Chinese gall leaven processing method capable of avoiding aspergillus flavus

Abstract

The invention discloses a Chinese gall leaven processing method capable of avoiding aspergillus flavus. The Chinese gall leaven processing method comprises the following steps: 1) taking Chinese gall, cleaning, drying and crushing, sieving with a 40-80-mesh sieve, disinfecting and sterilizing, and cooling to room temperature for later use; 2) preparing a tannase purified solution for later use; 3) adding purified water into green tea, decocting into juice, filtering out tea residues, concentrating and cooling to room temperature for later use; 4) mixing the gallnut powder, the tannase purified liquid, the green tea thick juice and the activated carbon powder condensed liquid in a sealed reaction kettle; (5) after the reaction is finished, extracting reaction liquid in the reaction kettle, treating the reaction liquid in a water bath at 100 DEG C for 5-10 minutes, and cooling to room temperature, and (6) freeze-drying the reaction liquid, compressing powder obtained after freeze-drying into blocks, namely a processed Chinese gall leaven product, and sealing and storing the Chinese gall leaven product.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine preparation, in particular to a method for preparing Baiyaojian for avoiding aflatoxin. Background Art

[0002] Baiyaojian, also known as Zhibaiyaojian and Baicaojian, is a dried block made by fermenting gallnut with tea, distiller's grains or distiller's yeast. Its main ingredient is gallic acid produced by the hydrolysis of hydrolyzable tannins in gallnut. It has the effects of clearing away heat and resolving phlegm, promoting salivation and quenching thirst. It can be used clinically to treat lung heat cough, wind-fire toothache, oral and tongue erosion, chronic dysentery and prolapse of the anus.

[0003] The traditional preparation of Baiyaojian is completed by mixed solid fermentation of multiple microorganisms existing in nature. The fermentation strains are unstable and mixed with invalid strains, which may cause large fluctuations in the gallic acid concentration in the fermentation matrix of Baiyaojian, affecting the quality stability of Baiyaojian.

[0004] Jiuqu (drinking yeast), the raw material of Baiyaojian (Baiyaojian), is highly susceptible to contamination with Aspergillus flavus. Aflatoxin B1, produced by aflatoxinase, is extremely toxic and can induce teratogenic, carcinogenic, and mutagenic effects in humans. Aflatoxin testing is a crucial component of Baiyaojian's quality control. To ensure that aflatoxin levels in the final Baiyaojian product meet quality standards, strict quality control is required during the fermentation process. Quality control studies for Baiyaojian include testing for moisture, total ash, acid-insoluble ash, aflatoxins, and microscopic characteristics. This suggests a risk of aflatoxin contamination during the production and storage of Baiyaojian, necessitating quality control measures to prevent levels exceeding safety standards. Although there is no direct evidence that aflatoxin is invariably produced during the fermentation process, the prevalence and harmful effects of aflatoxins make quality control of Baiyaojian challenging. Summary of the Invention

[0005] The object of the present invention is to provide a method for preparing Baiyaojian without using biological fermentation technology, without using distiller's grains as raw materials, and preventing the occurrence of aflatoxin.

[0006] The present invention is achieved through the following technical solution: a method for preparing Baiyaojian to avoid aflatoxin, comprising the following steps: (1) Wash and dry the gallnuts, crush them, sieve them through a 40-80 mesh sieve, sterilize them, and cool them to room temperature for later use; (2) preparing tannase purified solution for later use; (3) Take green tea and add purified water to boil into juice, filter out the tea residue, concentrate and cool to room temperature for later use; (4) Mix the gallnut powder, tannase purified solution, green tea concentrate, and activated carbon powder concentrate in a sealed reactor, maintain stirring at a speed of 30 rad / min to 60 rad / min, maintain the temperature at 35°C to 45°C, and the reaction time is 3h to 6h. The initial pH is 6.5. (5) After the reaction is completed, extract the reaction liquid in the reactor, treat it in a water bath at 100°C for 5-10 minutes, and then cool it to room temperature; (6) The reaction solution is freeze-dried, and the powder obtained after freeze-drying is compressed into blocks to form the finished Baiyaojian product, which is then stored in a ventilated and dry place.

[0007] The main active ingredient of Baiyaojian is gallic acid, which is converted from hydrolyzable tannins in gallnut through microbial fermentation. It has trypanocidal, anti-inflammatory, antibacterial, antitumor, and antiviral activities. The efficacy of Baiyaojian is generally determined by detecting the gallic acid content in Baiyaojian. Microbial fermentation essentially uses tannase produced by microorganisms to convert hydrolyzable tannins in gallnut into gallic acid. Although this technical solution directly uses tannase to mix with gallnut, it is different from the simple extraction of gallic acid from Schisandra chinensis. It also adds concentrated tea juice as an auxiliary ingredient. It is necessary to simulate the fermentation process of distiller's yeast to a certain extent to obtain a complete Baiyaojian, rather than simply gallic acid. Therefore, the use of tannase alone cannot achieve this technical effect, and tannase has no effect on tea juice. During the experiment, the inventor added a small amount of activated carbon powder, which was originally used to enhance disinfection and adsorption of escaping gases. As a result, the state of the reaction solution finally obtained was able to play the anti-inflammatory, analgesic, and cough-relieving and expectorant effects of Baiyaojian. By subsequently freeze-drying the reaction mixture, a Baiyaojian (Baiyaojian) essentially identical to the traditional Baiyaojian was produced. The inventors' analysis suggests that the loose pores in the activated carbon adsorbed the hydrolyzable tannins from Schisandra chinensis and the catechols from the concentrated tea juice, allowing the tannase enzyme to perform a fermentation-like enzymatic hydrolysis process. Alternatively, the activated carbon may have altered the morphology of the tannase enzyme or accelerated its enzymatic reaction, causing it to undergo a process similar to that of fermentation by koji. The exact role of the activated carbon remains to be further studied. In short, the inventors have developed a new method for preparing Baiyaojian. This method avoids the use of fermentation strains and instead simulates koji fermentation to produce the finished Baiyaojian. The entire process, devoid of biological fermentation and rigorously disinfected, essentially eliminates the infiltration of harmful bacteria like Aspergillus flavus, significantly improving the quality and safety of the Baiyaojian.

[0008] In order to better implement the method of the present invention, further, in the step (1), the washed gallnuts are placed in an oven at 70° C. for 48 hours for drying, and the dried gallnuts are crushed by a crusher.

[0009] In order to better implement the method of the present invention, further, in the step (1), the gallnut powder is disinfected and sterilized with high-temperature steam at 250°C to 300°C.

[0010] In order to better implement the method of the present invention, further, in step (2), the preparation process of the tannase purified solution includes the following steps: (2.1) Activate the preserved Aspergillus tubingensis on an agar slant; (2.2) Prepare the culture medium and add it to a shake flask. Inoculate the activated Aspergillus tubingensis into the shake flask containing the culture medium for propagation and expansion. (2.3) Transfer the Aspergillus tubingensis grown in the shake flask into a water-tight constant temperature incubator at 37°C and ferment for 60-90 hours. (2.4) After fermentation, add pH 6.5 tartaric acid-sodium tartrate buffer, place on a shaker and shake for 1-2 hours, filter, and collect the crude tannase enzyme solution; (2.5) The crude enzyme solution is purified by the layered precipitation method to obtain purified tannase solution.

[0011] To better implement the method of the present invention, further, the preparation process of the culture medium in step (2.2) is to use bran, glucose, and soybean meal powder as substrates of the fermentation medium, add an aqueous solution of inorganic salts, and then sterilize and cool.

[0012] To better implement the method of the present invention, further, in the culture medium, bran, glucose, and soybean meal powder are uniformly mixed in a mass ratio of 1:2:1, the culture medium substrate and the aqueous solution added with inorganic salts are in a mass ratio of 3:5, and each liter of the aqueous solution of inorganic salts contains 5g~10g of NH4H2PO4, 2g~4g of K2HPO4, 1g~3g of MgSO4·7H2O, and 1g~3g of CaCl2.

[0013] To better implement the method of the present invention, further, in the step (2.5), the specific process of purifying the crude enzyme solution by the layered precipitation method is as follows: adding a saturated sodium chloride aqueous solution to the crude enzyme solution, filtering and collecting the precipitate, dissolving the precipitate with tartaric acid-sodium tartrate buffer, and performing chromatography on a chromatography column to obtain a purified tannase solution.

[0014] Compared with the prior art, the present invention has the following advantages and beneficial effects: The invention discloses a novel preparation method for Baiyaojian, which does not involve fermentation bacteria. Tannin enzyme is directly added and mixed with gallnut and concentrated tea juice, and activated carbon powder is added, and a similar fermentation method with yeast is used to obtain the finished Baiyaojian. The whole process does not involve biological fermentation, has a strict disinfection process, and basically prevents the mixing of harmful bacteria such as aflatoxin, thereby greatly reducing the difficulty of quality control of Baiyaojian. The gallic acid content in the prepared Baiyaojian finished product exceeds the gallic acid content in the traditional Baiyaojian, and the process has good repeatability. The gallic acid content in each finished product is relatively stable. DETAILED DESCRIPTION

[0015] In order to make the purpose, process conditions and advantages of the present invention more clear, the present invention is further described in detail in conjunction with the following embodiments. However, the embodiments of the present invention are not limited thereto. Without departing from the above-mentioned technical ideas of the present invention, various replacements and changes are made according to common technical knowledge and customary means in this field, which should be included in the scope of the present invention. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0016] Example 1: The present embodiment provides a method for preparing Baiyaojian to prevent aflatoxin, comprising the following steps: (1) Wash 500 g of gallnuts and bake them in an oven at 70°C for 48 hours to dry them. Use a grinder to sieve the dried gallnut powder through a 60-mesh sieve. Sterilize the gallnut powder with 270°C high-temperature steam and cool it to room temperature for later use. (2) preparing tannase purified solution for later use; (3) Take green tea and add purified water to boil into juice, filter out the tea residue, concentrate and cool to room temperature for later use; (4) Gallnut powder, tannase purified solution, green tea concentrate, and activated carbon powder concentrate were mixed in a sealed reactor, stirred at a speed of 50 rad / min, maintained at a temperature of 40°C, reacted for 4.5 h, and had an initial pH of 6.5. The enzyme concentration was 25 U / g, and the amount of activated carbon powder contained in each liter of reaction solution did not exceed 20 g. (5) After the reaction is completed, the reaction liquid in the reactor is extracted, treated in a water bath at 100°C for 8 minutes, and then cooled to room temperature. The purpose of this step is mainly to inactivate tannase and prevent the active tannase from causing adverse effects on the final Baiyaojian product; (6) Freeze-dry the reaction solution, and compress the powder obtained after freeze-drying into blocks to obtain the finished Baiyaojian product. Since the final product contains inactivated tannase, even after freeze-drying into powder, the inactivated tannase can also serve as a bonding and curing agent to ensure that the final Baiyaojian product can be directly compressed into blocks. The Baiyaojian product is stored in a ventilated and dry place. High performance liquid chromatography was used to detect the content of gallic acid in the prepared Baiyaojian product, and the final measured gallic acid content was 49.5%. The prepared Baiyaojian met the requirement that the gallic acid content should be no less than 30.0%.

[0017] Example 2: In the above embodiment, this embodiment further defines that in step (2), the preparation process of the tannase purified solution includes the following steps: (2.1) Activate the preserved Aspergillus tubingensis on an agar slant; (2.2) Prepare a culture medium and add the culture medium to a shake flask. Inoculate activated Aspergillus tubingensis into the shake flask containing the culture medium for propagation and expansion. The culture medium uses bran, glucose, and soybean meal powder as fermentation substrates. After adding an inorganic salt aqueous solution, sterilize and cool. The bran, glucose, and soybean meal powder are uniformly mixed in a mass ratio of 1:2:1. The culture medium substrate and the inorganic salt aqueous solution are in a mass ratio of 3:5. Each liter of the inorganic salt aqueous solution contains 5g-10g of NH4H2PO4, 2g-4g of K2HPO4, 1g-3g of MgSO4·7H2O, and 1g-3g of CaCl2.

[0018] (2.3) Transfer the Aspergillus tubingensis grown in the shake flask into a water-tight constant temperature incubator at 37°C and ferment for 60-90 hours. (2.4) After fermentation, add pH 6.5 tartaric acid-sodium tartrate buffer, place on a shaker and shake for 1-2 hours, filter, and collect the crude tannase enzyme solution; (2.5) The crude enzyme solution is purified by a layered precipitation method to obtain a purified tannase solution. The specific process of purifying the crude enzyme solution by the layered precipitation method is as follows: a saturated sodium chloride aqueous solution is added to the crude enzyme solution, the precipitate is filtered out and collected, and the precipitate is dissolved in a tartaric acid-sodium tartrate buffer solution, and the precipitate is subjected to chromatography on a chromatography column to obtain a purified tannase solution.

[0019] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the claims and their equivalents.

Claims

1. A method for preparing Baiyaojian to avoid Aspergillus flavus, characterized in that: The following steps are involved: (1) Wash the Chinese gallnut, dry and crush it, pass it through a 40-80 mesh sieve, sterilize it, and cool it to room temperature for later use; (2) preparing tannase purified solution for later use; (3) Take green tea and add purified water to boil into juice, filter out the tea residue, concentrate and cool to room temperature for later use; (4) Mix the gallnut powder, tannin purified solution, green tea concentrate, and activated carbon powder concentrate in a sealed reactor, keep stirring at a speed of 30 rad / min to 60 rad / min, keep the temperature at 35°C to 45°C, the reaction time is 3h to 6h, and the initial pH is 6.5; (5) After the reaction is completed, extract the reaction liquid in the reactor, treat it in a 100°C water bath for 5 min to 10 min, and then cool it to room temperature; (6) freeze-drying the reaction solution, and compressing the powder obtained after freeze-drying into blocks to obtain the finished Baiyaojian product. The Baiyaojian product is stored in a ventilated and dry place.

2. The method for preparing Baiyaojian for avoiding aflatoxin according to claim 1, characterized in that: In the step (1), the washed gallnuts are placed in an oven at 70° C. for 48 hours for drying, and the dried gallnuts are crushed by a crusher.

3. A method for preparing Baiyaojian to avoid Aspergillus flavus according to claim 1 or 2, characterized in that: In the step (1), the gallnut powder is sterilized by using high-temperature steam at 250° C. to 300° C.

4. A method for preparing Baiyaojian for avoiding Aspergillus flavus according to claim 1 or 2, characterized in that: In the step (2), the preparation process of the purified tannase solution comprises the following steps: (2.1) Activating the preserved Aspergillus tubingensis on an agar slants; (2.2) preparing a culture medium and adding the culture medium into a shake flask, inoculating the activated Aspergillus tubingensis into the shake flask containing the culture medium for propagation and expansion; (2.3) Transfer the Aspergillus tubingensis grown in the shake flask into a water-tight constant temperature incubator, provide a constant temperature of 37°C, and ferment for 60h~90h; (2.4) After the fermentation is completed, add pH 6.5 tartaric acid-sodium tartrate buffer, place on a shaker for 1h~2h, filter and collect the crude enzyme solution of tannin; (2.5) The crude enzyme solution is purified by the layered precipitation method to obtain tannase purified solution.

5. The method for preparing Baiyaojian for avoiding Aspergillus flavus according to claim 4, characterized in that: The preparation process of the culture medium in step (2.2) is to use bran, glucose and soybean meal powder as substrates of the fermentation medium, add an aqueous solution of inorganic salts, and then sterilize and cool.

6. The method for preparing Baiyaojian for avoiding aflatoxin according to claim 5, characterized in that: In the culture medium, bran, glucose and soybean meal powder are uniformly mixed in a mass ratio of 1:2:1, the culture medium substrate and the aqueous solution with added inorganic salts are in a mass ratio of 3:5, and each liter of the aqueous solution of inorganic salts contains 5g~10g of NH4H2PO4, 2g~4g of K2HPO4, 1g~3g of MgSO4·7H2O and 1g~3g of CaCl2.

7. The method for preparing Baiyaojian for avoiding aflatoxin according to claim 6, characterized in that: In the step (2.5), the specific process of purifying the crude enzyme solution by the layered precipitation method is as follows: adding a saturated sodium chloride aqueous solution to the crude enzyme solution, filtering out and collecting the precipitate, dissolving the precipitate with a tartaric acid-sodium tartrate buffer, and performing chromatography on a chromatography column to obtain a tannase purified solution.