia elata with a gastrodin content of 85 μg / mL, a pH value of 3.69, and an ethanol content of 0.28% (w / w) was degassed under a vacuum of 0.04 MPa and a temperature of 50℃. After degassing, the fermentation was terminated by sterilization at 121℃ for 5 seconds to produce Gastrodia elata enzyme liquid. This Gastrodia elata enzyme liquid has a unique flavor and no unpleasant odor.

[0107] Example 4

[0108] The method for preparing gastrodia enzyme in this embodiment includes the following steps: fresh gastrodia is washed, pulped, enzyme-inactivated, cooled, ultrasonically extracted, enzymatically hydrolyzed, filtered, homogenized, sterilized once, inoculated for fermentation, degassed, and sterilized a second time to obtain gastrodia enzyme liquid.

[0109] In the pulping step, the material-to-water ratio is 1:8, the water temperature is 90℃, the pulper is circulated twice using a colloid mill, and pulped for 5 minutes using a high-shear mill at a speed of 2800 rpm.

[0110] The enzyme inactivation step was performed at 85℃ for 5 minutes.

[0111] In the ultrasonic extraction step, ultrasonic extraction was performed at 20 kHz for 10 min, and the temperature of the extract was kept below 50 ℃.

[0112] In the enzymatic hydrolysis step, 5 U / g (based on the weight of fresh Gastrodia elata) of thermoresistant α-amylase was added at 98℃, and the temperature was maintained for 60 min. The temperature was then lowered to 80-70℃, 10 U / g (based on the weight of fresh Gastrodia elata) of saccharifying enzyme was added, and the temperature was maintained for 60 min. The temperature was then lowered to 55-50℃, 2 U / g (based on the weight of fresh Gastrodia elata) of pectinase was added, and the temperature was maintained for 30 min to obtain the Gastrodia elata enzymatic hydrolysate.

[0113] In the steps of filtration, homogenization, and primary sterilization, the gastrodia hydrolysate is filtered through a 300-mesh filter and then 1% glucose (based on the weight of the gastrodia hydrolysate) is added. After two-stage high-pressure homogenization at 20MPa, it is sterilized at a high temperature of 90-95℃ for 1 hour.

[0114] In the inoculation and fermentation step, the sterilized Gastrodia elata hydrolysate is cooled to 37℃, and 120U / T of enzyme fermentation agent (Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus acidophilus) (based on the weight of the sterilized hydrolysate) and 0.02% of baker's yeast (based on the weight of the sterilized Gastrodia elata hydrolysate) are added. Fermentation begins after stirring for 15-20 minutes, with the temperature controlled at 35-38℃ and the fermentation time being 12 hours.

[0115] In the degassing and secondary sterilization steps, the fermentation broth of Gastrodia elata with a gastrodin content of 68 μg / mL, a pH value of 3.96, and an ethanol content of 0.18% (w / w) was degassed under a vacuum of 0.04 MPa and a temperature of 50°C. After degassing, the fermentation was terminated by sterilization at 121°C for 5 seconds to produce Gastrodia elata enzyme liquid. This Gastrodia elata enzyme liquid has a unique flavor and no unpleasant odor.

[0116] Example 5

[0117] This embodiment illustrates one application of the gastrodia enzyme prepared according to the present invention in functional foods: The gastrodia enzyme prepared in Example 1 is weighed out according to the following ratio: 45 parts gastrodia enzyme, 15 parts kudzu root, 10 parts poria cocos, and 300 parts water. First, kudzu root, poria cocos, and water are boiled and kept at a gentle boil for 30 minutes. After filtering with a 300-mesh filter, gastrodia enzyme is added to the liquid and stirred evenly to prepare gastrodia elata and kudzu root decoction oral liquid.

[0118] Comparative Example 1

[0119] The only difference between the preparation method of Gastrodia elata enzyme in Comparative Example 1 and Example 1 is that the enzyme inactivation operation was not performed after pulping.

[0120] Comparative Example 2

[0121] The only difference between the preparation method of Gastrodia elata enzyme in Comparative Example 2 and Example 1 is that ultrasonic extraction was not performed.

[0122] Comparative Example 3

[0123] The only difference between the preparation method of Gastrodia elata enzyme in Comparative Example 3 and Example 1 is that no enzymatic hydrolysis was performed.

[0124] Comparative Example 4

[0125] The difference between the preparation method of Gastrodia elata enzyme in Comparative Example 4 and Example 1 is that only enzyme fermentation agents (Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus acidophilus) are added in the inoculation and fermentation step.

[0126] Table 1. Sensory Indicators (Scores) of Gastrodia Enzyme in Examples and Comparative Examples

[0127] Color Organizational Form taste odor impurities total standard 10 25 20 25 20 100 Example 1 10 24 18 25 18 95 Example 2 9 15 19 24 12 79 Example 3 8 24 16 20 18 86 Example 4 6 24 12 20 18 80 Comparative Example 1 10 22 18 24 18 92 Comparative Example 2 10 22 19 24 18 93 Comparative Example 3 10 18 16 16 12 72 Comparative Example 4 9 24 18 10 16 77

[0128] Table 2. Determination of gastrodin and total acid content in gastrodin enzymes from Examples and Comparative Examples.

[0129]

[0130] Table 3. Ethanol content in Gastrodia elata enzymes from the examples and comparative examples.

[0131]

[0132]

[0133] Table 4. pH value determination during the preparation of Gastrodia elata enzyme in the examples and comparative examples.

[0134]

[0135] Based on the test data in Tables 1-4 regarding the overall sensory evaluation of gastrodin enzyme, gastrodin content, total acid content, ethanol content, and pH changes during fermentation, it can be shown that the gastrodin enzyme prepared through Examples 1-4 can maintain a balance between low ethanol content, high gastrodin content, good flavor, and low fermentation cost, which has significant advantages compared to the comparative examples and can effectively meet the high standards of customers and the market.

[0136] Examples 1-4 all follow the method of preparing gastrodia enzyme by washing, pulping, enzyme inactivation, cooling, ultrasonic extraction, enzymatic hydrolysis, filtration, homogenization, primary sterilization, inoculation fermentation, degassing, and secondary sterilization of fresh gastrodia. In the inoculation fermentation step, enzyme fermentation agent and baker's yeast are added for symbiotic fermentation. The resulting gastrodia enzyme has an ethanol content of less than 0.32g / 100g, a gastrodin content of more than 68μg / mL, and a total acid content (calculated as lactic acid) of more than 8.12g / L. Furthermore, short-cycle balanced fermentation can be achieved during the fermentation process, reducing fermentation costs.

[0137] Compared with Example 1, Comparative Example 1 did not involve enzyme inactivation after pulping. This resulted in the rapid activation of β-glycosidic bonds in the fresh Gastrodia elata after pulping, accelerating the decomposition of gastrodin. Consequently, the gastrodin content in the Gastrodia elata enzyme prepared in Comparative Example 1 was 50.5% lower than that in Example 1. Compared with Example 1, Comparative Example 2 did not undergo ultrasonic extraction, only pulping extraction. As gastrodin could not be fully extracted at this time, the gastrodin content in the obtained Gastrodia elata enzyme was 17.7% lower than that in Example 1. Compared with Example 1, Comparative Example 3 did not undergo enzymatic hydrolysis. In this case, the high-quality starch and polysaccharides in Gastrodia elata could not be effectively dissolved into the Gastrodia elata pulp. Furthermore, the pectin and starch in the Gastrodia elata peel rapidly increased the viscosity of the pulp upon heating, hindering the dissolution of undissolved Gastrodia elata starch and Gastrodia elata. The dissolution of polysaccharides and the increased viscosity of the original pulp also lead to the filtration of some gastrodin along with impurities and gastrodin peel residue during subsequent filtration steps, resulting in a lower gastrodin content in the gastrodin enzyme. Simultaneously, the pH of the enzymatically hydrolyzed gastrodin solution decreases by about 1 during this step, providing a suitable acidic environment for subsequent fermentation. In contrast, the pH of the unhydrolyzed gastrodin solution remains high before fermentation. Although the decisive factors affecting pH during fermentation are the amount of enzyme starter (Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus acidophilus) and the concentration of monosaccharides in the solution (from enzymatically hydrolyzed gastrodin and added glucose), the gastrodin solution without the initial enzymatic hydrolysis process results in a lower pH during later fermentation compared to the enzymatically hydrolyzed gastrodin. The pH value of the liquid changes more slowly, affecting the fermentation effect and reducing the fermentation efficiency. Furthermore, the absence of an enzymatic hydrolysis step will seriously affect the total acid content in the finished gastrodia elata enzyme product. The total acid (calculated as lactic acid) in the gastrodia elata enzyme prepared in Comparative Example 3 was only 7.25 g / L, which did not meet the relevant industry standard of ≥8 g / L. Compared with Example 1, Comparative Example 4 did not add baker's yeast for symbiotic fermentation. The addition of baker's yeast can utilize the limited glucose in the system to quickly activate and carry out aerobic fermentation, producing a large amount of carbon dioxide and a small amount of aroma substances, so that the fermentation liquid quickly enters an anaerobic state. At this time, the monosaccharides (mainly glucose) in the system have been consumed or the concentration is extremely low, which is just right for the enzyme starter (Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus acidophilus). In an anaerobic fermentation environment, the fermentation process enters the true fermentation stage, producing lactic acid, esters, ketones, and aldehydes. This effectively prevents the fermentation pathway and production of ethanol. At the same time, the aroma compounds produced can perfectly mask the unique unpleasant flavor of Gastrodia elata. However, without the addition of bread yeast, balanced fermentation will not occur. In this case, yeast will ferment independently or become the dominant bacteria at a certain stage during the fermentation of Gastrodia elata enzyme, which will change the yeast fermentation pathway. This will produce a large amount of ethanol while severely inhibiting the activity of lactic acid bacteria. The resulting Gastrodia elata enzyme will have a distinct alcoholic taste, and the acidity of the Gastrodia elata enzyme will be greatly reduced, significantly affecting the flavor. In Comparative Example 4, the ethanol content of Gastrodia elata enzyme is >0.5 (g / 100g), which does not meet the relevant national or industry food safety standards.

[0138] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing gastrodia enzyme, characterized in that, Includes the following steps: Fresh Gastrodia elata is obtained by washing, pulping, enzyme inactivation, cooling, ultrasonic extraction, enzymatic hydrolysis, filtration, homogenization, primary sterilization, inoculation fermentation, degassing, and secondary sterilization. In the pulping step, the material-to-water ratio is 1:3-8, the water temperature is 90-100℃, the pulper is circulated twice with a colloid mill, and pulped for 5 minutes with a high shear mill at a speed of 2800-3800 rpm. The enzyme inactivation step is performed at a temperature of 85-98℃ for 5-12 minutes. The enzymatic hydrolysis step is as follows: heat the Gastrodia elata extract to 88-98℃, add 5-20 U / g of thermoresistant α-amylase, keep warm for 50-70 min, cool down to 70-80℃, add 10-25 U / g of saccharifying enzyme, keep warm for 50-70 min, cool down to 50-55℃, add 2-10 U / g of pectinase, keep warm for 20-40 min to obtain the Gastrodia elata enzymatic hydrolysate; The inoculation and fermentation step involves cooling the sterilized enzyme hydrolysate to 32-38℃, adding 120-300 U / T of enzyme fermentation agent and 0.01-0.02% of baker's yeast by weight of the sterilized enzyme hydrolysate, stirring for 15-20 min and then starting fermentation. Fermentation is carried out at a temperature of 35-38℃ for 12-72 h to obtain Gastrodia elata fermentation liquid. The enzyme fermentation agent is a mixture of Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus acidophilus.

2. The preparation method according to claim 1, characterized in that, The ultrasonic extraction step involves ultrasonic extraction at 20 kHz for 10-40 min to obtain Gastrodia elata extract, with the temperature of the extract maintained below 50℃.

3. The preparation method according to claim 1, characterized in that, The filtration step involves filtering the Gastrodia elata enzymatic hydrolysate through a 100-300 mesh screen; the homogenization step involves adding 1-4% glucose by weight of the Gastrodia elata enzymatic hydrolysate to the filtrate and homogenizing it under two stages of high pressure at 20 MPa; the primary sterilization step involves sterilizing the hydrolysate at 90-95℃ for 1-1.1 h to obtain sterilized enzymatic hydrolysate.

4. The preparation method according to claim 1, characterized in that, The degassing step involves degassing the Gastrodia elata fermentation liquid under a vacuum of 0.04 MPa and a temperature of 50-60℃. The secondary sterilization step involves sterilizing the degassed Gastrodia elata fermentation liquid at 121℃ for 5 seconds to terminate fermentation.

5. Gastrodia elata enzyme prepared by the preparation method according to any one of claims 1 to 4.

6. The application of the gastrodia enzyme according to claim 5 in food, characterized in that, The food products include any one of the following: oral liquids, health products, beverages, dairy products, candies, biscuits, prepared dishes, and condiments.

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