WO2021110101A1 - Method for preparing deapioplatycodin d and application thereof - Google Patents

Abstract

Provided is a method for preparing deapioplatycodin D, which is characterized in that two or more strains are used for simultaneous preparation, comprising a pichia kudriavzevii strain and a bacillus velezensis strain, so as to prepare the method for preparing deapioplatycodin D. Using two strains of different conversion mechanisms and converting platycodin by using a means of mixed fermentation effectively increases the yield of deapioplatycodin D.

Description

A kind of preparation method and application of de-celery platycodin D

This application claims the priority of the Chinese patent application filed with the Chinese Patent Office on December 03, 2019, the application number is CN201911224253.6, and the invention title is "a preparation method and application of decelery platycodin D", which The entire content is incorporated into this application by reference.

Technical field

The invention relates to a preparation method and application of decelerylated platycodin D, and relates to a preparation method of decelerylated platycodin D by adopting a mixed strain containing Pichia kudriazwei strain and Bacillus velezia, and Its application.

Background technique

Platycodon grandiflorum saponins can treat hyperlipidemia, hypertension, diabetes, obesity, etc. The main biologically active ingredient is platycosides. According to investigations, more than 30 kinds of platycodin and a variety of sapogenins have been isolated and identified. Platycodin D (Platycodin D), abbreviated as PD, is a white crystalline powder with a density of 1.56g/cm ³ and a melting point of 228～ At 237℃, the molecular formula is C ₅₇ H ₉₂ O ₂₈ , the relative molecular weight is 1224.58, and the water solubility is good. Platycodin D is a disaccharide chain saponin of platycodin, and its sugar group is connected to the C-3 and C-28 positions of the nucleus. The 2010 edition of "Chinese Pharmacopoeia" stipulates that the mass fraction of Platycodin D in Platycodon grandiflorum is 0.1%. Platycodin D is the most representative among the biological activities of platycodin. It shows strong cytotoxicity to tumor cell lines and can be used as a drug for the treatment of cancer. WIE et al. treated with the crude enzyme extract of Aspergillus niger to improve Platycodin D, significantly reducing the cytotoxicity and erythrocyte hemolytic toxicity of V79-4 cells (Chinese hamster lung fibroblasts), and increasing biological activity And improve the sensory value. Deapio-platycodin D (deapio-platycodin D), abbreviated as (dPD), white or off-white powder, molecular formula C ₅₂ H ₈₄ O ₂₄ , relative molecular weight 1093.22, good water solubility. Compared with Platycodin D, its molecular structure is one less celery sugar. Studies have found that de-celery Platycodon grandiflorum soap D can inhibit pancreatic lipase activity and anti-tumor activity.

At present, platycodin production methods include chemical methods, enzyme preparation methods and microbial transformation. The chemical method produces a series of side reactions and chemical residues. The chemical reaction is time-consuming, low in selectivity and low in conversion during the preparation process. The enzyme preparation method is fast and efficient, and is gradually regarded as an ideal method for preparing active substances. However, the production cost of enzyme preparations is high and the storage conditions are harsh, and they cannot be used in large quantities in a short period of time. Therefore, many scholars have gradually invested in the preparation of platycodin by microbial transformation. Lee et al. screened out a microbial strain that can produce Platycodin D from nuruk and was identified as Cyberlindnera fabianii. Ling et al. used recombinant lactic acid bacteria with β-glucosidase activity to ferment to produce ginsenoside Rg ₃ (S) and compound K. Through permeabilization and mixed fermentation, the conversion yields reached 61% and 70%, respectively.

The Chinese name of Pichia kudriavzevii is Pichia kudriavzevii. Domestic scholars have found that the Pichia kudriavzevii strain ZJPH0802 can transform curcumin well and obtain two conversion products, hexahydrocurcumin and tetrahydrocurcumin. Vegetarian. The main product is tetrahydrocurcumin, and experiments by Natio and others have proved that the antioxidant capacity of tetrahydrocurcumin is greater than that of hexahydrocurcumin and curcumin. From this we can see that this strain has a good research prospect. Fan Guangsen et al. screened out Pichia kudriazwei from Luzhou-flavor Daqu. This strain not only produces high phenethyl alcohol, but also can produce nearly 30 flavors in the bean sprout juice medium. Therefore, the strain has broad application prospects in liquor and has good market value. Gilberto V.M and others also found that Pichia kudriazweis strains LPB06 and LPB07 fermented cocoa beans to make cocoa beans have a better color and richer aroma composition, which can be used to adjust the flavor of cocoa beans. It can be seen that this strain has a bright future in improving flavor. Another scholar screened out Pichia kudriazwei from fermented grains in Africa and found that the strain has folate biosynthesis genes, which can not only produce folate and phytase, but also can be passaged under the conditions of the human gastrointestinal tract. Survival, has adhesion to intestinal cells, has good probiotic potential, can directly exert potential beneficial effects in the host's body, and increase its impact on the host's health. Therefore, the strain has broad prospects in the research and development of improving the nutritional value of food through natural fermentation and the preparation of probiotic fermentation tanks.

The Chinese name of Bacillus velezensis is Bacillus velezensis. Recent studies have reported that this strain is widely used in bacteriostasis. Bacillus velezensis has been isolated from Dioscorea zingiberensis plants and has certain resistance to pathogens. Bacterial activity. Yang Shengqing proved that Bacillus Velez has a broad antibacterial effect and does not infect plants within a certain dose. Its fermentation broth is effective in preventing and controlling early tomato blight. Liu, G et al. proved that Velez Bacillus LS69 exhibits antagonistic activity against a variety of pathogenic bacteria. The genomic analysis and identification of the strain found that the strain contains a series of genes involved in enhancing plant growth and triggering plant immunity, in order to clarify its biological Provide a basis for prevention and control mechanisms and promote its application in the future. SaadA.M. et al. used the response surface method to optimize the extracellular polysaccharide production conditions of Bacillus Velez KY498625, and the results showed that the molecular weight of the purified EPS was 1.29×10 ⁵ Da and the number average molecular weight was 1.14×10 ⁵ Da, mainly composed of glucose. Composed of mannose and galactose, the production of EPS under the optimal conditions obtained through data analysis successfully increased the productivity from the initial EPS output to 4.1 to 7.6g/L.

At present, researches on the biotransformation of saponins at home and abroad have found that the transformation pathway of a single strain is limited and the saponin resources cannot be fully utilized. Therefore, many scholars in the field of biotransformation have begun to use mixed enzyme systems and mixed strains for biotransformation research. The effect is ideal and excellent. In the traditional single strain transformation. Li, Ling et al. used recombinant lactic acid bacteria with two different transformation pathways to transform ginsenosides, and achieved high-efficiency transformation of ginsenosides by means of inducing enzyme production, permeabilization and crushing treatment, and mixed fermentation. Using the fermentation characteristics of the strain to biotransform platycodon saponins can not only realize the safe and environmentally friendly production of platycodon saponins, but also make full use of platycodon resources to convert the platycodin with low biological activity into saponins with high biological activity. The effect of turning waste into treasure.

Summary of the invention

In view of the shortcomings in the prior art, the purpose of the present invention is to provide a strain of Pichia kudriazwei, which is preserved in the General Microbiology Center of the Chinese Microbial Culture Collection Management Committee, and the preservation date is June 17, 2019. The deposit number is CGMCC No.17939.

The method for preparing decelerylated Platycodin D from the Pichia Kudriazwei strain of the present invention includes the following steps:

Cultivation steps: the culture of the Pichia azweed yeast selects YPD liquid medium and yeast extract peptone dextrose agar medium (YPD);

Seed culture steps: the colonies are inoculated in 20mL liquid medium, placed at 30℃, 110r/min, shaking culture for 14h-18h, as the seed culture solution;

Transformation step: add 2% inoculum to 0.1mg/mL saponin standard solution and transform at 30℃ for 24h～72h.

The preparation method of the present invention is characterized in that, during the bioconversion process, the fermentation temperature is controlled at 30°C.

The present invention also provides a preparation method of de-celery platycodin D, which is characterized in that it comprises the steps of using two or more strains to prepare by mixed fermentation.

In the method of the present invention, the strains are Pichia kudriavzevii strains and Bacillus velezensis (Bacillus velezensis), and the Pichia kudriavzevii strains are deposited in China General Microbiology Center of the Microbial Culture Collection Management Committee, preservation address: Beijing, China, the preservation date is June 17, 2019, and the preservation number is CGMCC No.17939; the Bacillus velez strains are deposited in the China Microbial Culture Collection Management Committee General Microbiology Center, preservation address: Beijing, China, preservation date is June 17, 2019, preservation number is CGMCC No.17938.

In the preparation method of the present invention, during the biological transformation process, the fermentation temperature is controlled at 30-37°C.

In the preparation method of the present invention, the fermentation temperature is 37°C.

The present invention further provides an application of decelery Platycodin D in the preparation of anti-tumor drugs, anti-rheumatoid arthritis drugs, anti-obesity drugs, kidney-protecting drugs, liver-protecting drugs, immune-regulating drugs, and anti-inflammatory drugs. Application in diabetes drugs, analgesic drugs, and enzyme inhibitor drugs.

The inventors of the present invention used two strains with different transformation mechanisms for the first time to transform platycodin by mixed fermentation, which effectively increased the yield of platycodin D and de-celerylated platycodin D. Using the fermentation characteristics of the strain to biotransform platycodon saponins can not only realize the safe and environmentally friendly production of platycodon saponins, but also make full use of platycodon resources to convert the platycodin with low biological activity into saponins with high biological activity. The effect of turning waste into treasure. The present invention provides two strains with different transformation modes and a method for preparing platycodin D and decelerylated platycodin D by using the two strains to perform mixed fermentation.

Kudriazwei Pichia yeast converts Platycodon grandiflorum saponin E into Platycodon grandiflorum saponin D _{3 and} then converts Platycodon grandiflorum saponin D ₃ into Platycodon grandiflorum saponin D (PE→PD ₃ →PD), and converts decelerylated Platycodon grandiflorin E into decelery The sugar platycodin D _{3 is} then transformed into de-celerylated platycodin D (dPE → dPD ₃ → dPD), _{and the conversion speed of dPD 3} and PD ₃ is significantly higher than that of PE and dPE; Bacillus Velez can directly convert the platycodin E is transformed into platycodin D (PE→PD), and the de-celerylated platycodon saponin E is transformed into de-celerylated platycodon saponin D. The transformation rate of Pichia kudriazwei is high, but the transformation speed is slow, while the transformation rate of Bacillus velez is fast, but the transformation rate is lower than that of Pichia kudriazwei, so it is fully utilized from Platycodin resources From a perspective, you can try to transform a mixed strain.

Using the fermentation characteristics of different strains to carry out mixed fermentation and biotransformation of Platycodon grandiflorum saponins can not only realize the safe and environmentally friendly production of Platycodon grandiflorum saponins, but also make full use of Platycodon grandiflorum resources to convert the internally contained platycodin with low biological activity into high biological activity. Saponins achieve the effect of turning waste into treasure.

Description of the drawings

Figure 1 shows the change of saponins content during the fermentation of Platycodon grandiflorum by Pichia Kudriazwei at 30°C;

Figure 2 shows the change of saponin content during the fermentation of Platycodon grandiflorum by Pichia Kudriazwei at 37°C;

Figure 3 shows the change of saponin content during the fermentation of Platycodon grandiflorum by Velez Bacillus at 37°C;

Figure 4 shows the change of saponin content during the fermentation of Platycodon grandiflorum by Bacillus Velez at 30°C;

Figure 5 shows the changes of saponins during the fermentation of Platycodon grandiflorum by Bacillus Velez;

Figure 6 shows the change of saponin content in the process of fermenting Platycodon grandiflorum at 30°C with mixed strains;

Figure 7 shows the change of saponin content during the fermentation of Platycodon grandiflorum by mixed strains.

Biological preservation instructions

Pichia kudriavzevii SK-P1, deposited in the General Microbiology Center of the China Microbial Culture Collection and Management Committee on June 17, 2019, the address is No. 3, Beichen West Road, Chaoyang District, Beijing No., the deposit number is CGMCC No.17939;

Bacillus velezensis SK-P1, deposited in the General Microbiology Center of China Microbial Culture Collection and Management Committee on June 17, 2019, the address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, with the preservation number It is CGMCC No. 17938.

Detailed ways

In order to enable those skilled in the art to more clearly understand the objectives, technical solutions, and advantages of the present invention, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, but not used to limit the present invention.

The Pichia kudriazwei strain of the present invention is preserved in the General Microbiology Center of China Microbial Species Collection Management Committee, the preservation date is June 17, 2019, and the preservation number is CGMCC No.17939.

The method for preparing Platycodin D using the above-mentioned Pichia kudriazwei strain in the present invention includes the following steps: a culturing step: the culture of the Pichia azwei yeast selects YPD liquid medium and yeast extract peptone glucose agar Medium (YPD); seed culture steps: colonies are inoculated in 20mL liquid medium, placed at 30℃, 110r/min and shake culture for 14h-18h, as seed culture solution; transformation step: 2% (volume fraction) The inoculum was added to 0.1mg/mL saponin standard solution and transformed at 30℃ for 24h～72h.

In the present invention, the Pichia azweed is a strain of Pichia kudriazwe.

In the present invention, the saponin standard solution includes a de-celerylated Platycodon E standard solution.

In the preparation method of the present invention, during the biological transformation process, the fermentation temperature is controlled at 30°C to 37°C, preferably the temperature is controlled at 30°C.

The present invention further provides a preparation method of Platycodon grandiflorum saponin D, using the following mixed strains; including Pichia kudriazwei strain and Bacillus velezia, and the Pichia kudriazwei strain is preserved At the General Microbiology Center of the China Microbial Culture Collection Management Committee, the preservation date is June 17, 2019, and the deposit number is CGMCC No.17939; the Bacillus velez strain is deposited at the General Microbiology Center of the China Microbial Culture Collection Management Committee , The preservation date is June 17, 2019, and the preservation number is CGMCC No.17938.

The present invention screened and obtained Pichia kudriazweed extract from the fermented Platycodon grandiflorum of the spicy cabbage sample. Compared with the unfermented group, the fermented Platycodon grandiflorum extract of this strain significantly increased the PD content, and the conversion rate was 46.44%, and The strain has no effect on human health, can be used to ferment Platycodon grandiflorum extract to increase the PD content, and has a good prospect in food fermentation and the preparation of starter. The Pichia Kudriazwei strain in this patent is derived from kimchi.

The invention separates and screens the fermented broth of the spicy cabbage sample to obtain Bacillus belez. After the strain fermented the Platycodon grandiflorum extract, compared with the unfermented group, the PD content was significantly increased, and the conversion rate was 48.92%. It has not been reported that this strain is pathogenic to humans, so it can be used for microbial fermentation to transform Platycodin D.

In the bioconversion process of the preparation method of the present invention, the fermentation temperature is controlled at 30-37°C, and when the mixed strain is used to prepare decelery Platycodin D, the fermentation temperature is preferably 37°C.

The present invention provides an application of decelerylated Platycodon D in the preparation of anti-tumor drugs, anti-rheumatoid arthritis drugs, anti-obesity drugs, kidney-protecting drugs, liver-protecting drugs, immune-regulating drugs, and anti-diabetic drugs. Drugs, analgesic drugs, enzyme inhibitor drugs.

The inventors of the present invention used two strains with different transformation mechanisms for the first time to transform platycodin by mixed fermentation, which effectively increased the yield of de-celerylated platycodin D. Using the fermentation characteristics of the strain to carry out biotransformation of platycodon saponins can not only realize the safe and environmentally friendly production of platycodin saponins, but also make full use of platycodon resources to convert the internally contained platycodin with low biological activity into organisms. Saponins with high activity have the effect of turning waste into treasure.

The present invention inoculates the seed culture solution of the two strains of Pichia azweir and Bacillus velezia in an inoculum of 2% (volume fraction) into an Erlenmeyer flask containing 50 mL of Platycodon grandiflorum water extract, and shakes the culture at 110 r/min At 72h, samples were taken at regular intervals (0, 4, 8, 12, 24, 36, 48, 60, 72h) during the fermentation process, and the content of six platycodins was determined by HPLC.

In the present invention, the volume ratio of the seed culture liquid of the Pichia azweed strain and the seed culture liquid of the Bacillus velezia strain is preferably 1:1; the seed culture of the Pichia azway strain The number of effective viable bacteria in the seed culture solution and the seed culture solution of Bacillus velezia strain is preferably 1×10 ⁶ cfu/mL, respectively.

Table 1 Setting of fermentation group

Mixed strains: Pichia Kudriazwei, Bacillus velezia

Mixed strains: Pichia Kudriazwei, Bacillus velezia

The HPLC detection conditions are as follows:

Device model: Agilent 1200RRLC-G6410B

Chromatographic column: Zorbax C18 column (150mm×2.1mm, 3.5μm particle size); HPLC conditions: mobile phase water (A)-acetonitrile (B); gradient elution: 0～6min: 10%～15% B, 6～ 50min: 15%～25%B, 50～60min: 25%～47.5%B; flow rate: 0.2mL/min; temperature: room temperature; detection wavelength: 210nm; injection volume 1.0μL.

MS conditions: ESI-negative ion mode, electrospray ion source, 4KV ion injection voltage, -15V capillary voltage, 300°C capillary temperature, scan times: 500, total ion chromatogram: 100～1000m/z.

The present invention is the first to discover that two strains have different transformation pathways for de-celylated platycodon saponins. As follows, the present invention provides a basis for the microbial production of de-celylated platycodon saponins D.

In the present invention, the calculation method for the conversion rate of de-celery platycodin is as follows:

Conversion rate of celeryside platycodin saponins%=(Y-X)/Y×100%

In the formula: Y- content before fermentation (mg/mL)

X-content after fermentation (mg/mL)

The SPSS 17.0 software was used to process the test results, and the Duncan method was used for multiple comparisons, and the test results were expressed as mean ± standard deviation.

In the present invention, the 30°C fermentation results of Pichia kudriazwei are described in detail as follows:

In the present invention, Pichia kudriaz has the presence of (PE → PD ₃ → PD), (dPE → dPD ₃ → dPD), (PD ₃ → PD) and (dPD ₃ → dPD). ) Transformation pathway.

Table 2 Changes of saponins content during the fermentation of Platycodon grandiflorum by Pichia kudriazwei at 30℃

Figure 1 and Table 2 show the changes of saponin content during the fermentation of Platycodon grandiflorum by Pichia kudriazwei at 30°C. As can be seen from Figure 1, during the fermentation of Platycodon grandiflorum by Pichia kudriazwei at 30°C, The dPE, PE, and dPD ₃ in the fermentation broth of Platycodon grandiflorum showed a downward trend. The PD ₃ first rose and then fell. The content of dPD and PD showed an upward trend. Among them, the content of dPE and PE did not change significantly within 0-24h, and after 24h Rapid decline, dPE was not detected after 48h and PE after 60h. The conversion rate reached 100%, and all conversion may have been completed. This may be because the platycodon grandiflorum extract contains sufficient platycodon grandiflorum polysaccharide as a carbon source for Kudri The growth and utilization of Pichia azweed is caused by the consumption of polysaccharides in the later stage of fermentation and the production of bioconverting enzymes to hydrolyze Platycodon saponins. dPD ₃ showed a slow downward trend from 0 to 48 hours, and rapidly decreased within 48 to 60 hours. After 72 hours, there was still a residual content of 0.0023 mg/mL. The content of PD ₃ did not change significantly within 0 to 36 hours, and in 36 to 48 hours The internal content increased significantly (P<0.01), which was caused by the decomposition and transformation of PE. After 48h, the content gradually decreased, and the conversion produced PD. After 72h, there was still a residual content of 0.0229mg/mL, which showed that the content of PD was 0.0229mg/mL. _{There is still room for transformation of 3} , and further research on the enzyme production conditions of the strain is needed; the content of dPD and PD at the end of the fermentation has been extremely significantly increased (P<0.01), and the content of dPD after 72h is 0.054mg/mL Compared with the initial 0.0462mg/mL, the content of PD is 0.2445mg/mL, which is 8.86% higher than the initial 0.2246mg/mL.

Table 3 Changes of saponins content during the fermentation of Platycodon grandiflorum by Pichia Kudriazwei at 37℃

Figure 2 and Table 3 show the change of saponin content during the fermentation of Platycodon grandiflorum by Pichia kudriazwei at 37°C. It can be seen from Figure 2 that Pichia kudriazwei fermented during the fermentation of Platycodon grandiflorum at 37°C. The change trend of Platycodin content in the liquid is similar to that of 30°C fermentation conditions, but the dPE and PE content began to decrease after 36h. Compared with 30°C fermentation conditions, it was delayed by 12h. In addition _{, the changes in dPD 3} and PD ₃ contents were not obvious. This indicates that the fermentation pathway of Pichia kudriazwei at 37°C is not efficient. This may be due to the low activity of the invertase produced at 37°C; it is similar to fermentation at 30°C. Compared with that, 30°C is more suitable for the transformation of Platycodin by Pichia Kudriazwei.

Table 4 Changes of saponins content during the fermentation of Platycodon grandiflorum by Bacillus Velez at 37℃

Figure 3 and Table 4 show the changes of saponin content during the fermentation of Platycodon grandiflorum by Bacillus Velez at 37°C. As can be seen from Figure 3, during the fermentation of Platycodon grandiflorum by Bacillus Velez at 37°C, the fermentation broth of Platycodon grandiflorum, dPE and PE showed a downward trend. The _{content of dPD 3} and PD _{3 did} not change significantly, and the content of dPD and PD showed an upward trend. Among them, the content of dPE and PE declined rapidly within 24 hours, and dPE was not detected after 24 hours. The conversion may have been completed. Analysis of the changes of dPE and PE content indicates that the platycodin converting enzyme of Bacillus Velez may be produced in the early stage of fermentation and will not be affected by the carbon source in platycodon; the content of dPD and PD in the later stage of fermentation are respectively 0.0434mg/mL , 0.2847mg/mL, compared with 0.0418mg/mL, 0.2560mg/mL before fermentation increased by 3.83% and 11.21%, respectively.

Table 5 Changes of saponins content during the fermentation of Platycodon grandiflorum by Bacillus Velez at 30℃

Note: The same column with the same letters indicates that the difference is not significant (P>0.05), the difference in uppercase letters indicates that the difference is extremely significant (P<0.01), and the difference in lowercase letters indicates a significant difference (P<0.05), the same in the table below.

Figure 4 and Table 5 show the change of saponins content during the fermentation of Platycodon grandiflorum by Bacillus Velez at 30°C. Figure 4 shows that the dPE in Platycodon grandiflorum fermentation broth during the fermentation of Platycodon grandiflorum by Bacillus Velez at 30°C The content of PE decreased rapidly within 8-12h, and dPE was not detected after 12h, and may have been completely converted; the content of dPD and PD decreased in the later stage of fermentation. Compared with fermentation at 37℃, 37℃ is more suitable for Kudri Transformation of Platycodin by Pichia pastoris.

In addition, during the fermentation of Bacillus Velez at 30°C and 37°C, a gradual increase in the chromatographic peak area was observed. The figure shows the change of saponin during the fermentation of Bacillus bellis, as shown in Figure 5. Between dPD and PD ₃ , and as the peak area of the unknown peak increases, the area _{of other peaks near dPD 3} gradually decreases. It is speculated that this may be a change in the content of other saponins, and the biotransformation of platycodin by Bacillus Velez may be There are other transformation mechanisms that require further experimental research.

Table 6 Changes of saponin content during the fermentation of Platycodon grandiflorum by mixed strains at 30℃

Figure 6 and Table 6 show the change of saponin content during the fermentation of Platycodon grandiflorum by the mixed strains at 30°C. It can be seen from Figure 6 that during the fermentation of Platycodon grandiflorum by the mixed strains at 37°C, the dPE and PE in the Platycodon grandiflorum fermentation broth showed a downward trend. The _{content of dPD 3} fluctuated in the first and middle stages of fermentation, and showed an upward trend in the latter stage of fermentation. The _{content of PD 3} showed an upward trend, the content of dPD showed an upward trend, and the content of PD first declined and then increased; among them, dPE and PE were within 24 to 36 hours. Rapid decline, there is still residue at the end of the fermentation; dPD ₃ began to rise after 24h, and PD ₃ began to rise after 12h, and there was no decline. Combined with the analysis of the fermentation results of Pichia kudriazwei at 37℃, Kudris Pichia azweed may have been inhibited to a certain extent during the fermentation of the mixed strain at 37°C, resulting in the _{inability to decrease the content of dPD 3} and PD _{3; the} content of dPD and PD were 0.0562 mg/mL and 0.3213 mg/mL at the later stage of fermentation. mL, compared with 0.0458mg/mL and 0.2960mg/mL before fermentation, which increased by 22.71% and 8.55%, respectively. Therefore, the condition of 37℃ can be used for the fermentation of mixed strains to transform Platycodin.

Table 7 Changes of saponin content during the fermentation of Platycodon grandiflorum by mixed strains at 37℃

Figure 7 and Table 7 show the changes of saponin content during the fermentation of Platycodon grandiflorum by the mixed strains. It can be seen from Figure 7 that during the process of fermenting Platycodon grandiflorum by the mixed strains at 30°C, the dPD content in the Platycodon grandiflorum fermentation broth fluctuated and the PD content was almost unchanged. The content of dPE and PE showed a downward trend, and the _{content of dPD 3} showed a fluctuating change. The _{content of PD 3} increased first and then decreased. The content of dPE and PE did not change significantly within 4 hours (P>0.05), and the content of dPE within 4 hours After 24h, it was not detected, and it may have been completely transformed. PE decreased slowly within 4-8h, and rapidly decreased within 12-24h. The changes in the content of dPE and PE in the early stage indicated that Velez Bacillus was involved in the early stage of fermentation. Saponin conversion; dPD ₃ content increased in 8-12h, but there was no significant difference (P>0.05), PD ₃ content increased significantly in 12-36h (P<0.01), and then slowly decreased in 36-48h ( P<0.01), which indicates that Pichia kudriazweis participates in the saponin conversion in the middle and late stages of fermentation; compared with 30°C, 37°C is more suitable for the fermentation of mixed strains.

The present invention uses two strains with different transformation mechanisms for the first time to transform platycodin by means of mixed fermentation, which effectively improves the yields of platycodin D and de-celerylated platycodin D. It provides a theoretical basis for the microbial production of platycodin D and de-celerylated platycodin D, and the mixed fermentation of platycodon with Pichia kudriazwei and Bacillus velezia. According to the present invention, the transformation path of a single strain is limited, and saponin resources cannot be fully utilized, which is significantly better than traditional single strain transformation. Using the fermentation characteristics of the strain to biotransform platycodon saponins can not only realize the safe and environmentally friendly production of platycodon saponins, but also make full use of platycodon resources to convert the platycodin with low biological activity into saponins with high biological activity. The effect of turning waste into treasure.

The present invention uses Pichia Kudriazwei and Bacillus velezia to ferment the platycodin aqueous extract, respectively monitors the biotransformation of platycodin by individual strains and mixed strains at 30°C and 37°C. The fermentation of Platycodon grandiflorin by Pichia kudriazwei at 30°C is better than 37°C, dPD increased by 16.88%, PD increased by 8.86%; Bacillus Velezia fermented Platycodin at 37°C better At 30°C, dPD increased by 3.83% and PD increased by 11.21%; the two strains were better than 30°C in fermentation of platycodin at 37°C, dPD increased by 22.71%, PD increased by 8.55%, mixed fermentation mode It can be used as a way to transform platycodin.

In the present invention, the transformation mechanism of Pichia azweed is (PE→PD ₃ →PD) and (dPE→dPD ₃ →dPD), _{and the conversion speed of dPD 3} and PD ₃ is significantly higher than that of PE and dPE; The transformation mechanism of Bacillus velezensis is (PE→PD) and (dPE→dPD), and the transformation speed is fast. Considering the full utilization of platycodin resources, it is possible to try mixed strain transformation.

According to the present invention, the selection of the most suitable transformation resources for Platycodon grandiflorum: the moisture, total ash, alcohol extract and Platycodon grandiflorum saponin D in twelve different regions of Platycodon grandiflorum all meet the requirements of the pharmacopoeia; the crude protein content is between 4.38% and 20.13% The crude fiber content is between 3.80% and 12.70%; the polysaccharide content is between 18.86% and 51.00%%; the total flavonoid content is between 0.22% and 0.54%; the total phenol content is between 0.64% and 0.96%; each The contents of dPD, PD and total saponins in Platycodon grandiflorum in different regions are quite different. The content of Platycodon grandiflorum in Anhui Dabie Mountain is the highest, with a value of 11.88%, followed by Zibo in Shandong and Changbai Mountain in Jilin, with 11.25% and 11.00% respectively; Platycodin D in Zhejiang The highest content is 0.8212%, followed by Jilin Changbaishan Platycodon with a value of 0.6280%; the highest content of de-celerylated Platycodon grandiflorum is Zhejiang Platycodon with a value of 0.1196%, followed by Guangdong and Jilin Platycodon with a value of 0.0793%, respectively. 0.0777%; According to the comprehensive consideration of polysaccharides, total saponins and six saponins, Jilin Changbai Mountain Platycodon grandiflorum was selected as the biotransformation material.

According to the present invention, the fermentation of Platycodon grandiflorum by Pichia kudriazwei at 30°C is better than 37°C, dPD increased by 16.88%, PD increased by 8.86%; Velez Bacillus was better than that at 37°C At 30℃, dPD increased by 3.83% and PD increased by 11.21%; the mixed fermentation of the two strains at 37℃ was better than 30℃, dPD increased by 22.71%, PD increased by 8.55%, and it was determined that the mixed fermentation mode could be used as platycodin conversion One way.

In the present invention, the following experimental materials are used for testing. The strains of Bacillus velezia and Pichia kudriazwei were screened by our laboratory, and Platycodon grandiflorum tablets were purchased from Hebei Quantai Pharmaceutical Co., Ltd.

The experimental reagents used in the present invention were purchased from Tianjin Komiou Chemical Reagent Co., Ltd., including sodium nitrite, potassium ferricyanide, trichloroacetic acid, iron trichloride, sodium hydroxide, disodium hydrogen phosphate, and dihydrogen phosphate Sodium, ethanol, aluminum nitrate, folinol, sodium carbonate, salicylic acid, hydrogen peroxide, ferrous sulfate, etc.; Tris-HCl buffer, pyrogallol, ascorbic acid were purchased from Beijing Soleibao Technology Co., Ltd.; rutin standard Products and DPPH were purchased from Shanghai Yuanye Biological Technology Co., Ltd. The gallic acid standard was purchased from Shanghai Macleans Biochemical Technology Co., Ltd. The total antioxidant capacity kit was purchased from Nanjing Jiancheng Institute of Biological Engineering.

Determination of total antioxidant capacity

Prepare reagent two (R2) and reagent three (R3) application solutions according to Table 8. Accurately measure 0.1 mL of the sample solution to be tested, configure the measuring tube and control tube according to the steps in Table 9, with a concentration of 0.5 mg/mL, 1 mg/mL , 2mg/mLVC solution is used as a positive control.

Table 8 Reagent composition and preparation

Table 9 Operation steps for determination of total antioxidant capacity

Note: a is the volume of the sample to be tested, mL.

After configuration, mix well and let stand for 10 minutes. Using distilled water as a blank, the absorbance of each tube was measured at 520nm.

The calculation formula of total antioxidant capacity (U/mL):

ODU-measure the absorbance value of the tube;

ODC- the absorbance value of the control tube;

V- the total volume of the reaction solution (mL) during measurement;

V0-sample sampling volume (mL);

M-the dilution factor of the sample.

DPPH·Determination of clearance rate

Weigh 5mg DPPH accurately, dissolve it with absolute ethanol, and dilute it into a 100mL volumetric flask, dilute the sample solution 2 times, 4 times, 6 times, 8 times with three-distilled water, and configure to 50mg/mL, 25mg/mL , 12.5mg/mL, 8.33mg/mL, 6.25mg/mL sample solutions of different concentrations, respectively take 2mL of sample solutions of different concentrations, and add 2mLDPPH solution, with absolute ethanol as a control, the blank group is absolute ethanol, Distilled water each 2mL, shake well, stand for 30min in the dark at room temperature, use Vc as a positive control, perform 3 parallel tests, measure the absorbance at a wavelength of 517nm.

Calculation formula:

DPPH·Clearance rate=[1-(Ai-Aj)/A0]×100%,

A0 is the absorbance value without scavenger (add absolute ethanol)

Ai is the absorbance value of adding scavenger

Aj is the absorbance of sample solution + absolute ethanol

Determination of Scavenging Rate of Hydroxyl Radical in Salicylic Acid System

Dilute the sample solution according to the above method, take 0.5mL of the sample solution of different concentrations into the test tube, add in order, 0.6mL of 8mmol/L ferrous sulfate solution, 0.5mL of 20mmol/L hydrogen peroxide and 3mmol/L of salicylic acid (Dissolved in absolute ethanol) 2mL, mix well, put in a water bath, react at 37°C for 30 minutes, and cool with running water. Use absolute ethanol as the control group, deionized water instead of salicylic acid as the blank group, and use Vc as the positive control to do three parallel experiments. The absorbance was measured at a wavelength of 510nm.

Calculate the clearance rate as follows:

Clearance rate (%)=[1-(Ai-Aj)/A0]×100%,

A0 is the absorbance value without scavenger (add absolute ethanol)

Ai is the absorbance value of adding scavenger

Aj is the absorbance value of the blank group

Determination of superoxide anion scavenging rate

Prepare sample solutions of different concentrations according to the above method, take 0.1mL of sample solutions of different concentrations, add 2.8mL of 0.05mol/LTris-HCl buffer (pH=8.2), 0.1mL 3mmol/L pyrogallol, and replace with distilled water The sample solution was used as the control group, and the blank group was 2.8 mL Tris-HCl buffer, 0.1 mL distilled water, 0.1 mL 0.01 mol/L HCl, and Vc was used as a positive control, and three parallel experiments were performed. After starting the reaction for 30 seconds, the absorbance was measured at a wavelength of 320 nm, and the absorbance value was recorded every 30 seconds, and the reaction was conducted for 5 minutes.

Calculation formula:

Oxidation rate = (last recorded value-first recorded value)/5

Clearance rate (%) = (A0-A1)/A0×100

In the formula: A0 is the absorbance value without scavenger (adding distilled water)

A1 is the absorbance value of adding scavenger.

Determination of reducing power

Prepare sample solutions of different concentrations according to the above method, respectively take 1mL of sample solutions of different concentrations in a test tube, add 2.5mL each of pH=6.6 phosphate buffer and 0.3% potassium ferricyanide solution, mix and place In a water bath, incubate at a constant temperature of 55°C for 20 minutes, then take it out, quickly cool, add 2.5mL of 10% trichloroacetic acid solution, 3000rpm, centrifuge for 10min, take 2.5mL of supernatant, add 2mL of distilled water, 0.5mL of 0.1% mass fraction Mix the FeCl 3 solution thoroughly. After standing for 10 minutes, measure the absorbance value at a wavelength of 700 nm. The larger the absorbance value, the stronger the reducing power. The distilled water group was used as the blank group, and Vc was the positive control, and three parallel experiments were performed.

Calculation formula:

Reducing power = absorbance of sample-absorbance of blank control group

Total antioxidant capacity

Table 10 The effect of fermentation on the total antioxidant capacity of Platycodon grandiflorum extract

Note: The same letters on the shoulders of the same industry indicate that the difference is not significant (P>0.05), and different letters indicate significant differences (P<0.05).

As shown in Table 10 above, the total antioxidant capacity of the 50 mg/mL unfermented group and the Pichia Kudriazwei fermentation group is equivalent to 1 mg/mL ascorbic acid, while the total antioxidant capacity of the 50 mg/mL Velez Bacillus fermented group Stronger than 1mg/mL of ascorbic acid, it can be seen that microbial fermentation improves the total antioxidant capacity of Platycodon grandiflorum extract.

DPPH·Removal ability

Table 11 The effect of fermentation on the DPPH scavenging ability of Platycodon grandiflorum extract

Note: The same column with the same letter indicates that the difference is not significant (P>0.05), and the different letter indicates the difference is significant (P<0.05)

As shown in Table 11, both the fermented group and the unfermented group had an increase in the scavenging ability of DPPH· with increasing concentration, which was concentration-dependent. When the concentration was 50 mg/mL, the scavenging ability of each liquid reached the maximum. The clearance rate of Bacillus velezia fermentation group was the highest, as high as 90.73%, the Pichia kudriazwei fermentation group was 85.16%, and the unfermented group was 80.23%. At the same concentration, the strength of each liquid on DPPH·clearance is as follows: Velez Bacillus fermented group>Pichia kudriazwe fermented group>non-fermented group. There are significant differences between the groups, showing that the fermentation is significant Improved the ability of Platycodon grandiflorum extract to remove DPPH·.

Salicylic acid hydroxyl scavenging ability

Table 12 The effect of fermentation on the hydroxyl scavenging ability of Platycodon grandiflorum extract

Note: The same column with the same letter indicates that the difference is not significant (P>0.05), and the different letter indicates the difference is significant (P<0.05)

It can be seen from Table 12 above that both the fermented group and the unfermented group have a certain ability to scavenge hydroxyl radicals, and as the concentration increases, the ability to scavenge hydroxyl groups also increases. When the concentration is 50mg/mL, both the solution concentration reaches the maximum, the hydroxyl scavenging ability of each liquid also reaches the maximum. Among them, the Bacillus Velez fermentation group has the largest clearance rate, which is 70.28%, and the Pichia pastoris fermentation group is the largest. It was 61.54%, and the unfermented group was 57.30%. At the same concentration, the hydroxyl scavenging ability is in the order of: Velez Bacillus fermented group>Kudriazwe Pichia pastoris fermented group>non-fermented group. Compared with the unfermented group, there is no significant difference between the Pichia kudriazwei fermentation group and the unfermented group. Compared with the unfermented group and the Pichia kudriazwei group, the difference between the groups is significant. . It can be seen that the fermented Bacillus Velez strain significantly improves the ability of Platycodon grandiflorum extract to scavenge hydroxyl free radicals.

Superoxide anion scavenging ability

Table 13 The effect of fermentation on the scavenging ability of Platycodon grandiflorum extract on superoxide anions

Note: The same column with the same letter indicates that the difference is not significant (P>0.05), and the different letter indicates the difference is significant (P<0.05)

It can be seen from Table 13 that the superoxide anion scavenging capacity of the fermented group and the unfermented group increased with the increase of the concentration. There is a positive correlation with the concentration. The scavenging ability of each liquid superoxide anion at the same concentration is in the order: Bacillus velezia fermentation group>Pichia kudriazwei fermentation group>non-fermented group. At a concentration of 50 mg/mL, the clearance rate of Bacillus velezia fermentation group was 63.28%, that of Pichia kudriazwei fermentation group was 54.68, and that of unfermented group was 47.74%. Compared with the unfermented group, the fermented group has significant differences between the groups. It can be seen that the superoxide anion scavenging ability of the Platycodon grandiflorum extract will also be significantly improved after the microbial fermentation.

Reducing power

Table 14 The influence of fermentation on the reducing power of Platycodon grandiflorum extract

Note: The same column with the same letter indicates that the difference is not significant (P>0.05), and the different letter indicates the difference is significant (P<0.05)

As shown in Table 14, the reducing power of both the fermented group and the non-fermented group increases with the increase of the concentration. At the same concentration, the reducing power of each liquid is in the order of: Velez Bacillus fermented group>Kudriaz Pichia pastoris fermentation group>non-fermented group. The difference between the Bacillus velezia group and the Pichia kudriazwei Pichia fermentation group was not significant, but compared with the unfermented group, the difference between the groups was significant. It can be seen that the reducing power of Platycodon grandiflorum extract after microbial fermentation is also There will be a significant improvement.

According to the present invention, the total flavonoids and total phenol content of Bacillus velezia and Pichia kudriazwei fermented Platycodon grandiflorum extract were significantly increased. Wang Xing et al. measured phenols and other substances in the fermentation process of blueberry wine. It was found that during the fermentation process of blueberry wine, the content of total phenols and total flavonoids increased first and then decreased; however, they were significantly higher than the initial content. Chen Caiyun uses Natto bacteria to ferment Sophora rice to increase the content of rutin and quercetin. However, the contents of total flavonoids and total phenols in each liquid in this experiment are low whether fermented or unfermented. According to literature review, the content of flavonoids extracted from Platycodon grandiflorum is less than that of alcohol extracts. Flavonoids are more soluble in alcohol reagents, so the content will be lower . Qian Hua et al. also showed that antioxidant active substances such as phenols and flavonoids are mostly present in alcohol extracts.

In the aspect of using microbial fermentation to improve in vitro antioxidant capacity, Yang Xuejuan used Bacillus natto for solid fermentation of soybean meal, which proved that the soybean meal extract after fermentation not only has higher activity and reducing power in scavenging DPPH free radicals and superoxide anions in vitro And the ability to inhibit lipid peroxidation, and also has a higher antioxidant capacity in the body. From this, we can infer that the fermented extract may be due to the increase or production of a variety of substances with antioxidant activity, so that the antioxidant capacity has been significantly improved.

The present invention inoculates Bacillus velez and Pichia Kudriazwei into the extract of Platycodon grandiflorum, carries out microbial fermentation, and compares the extract with the unfermented extract to study the effect of microbial fermentation on the antioxidant capacity of the extract of Platycodon grandiflorum. influences. The results are as follows:

1. After microbial fermentation, the content of total flavonoids and total phenols in Platycodon grandiflorum extract increased significantly. The total antioxidant capacity of 50 mg/mL Platycodon grandiflorum extract and Pichia Kudriazwei fermentation group was equivalent to 1 mg/mL ascorbic acid. The total antioxidant capacity of 50mg/mL Bacillus velez fermentation broth is stronger than that of 1mg/mL ascorbic acid solution.

2. By investigating the scavenging ability and reducing power of DPPH·, hydroxyl free radicals, superoxide anion, and studying its anti-oxidation ability in vitro, it was found that the free radical scavenging ability and reducing power of the fermented group and the unfermented group increased with the solution concentration Large and increase, and are positively correlated. Two of the fermented groups have higher antioxidant capacity than the unfermented group. After Bacillus Velez fermented, the in vitro antioxidant capacity of Platycodon grandiflorum extract was significantly improved. It shows that microbial fermentation can significantly improve the in vitro antioxidant capacity of Platycodon grandiflorum extract.

The invention can be used in production in a short time with low cost and low storage conditions. Those skilled in the art can easily understand that the above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modification, equivalent replacement and improvement, etc. made within the spirit and principle of the present invention, All should be included in the protection scope of the present invention.

Claims (8)

1. A strain of Pichia kudriazvii, characterized in that it is deposited in the General Microbiology Center of China Microbial Species Collection Management Committee, the preservation date is June 17, 2019, and the preservation number is CGMCC No.17939.
2. A method for preparing de-celerylated Platycodin D by using the Pichia Kudriazwei Pichia strain according to claim 1, comprising the following steps:

   Cultivation steps: the culture of the Pichia azweed yeast selects YPD liquid medium and yeast extract peptone glucose agar medium;

   Seed culture steps: the colonies are inoculated in 20mL liquid medium, placed at 30℃, 110r/min, shaking culture for 14h-18h, as the seed culture solution;

   Transformation steps: add 2% inoculum to 0.1mg/mL saponin standard solution, transform at 30℃ for 24h～72h;

   The colony includes the colony of the Pichia kudriazwe strain.
3. The method of claim 2, wherein the colony comprises a colony of the Pichia kudriazwe strain.
4. The preparation method according to claim 3, wherein the strains are Pichia kudriazweis strain and Bacillus velezia, and the Pichia kudriazweis strain is preserved in the Chinese microorganism strain The General Microbiology Center of the Species Collection Management Committee, the preservation date is June 17, 2019, and the preservation number is CGMCC No.17939; the Bacillus velez strain is deposited in the General Microbiology Center of the China Microbial Species Collection Management Committee, and the preservation date is On June 17, 2019, the deposit number was CGMCC No.17938.
5. The preparation method according to claim 3, wherein the temperature of the fermentation is controlled at 30-37°C.
6. The preparation method according to claim 4, wherein the fermentation substrate of the mixed fermentation comprises an aqueous extract of Platycodon grandiflorum.
7. The preparation method according to claim 6, characterized in that the seed culture of the two strains of Pichia kudriazwei and Bacillus velezia is inoculated into the water extract of Platycodon grandiflorum at an inoculum of 2%.
8. The decelerylated platycodin D prepared by the preparation method of any one of claims 2-7 is used in the preparation of anti-tumor drugs, anti-rheumatoid arthritis drugs, anti-obesity drugs, kidney-protecting drugs, liver-protecting drugs, Application in immune-regulating drugs, anti-diabetic drugs, analgesic drugs or enzyme inhibitor drugs.

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