New application of compounding pediococcus pentosaceus and aspergillus aculeatus and preparation method of indigo naturalis

Abstract

The invention provides application of aspergillus aculeatus in fermentation processing of traditional Chinese medicine indigo naturalis. The invention further provides application of the combined use of the pediococcus pentosaceus and the aspergillus aculeatus in fermentation processing of the traditional Chinese medicine indigo naturalis. The combination of the aspergillus aculeatus and the pediococcus pentosaceus has obvious advantages in the fermentation of the indigo naturalis. The invention also provides a preparation method of indigo naturalis. In the aspect of an enzyme system, beta-glucosidase generated by the aspergillus aculeatus and the pediococcus pentosaceus can promote dissolution of an indigo precursor substance indole glycoside. In addition, other specific enzymes, such as glucoside hydrolase or oxidoreductase and the like, can also be generated, complex components in indigo naturalis are further decomposed through mutual cooperation of the two enzymes, more precursor substances are released, generation and accumulation of indigo blue and indirubin are jointly promoted, and the content of indigo blue and indirubin is increased. In addition, growth and metabolism of harmful microorganisms can be inhibited, harmful substances possibly generated in the indigo naturalis fermentation process are reduced, and the quality and safety of indigo naturalis products are improved.

Description

Technical Field

[0001] The invention relates to a new application of a compound of Pediococcus pentosaceus and Aspergillus aculeatus, and a method for preparing Indigo Naturalis. Background Art

[0002] Indigo is a dry powder, agglomerate or granule obtained by soaking and fermenting the leaves or stems of Baphicacanthuscusia (Nees) Bremek. of the Acanthaceae family, Polygonum tinctorium Ait. of the Polygonaceae family or Isatis indigotica Fort. of the Cruciferae family, and then beating the indigo with lime and refining with water. Indigo is widely used in the field of traditional medicine. In the theory of traditional Chinese medicine, Indigo has the effects of clearing away heat and detoxifying, cooling blood and eliminating spots, purging fire and calming the nerves. It is often used to treat various diseases such as fever and spots, vomiting and bleeding due to blood heat, coughing and bleeding due to chest pain, mouth sores, mumps, throat paralysis, and epilepsy in children. It is cold in nature and salty in taste, and belongs to the liver meridian. It can play a unique medicinal role through the combination of various prescriptions.

[0003] In modern medical research, indigo has also shown important value. Indigo and indirubin, as the main active ingredients of indigo, have significant pharmacological activity. Indigo has certain antibacterial and anti-inflammatory effects, can inhibit the growth and reproduction of a variety of pathogens, and has potential application prospects in the treatment of infectious diseases. Indirubin has been found to have anti-tumor activity. It can exert anti-cancer effects through multiple pathways such as intervening in the proliferation of tumor cells, inducing tumor cell apoptosis, and inhibiting tumor angiogenesis. It has attracted much attention in the treatment of cancers such as leukemia. In addition, in other fields, indigo is also used in the printing and dyeing industry. Its natural pigment components can be used for fabric dyeing, and it has the advantages of environmental protection and unique color. In the field of cosmetics, indigo extracts are added to skin care products because of their antioxidant properties, which helps to delay skin aging and improve skin health. Indigo and indirubin have become the core supporting components of the medicinal and other application values ​​of indigo with their unique chemical structure and biological activity, and they are of irreplaceable importance in promoting the development and application of indigo-related products.

[0004] At present, the content of indigo and indirubin in Indigo is still at a low level. Factors such as its growth environment and harvesting season will affect the content of the active ingredients of the plant source itself. Even after traditional processing, it is difficult to significantly increase the content of these two ingredients, and it is difficult to meet the growing demand for high-content indigo and indirubin in the pharmaceutical and related industries. Indigo is a Chinese herbal medicine obtained from a series of processing such as fermentation and indigo beating from the plant Indigo. However, in the processing and preparation of Indigo, the increase in the content of indigo and indirubin faces many severe challenges. The fermentation process has obvious limitations. It mostly relies on natural fermentation or a relatively extensive fermentation method, and it is difficult to accurately control the fermentation process, which makes the stability and consistency of the fermentation reaction poor, resulting in great fluctuations in the production of indigo and indirubin, and it is difficult to reach the ideal content level. In addition, the fermentation cycle of the traditional fermentation process is often long, which not only reduces production efficiency, but also increases the risk of contamination and production costs. Summary of the invention

[0005] In view of the above-mentioned difficulties in the processing and preparation of Indigo Naturalis, the present invention utilizes microorganisms to perform biological fermentation to increase the content of effective ingredients such as indigo and indigotin in Indigo Naturalis slices.

[0006] The invention provides a use of Pediococcus pentosaceus and Aspergillus aculeatus in the fermentation and processing of a traditional Chinese medicine, wherein the registration number of the Pediococcus pentosaceus in the China Industrial Microbiological Culture Collection Administration Center is CICC 22227, and the registration number of Aspergillus aculeatus in the China Industrial Microbiological Culture Collection Administration Center is CICC 2653.

[0007] The invention provides a method for preparing Indigo Naturalis, which is prepared by fermenting and processing by compounding Pediococcus pentosaceus and Aspergillus aculeatus, and comprises the following steps:

[0008] a. Selection and pretreatment of raw materials: Using Indigofera ovata as raw material, the stems and leaves are collected for fermentation;

[0009] b. Feeding: Put the stems and leaves of Indigofera tinctoria obtained in step a into a fermentation tank, and add tap water at a material ratio of 1:12;

[0010] c. Inoculate the fermentation bacteria: Pour the bacterial solution of Pediococcus pentosaceus / Aspergillus aculeatus into the fermentation tank at an inoculation rate of 3-7% and mix evenly;

[0011] d. Fermentation: The fermentation tank inoculated with the fermentation liquid is fermented at a constant temperature indoors at a temperature of 25-35°C and a pH value of 5-7;

[0012] e. Beating indigo: adding lime milk and carbonated water to the liquid fermented in step d, and introducing compressed air to beat indigo;

[0013] f. Drying: Dry and crush the thick indigo paste obtained after the indigo is beaten in step e to obtain a finished product.

[0014] Wherein, the bacterial liquid concentration of Pediococcus pentosaceus described in step c is 10 7 -10 8 CFU / mL, the concentration of Aspergillus aculeatus was 4×10 6 CFU / mL; the inoculation volume ratio was 3:2 for Pediococcus pentosaceus and Aspergillus aculeatus.

[0015] Wherein, the bacterial solution of Pediococcus pentosaceus / Aspergillus aculeatus described in step c is inoculated at a rate of 5%.

[0016] Wherein, the fermentation conditions described in step d are: fermentation temperature 30°C, pH value 7; fermentation time: 36h.

[0017] The invention provides use of Pediococcus pentosaceus / Aspergillus aculeatus in a synthesis process for degrading indole glycosides and converting indigo. The registration number of Pediococcus pentosaceus in the China Industrial Microbiological Culture Collection Administration Center is CICC 22227, and the registration number of Aspergillus aculeatus in the China Industrial Microbiological Culture Collection Administration Center is CICC 2653.

[0018] The invention provides a novel preparation process of soaking and fermenting Indigo Naturalis, which improves the yield of indigo and indirubin and shortens the soaking and fermentation time of Indigo Naturalis (from 72 hours to 36 hours) by compounding Pediococcus pentosaceus with Aspergillus aculeatus.

[0019] The combination of Aspergillus aculeatus and Pediococcus pentosaceus has significant advantages in the fermentation of indigo naturalis. In terms of enzyme system, the β-glucosidase produced by Aspergillus aculeatus and Pediococcus pentosaceus can promote the dissolution of indigo precursor indole glycosides. In addition, other specific enzymes may also be produced, such as glycoside hydrolases or oxidoreductases, which cooperate with each other to further decompose the complex components in indigo naturalis, release more precursors, and jointly promote the production and accumulation of indigo and indirubin, and increase their content.

[0020] In terms of metabolites, Pediococcus pentosaceus produces organic acids such as lactic acid during fermentation, which lowers the pH value of the fermentation environment. The acidic environment formed is not only conducive to the growth and reproduction of Aspergillus aculeatus, enabling it to function better, but may also have a positive effect on the transformation and extraction of certain components in Indigo Naturalis, thereby promoting the increase of indigo and indirubin content; at the same time, the intermediate metabolites produced by the two strains during the fermentation process can serve as each other's nutrients or growth factors, promote each other's growth and metabolism, enhance the overall fermentation effect, and thereby increase the yield of indigo and indirubin.

[0021] The combined use can also enhance the stability and controllability of fermentation. The two strains can form a relatively stable microbial ecosystem in the fermentation system. The interaction and restriction can reduce the contamination and growth opportunities of foreign bacteria, reduce the unstable factors in the fermentation process, make the fermentation process more stable and controllable, and help to achieve a stable increase in the content of indigo and indirubin. In large-scale industrial production, in the face of fluctuations in fermentation conditions, the two strains in the combined fermentation system can better cope with environmental changes through their own adaptability and mutual regulation ability, maintain the normal progress of fermentation and the stable production of products. In addition, it can inhibit the growth and metabolism of harmful microorganisms, reduce the harmful substances that may be produced during the fermentation of indigo, and improve the quality and safety of indigo products. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Effect of inoculation time of two bacteria on enzyme production;

[0023] Figure 2 Effect of the inoculation ratio of the two bacteria on enzyme production;

[0024] Figure 3 (A) Effect of temperature on enzyme production in mixed fermentation, (B) Effect of initial pH on enzyme production in mixed fermentation, (C) Effect of inoculation size on enzyme production in mixed fermentation;

[0025] Figure 4 (A) Changes of indole glycosides at different fermentation time periods, (B) Changes of indigo at different fermentation time periods, (C) Changes of indirubin at different fermentation time periods. DETAILED DESCRIPTION

[0026] Embodiment 1 Preparation method of Indigo Naturalis of the present invention

[0027] (1) Selection and pretreatment of raw materials: The indigofera tinctoria grown in Lushan, Ya'an was used as raw material, and the stems and leaves were collected for fermentation;

[0028] (2) feeding: putting the stems and leaves of Indigofera tinctoria obtained in step (1) into a fermentation tank, and adding tap water at a material ratio of 1:12;

[0029] (3) Inoculation of fermentation bacteria: Pediococcus pentosaceus and Aspergillus aculeatus bacterial suspensions (concentrations of 1×10 7 -10 8 CFU / mL, 4×10 6 CFU / mL) was poured into the fermentation tank at an inoculum volume of 5% and mixed evenly;

[0030] (4) Fermentation: The fermentation tank inoculated with the fermentation liquid was placed in a constant temperature fermentation room at a temperature of 30° C. and a pH value of 7 for a total of 36 hours;

[0031] (5) beating the indigo: adding lime milk and carbonated water to the liquid fermented in step (4), and introducing compressed air to be beating the indigo;

[0032] (6) Drying: Drying and crushing the thick indigo paste obtained after the indigo extraction in step (5) to obtain a finished product.

[0033] Among them, the strain preservation method is to mix glycerol and physiological saline in a ratio of 1:1 to form a glycerol solution with a final concentration of 50%. When in use, mix at a ratio of 50% glycerol: bacterial solution = 1:1, so that the final glycerol concentration is 20-30% to prevent the glycerol concentration from being too high or too low, which is not conducive to the preservation of the strain. Transfer the glycerol strain to a -80℃ refrigerator for long-term freezing. When the strain needs to be taken, place the cryopreservation tube in a 37℃ water bath for rapid thawing until the strain is completely thawed, at which time the strain can be taken out for activation.

[0034] Example 2 Determination of the inoculation order of the present invention

[0035] Determination of inoculation order Three groups of experiments were set up: ① Pediococcus pentosaceus (1×10 8 CFU / mL) were inoculated into MRS liquid medium at a 1% inoculation rate, incubated at 30°C for 12 h, and then inoculated with 1% Aspergillus aculeatus (4×10 6 CFU / mL) and co-cultured for 24h; ② Pediococcus pentosaceus (1×10 8 CFU / mL) with a 1% inoculum and 1% Aspergillus aculeatus (4×10 6 CFU / mL) were inoculated into MRS liquid culture medium and cultured at 30℃ for 24h; ③ Aspergillus aculeatus (4×10 6 CFU / mL) were inoculated into MRS liquid medium at a 1% inoculation amount, incubated at 30°C for 12 h, and then inoculated with 1% Pediococcus pentosaceus (1×10 8 CFU / mL) and co-cultured for 24 h. The inoculation order of the strains was confirmed by measuring the β-glucosidase activity.

[0036] from Figure 1 It can be seen that there are obvious differences in the enzyme activity of the three groups of experiments. Among them, the enzyme activity of group ② is the highest, followed by group ③, and the enzyme activity of group ① is relatively the lowest. The experimental results also show that the inoculation order has a significant effect on the activity of β-glucosidase. The enzyme activity reaches the highest when the two strains are inoculated at the same time, while the enzyme activity is reduced when one strain is inoculated first and then the other strain is inoculated, and the enzyme activity of Aspergillus aculeatus inoculated first and then Pediococcus pentosaceus is lower than that of Pediococcus pentosaceus inoculated first and then Aspergillus aculeatus.

[0037] When P. pentosaceus and A. aculeatus are inoculated at the same time, the two strains may interact with each other at the early stage of growth and form a synergistic relationship. It is speculated that P. pentosaceus secretes certain metabolites, which can provide favorable conditions for the growth and enzyme production of A. aculeatus at an early stage, and vice versa. The metabolic pathways of the two strains may complement each other and jointly promote the synthesis and secretion of β-glucosidase, resulting in the highest enzyme activity. In addition, when inoculated at the same time, the two strains may compete and utilize nutrients in the culture medium more effectively. They may optimize the distribution and utilization efficiency of nutrients in some way, allowing the strains to grow better and produce enzymes. For example, P. pentosaceus may change the microenvironment of the culture medium, making it easier for A. aculeatus to obtain certain key nutrients, thereby promoting enzyme production.

[0038] Example 3 Determination of the inoculation ratio of the present invention

[0039] The number of live bacteria of Pediococcus pentosaceus and Aspergillus aculeatus was inoculated into MRS liquid culture medium at a ratio of 1:4, 2:3, 3:2, and 4:1, wherein the concentration of 1% Aspergillus aculeatus was (4×10 6 CFU / mL), and the concentration of 1% Pediococcus pentosaceus is (1×10 8 The total inoculation amount of 1000 CFU / mL was 5%. The culture was kept static at 30°C and the enzyme activity of the strain was measured after 24 hours.

[0040] Depend on Figure 2It can be seen that with the change of the inoculation ratio of Pediococcus pentosaceus and Aspergillus aculeatus, the enzyme activity of the strain showed a trend of first increasing and then decreasing. When the inoculation ratio was 3:2, the enzyme activity of the strain reached the maximum value, which was significantly higher than the enzyme activity at other inoculation ratios. When the inoculation ratio was 0:5 (only Aspergillus aculeatus was inoculated) and 5:0 (only Pediococcus pentosaceus was inoculated), the enzyme activity was relatively low, and lower than the enzyme activity at other mixed inoculation ratios. The two bacteria will have a synergistic and competitive balance phenomenon. The two bacteria have a synergistic effect within a certain range, such as complementary metabolic pathways. The best balance is reached when the inoculation ratio is 3:2, and the strongest synergy promotes enzyme synthesis; deviating from this ratio, the synergy is weakened and the competition is enhanced. One bacterium inhibits the growth and metabolism of the other bacterium, affecting enzyme production. Different ratios affect nutrient consumption and distribution. At 3:2, the two bacteria efficiently utilize nutrients, and the growth and metabolism of the strain are good, which is conducive to enzyme synthesis; when the ratio is unbalanced or only one bacterium is inoculated, the nutrient utilization is limited or the metabolic pathway is blocked, and the enzyme activity is low. Improper mixed inoculation ratios will cause excessive nutrient consumption or accumulation, affecting strain growth and enzyme production. In addition, the metabolites produced by the growth of the two bacteria affect themselves or each other. At a ratio of 3:2, the products promote each other to form a microenvironment that is conducive to enzyme synthesis; at other ratios, product accumulation or deficiency will inhibit enzyme synthesis. For example, if there is too much of one bacteria, its product accumulation is not conducive to the growth and enzyme synthesis of the other bacteria. Therefore, the mixed bacterial inoculation volume ratio of Pediococcus pentosaceus to Aspergillus aculeatus was selected in this experiment as 3:2.

[0041] Example 4 Fermentation enzyme production condition optimization test

[0042] The single-factor rotation method was used to examine the enzyme production of the strains one by one, including the inoculation amount, temperature, and initial pH. The initial fermentation conditions were: inoculation amount 4% (Pediococcus pentosaceus: Aspergillus aculeatus = 3:2), temperature 30℃, pH7, and rotation speed 150r / min. In the optimal pH test, the initial pH value of the basal culture medium was adjusted to 4, 5, 6, 7, 8, and 9; in the optimal temperature test, the strain was inoculated into the basal culture medium and placed in a constant temperature incubator at 15, 20, 25, 30, 35, 40, 45, and 50℃, respectively. In the optimal inoculation amount test, other initial fermentation conditions remained unchanged, and the total inoculation amount of 1%, 3%, 5%, 7%, and 9% was taken for investigation. The bacterial solution was filtered through sterile gauze, and the filtrate was heated at 8000r·min -1 After centrifugation for 20 min, the supernatant was collected and the β-glucosidase activity was determined. Each treatment was repeated 3 times.

[0043] Depend on Figure 3A It can be seen that when cultured at low temperatures (such as 20°C), the metabolic rate of microorganisms slows down, and the synthesis rate of enzymes and the catalytic activity of enzymes are inhibited. This results in low enzyme activity, a decrease in the ability of its molecules to bind to substrates, and a slowdown in the rate of biochemical reactions in microbial cells. At around 30°C, the active center of the enzyme has a strong ability to bind to the substrate, the microbial metabolic activity is vigorous, and the enzyme synthesis and catalytic efficiency reach the best balance. At this time, the enzyme activity is as high as 24.84U / mL. However, high temperatures (such as 40°C and above) will denature the enzyme protein, destroy the spatial structure of the enzyme, and make it inactive. At the same time, high temperatures may damage microbial cells, affecting their normal physiological functions and the synthesis and secretion of enzymes.

[0044] Depend on Figure 3 B shows that the optimum initial pH for fermentation is 7. At this time, the spatial structure of the enzyme is stable, the active center can efficiently bind to the substrate, and the biochemical reaction in the microbial cell proceeds smoothly, which is conducive to the synthesis and catalysis of the enzyme, so the enzyme activity is the highest. However, too low or too high pH will change the charge distribution and spatial structure of the enzyme protein, causing the conformation of the enzyme active center to change, resulting in a decrease in the ability of the enzyme to bind to the substrate and a decrease in enzyme activity. At the same time, extreme pH will affect the acid-base balance and metabolic pathways in the microbial cell, thereby affecting the synthesis and secretion of the enzyme.

[0045] Depend on Figure 3 C shows that the optimum inoculation amount for mixed fermentation is 5%, and the enzyme activity is 26.80U / mL. When the number of microorganisms of the two strains is moderate, it can not only ensure rapid growth and reproduction in the initial stage of fermentation to form a certain bacterial density, but also reasonably utilize the nutrients and oxygen in the culture medium. The accumulation of metabolites will not significantly inhibit microbial growth and enzyme synthesis, thereby achieving the highest enzyme activity.

[0046] Therefore, the optimized conditions of mixed fermentation in this experiment are: temperature is 30°C, initial pH is 7, and total inoculation amount is 5%.

[0047] Example 5 Comparison between mixed fermentation and original fermentation

[0048] (1) Sample collection

[0049] This experiment is a comparative test, one of which is the original fermentation without adding Pediococcus pentosaceus and Aspergillus aculeatus (NF group), and the other is a mixed fermentation group (MSF group) with Pediococcus pentosaceus and Aspergillus aculeatus added during fermentation (activated under optimized fermentation conditions, temperature 30°C, pH 7, total inoculation volume 5%). Both groups used the same initial fermentation conditions for constant temperature immersion fermentation simulation experiments, and the material ratio was set to 1:12. The fermentation broth was collected from the fermentation barrels of the same batch at 6, 12, 18, 24, 30, 36, 48, 60, and 72 hours of fermentation, respectively. The volume of the fermentation broth collected each time was 50 ml, and 3 sets of parallel samples were set for sampling at each time point.

[0050] (2) Determination of indigo content

[0051] Determination of indole glycoside content

[0052] The absorbance of the mixed fermentation broth and the original fermentation broth at different time periods was measured by UV-visible spectrophotometer at a wavelength of 231 nm. The absorbance range was controlled at 0.2-0.8. Each group was tested three times in parallel, and the content of indole glycosides in the fermentation broth at different time periods was calculated using a standard curve.

[0053] Determination of indigo content

[0054] Refer to the indigo content determination method under the Indigo Naturalis item in the 2020 edition of the Chinese Pharmacopoeia (Volume 1).

[0055] Preparation of reference solution: Take 2.5 mg of indigo reference, accurately weigh it, place it in a 250 mL volumetric flask, add about 220 mL of 2% chloral hydrate solution in chloroform (take chloral hydrate, place it in a silica gel desiccator for 24 h, weigh 2.0 g, add chloroform to 100 mL, place it until it becomes turbid, then dehydrate it with anhydrous sodium sulfate, filter it, and you will get it), ultrasonically treat it (power 250 W, frequency 33 kHz) for 1.5 h, cool it, add 2% chloral hydrate solution in chloroform to the scale, shake it well, and you will get it (each 1 mL contains 10 μg of indigo).

[0056] Preparation of test solution: Take the fermentation freeze-dried sample powder collected at different time points, accurately weigh it, put it in a 10mL volumetric flask, add about 220mL of 2% chloral hydrate solution in chloroform, ultrasonically treat (power 250W, frequency 33kHz) for 30min, cool, add 2% chloral hydrate solution in chloroform to the scale, shake well, filter, and take the filtrate to obtain.

[0057] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel as filler; methanol-water (75:25) as mobile phase; detection wavelength at 606 nm; column temperature: 35 °C; flow rate: 1.0 mL min -1 The number of theoretical plates calculated based on the indigo peak should not be less than 1800.

[0058] Determination method: Accurately pipette 10 μL of reference solution and test solution respectively, inject into LC-10AVP high performance liquid chromatograph (Shimadzu Corporation, Japan), and determine.

[0059] Determination of indirubin content

[0060] Refer to the indigo carmine content determination method under the Indigo Naturalis item in the 2020 edition of the Chinese Pharmacopoeia (Volume 1).

[0061] Preparation of reference solution: Take 2.5 mg of indigo red reference, accurately weigh it, place it in a 50 mL volumetric flask, add about 45 mL of N, N-dimethylformamide, and dissolve it by ultrasonic treatment (power 250 W, frequency 33 kHz). Let it cool, add N, N-dimethylformamide to the scale, and shake well; accurately measure 10 mL, place it in a 100 mL volumetric flask, add N, N-dimethylformamide to the scale, shake well, and obtain (each 1 mL contains 5 μg of indigo red).

[0062] Preparation of test solution: Take the fermentation freeze-dried sample powder collected at different time points, accurately weigh it, put it in a 25mL volumetric flask, add about 20mL of N,N-dimethylformamide, ultrasonically treat (power 250W, frequency 33kHz) for 30min, cool, add N,N-dimethylformamide to the scale, shake well, filter, and take the filtrate to obtain.

[0063] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel as filler; methanol-water (75:25) as mobile phase; detection wavelength at 292 nm; column temperature: 35 °C; flow rate: 1.0 mL min -1 The number of theoretical plates calculated based on the indigo peak should not be less than 3000.

[0064] (4) Experimental results

[0065] from Figure 4 Analogous to the data trend in A, at the beginning of fermentation (6-12h), the dissolution amount of some components related to the fermentation of Ipomoea indigofera leaves (such as indole glycoside analogues) in the mixed fermentation group may be slightly higher than that in the original fermentation, but the overall content of related components is low. In the middle of fermentation (12-48h), the content of related fermentation products (indigo, indigotin and other related substances) in the mixed fermentation group may increase rapidly and may reach a peak in advance. Figure 4 B. Figure 4 In C, the MSF group may make the key components reach a higher concentration within a specific time. The indigo content of the MSF group reached a peak of about 80.25μg / mL at 36 hours, and the peak value of the NF group (45.51μg / mL) was lower than that of the MSF group. Figure 4In C, the indirubin content increased rapidly between 12 and 36 hours. The MSF group reached a peak of about 6.38 μg / mL at 36 hours, and the peak of the NF group (2.26 μg / mL) was lower than that of the MSF group. In the later stage of fermentation (48-72h), although the content of related fermentation products in the mixed fermentation group decreased, the content of key fermentation products produced during the entire fermentation process was significantly higher than that of the original fermentation. In general, MSF (mixed fermentation) promotes the production of indole glycosides, indigo and indirubin during the fermentation process. Compared with NF (original fermentation), MSF can make these three components reach higher peaks faster during the fermentation process and maintain a higher content in the later stage of fermentation.

[0066] In summary, this application innovatively proposes a method for soaking and fermenting indigo using Pediococcus pentosaceus and Aspergillus aculeatus for mixed bacterial compounding. This study has many scientific significances. First, it optimizes the preparation process of indigo, clarifies the key fermentation parameters, provides scientific basis and technical support for industrial production, helps to improve the yield and quality of indigo, reduce production costs, and promote the development of the indigo industry; second, through the study of inoculation sequence and proportion, it deeply explores the synergistic mechanism between microorganisms, enriches the theoretical knowledge in the field of microbial fermentation, and provides a reference for the development of other microbial mixed fermentation systems; third, the study of the content determination and change law of components during the fermentation process helps to establish standards and methods for indigo quality control, ensure stable product quality, and also provide basic data for further research on the pharmacological effects and efficacy of these components; fourth, the traditional indigo preparation process is combined with modern microbial fermentation technology and analytical detection technology to realize the modernization and scientificization of traditional Chinese medicine preparation technology, and provides new ideas and methods for the development and innovation of the Chinese medicine industry, which is of great significance to promoting the modernization of Chinese medicine.

Claims

1. Use of Aspergillus aculeatus in the fermentation and processing of the traditional Chinese medicine Indigo Naturalis, wherein the registration number of the Aspergillus aculeatus in the China Industrial Microbiological Culture Collection Center is CICC 2653.

2. The purpose of using Pediococcus pentosaceus and Aspergillus aculeatus in the fermentation and processing of the traditional Chinese medicine Indigo Naturalis. The registration number of Pediococcus pentosaceus in the China Industrial Microbiological Culture Collection is CICC22227, and the registration number of Aspergillus aculeatus in the China Industrial Microbiological Culture Collection is CICC 2653.

3. A method for preparing Indigo Naturalis, characterized in that: It is prepared by fermenting Pediococcus pentosaceus and Aspergillus aculeatus, and includes the following steps: a. Selection and pretreatment of raw materials: Using Indigofera ovata as raw material, the stems and leaves are collected for fermentation; b. Feeding: Put the stems and leaves of Indigofera tinctoria obtained in step a into a fermentation tank, and add tap water at a material ratio of 1:12; c. Inoculate the fermentation bacteria: Pour the bacterial solution of Pediococcus pentosaceus / Aspergillus aculeatus into the fermentation tank at an inoculation rate of 3-7% and mix evenly; d. Fermentation: The fermentation tank inoculated with the fermentation liquid is fermented at a constant temperature indoors at a temperature of 25-35°C and a pH value of 5-7; e. Beating indigo: adding lime milk and carbonated water to the liquid fermented in step d, and introducing compressed air to beat indigo; f. Drying: Dry and crush the thick indigo paste obtained after the indigo is beaten in step e to obtain a finished product.

4. The method for preparing Indigo Naturalis according to claim 3, wherein: The bacterial solution concentration of Pediococcus pentosaceus described in step c is 10 7 -10 8 CFU / mL, the concentration of Aspergillus aculeatus was 4×10 6 CFU / mL; the inoculation volume ratio was 3:2 for Pediococcus pentosaceus and Aspergillus aculeatus.

5. The method for preparing Indigo Naturalis according to claim 3, characterized in that: The bacterial solution of Pediococcus pentosaceus / Aspergillus aculeatus described in step c is inoculated at a rate of 5%.

6. The method for preparing Indigo Naturalis according to claim 3, characterized in that: The fermentation conditions described in step d are: fermentation temperature 30°C, pH value 7; fermentation time: 36h.

7. Use of Pediococcus pentosaceus / Aspergillus aculeatus in a synthetic process for degrading indole glycosides and converting indigo, wherein the registration number of Pediococcus pentosaceus in the China Industrial Microbiological Culture Collection is CICC 22227, and the registration number of Aspergillus aculeatus in the China Industrial Microbiological Culture Collection is CICC 2653.

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