New application of pediococcus pentosaceus and preparation method of indigo naturalis

Abstract

The invention provides application of pediococcus pentosaceus in fermentation processing of traditional Chinese medicine indigo naturalis. The invention provides a preparation method of indigo naturalis. According to the preparation method, pediococcus pentosaceus is adopted for fermentation and processing. Specific pediococcus pentosaceus is carefully screened out, the biological characteristics of the pediococcus pentosaceus and the adaptability of the pediococcus pentosaceus to natural indigo fermentation are deeply studied, and the fermentation process is optimized, so that the content of indigo blue and indirubin in the natural indigo fermentation process is remarkably and stably increased; the defects of an existing indigo naturalis fermentation technology in the aspect of increasing the content of indigo blue and indirubin are overcome, a new path and a new method for processing and preparing indigo naturalis are developed, and the method has wide application prospects and remarkable economic and social benefits.

Description

Technical Field

[0001] The invention relates to a new use of Pediococcus pentosaceus 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 Baphicacanthus cusia (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 use of Pediococcus pentosaceus in fermentation and processing of traditional Chinese medicine Indigo Naturalis. The registration number of the Pediococcus pentosaceus in the China Industrial Microbiological Culture Collection Center is CICC22227.

[0007] The invention provides a method for preparing Indigo Naturalis, which is prepared by fermentation with Pediococcus pentosaceus, 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 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.

[0015] Wherein, the bacterial solution of Pediococcus pentosaceus 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 6; fermentation time: 3 days.

[0017] The present invention also provides the use of Pediococcus pentosaceus in the synthesis process of degrading indole glycosides and converting indigo. The registration number of the Pediococcus pentosaceus in the China Industrial Microbiological Culture Collection Center is CICC 22227.

[0018] The present invention provides a synthesis process of indigo and indirubin, which uses indole glycoside as a substrate and uses Pediococcus pentosaceus CICC 22227 enzyme to degrade and transform indigo. The synthesis route is:

[0019] (a) Pediococcus pentosaceus produces β-glucosidase at a temperature of 30°C, an initial pH of 6, an inoculum size of 5%, and a rotation speed of 180 r / min. The enzyme degrades indole glycosides as substrates to obtain indigo intermediates indoxyl and glucose;

[0020]

[0021] (b) hydrolyzing the obtained indoxyl to produce indoxyl ketone by undergoing keto-tautomerism in an inorganic salt liquid medium;

[0022]

[0023] (c) In the presence of sufficient oxygen, indoxyl is further oxidized to indoledione;

[0024]

[0025] (d) Two molecules of indole ketone dimerize directly to form water-insoluble indigo under the action of oxygen;

[0026]

[0027] (e) One molecule of indoledione and one molecule of indolone can generate indigo red under the action of oxygen;

[0028]

[0029] Wherein, the time of the degradation conversion is 6 hours.

[0030] The inorganic salt liquid culture medium comprises: 1 g ammonium nitrate, 0.1 g calcium chloride, 0.5 g dipotassium hydrogen phosphate, 0.02 g ferric chloride, 0.5 g potassium dihydrogen phosphate, 0.05 g yeast extract, 0.5 g magnesium sulfate heptahydrate, 1 g sodium chloride, and pH 7.0±0.2.

[0031] The present invention aims to achieve a significant and stable improvement in the content of indigo and indirubin during the fermentation of Indigo by carefully screening out specific Pediococcus pentosaceus, and in-depth research on its biological characteristics and compatibility with Indigo fermentation, combined with the optimization of the fermentation process. The Pediococcus pentosaceus strain produces β-glucosidase for the soaking and fermentation of Indigo, thereby promoting the dissolution of indigo precursor indole glycosides and the increase in the content of indigo and indirubin. This not only helps to improve the quality and efficacy of Indigo products, promote the modernization of the traditional Chinese medicine industry, but also provides reliable and efficient technical support for the industrial large-scale production of high-value-added Indigo fermented products, fills the shortcomings of existing Indigo fermentation technology in improving the content of indigo and indirubin, and opens up new paths and methods for the preparation and processing of Indigo, which has broad application prospects and significant economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 (A) β-glucosidase production of 16 strains, (B) enzyme activity evaluation of 16 strains;

[0033] Figure 2 (A) Morphology and microscopic observation of five strains, (B) Physiological and biochemical characteristics of microbial strains, (C) Phylogenetic tree constructed by 16S rDNA sequences of bacteria, (D) Phylogenetic tree constructed by ITS sequences of fungi;

[0034] Figure 3 (A) Growth curves of five strains, (B) Effect of temperature on the stability of β-glucosidase produced by strains, (C) Effect of pH on the stability of β-glucosidase produced by strains, (D) Affinity of indole glycosides with β-glucosidase produced by strains;

[0035] Figure 4 (A) Effect of temperature on enzyme production in MZ-4 fermentation, (B) Effect of initial pH on enzyme production in MZ-4 fermentation, (C) Effect of inoculation amount on enzyme production in MZ-4 fermentation;

[0036] Figure 5 (A) Establishment of the absorption wavelength of indole glycosides, (B) Establishment of the standard curve of indole glycosides;

[0037] Figure 6 (A) The degradation rate of indole glycosides by MZ-4 enzymes, (B) The content of indigo converted by MZ-4, (C) The content of indigo red converted by MZ-4;

[0038] Figure 7 (A) Changes in pH during different fermentation periods, (B) Changes in dissolved oxygen during different fermentation periods, (C) Changes in redox potential during different fermentation periods;

[0039] Figure 8 (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

[0040] Embodiment 1 Preparation method of Indigo Naturalis of the present invention

[0041] (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;

[0042] (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;

[0043] (3) Inoculation of fermentation bacteria: The bacterial solution of Pediococcus pentosaceus CICC 22227 (concentration of 10 7 -10 8 CFU / mL) was poured into the fermentation tank at an inoculum volume of 5% and mixed evenly;

[0044] (4) Fermentation: The fermentation tank inoculated with the fermentation liquid is placed in a constant temperature fermentation room at a temperature of 30° C. and a pH value of 6 for a total of 3 days;

[0045] (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;

[0046] (6) Drying: Drying and crushing the thick indigo paste obtained after the indigo extraction in step (5) to obtain a finished product.

[0047] 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.

[0048] Example 2 Strain screening test in the preparation method of Indigo Naturalis of the present invention

[0049] The indigofera leaf fermentation liquid of the present invention is collected from the indigofera leaf fermentation tank of Sichuan Ya'an Xunkang Pharmaceutical Co., Ltd. The fermentation is carried out by adding 10 times the amount of water, and the soaking fermentation lasts for 3 days. At 10 o'clock every morning, the fermentation tank is fixed with an emptying sampler at a position about 30 cm below the water surface of the indigo soaking tank. 500 ml of fermentation liquid is collected each time, and the fermentation liquid is repeated 3 times, and the collected fermentation liquid is subjected to low-speed centrifugation treatment (4500X, 10min), and the precipitate is collected.

[0050] 1. Screening of dominant bacteria producing β-glucosidase

[0051] (1) Isolation and purification of indigofermentation bacteria

[0052] The collected fermentation precipitate was placed in a 30°C constant temperature incubator to revive the bacteria, and then the bacterial solution was diluted by gradient dilution (10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 ). Pipette 200 μL of the dilution solution and place it on solid culture medium (bacteria: nutrient broth medium, fungi: potato glucose water medium) for coating. Repeat 3 times for each concentration. Place it in a constant temperature incubator (bacteria 35℃, fungi 28℃) and culture for 3-5 days. Pick out the grown bacteria and continuously transfer them to NA and PDA culture medium until a purified strain is obtained.

[0053] (2) Initial screening of β-glucosidase-producing endophytes

[0054] The purified blue fermentation bacteria are streaked onto the solid medium of aesculin and cultured for a period of time to observe the color change. If the medium near the strain streaking line turns black, the strain can produce β-glucosidase. This screening method mainly uses the color development principle of aesculin, that is, 6,7-dihydroxycoumarin (also known as aesculin) generated after aesculin is hydrolyzed by β-glucosidase can react with iron ions, and dark brown substances will be generated around the colonies on the culture medium.

[0055] (3) Rescreening of β-glucosidase-producing endophytes

[0056] The bacteria that showed color reaction on the aesculin culture medium were inoculated into the nutrient broth culture medium and cultured at 35°C and 150r / min, and the fungi were inoculated into the potato glucose water culture medium and cultured at 28°C and 150r / min. The cultured strain fermentation liquid was centrifuged at 4°C and 8,000g for 10 minutes, and the supernatant was taken as the crude enzyme liquid. The β-glucosidase activity was determined by the p-ngp method (about 2 ml of p-nitrophenyl β-D-pyranoglucoside (5 mM PNPG, stored in 0.02 m sodium acetate buffer (pH 4) and added to 0.5 ml of culture filtrate. After incubation in a 37°C water bath for 30 min, the reaction was terminated by adding 2 ml of 0.2 M sodium borate buffer (pH 10). The release of p-nitrophenol was read at 400 nm using a UV spectrophotometer. Definition of β-glucosidase activity: 1 nmol of p-nitrophenol produced per minute by the sample at 37°C is defined as one enzyme activity unit U.) and five strains with higher enzyme activity were selected as potential strains.

[0057] (4) Physiological and biochemical tests, molecular biological identification of strains

[0058] Relevant physiological and biochemical tests were carried out on the strains producing β-glucosidase according to the Bergey's Manual of Bacterial Identification and the Manual of Identification of Common Bacterial Systems.

[0059] The strain was inoculated into CM liquid medium and placed in a constant temperature shaker at 30℃ and 200r / min for 2 days; the hyphae in the CM liquid medium were filtered and collected for genomic DNA extraction; the internally transcribed spacer (ITS) of the strain was amplified using primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCGCCTTATTGATATGC-3'); DNA was detected by agarose gel electrophoresis and placed in a gel imaging system to analyze the electrophoresis results; the DNA gel was recovered and the recovered fragments were connected and transformed with vectors; the same pair of primers were used for polymerase chain reaction (PCR) amplification and gel agarose electrophoresis detection to verify whether the target gene was lost. The bacterial solution with the target band was sent to Chengdu Luoning Biotechnology Co., Ltd. for sequencing. After sequencing, the obtained sequence was compared with the sequence in GenBank by BLAST.

[0060] The purified single colony was picked and inoculated into nutrient broth liquid culture medium and cultured in a shaking incubator at 35℃ and 150r / min for 24h. After centrifugation at 4℃ and 5 000r / min for 5min, the precipitated bacteria were taken and the DNA of the strain was extracted according to the operation steps of the bacterial DNA extraction kit. The 16S rRNA universal primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTACGACTT-3′) were used as forward and reverse primers for amplification, respectively. The fungus was inoculated into potato glucose water culture medium and cultured in a shaking incubator at 28℃ and 150r / min for 5d. After centrifugation at 4℃ and 8000r / min for 5min, the bacteria were collected, quickly ground into powder by adding liquid nitrogen, and the fungal DNA in the fermentation broth was extracted according to the operation steps of the fungal DNA extraction kit. The fungal universal primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGAATGC-3′) were used for amplification. The PCR products were checked by 1.0% agarose gel electrophoresis, and the PCR products with bands were sent to Chengdu Luoning Biotechnology Co., Ltd. for sequencing. The sequencing results of bacteria and fungi were compared with the known model strain sequences in the EzBioCloud database (https: / / eztaxon-e.ezbiocloud.net) and the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and the strain sequences with higher similarity were selected. The phylogenetic tree was constructed using the neighbor-joining method of MEGA7.0 software, and the check value bootstrap was set to 1 000. The constructed evolutionary tree file was uploaded to the iTol website (https: / / itol.embl.de) to improve the evolutionary tree.

[0061] (5) Experimental results

[0062] 98 strains of single bacteria were isolated and purified from the fermentation broth of Malan collected from Ya'an Xunkang Pharmaceutical Factory. After activation, the function of the substrate aesculin was used to perform a preliminary screening of β-glucosidase. It was found that 16 of the screening plates had black hydrolysis circles ( Figure 1 A), indicating that these 16 strains have β-glucosidase activity. Subsequently, rescreening was performed to measure the enzyme activity of the fermentation supernatant, and finally 5 strains with high and stable enzyme activity (enzyme activity ≥ 7.5U / mL) were screened out from the 16 strains. The results are as follows Figure 1 As shown in B, the five strains with higher β-glucosidase production are: MZ-4, X-8, YY-1, TR-7, and YDG-1.

[0063] like Figure 2 As shown in A, after plate streaking separation, the four bacterial strains were observed to have round colonies with a smooth, white, protruding surface that looked moist and shiny with clear edges. The fungal strains were round colonies with dull luster, dry texture, intact edges and black spores. After Gram staining, YY-1, YDG-1X-8 bacteria were short rod-shaped, Gram-negative bacteria, while the MZ-4 strain was spherical under a microscope and Gram-positive. In addition, the results of the microbial glucose / lactose glycolysis, methyl red, VP test, etc. are shown in the figure below. Figure 2 As shown in B, most strains are able to utilize carbohydrate compounds and produce biological enzymes such as oxidase and protease.

[0064] The purified strains were sequenced and analyzed using 16S / ITS gene fragments. The sample DNA sequence genes obtained after PCR amplification were compared in the GenBank database. The sequence identification and analysis standards were query cover ≥ 95.00% and Per.Indent ≥ 99.00%. Figure 2 As shown in C, after sequencing, BLAST found that the sequence of bacteria YY-1 matched that of Kluyvera georgiana (ATCC51702), strain YDG-1 was similar to Klebsiella variicola (CICC24980), X-8 matched that of Enterobacter quasiroggenkampii (Taxonomy ID: 2497436), and after identification, MZ-4 was consistent with the sequence of Pediococcus pentosaceus (CICC 22227). The phylogenetic tree of strain TR-7 is shown in Figure 2 D. The sequence of fungus TR-7 matches Aspergillus aculeatus (CICC2653) by more than 98%. Therefore, based on the morphological characteristics and ITS sequence homology analysis results, it was determined to be Aspergillus aculeatus.

[0065] 2. Investigation of enzyme production properties

[0066] (1) Strain growth curve

[0067] The bacterial suspension was inoculated into 200 mL of liquid culture medium at a 1% inoculum size and cultured at 30°C and 180 r / min in a shaking incubator under conventional culture conditions. A bacterial liquid sample was taken every 2 h during the culture process and the absorbance was measured at D600 nm. The measurement was repeated for 3 groups and the growth curves of the strains were plotted to determine the optimal activation point.

[0068] (2) Effect of temperature on β-glucosidase activity

[0069] The reaction temperature of the enzyme is a variable for evaluating the thermal activity of the enzyme. The enzyme reaction mixture (pH 6.0) was incubated at different temperatures (30, 40, 50, 60, 70, 80, 90°C) for 40 minutes, and the β-glucosidase activity was determined using the p-nitrophenyl glucoside hydrolysis method to determine the optimal reaction temperature. At the same time, the crude enzyme solution was placed in the above temperature gradient and incubated at the same temperature for 2 hours, and the β-glucosidase activity was determined. The highest enzyme activity was 100%, and the relative enzyme activity at different reaction temperatures was calculated. The calculation formula is as follows: Relative enzyme activity (%) = A / A0×100%.

[0070] (3) Effect of pH on β-glucosidase activity

[0071] The pH of the enzyme reaction mixture system was set at 50°C (optimum temperature) and incubated for 40 min under different pH conditions (3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0). The enzyme activity of the crude enzyme solution in different pH buffers was measured, and the relative enzyme activity under different pH conditions was calculated to determine the effect of pH on enzyme activity. Each experiment was performed three times.

[0072] (4) Enzyme reaction kinetic parameters

[0073] Under the optimal reaction conditions, the enzyme activity of β-glucosidase produced by the five strains was measured at different substrate concentrations of 0.1-2.0 mmol / L using indole glycoside as the substrate. The measurement results were combined with the Michaelis-Menten equation and the double reciprocal plot method to calculate the reaction kinetic parameter Km, and the affinity of the enzymes produced by the five strains for indole glycoside was compared.

[0074] (5) Experimental results

[0075] like Figure 3 As shown in A, the figure uses optical density (OD600) to measure the growth of strains such as YY-1, X-8, YGD-1, MZ-4, and TR-7 within 24 hours. From 0 to 4 hours, the strains are in the delayed phase, with low OD600 values ​​and slow growth; from 4 to 12 hours, YY-1 and other four strains grow exponentially, and from 12 to 24 hours, they enter the stable phase, with stable OD600 values. At this time, cell growth and death are balanced, nutrients are consumed, and waste accumulates. In the logarithmic growth phase, YY-1 and other four strains grow faster than TR-7. The OD600 values ​​of YY-1 and MZ-4 in the stable phase are slightly higher, and their maximum biomass is relatively high, which may be related to the physiological characteristics of the strains.

[0076] like Figure 3As shown in Figure B, the activity of β-glucosidase is relatively low at low temperature; with the increase of temperature, the activity of enzyme gradually increases, reaches the peak value and then begins to decline; the optimum temperature of enzyme produced by each strain is different, the optimum temperature of enzyme produced by YY-1 and X-8 strains is about 60℃, at which the activity of enzyme is close to 100%, the optimum temperature of enzyme produced by YDG-1 and MZ-4 strains is about 40℃, the activity of enzyme is the highest, and the activity of TR-7 reaches 100% when the temperature reaches 50℃. Combined with the temperature control range of industrial production of Indigo Naturalis, the β-glucosidase produced by YDG-1 and MZ-4 strains may be more suitable for the temperature of Indigo Naturalis soaking and fermentation process, and maintain a higher enzyme activity.

[0077] like Figure 3 As shown in Figure C, the optimum pH of β-glucosidase produced by different strains is different. The enzymes produced by strains YY-1 and YDG-1 reach the highest enzyme activity at around pH = 8, close to 100%. The enzymes produced by X-8 and MZ-4 reach their peak at a pH of 6, and the enzyme produced by TR-7 has a higher enzyme activity in a slightly acidic environment (around pH = 5). Subsequent experiments are needed to carry out and the pH range (5.5-7.5) of the indigo naturalis soaking and fermentation stage. The enzymes produced by strains X-8 and MZ-4 may be more suitable for use in the indigo naturalis soaking and fermentation environment, and can maintain a higher enzyme activity.

[0078] In addition, by comparing the Michaelis constant (Km) of the binding of β-glucosidase produced by the five strains to indole glycosides, there are significant differences in their kinetic characteristics ( Figure 3 D). YY-1 and YDG-1 have weak binding to indole glycosides, while the enzymes produced by X-8, MZ-4 and TR-7 have good affinity to indole glycosides, among which TR-7 has the highest affinity to indole glycosides. Based on the above considerations, MZ-4 was finally selected as the target strain for subsequent indole fermentation.

[0079] 3. Optimization of enzyme production conditions for MZ-4 strain activation

[0080] (1) Optimization test of activation enzyme production conditions

[0081] 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 factors. The initial fermentation conditions were: inoculation amount 4%, temperature 30°C, pH 7, and culture at a rotation speed of 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 cultured in a constant temperature incubator at 15, 20, 25, 30, 35, 40, 45, and 50°C. In the optimal inoculation amount test, other initial fermentation conditions remained unchanged, and the inoculation amounts of 1%, 3%, 5%, 7%, and 9% were taken for investigation. The bacterial solution was filtered through sterile gauze, and the filtrate was cultured 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.

[0082] (2) Experimental results

[0083] Depend on Figure 4 A It can be seen that the activity of β-glucosidase produced by MZ-4 increases with the increase of temperature. When the temperature is 30℃, the enzyme activity reaches the maximum value, which is about 17.14U / mL. When the temperature is higher than 30℃, the enzyme activity begins to decrease significantly. Too high temperature will cause MZ-4 to reproduce and age rapidly, and reduce the ability of the enzyme to perform degradation. Therefore, it is preliminarily speculated that the optimum temperature for MZ-4 enzyme production is 30℃.

[0084] Depend on Figure 4 B shows that with the increase of initial pH, the enzyme activity of MZ-4 shows a trend of first increasing and then decreasing. When the initial pH value is lower than 6, it is not conducive to the growth and reproduction of MZ-4 bacteria, making it impossible to produce enzymes. When the pH value is 6, the enzyme activity reaches its maximum value, which is about 17.47U / mL. When the pH value is higher than 6, the enzyme activity begins to gradually decrease. This is mainly because the excessively high initial pH value restricts the growth and metabolism of MZ-4, resulting in the inability of the produced enzyme to adapt to the environment and the reduced activity. Therefore, it is preliminarily speculated that the optimal initial pH value for MZ-4 enzyme production is about 6.

[0085] Depend on Figure 4 C shows that when the inoculation amount of the MZ-4 strain increases gradually, the activity of the produced enzyme increases accordingly, because the increase in the number of strains will accelerate the expression of enzyme genes, promote the expression and activity of the enzyme, and when the inoculation amount is 5%, the enzyme activity reaches a peak of about 16.60U / mL. However, after the inoculation amount exceeds 5%, the enzyme activity begins to decline. This may be due to factors such as fierce competition for nutrients, large accumulation of metabolites, and changes in cell-to-cell interactions, which lead to reduced enzyme activity. In summary, 5% is the optimal inoculation amount for enzyme production by this strain.

[0086] Finally, the conditions for activation and enzyme production of the MZ-4 strain were screened out as follows: temperature 30°C, initial pH 6, and inoculation amount 5%.

[0087] Example 3 Experiment on the degradation of indole glycosides by MZ-4 and its conversion into indigo

[0088] The synthetic route is:

[0089] (a) Pediococcus pentosaceus produces β-glucosidase at a temperature of 30°C, an initial pH of 6, an inoculum size of 5%, and a rotation speed of 180 r / min. The enzyme degrades indole glycosides as substrates to obtain indigo intermediates indoxyl and glucose;

[0090]

[0091] (b) the indoxyl obtained by hydrolysis undergoes keto-tautomerism in an inorganic salt liquid medium (ammonium nitrate 1 g, calcium chloride 0.1 g, potassium dihydrogen phosphate 0.5 g, ferric chloride 0.02 g, potassium dihydrogen phosphate 0.5 g, yeast extract 0.05 g, magnesium sulfate heptahydrate 0.5 g, sodium chloride 1 g, pH 7.0±0.2) to produce indoxyl ketone;

[0092]

[0093] (c) In the presence of sufficient oxygen, indoxyl is further oxidized to indoledione;

[0094]

[0095] (d) Two molecules of indole ketone dimerize directly to form water-insoluble indigo under the action of oxygen;

[0096]

[0097] (e) One molecule of indoledione and one molecule of indole ketone can produce indigo carmine under the action of oxygen.

[0098]

[0099] The time of the degradation transformation is 6h.

[0100] The isolated MZ-4 strain was inoculated from the glycerol tube into an LB solid culture medium plate at 30°C for activation, and then transferred to an LB liquid culture medium test tube for culture to obtain a seed solution. The activated seed solution was transferred to an inorganic salt liquid culture medium containing 2% indole glycoside as the sole carbon source according to the optimized fermentation conditions, and the culture solution without the strain was used as a control. The culture was placed in a constant temperature culture shaker. Samples were taken every 1 hour (a total of 12 hours). Each group was repeated three times.

[0101] (1) Determination of the optimal absorption wavelength of indole glycosides

[0102] A 100 mg / L indole glycoside standard solution was prepared, and pure water was used as a blank control. The absorbance of the indole glycoside standard solution was measured using a UV-visible spectrophotometer within a wavelength range of 200 to 800 nm to determine the optimal absorption wavelength.

[0103] (2) Draw a standard curve

[0104] Weigh 5 mg of indole glycoside standard, add ultrapure water to a 10 mL volumetric flask, dissolve under ultrasonic oscillation for 30 min, filter with a 0.45 μm microporous filter membrane to obtain a filtrate, dilute and prepare standard solutions of indole glycoside with a mass concentration of 10, 20, 30, 40, 50, 60 and 70 μg / mL, place in an ultraviolet spectrophotometer, and measure at the confirmed maximum absorption wavelength of indole glycoside. Repeat the measurement 3 times to control the absorbance range between 0.2 and 0.8, and perform regression calculation with the reference substance (μg / mL) as the horizontal coordinate (x) and the absorbance as the vertical coordinate (y).

[0105] (3) Determination of strain degradation rate

[0106] The absorbance of the MZ-4 strain and its control group at different time periods was measured at a wavelength of 231 nm using a UV-visible spectrophotometer. Each group was repeated three times. The indole glycoside content was calculated using the standard curve, and the degradation rate of the strain was calculated using formula (1).

[0107] Indole glycoside degradation rate (%) = (AB) / A*100%

[0108] Wherein: A is the content of indole glycoside (μg / mL), and B is the residual amount of indole glycoside (μg / mL).

[0109] (4) Determination of indigo and indirubin content

[0110] Determination of indigo content

[0111] Refer to the indigo content determination method under the Indigo Naturalis item in the 2020 edition of the Chinese Pharmacopoeia (Volume 1).

[0112] 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).

[0113] 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.

[0114] 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.

[0115] 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.

[0116] Determination of indirubin content

[0117] Refer to the indigo carmine content determination method under the Indigo Naturalis item in the 2020 edition of the Chinese Pharmacopoeia (Volume 1).

[0118] 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).

[0119] 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.

[0120] 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.

[0121] (5) Experimental results

[0122] Depend on Figure 5A shows that when the full wavelength of indole glycosides is scanned in the range of 200-800 nm, three obvious absorption peaks are found at 231, 247, and 263 nm, which are within the concentration range of the standard curve and the maximum absorption wavelength of indole glycosides reported in relevant literature is 230 nm. Finally, 231 is selected as the detection wavelength of indole glycosides. Linear regression analysis is performed on the concentration of the reference substance, and the regression equation y=0.0157x+0.124(R 2 =0.999 6), x has a good linear relationship in the range of 10 to 70 μg / mL ( Figure 5 B).

[0123] Under the conditions of optimized activation and enzyme production, the effects of decreasing indole glycosides in the MZ-4 strain at different times were compared. Figure 6 A shows that indole glycosides remained almost unchanged within 2 hours and no degradation occurred, which may be due to the slow growth of the MZ-4 strain, which needs time to adapt to the system. However, within 2-6 hours after the addition of the strain, the microorganisms multiplied and grew in large numbers, and were able to utilize and consume a large amount of indole glycosides, and their concentration began to drop rapidly. Within 6-12 hours, the indole glycoside content in the solution was almost not degraded, which may be due to the fact that the indole products produced would affect the further degradation of indole glycosides by the MZ-4 strain. In the end, the degradation rate of indole glycosides by MZ-4 exceeded 83.7%. In addition, Figure 6 B. Figure 6 C shows that the content of indigo and indirubin of MZ-4 strain gradually increases with time, and reaches the maximum value at 6h, with indigo being 56.63μg / mL and indirubin being 2.26μg / mL. This indicates that MZ-4 strain not only has a high degradation effect on indole glycosides in water, but also has a strong ability to transform indigo components, and has great application prospects.

[0124] Example 4 Comparison of MZ-4 fermentation and original fermentation

[0125] (1) Sample collection

[0126] This experiment is a comparative experiment, one of which is the original fermentation without adding MZ-4 (NF group), and the other group adds MZ-4 during fermentation ((MZ-4) F group) (activated under the optimized fermentation conditions). 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 liquid was collected from the fermentation barrels of the same batch at 6, 12, 18, 24, 30, 36, 48, 60, and 72 hours of fermentation, and the volume of the fermentation liquid collected each time was 50 ml, and 3 sets of parallel samples were set for sampling at each time point.

[0127] (2) Determination of physical and chemical parameters

[0128] A portable pH meter, a redox potential meter, and a portable dissolved oxygen meter were used to measure the changes in pH, dissolved oxygen content, and redox potential of the two groups during the fermentation process at each time point in real time, and the measurements were carried out in parallel three times.

[0129] (3) Determination of indigo content

[0130] Determination of indole glycoside content

[0131] The absorbance of MZ-4 fermentation broth and 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.

[0132] Determination of indigo content

[0133] Refer to the indigo content determination method under the Indigo Naturalis item in the 2020 edition of the Chinese Pharmacopoeia (Volume 1).

[0134] 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).

[0135] 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.

[0136] 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.

[0137] 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.

[0138] Determination of indirubin content

[0139] Refer to the indigo carmine content determination method under the Indigo Naturalis item in the 2020 edition of the Chinese Pharmacopoeia (Volume 1).

[0140] 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).

[0141] 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.

[0142] 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.

[0143] (4) Experimental results

[0144] In order to better compare the advantages of MZ-4 strain added to the indigofermentation and the original natural fermentation, the pH value, dissolved oxygen content and redox potential of the two fermentation systems were tested at different fermentation time periods. The changes of pH value, dissolved oxygen content and redox potential of the fermentation system over time are shown in Figure 2. Figure 7 shown.

[0145] During the fermentation of Indigofera ovata, the pH values ​​of the two fermentation systems changed significantly ( Figure 7 A). In the first 12 hours of the fermentation of Indigofermenta indigofermenta, the pH value of the fermentation system decreased slowly, but as the fermentation time went on, the pH value of the fermentation system decreased rapidly, and finally reached the lowest value at 48 hours. Compared with the original fermentation, the pH of the fermentation with the addition of the MZ-4 strain was lower overall, and the pH value of the final fermentation system was between 6.0 and 6.5, and the fermentation liquid was weakly acidic. This indicates that the addition of the MZ-4 strain makes the fermentation system more conducive to the dissolution and fermentation of indole glycosides in Indigofermenta indigofermenta.

[0146] After testing, as the fermentation progressed, the dissolved oxygen concentration of both fermentation methods gradually decreased ( Figure 7B). In the early stage of fermentation (before 12h), there is a high level of dissolved oxygen in the fermentation broth, indicating that the fermentation environment is in an aerobic state. In the middle stage of fermentation (12h-48h), the dissolved oxygen concentration of the fermentation broth drops rapidly, indicating that the growth and activity of microorganisms cause a large amount of oxygen to be consumed, and the fermentation environment gradually changes from an aerobic state to an anaerobic state. In the late stage of fermentation (48h-72h), the dissolved oxygen in the fermentation broth is lower than 0.2mg / L, and the microorganisms mainly carry out anaerobic fermentation. In addition, the fermentation speed of the solution fermented with MZ-4 is improved compared with the original fermentation.

[0147] The redox potential of the fermentation system dropped sharply within the first 30 hours of fermentation. As the fermentation time went on, the redox potential of the fermentation system eventually stabilized, with the potential between -400mV and -500mV ( Figure 7 C). The results showed that, compared with the original fermentation, adding MZ-4 strain to the fermentation could effectively advance the fermentation and shorten the fermentation time.

[0148] The changes of the contents of different components (indole glycosides, indigo, indirubin) in the NF group and the (MZ-4)F group during the fermentation process are shown in Figure 2. Figure 8 As shown. In the early stage of fermentation (before 12h), the dissolution of indole glycosides was the main factor, and the content of indigo and indirubin was low, and the content of indole glycosides in the fermentation with MZ-4 was slightly higher than that in the original fermentation. In the middle stage of fermentation (12-48h), the content of indole glycosides, indigo and indirubin in both groups increased rapidly, indicating that indole glycosides as indigo precursors were further dissolved in the fermentation system, and indigo and indirubin were further produced under the action of microorganisms. Compared with the NF group, the (MZ-4) F group reached the peak value earlier, with the indole glycoside content reaching a peak of about 430μg / mL at 30h; the indigo content reached a peak of about 67μg / mL at 48h, an increase of 47.46%; the indirubin content reached a peak of about 3.57μg / mL at 36h, an increase of 58.19%. In the late fermentation period (48-72h), although the indole glycosides, indigo, and indirubin in both fermentation methods were decreasing, the content of the (MZ-4)F group was always higher than that of the NF group. In general, the content of indigo substances produced by adding the MZ-4 strain to the fermentation process was higher than that of the original fermentation without adding MZ-4. This shows that MZ-4 has a promoting effect on the production of indigo components during the fermentation process.

[0149] In summary, this application innovatively proposes a method for soaking and fermenting Indigo naturalis using the MZ-4 strain, which provides a new approach for Indigo naturalis fermentation; can effectively increase the content of indigo and indigotin in Indigo naturalis and enhance its medicinal value; optimizes the Indigo naturalis fermentation process, improves fermentation efficiency and product quality; shortens the fermentation cycle, improves production efficiency, and thereby reduces production costs.

Claims

1. Use of Pediococcus pentosaceus in the fermentation and processing of the traditional Chinese medicine Indigo Naturalis. The registration number of the Pediococcus pentosaceus in the China Industrial Microbiological Culture Collection Center is CICC 22227.

2. A method for preparing Indigo Naturalis, characterized in that: It is fermented and processed by Pediococcus pentosaceus, 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 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.

3. The method for preparing Indigo Naturalis according to claim 2, wherein: The bacterial solution concentration of Pediococcus pentosaceus described in step c is 10 7 -10 8 CFU / mL.

4. The method for preparing Indigo Naturalis according to claim 2, wherein: The bacterial solution of Pediococcus pentosaceus described in step c is inoculated at a rate of 5%.

5. The method for preparing Indigo Naturalis according to claim 2, wherein: The fermentation conditions described in step d are: fermentation temperature 30° C., pH 6; fermentation time: 3 days.

6. Use of Pediococcus pentosaceus in the synthesis process of degrading indole glycosides and converting indigo, wherein the registration number of Pediococcus pentosaceus in the China Industrial Microbiological Culture Collection Center is CICC22227.

7. A process for synthesizing indigo and indirubin, characterized in that: It uses indole glycoside as substrate and converts indigo through the enzyme degradation of Pediococcus pentosaceus CICC 22227. Its synthetic route is: (a) Pediococcus pentosaceus produces β-glucosidase at a temperature of 30°C, an initial pH of 6, an inoculum size of 5%, and a rotation speed of 180 r / min. The enzyme degrades indole glycosides as substrates to obtain indigo intermediates indoxyl and glucose; (b) hydrolyzing the obtained indoxyl to produce indoxyl ketone by undergoing keto-tautomerism in an inorganic salt liquid medium; (c) In the presence of sufficient oxygen, indoxyl is further oxidized to indoledione; (d) Two molecules of indole ketone dimerize directly to form water-insoluble indigo under the action of oxygen; (e) One molecule of indoledione and one molecule of indolone can generate indigo red under the action of oxygen; 8. The synthesis process according to claim 7, characterized in that: The time of the degradation transformation is 6h.

9. The synthesis process according to claim 7, characterized in that: The inorganic salt liquid culture medium comprises: 1 g ammonium nitrate, 0.1 g calcium chloride, 0.5 g dipotassium hydrogen phosphate, 0.02 g ferric chloride, 0.5 g potassium dihydrogen phosphate, 0.05 g yeast extract, 0.5 g magnesium sulfate heptahydrate, 1 g sodium chloride, and pH 7.0±0.2.

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