Preparation method of fermented pinellia tuber

Abstract

The invention relates to the technical field of traditional Chinese medicines, in particular to a preparation method of fermented pinellia tuber, which comprises the following specific steps: S1, crushing rhizoma pinelliae preparata and medicated leaven into fine powder; s2, preparing ginger juice; s3, dissolving alum in a proper amount of water, adding the ginger juice, uniformly mixing, and uniformly mixing with the rhizoma pinelliae preparata, the medicated leaven, the flour and the raw bran which are crushed into fine powder; s4, knocking out an ochratoxin synthetic gene (otaA) from the aspergillus oryzae CICC 40186 through CRISPR-Cas9, and transferring the ochratoxin synthetic gene (otaA) into a lactic dehydrogenase gene (ldhL) of lactobacillus plantarum; s5, preparing a compound bacterium suspension; s6, placing the inoculated material in a solid state fermentation reactor; s7, vacuum freeze drying and variable-temperature drying are combined; the consistency of components of different batches of fermented pinellia can be guaranteed, the risk of infectious microbe contamination can be avoided, ochratoxin can be completely eradicated, the fermentation period can be greatly shortened, the overall production efficiency can be improved, the content of the key active component gamma-aminobutyric acid can be greatly improved, and the stability between batches is good.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine, and particularly relates to a preparation method of pinellia ternate cake. BACKGROUND

[0002] Pinellia ternate cake is a traditional Chinese medicinal material. The product is prepared from pinellia ternate, flour and ginger juice. It has the functions of relieving cough and reducing phlegm, and treating food accumulation and diarrhea.

[0003] The existing traditional pinellia ternate cake is mostly prepared by natural fermentation, that is, by relying on natural microbial communities in the natural environment (such as mold, yeast and bacteria in the air). Although the fermentation can be completed and pinellia ternate cake can be prepared, the natural fermentation has the following disadvantages: 1. The fermentation bacterial population is too dependent on the production environment (such as workshop air, equipment and microorganisms carried by operating personnel), and the composition of different batches of bacterial populations is significantly different, so that the consistency of the components of different batches of pinellia ternate cake cannot be guaranteed; 2. The risk of contamination by miscellaneous bacteria is high, and the safety cannot be guaranteed, and there is a risk of contamination by ochratoxin; 3. The fermentation period is long, and the efficiency is low; 4. The content of the effective component (such as gamma-aminobutyric acid) is low and unstable. SUMMARY

[0004] The present application aims to solve the problems in the prior art. The preparation method of pinellia ternate cake can guarantee the consistency of the components of different batches of pinellia ternate cake, avoid the risk of contamination by miscellaneous bacteria, eliminate the production of ochratoxin, greatly shorten the fermentation period, improve the overall production efficiency, greatly increase the content of the key active component gamma-aminobutyric acid and improve the stability between batches.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: a preparation method of pinellia ternate cake, comprising the following specific steps:

[0006] S1, grinding the pinellia ternate and the wheat bran into fine powder, wherein the proportion of the pinellia ternate and the wheat bran that can pass through a No. 5 sieve and a No. 6 sieve is not less than 95%;

[0007] S2, preparing ginger juice;

[0008] S3, dissolving the white alum in water, mixing the ginger juice, and then mixing the ground pinellia ternate and the wheat bran with flour and bran, and forming the mixture into block-shaped fermentation blocks;

[0009] S4, knocking out the ochratoxin synthesis gene (otaA) of Aspergillus oryzae CICC 40186 by CRISPR-Cas9 and transferring the lactic acid dehydrogenase gene (ldhL) of Lactobacillus plantarum to obtain a recombinant strain AO-LDH;

[0010] S5, preparing a complex bacteria suspension, and spraying the complex bacteria suspension on the to-be-fermented mass, the complex bacteria suspension comprising the recombinant strain AO-LDH, Lactobacillus plantarum CICC 23128, and Saccharomyces cerevisiae CICC 31080;

[0011] S6, placing the inoculated material in a solid-state fermentation reactor, dynamically adjusting the temperature to 30-35℃, the humidity to 60-80%, and the ventilation volume to 0.05-0.2 L / min·kg, and fermenting until the pH stabilizes at 4.2-4.5;

[0012] S7, adopting vacuum freeze drying combined with variable-temperature drying, pre-freezing at -35 to -40℃ for 1.5-2h, and then drying at 40-45℃ and 9-10 Pa until the moisture content is ≤12%.

[0013] Further improvement of the above scheme is that the step S2 specifically comprises the following steps:

[0014] The ginger juice is extracted by using cellulase-pectinase complex enzymolysis combined with pulsed ultrasonic extraction, the cellulase is added in an amount of 0.2-0.5% w / w, and the pectinase is added in an amount of 0.1-0.3% w / w, wherein the enzymolysis conditions are 45-50℃, pH 5.0-5.5 for 30-40min, and the ultrasonic parameters are 35-40kHz, 450-500W pulsed for 20-25min.

[0015] Further improvement of the above scheme is that the inoculation amount of the complex bacteria suspension in the step S5 is 3-8% v / w, the spore number of the recombinant strain AO-LDH is ≥1×10 6 CFU / g, the viable bacterial count of Lactobacillus plantarum is ≥1×10 7 CFU / mL, and the viable bacterial count of the yeast is ≥1×10 6 CFU / mL.

[0016] Further improvement of the above scheme is that the step S6 comprises three-stage regulation, specifically as follows:

[0017] Adaptation period 0-12h: control the temperature to 30-32℃, the humidity to 70-75%, and intermittent ventilation (ventilation 3-5min / h);

[0018] Logarithmic phase 12-36h: control the temperature to 32-34℃, the humidity to 75-80%, and continuous ventilation 0.2-0.25 L / min·kg;

[0019] Stationary phase 36-48h: control the temperature to 33-35℃, the humidity to 60-65%, and reduce the ventilation volume to 0.05-0.1 L / min·kg.

[0020] A further improvement of the above scheme is that the drying process of step S7 further comprises spraying 0.1-0.3% nano-SiO2 moisture-proof coating in the variable-temperature drying stage.

[0021] A further improvement of the above scheme is that the spraying of the complex bacteria suspension on the fermentation lumps in step S5 uses an electrostatic spraying device with an atomized particle size of 20-50 μm, the voltage of the electrostatic spraying is 5-8 kV, and the spraying rate is 3-5 mL / min.

[0022] A further improvement of the above scheme is that the spraying of the complex bacteria suspension on the fermentation lumps in step S5 is sprayed twice, the first spraying is at the beginning of fermentation and the inoculation amount is 60-70%, and the remaining 30-40% inoculation amount is supplemented after 10-12 h of fermentation, and after each spraying, sterile breathable film is immediately covered, and ventilation is started after 2-3 h of standing.

[0023] A further improvement of the above scheme is that in step S4, the Aspergillus oryzae CICC 40186 is knocked out of the ochratoxin synthesis gene (otaA) by CRISPR-Cas9 and the lactate dehydrogenase gene (ldhL) of Lactobacillus plantarum is introduced to obtain a recombinant strain AO-LDH, which specifically comprises the following steps:

[0024] S41, design sgRNA targeting otaA gene;

[0025] S42, construct CRISPR-Cas9 editing vector containing sgRNA and hSpCas9 gene;

[0026] S43, clone Lactobacillus plantarum ldhL gene and connect to Aspergillus oryzae GAPDH site homologous recombination vector;

[0027] S44, co-transform the above vector into Aspergillus oryzae CICC 40186 by Agrobacterium mediation;

[0028] S45, screen the transformants on PDA plates containing 0.3-0.5 mol / L Al 3 + and 100 μg / mL hygromycin;

[0029] S46, verify otaA knockout and ldhL expression by PCR and Western blot.

[0030] A further improvement of the above scheme is that the complex bacteria suspension in step S5 further comprises 0.1-0.5% trehalose, 0.05-0.15% polyvinylpyrrolidone, 2-5 mM CaCl2, and 0.01-0.05% ascorbic acid.

[0031] A further improvement of the above scheme is that the preparation of the complex bacteria suspension in step S5 specifically comprises the following steps:

[0032] S51, after the recombinant strain AO-LDH is washed with sterile normal saline, it is suspended in a solution containing 0.1-0.5% trehalose to obtain a first bacterial suspension;

[0033] S52, centrifugal collection of activated culture of Lactobacillus plantarum CICC 23128 and Saccharomyces cerevisiae CICC 31080 is carried out, and the bacterial body is resuspended in a buffer containing 2-5mM CaCl2 to obtain a second bacterial suspension;

[0034] S53, the first bacterial suspension and the second bacterial suspension are mixed, and 0.05-0.15% polyvinylpyrrolidone and 0.01-0.05% ascorbic acid are added to obtain the composite bacterial suspension.

[0035] The preparation method of the half summer cake has the advantages that:

[0036] S1, the clean pinellia ternata and the six gods cake are crushed into fine powder, and the clean pinellia ternata and the six gods cake crushed into fine powder can pass through a five-hole sieve and the proportion of passing through a six-hole sieve is not less than 95%;

[0037] S2, ginger juice is prepared;

[0038] S3, the alum is dissolved in water, the ginger juice is added and mixed, and then the clean pinellia ternata and the six gods cake crushed into fine powder, flour and raw bran are mixed, and the mixture is made into a block-shaped fermentation mass;

[0039] S4, the aspergillus oryzae CICC 40186 is knocked out of the ochratoxin synthesis gene (otaA) by CRISPR-Cas9 and the lactate dehydrogenase gene (ldhL) of lactobacillus plantarum is introduced to obtain a recombinant strain AO-LDH;

[0040] S5, a composite bacterial suspension is prepared, and the composite bacterial suspension is sprayed on the fermentation mass, the composite bacterial suspension comprising the recombinant strain AO-LDH, lactobacillus plantarum CICC 23128 and saccharomyces cerevisiae CICC 31080;

[0041] S6, the inoculated material is placed in a solid-state fermentation reactor, the temperature is dynamically adjusted to 30-35℃, the humidity is dynamically adjusted to 60-80%, and the ventilation is dynamically adjusted to 0.05-0.2L / min·kg, and the fermentation is carried out until the pH is stable at 4.2-4.5;

[0042] S7, vacuum freeze drying combined with variable temperature drying is adopted, pre-freezing at-35 to-40℃ for 1.5-2h, and then drying at 40-45℃, 9-10Pa to moisture≤12%;

[0043] The high-activity amylase and protease are secreted by the recombinant strain AO-LDH to decompose starch and protein in pinellia ternate and release precursor substances; the synthesis ability of ochratoxin is eliminated by knocking out the ochratoxin synthesis gene (otaA); the exogenous lactate dehydrogenase is expressed by introducing the lactate dehydrogenase gene (ldhL) of lactobacillus plantarum to convert pyruvic acid into lactic acid, so that the pH of the fermentation system is rapidly reduced to 4.2-4.5; lactic acid is metabolized by lactobacillus plantarum CICC 23128, which not only can reduce the pH value in cooperation with the recombinant strain AO-LDH, but also can inhibit pathogenic bacteria such as escherichia coli; phenylethanol and ethyl acetate are metabolized by saccharomyces cerevisiae CICC 31080, which not only can synthesize aroma substances, but also can consume residual oxygen to promote the anaerobic metabolism of the recombinant strain AO-LDH, and can convert part of lactic acid into acetic acid to maintain the pH stability at 4.2-4.5; by using the gene editing recombinant strain AO-LDH and the compound microbial flora of lactobacillus plantarum CICC 23128 and saccharomyces cerevisiae CICC 31080, the problem of randomness and uncontrollability of natural fermentation can be avoided, the consistency of the components of pinellia ternate can be ensured, the risk of contamination by miscellaneous bacteria can be avoided, the production of ochratoxin can be eliminated, the fermentation period can be greatly shortened, the overall production efficiency can be improved, the content of the key active ingredient gamma-aminobutyric acid can be greatly improved, and the batch stability is good. DETAILED DESCRIPTION

[0044] The application is further described below, and the application comprises the following specific steps:

[0045] S1, the pinellia ternate and the six gods are crushed into fine powder, and the pinellia ternate and the six gods crushed into fine powder can pass through a five-hole sieve and a six-hole sieve at a ratio of not less than 95%;

[0046] S2, preparing ginger juice;

[0047] S3, dissolving the white alum in water, adding the ginger juice, mixing, then mixing with the pinellia ternate and the six gods crushed into fine powder, flour and raw bran to form a block-shaped fermentation mass;

[0048] S4, knocking out the ochratoxin synthesis gene (otaA) of aspergillus oryzae CICC 40186 by CRISPR-Cas9 and introducing the lactate dehydrogenase gene (ldhL) of lactobacillus plantarum to obtain the recombinant strain AO-LDH;

[0049] S5, preparing a compound bacteria suspension, and spraying the compound bacteria suspension on the fermentation mass, wherein the compound bacteria suspension comprises the recombinant strain AO-LDH, lactobacillus plantarum CICC 23128 and saccharomyces cerevisiae CICC 31080;

[0050] S6, placing the inoculated material in a solid-state fermentation reactor, dynamically adjusting the temperature to 30-35 DEG C, the humidity to 60-80%, and the ventilation to 0.05-0.2 L / min·kg, and fermenting until the pH stabilizes at 4.2-4.5;

[0051] S7, using vacuum freeze drying combined with variable temperature drying, pre-freezing at -35 to -40 DEG C for 1.5-2 hours, and then drying at 40-45 DEG C and 9-10 Pa until the moisture content is less than or equal to 12%.

[0052] The recombinant strain AO-LDH secretes high-activity amylase and protease to decompose starch and protein in Pinellia ternate, release the precursor of the medicinal effect, eliminate the ability to synthesize ochratoxin by knocking out the ochratoxin synthesis gene (otaA), express exogenous lactate dehydrogenase by introducing the lactate dehydrogenase gene (ldhL) of Lactobacillus plantarum, convert pyruvic acid into lactic acid, rapidly reduce the pH of the fermentation system to 4.2-4.5, and produce lactic acid through the metabolism of Lactobacillus plantarum CICC 23128, which not only reduces the pH value in cooperation with the recombinant strain AO-LDH, but also inhibits pathogenic bacteria such as Escherichia coli, produces volatile components such as phenethyl alcohol and ethyl acetate through the metabolism of Saccharomyces cerevisiae CICC 31080, not only synthesizes aroma substances, but also consumes residual oxygen, promotes the anaerobic metabolism of the recombinant strain AO-LDH, and also converts part of the lactic acid into acetic acid to maintain the pH at 4.2-4.5. The present application uses gene editing to recombine the strain AO-LDH and the complex microbial flora of Lactobacillus plantarum CICC 23128 and Saccharomyces cerevisiae CICC 31080 in a ratio, and directional inoculation and fermentation, which can avoid the randomness and uncontrollability of natural fermentation, ensure the consistency of the components of Pinellia ternate in different batches, avoid the risk of contamination by miscellaneous bacteria and eliminate the production of ochratoxin, greatly shorten the fermentation period and improve the overall production efficiency, greatly improve the content of the key active ingredient gamma-aminobutyric acid and have good batch-to-batch stability.

[0053] The step S2 specifically comprises the following steps:

[0054] The cellulase-pectinase complex enzymolysis combined with pulse ultrasonic is used to extract ginger juice, the cellulase is added in an amount of 0.2-0.5% w / w, the pectinase is added in an amount of 0.1-0.3% w / w, the enzymolysis conditions are 45-50 DEG C, pH 5.0-5.5 for 30-40 min, and the ultrasonic parameters are 35-40 kHz, 450-500 W pulse for 20-25 min; compared with the traditional pressing method for extracting ginger juice, the cellulase is used to specifically degrade the cell wall cellulose, the pectinase is used to target the pectin layer, the double enzymes are used to improve the dissolution rate of the effective components, and the cavitation effect generated by the pulse ultrasonic greatly improves the cell wall breaking efficiency, so that the overall time for extracting ginger juice is shortened, and the ginger juice extraction rate is greatly improved.

[0055] The inoculation amount of the complex bacteria suspension in the step S5 is 3-8% v / w, the spore number of the recombinant strain AO-LDH is greater than or equal to 1*10 6 CFU / g, the initial activity of amylase is greater than or equal to 300 U / g, the viable bacterial number of Lactobacillus plantarum is greater than or equal to 1*10 7 CFU / mL, the pathogenic bacteria such as Escherichia coli can be better inhibited, and the viable bacterial number of yeast is greater than or equal to 1*10 6 CFU / mL, residual oxygen can be better consumed, and anaerobic metabolism of the recombinant strain AO-LDH is promoted.

[0056] The step S6 includes three-stage regulation, and the specific regulation is as follows:

[0057] The adaptation period is 0-12 h, the temperature is controlled to be 30-32 DEG C, the humidity is controlled to be 70-75%, and intermittent ventilation (ventilation 3-5 min / h) is performed;

[0058] The logarithmic phase is 12-36 h, the temperature is controlled to be 32-34 DEG C, the humidity is controlled to be 75-80%, and continuous ventilation 0.2-0.25 L / min kg is performed;

[0059] The stationary phase is 36-48 h, the temperature is controlled to be 33-35 DEG C, the humidity is controlled to be 60-65%, and the ventilation amount is reduced to 0.05-0.1 L / min kg;

[0060] The adaptation period regulation is more conducive to improving the spore germination rate of Aspergillus oryzae, the stepwise temperature rise can reduce the stress death of the bacteria, and the oxygen regulation from intermittent to continuous to reduction can accurately match the metabolic stage, so that the optimal fermentation environment condition can be realized more accurately through the three-stage cooperation.

[0061] The drying process of the step S7 also comprises spraying 0.1-0.3% nano-SiO2 moisture-proof coating in the variable-temperature drying stage, filling the material surface micro-cracks with nano-SiO2, forming a molecular sieve effect to block H2O molecules and allow O2 / CO2 to pass through, which can greatly reduce the surface mold adhesion rate.

[0062] The step S5 adopts an electrostatic spraying device with a 20-50 μm atomized particle size to spray the composite bacteria suspension on the to-be-fermented briquettes, the voltage of the electrostatic spraying is 5-8 kV, and the spraying rate is 3-5 mL / min; through the cooperation of accurate atomization and electrostatic adsorption, the problems of insufficient penetration, bacteria solution waste, and poor uniformity in the traditional inoculation mode are solved.

[0063] The step S5 comprises twice spraying of the composite bacteria suspension on the to-be-fermented briquettes, the first spraying is performed at the beginning of fermentation and the inoculation amount is 60-70%, and the remaining 30-40% inoculation amount is supplemented after 10-12 h of fermentation, the sterile breathable film is covered immediately after each spraying, and the ventilation is started after 2-3 h of standing; through the first inoculation, the dominant bacteria population can be quickly established, and the proportion of Aspergillus oryzae is more than 85%, and then through the second supplementation, the logarithmic phase demand is supplemented, which can reduce the overall bacteria usage and cost.

[0064] The step S4 comprises the following steps of obtaining a recombinant strain AO-LDH by knocking out the ochratoxin synthesis gene (otaA) of Aspergillus oryzae CICC 40186 through CRISPR-Cas9 and introducing the lactate dehydrogenase gene (ldhL) of Lactobacillus plantarum:

[0065] S41, designing an sgRNA targeting the otaA gene;

[0066] S42, constructing a CRISPR-Cas9 editing vector comprising sgRNA and hSpCas9 gene;

[0067] S43, cloning the ldhL gene of Lactobacillus plantarum and connecting it to the homologous recombination vector of the GAPDH site of Aspergillus oryzae;

[0068] S44, co-transforming the above-mentioned vector into Aspergillus oryzae CICC 40186 through Agrobacterium mediation;

[0069] S45, screening the transformants on PDA plates containing 0.3-0.5 mol / L Al 3 + and 100 μg / mL hygromycin;

[0070] S46, verifying the otaA knockout and ldhL expression through PCR and Western blot.

[0071] The complex bacteria suspension in the step S5 also comprises 0.1-0.5% trehalose, 0.05-0.15% polyvinylpyrrolidone, 2-5 mM CaCl2, 0.01-0.05% ascorbic acid; the trehalose plays the role of bacteria body protective agent, can play the roles of freeze-drying and spray protection, greatly improves the AO-LDH spore survival rate, the polyvinylpyrrolidone can prevent bacteria aggregation and reduce surface tension, so that the material penetration is enhanced, the CaCl2 can improve the acid tolerance of lactobacillus plantarum, the ascorbic acid can keep the superoxide dismutase (SOD) activity ≥85%, so that the fermentation can be further ensured better.

[0072] The preparation of the complex bacteria suspension in the step S5 specifically comprises the following steps:

[0073] S51, after the recombinant strain AO-LDH is washed with sterile normal saline, it is suspended in a solution containing 0.1-0.5% trehalose to obtain a first bacteria suspension;

[0074] S52, centrifugal collection of lactobacillus plantarum CICC 23128 and saccharomyces cerevisiae CICC 31080 activation culture bacteria, resuspended in a buffer containing 2-5 mM CaCl2 to obtain a second bacteria suspension;

[0075] S53, the first bacteria suspension and the second bacteria suspension are mixed, and 0.05-0.15% polyvinylpyrrolidone and 0.01-0.05% ascorbic acid are added to obtain the complex bacteria suspension.

[0076] The key parameters of the present application and the traditional natural fermentation process are compared as follows:

[0077] 1, comparison of basic process parameters

[0078]

[0079]

[0080] 2, comparison of product quality indicators

[0081]

[0082] Of course, the above-mentioned is only the preferred embodiment of the present application, so that the equivalent changes or modifications made according to the structure, features and principles described in the scope of the present application are included in the scope of the present application.

Claims

1. A method for preparing Pinellia ternata curd, characterized in that, The specific steps include the following: S1. Pulverize Pinellia ternata and Shenqu into fine powder, wherein all of the pulverized Pinellia ternata and Shenqu can pass through a No. 5 sieve and the proportion that can pass through a No. 6 sieve is not less than 95%. S2. Prepare ginger juice; S3. Dissolve alum in an appropriate amount of water, add ginger juice and mix well. Then mix it with finely powdered Pinellia ternata, Shenqu (a type of medicinal fermentation), flour, and raw bran. Make the mixture into blocks to be fermented. S4. The ochratoxin synthesis gene (otaA) of Aspergillus oryzae CICC 40186 was knocked out by CRISPR-Cas9 and transformed into the lactate dehydrogenase gene (ldhL) of Lactobacillus plantarum to obtain the recombinant strain AO-LDH. S5. Prepare a compound bacterial suspension and spray the compound bacterial suspension onto the fermentation agglomerates. The compound bacterial suspension contains recombinant strain AO-LDH, Lactobacillus plantarum CICC 23128 and Saccharomyces cerevisiae CICC 31080. S6. Place the inoculated material in a solid-state fermentation reactor, dynamically adjust the temperature to 30-35℃, humidity to 60-80%, and ventilation rate to 0.05-0.2L / min·kg, and ferment until the pH stabilizes at 4.2-4.5; S7. Vacuum freeze drying combined with variable temperature drying is adopted. First, it is pre-frozen at -35 to -40℃ for 1.5 to 2 hours, and then dried at 40 to 45℃ and 9 to 10 Pa until the moisture content is ≤12%.

2. The method for preparing Pinellia ternata according to claim 1, characterized in that: Step S2 specifically includes the following steps: Ginger juice was extracted using a combination of cellulase-pectinase enzymatic hydrolysis and pulsed ultrasound. The amount of cellulase added in the combined enzymatic hydrolysis was 0.2-0.5% w / w, and the amount of pectinase added was 0.1-0.3% w / w. The enzymatic hydrolysis conditions were 45-50℃ and pH 5.0-5.5 for 30-40 min, and the ultrasound parameters were 35-40 kHz and 450-500 W pulsed treatment for 20-25 min.

3. The method for preparing Pinellia ternata according to claim 2, characterized in that: The inoculation amount of the compound bacterial suspension in step S5 is 3-8% v / w, and the spore number of the recombinant strain AO-LDH is ≥1×10⁻⁶. 6 CFU / g, viable count of Lactobacillus plantarum ≥1×10 7 CFU / mL, viable yeast count ≥1×10⁻⁶ 6 CFU / mL.

4. The method for preparing Pinellia ternata according to claim 1, characterized in that: Step S6 includes three stages of regulation, as detailed below: Adaptation period 0-12h: control temperature 30-32℃, humidity 70-75%, intermittent ventilation (ventilation 3-5min / h); Logarithmic period 12–36 h: control temperature 32–34℃, humidity 75–80%, continuous ventilation 0.2–0.25 L / min·kg; Stabilization period 36-48h: control temperature at 33-35℃ and humidity at 60-65%, and reduce ventilation to 0.05-0.1L / min·kg.

5. The method for preparing Pinellia ternata according to claim 1, characterized in that: The drying process in step S7 also includes spraying a 0.1-0.3% nano-SiO2 moisture-proof coating during the variable temperature drying stage.

6. The method for preparing Pinellia ternata according to claim 1, characterized in that: In step S5, the compound bacterial suspension is sprayed onto the fermentation clumps using an electrostatic spraying device with an atomization particle size of 20–50 μm. The voltage of the electrostatic spray is 5–8 kV, and the spraying rate is 3–5 mL / min.

7. The method for preparing Pinellia ternata according to claim 6, characterized in that: In step S5, the compound bacterial suspension is sprayed on the fermentation clumps in two stages. The first spray is applied at the beginning of fermentation with an inoculum of 60-70%. The remaining 30-40% of the inoculum is applied after 10-12 hours of fermentation. After each spray, the clumps are immediately covered with a sterile, breathable membrane and allowed to stand for 2-3 hours before ventilation is started.

8. The method for preparing Pinellia ternata according to claim 1, characterized in that: Step S4, which involves knocking out the ochratoxin synthesis gene (otaA) from Aspergillus oryzae CICC 40186 using CRISPR-Cas9 and then transferring it into the lactate dehydrogenase gene (ldhL) of Lactobacillus plantarum to obtain the recombinant strain AO-LDH, specifically includes the following steps: S41. Design sgRNAs targeting the otaA gene; S42. Construct a CRISPR-Cas9 editing vector containing sgRNA and hSpCas9 genes; S43, clone the Lactobacillus plantarum ldhL gene and ligate it into the Aspergillus oryzae GAPDH site homologous recombination vector; S44. The above vector was co-transformed into Aspergillus oryzae CICC 40186 via Agrobacterium-mediated transformation. S45, containing 0.3-0.5 mol / L Al 3 Transformants were screened on PDA plates with + and 100 μg / mL hygromycin; S46. OtaA knockout and ldhL expression were verified by PCR and Western blot.

9. The method for preparing Pinellia ternata according to claim 1, characterized in that: The composite bacterial suspension in step S5 further includes 0.1-0.5% trehalose, 0.05-0.15% polyvinylpyrrolidone, 2-5 mM CaCl2, and 0.01-0.05% ascorbic acid.

10. The method for preparing Pinellia ternata according to claim 9, characterized in that: The preparation of the composite bacterial suspension in step S5 specifically includes the following steps: S51. After washing the recombinant strain AO-LDH with sterile physiological saline, it was suspended in a solution containing 0.1-0.5% trehalose to obtain the first bacterial suspension. S52. Centrifuge the activated cultures of Lactobacillus plantarum CICC 23128 and Saccharomyces cerevisiae CICC 31080 to collect the bacterial cells, and resuspend them in a buffer solution containing 2-5 mM CaCl2 to obtain a second bacterial suspension. S53. Mix the first bacterial suspension and the second bacterial suspension, and add 0.05-0.15% polyvinylpyrrolidone and 0.01-0.05% ascorbic acid to obtain the composite bacterial suspension.

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