WO2021110101A1 - Method for preparing deapioplatycodin d and application thereof - Google Patents
Method for preparing deapioplatycodin d and application thereof Download PDFInfo
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- WO2021110101A1 WO2021110101A1 PCT/CN2020/133631 CN2020133631W WO2021110101A1 WO 2021110101 A1 WO2021110101 A1 WO 2021110101A1 CN 2020133631 W CN2020133631 W CN 2020133631W WO 2021110101 A1 WO2021110101 A1 WO 2021110101A1
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- Prior art keywords
- pichia
- fermentation
- strain
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- platycodin
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Definitions
- 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.
- 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°C, 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.
- 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.
- dPD deapio-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.
- 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.
- 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 ⁇ -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.
- Pichia kudriavzevii The Chinese name of 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.
- 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.
- Bacillus velezensis 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.
- 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 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°C, 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°C 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.
- 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.
- the fermentation temperature is controlled at 30-37°C.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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°C, 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°C for 24h ⁇ 72h.
- 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°C, 110r/min and shake culture for 14h-18h, as seed culture solution; transformation step: 2% (volume fraction
- the Pichia azweed is a strain of Pichia kudriazwe.
- the saponin standard solution includes a de-celerylated Platycodon E standard solution.
- 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.
- 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.
- 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.
- 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.
- 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.
- HPLC detection conditions are as follows:
- Chromatographic column Zorbax C18 column (150mm ⁇ 2.1mm, 3.5 ⁇ m 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 1.0 ⁇ L.
- 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.
- the calculation method for the conversion rate of de-celery platycodin is as follows:
- 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.
- Pichia kudriaz has the presence of (PE ⁇ PD 3 ⁇ PD), (dPE ⁇ dPD 3 ⁇ dPD), (PD 3 ⁇ PD) and (dPD 3 ⁇ dPD). ) Transformation pathway.
- Figure 1 and Table 2 show the changes of saponin content during the fermentation of Platycodon grandiflorum by Pichia kudriazwei at 30°C.
- Figure 1 shows the changes of saponin content 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.
- 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.
- 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.
- 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.
- Figure 3 and Table 4 show the changes of saponin content during the fermentation of Platycodon grandiflorum by Bacillus Velez at 37°C.
- Figure 3 shows 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.
- 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.
- 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.
- 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.
- 37°C is more suitable for Kudri Transformation of Platycodin by Pichia pastoris.
- 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.
- the condition of 37°C can be used for the fermentation of mixed strains to transform Platycodin.
- 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 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.
- 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.
- 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.
- 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.
- 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%;
- Jilin Changbai Mountain Platycodon grandiflorum was selected as the biotransformation material.
- 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°C, dPD increased by 3.83% and PD increased by 11.21%; the mixed fermentation of the two strains at 37°C was better than 30°C, 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.
- 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.
- a is the volume of the sample to be tested, mL.
- V- the total volume of the reaction solution (mL) during measurement
- 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
- 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.
- 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
- 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.
- Oxidation rate (last recorded value-first recorded value)/5
- A0 is the absorbance value without scavenger (adding distilled water)
- A1 is the absorbance value of adding scavenger.
- the distilled water group was used as the blank group, and Vc was the positive control, and three parallel experiments were performed.
- 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.
- both the fermented group and the unfermented group had an increase in the scavenging ability of DPPH ⁇ with increasing concentration, which was concentration-dependent.
- 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%
- the unfermented group was 80.23%.
- 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 ⁇ .
- 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.
- concentration is 50mg/mL
- both the solution concentration reaches the maximum
- the hydroxyl scavenging ability of each liquid also reaches the maximum.
- the Bacillus Velez fermentation group has the largest clearance rate, which is 70.28%
- the Pichia pastoris fermentation group is the largest. It was 61.54%, and the unfermented group was 57.30%.
- 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.
- the reducing power of both the fermented group and the non-fermented group increases with the increase of the 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.
- 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.
- 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.
- 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:
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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
本申请要求于2019年12月03日提交中国专利局、申请号为CN201911224253.6、发明名称为“一种去芹糖桔梗皂苷D的制备方法及其应用”的中国专利申请的优先权,其全部内容通过引用结合在本申请中。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.
本发明涉及一种去芹糖桔梗皂苷D的制备方法及其应用,涉及采用含有库德里阿兹威毕赤酵母菌株和贝莱斯芽孢杆菌的混合菌株制备去芹糖桔梗皂苷D的制备方法及其应用。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.
桔梗皂苷具有治疗高脂血症、高血压、糖尿病、肥胖等功能,其中主要生物活性成分为三萜皂苷(platycosides)。据调查,目前已经有超过三十种桔梗皂苷及多种皂苷元已经被分离鉴定,桔梗皂苷D(Platycodin D),简称PD,性状为白色结晶粉末,密度1.56g/cm
3,熔点为228~237℃,分子式为C
57H
92O
28,相对分子量为1224.58,水溶性好。桔梗皂苷D是桔梗酸类双糖链皂苷,其糖基分别连接在母核C-3和C-28位上。2010版《中国药典》规定桔梗中桔梗皂苷D的质量分数为0.1%。在桔梗皂苷所表现的生物活性中,桔梗皂苷D最具有代表性。其对肿瘤细胞系表现出强烈的细胞毒性,可以作为治疗癌症的药物。WIE等通过黑曲霉(Aspergillus niger)的粗酶提取物处理,改良了桔梗皂苷D,显著减少了V79-4细胞(中国人仓鼠肺成纤维细胞)的细胞毒性和红细胞溶血毒性,增加了生物活性并提高了感官价值。去芹糖桔梗皂苷D(deapio-platycodin D),简称(dPD),白色或类白色粉末,分子式为C
52H
84O
24,相对分子量1093.22,水溶性好。其分子结构与桔梗皂苷D相比少一个芹菜糖。已有研究发现,去芹糖桔梗皂D具有抑制胰脂肪酶活性及抗肿瘤活性。
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.
目前,桔梗皂苷的生产方法有化学法、酶制剂法和微生物转化。化学法产生一系列副反应和化学残留,化学反应在制备过程中耗时、选择性低、转化率低。酶制剂法快速、高效,逐渐被认为是制备活性物质较为理想的方法。但是,酶制剂的制作成本高、保存条件苛刻,无法在短时间内大量应用于生产。因此,较多学者逐渐投入于微生物转化法制备桔梗皂苷。Lee等从酒曲(nuruk)中筛选出可以生产桔梗皂苷D的微生物菌株,经鉴定为Cyberlindnera fabianii。Ling 等利用具有β-葡萄糖苷酶活性的重组乳酸菌发酵生产人参皂苷Rg
3(S)和化合物K,通过透化处理和混合发酵,转化产率分别达到61%和70%。
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.
Pichia kudriavzevii中文名称叫库德里阿兹威毕赤酵母,国内学者研究发现,库德里阿兹威毕赤酵母菌株ZJPH0802能够较好的转化姜黄素,获得二种转化产物六氢姜黄素和四氢姜黄素。其中主要产物为四氢姜黄素,而Natio等实验证明了四氢姜黄素的抗氧化能力大于六氢姜黄素大于姜黄素,由此我们可以看出该菌株具有较好的研究前景。范光森等在浓香型大曲中筛选出库德里阿兹威毕赤酵母,该菌株不但产苯乙醇高,而且还能够在豆芽汁培养基中产近30种风味物质。所以该菌株在白酒中的应用前景广泛,具有较好的市场价值。Gilberto V.M等也发现库德里阿兹威毕赤酵母菌株LPB06和LPB07通过发酵可可豆,会使可可豆具有更好的色泽与更丰富的香气组成,可以用于可可豆的风味调节。可见该菌株在改善风味方面,前景大好。另有学者从非洲谷物发酵食品中筛选出库德里阿兹威毕赤酵母,发现该菌株存在叶酸生物合成基因,不但能够产叶酸和植酸酶,而且还能够在人类胃肠道的条件下传代生存,对肠细胞具有粘附力,具有良好的益生菌潜力,可直接在宿主体内发挥潜在的有益作用,增加其对宿主健康的影响。因此该菌株在研发通过天然发酵提高食品的营养价值及益生菌发酵罐的制备方面前景广泛。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.
Bacillus velezensis中文名称叫做贝莱斯芽胞杆菌,进来研究报道了该菌种在抑菌方面应用广泛,金疏桐等在盾叶薯蓣植物中分离得到贝莱斯芽胞杆菌,其具有一定的抗病原细菌活性。杨胜清证明了贝莱斯芽胞杆菌具有较广的抑菌作用,在一定剂量内对植物没有侵染,其发酵液在防治番茄早疫病方面效果较好。Liu,G等证明了贝莱斯芽胞杆菌LS69对多种致病细菌表现出拮抗活性,对其进行基因组分析鉴定发现该菌株含有一系列涉及增强植物生长和触发植物免疫的基因,为阐明其生物防治机制并促进其在未来的应用提供基础。SaadA.M.等利用响应面法优化贝莱斯芽胞杆菌KY498625的胞外多糖产生条件,结果表明纯化的EPS分子量为1.29×10
5Da,数均分子量为1.14×10
5Da,主要由葡萄糖,甘露糖和半乳糖组成,通过数据分析获得的最佳条件下EPS的生产成功地将生产率从初始EPS产量增加到4.1至7.6g/L。
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.
目前从国内外皂苷类生物转化的研究发现,单一菌株的转化途径有限,无法完全利用皂苷资源,因此对于生物转化方面很多学者开始利用混合酶体系、 混合菌株进行生物转化研究,其效果理想,优于传统的单一菌株转化。Li,Ling等利用两种不同转化途径的重组乳酸菌进行人参皂苷转化,通过诱导产酶、透化和破碎处理、混合发酵等手段实现了人参皂苷的高效转化。利用菌株发酵特性对桔梗皂苷进行生物转化,不仅可以实现对桔梗皂苷安全、环保的生产,还可以充分利用桔梗资源,使其内在含有的生物活性低的桔梗皂苷转化为生物活性高的皂苷,达到变废为宝的效果。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.
发明内容Summary of the invention
针对现有技术中存在的不足,本发明的目的在于提供一种库德里阿兹威毕赤酵母菌株,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏日期为2019年6月17日,保藏编号为CGMCC No.17939。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.
本发明所述的库德里阿兹威毕赤酵母菌株制备去芹糖桔梗皂苷D的方法,包括以下步骤:The method for preparing decelerylated Platycodin D from the Pichia Kudriazwei strain of the present invention includes the following steps:
培养步骤:所述阿兹威毕赤酵母的培养选用YPD液体培养基、酵母浸出粉胨葡萄糖琼脂培养基(YPD);Cultivation steps: the culture of the Pichia azweed yeast selects YPD liquid medium and yeast extract peptone dextrose agar medium (YPD);
种子培养步骤:菌落接种于20mL的液体培养基中,置于30℃,110r/min条件下震荡培养14h~18h,作为种子培养液;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;
转化步骤:2%接种量加入0.1mg/mL皂苷标准品溶液中,在30℃下转化24h~72h。Transformation step: add 2% inoculum to 0.1mg/mL saponin standard solution and transform at 30℃ for 24h~72h.
本发明所述的制备方法,其特征在于,生物转化过程中,发酵温度控制在30℃。The preparation method of the present invention is characterized in that, during the bioconversion process, the fermentation temperature is controlled at 30°C.
本发明还提供一种去芹糖桔梗皂苷D的制备方法,其特征在于,包括,采用两种以上的菌株混合发酵制备的步骤。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.
本发明所述的方法,所述菌株为以库德里阿兹威毕赤酵母菌株(Pichia kudriavzevii)和贝莱斯芽孢杆菌(Bacillus velezensis),所述库德里阿兹威毕赤酵母菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址:中国北京,保藏日期为2019年6月17日,保藏编号为CGMCC No.17939;所述贝莱斯芽孢杆菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址:中国北京,保藏日期为2019年6月17日,保藏编号为CGMCC No.17938。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.
本发明所述的制备方法,生物转化过程中,发酵温度控制在30~37℃。In the preparation method of the present invention, during the biological transformation process, the fermentation temperature is controlled at 30-37°C.
本发明所述的制备方法,所述发酵温度为37℃。In the preparation method of the present invention, the fermentation temperature is 37°C.
本发明进一步提供一种去芹糖桔梗皂苷D的应用,在制备抑制肿瘤药物、抑制类风湿关节炎药物、抗肥胖类药物、护肾类药物、护肝类药物、调节免疫活性类药物、抗糖尿病类药物、镇痛类药物、酶抑制类药物中的应用。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.
本发明的发明人首次利用不同转化机制的两种菌株,以混合发酵的方式转化桔梗皂苷,有效提高了桔梗皂苷D和去芹糖桔梗皂苷D的产率。利用菌株发酵特性对桔梗皂苷进行生物转化,不仅可以实现对桔梗皂苷安全、环保的生产,还可以充分利用桔梗资源,使其内在含有的生物活性低的桔梗皂苷转化为生物活性高的皂苷,达到变废为宝的效果。本发明提供不同转化方式的两种菌株及利用2种菌株进行混合发酵制备桔梗皂苷D和去芹糖桔梗皂苷D的方法。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.
库德里阿兹威毕赤酵母将桔梗皂苷E先转化成桔梗皂苷D
3再将桔梗皂苷D
3转化成桔梗皂苷D(PE→PD
3→PD),将去芹糖桔梗皂苷E转化成去芹糖桔梗皂苷D
3再转化成去芹糖桔梗皂苷D(dPE→dPD
3→dPD),其中对dPD
3和PD
3的转化速度显著高于PE、dPE;贝莱斯芽胞杆菌直接可以将桔梗皂苷E转化成桔梗皂苷D(PE→PD),将去芹糖桔梗皂苷E转化成去芹糖桔梗皂苷D。库德里阿兹威毕赤酵母的转化率高,但是转化速度慢,而贝莱斯芽胞杆菌转化速度快,但是转化率较库德里阿兹威毕赤酵母低,因此从桔梗皂苷资源充分利用的角度考虑,可以尝试进行混合菌株转化。
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.
利用不同菌株发酵特性对桔梗皂苷进行混合发酵生物转化,不仅可以实现对桔梗皂苷安全、环保的生产,还可以充分利用桔梗资源,使其内在含有的生物活性低的桔梗皂苷转化为生物活性高的皂苷,达到变废为宝的效果。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.
图1示出库德里阿兹威毕赤酵母30℃条件下发酵桔梗过程中皂苷含量的变化;Figure 1 shows the change of saponins content during the fermentation of Platycodon grandiflorum by Pichia Kudriazwei at 30°C;
图2示出库德里阿兹威毕赤酵母37℃条件下发酵桔梗过程中皂苷含量的变化;Figure 2 shows the change of saponin content during the fermentation of Platycodon grandiflorum by Pichia Kudriazwei at 37°C;
图3示出贝莱斯芽孢杆菌37℃条件下发酵桔梗过程中皂苷含量的变化;Figure 3 shows the change of saponin content during the fermentation of Platycodon grandiflorum by Velez Bacillus at 37°C;
图4示出贝莱斯芽孢杆菌30℃条件下发酵桔梗过程中皂苷含量的变化;Figure 4 shows the change of saponin content during the fermentation of Platycodon grandiflorum by Bacillus Velez at 30°C;
图5示出贝莱斯芽孢杆菌发酵桔梗过程中皂苷的变化;Figure 5 shows the changes of saponins during the fermentation of Platycodon grandiflorum by Bacillus Velez;
图6示出混合菌株30℃条件下发酵桔梗过程中皂苷含量的变化;Figure 6 shows the change of saponin content in the process of fermenting Platycodon grandiflorum at 30°C with mixed strains;
图7示出混合菌株发酵桔梗过程中皂苷含量的变化。Figure 7 shows the change of saponin content during the fermentation of Platycodon grandiflorum by mixed strains.
生物保藏说明Biological preservation instructions
库德里阿兹威毕赤酵母菌株(Pichia kudriavzevii)SK-P1,于2019年6月17日保藏于中国微生物菌种保藏管理委员会普通微生物中心,地址为北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC No.17939;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;
贝莱斯芽孢杆菌(Bacillus velezensis)SK-P1,于2019年6月17日保藏于中国微生物菌种保藏管理委员会普通微生物中心,地址为北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC No.17938。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.
为了使本领域技术人员更加清楚地明白本发明的目的、技术方案及优点,以下结合附图及实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明。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.
本发明的库德里阿兹威毕赤酵母菌株,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏日期为2019年6月17日,保藏编号为CGMCC No.17939。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.
本发明采用上述的库德里阿兹威毕赤酵母菌株制备桔梗皂苷D的方法,包括以下步骤:培养步骤:所述阿兹威毕赤酵母的培养选用YPD液体培养基、酵母浸出粉胨葡萄糖琼脂培养基(YPD);种子培养步骤:菌落接种于20mL的液体培养基中,置于30℃,110r/min条件下震荡培养14h~18h,作为种子培养液;转化步骤:2%(体积分数)接种量加入0.1mg/mL皂苷标准品溶液中,在30℃下转化24h~72h。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.
在本发明中,所述阿兹威毕赤酵母为库德里阿兹威毕赤酵母菌株。In the present invention, the Pichia azweed is a strain of Pichia kudriazwe.
在本发明中,所述皂苷标准品溶液包括去芹糖桔梗皂苷E标准品溶液。In the present invention, the saponin standard solution includes a de-celerylated Platycodon E standard solution.
本发明的制备方法,其生物转化过程中,发酵温度控制在30℃~37℃,优选温度控制在30℃。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.
本发明进一步提供一种桔梗皂苷D的制备方法,采用以下所述的混合菌株;包括库德里阿兹威毕赤酵母菌株和贝莱斯芽孢杆菌,所述库德里阿兹威毕赤酵母菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏日期为2019年6月17日,保藏编号为CGMCC No.17939;所述贝莱斯芽孢杆菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏日期为2019年6月17日,保藏编号为CGMCC No.17938。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.
本发明从辣白菜样品发酵的桔梗提取液中筛选得到了库德里阿兹威毕赤酵母,该菌株发酵桔梗提取液与未发酵组相比,显著提高了PD含量,转化率为46.44%,并且该菌株对人体健康不会造成影响,可用于发酵桔梗提取液提高PD含量,在食品发酵及发酵剂的制备方面有很好的前景。本专利中的库德里阿兹威毕赤酵母菌株来源于泡菜。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.
本发明从辣白菜样品的发酵液中分离筛选得到贝莱斯芽胞杆菌,该菌株发酵桔梗提取液后,与未发酵组相比,显著提高了PD含量,转化率为48.92%。目前尚未报到过该菌株对人体有致病性,故可用于微生物发酵转化桔梗皂苷D。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.
本发明所述的制备方法的生物转化过程中,发酵温度控制在30~37℃,在利用所述混合菌株制备去芹糖桔梗皂苷D时,发酵温度优选为37℃。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.
本发明提供一种去芹糖桔梗皂苷D的应用,在制备抑制肿瘤药物、抑制类风湿关节炎药物、抗肥胖类药物、护肾类药物、护肝类药物、调节免疫活性类药物、抗糖尿病类药物、镇痛类药物、酶抑制类药物中的应用。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.
本发明的发明人首次利用不同转化机制的两种菌株,以混合发酵的方式转化桔梗皂苷,有效提高了去芹糖桔梗皂苷D的产率。利用菌株发酵特性对去芹糖桔梗皂苷进行生物转化,不仅可以实现对去芹糖桔梗皂苷安全、环保的生产,还可以充分利用桔梗资源,使其内在含有的生物活性低的桔梗皂苷转化为生物活性高的皂苷,达到变废为宝的效果。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.
本发明将阿兹威毕赤酵母、贝莱斯芽胞杆菌两菌株种子培养液以2%(体积分数)的接种量接种到装有50mL桔梗水提液的锥形瓶中,110r/min震荡培养72h,发酵过程定期(0、4、8、12、24、36、48、60、72h)取样,HPLC测定六种桔梗皂苷含量。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.
在本发明中,所述阿兹威毕赤酵母菌株的种子培养液和贝莱斯芽胞杆菌菌株的种子培养液的体积比优选为1:1;所述阿兹威毕赤酵母菌株的种子培养液和贝莱斯芽胞杆菌菌株的种子培养液中有效活菌数分别优选为1×10
6cfu/mL。
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.
表1发酵组的设定Table 1 Setting of fermentation group
混合菌株:库德里阿兹威毕赤酵母、贝莱斯芽胞杆菌Mixed strains: Pichia Kudriazwei, Bacillus velezia
混合菌株:库德里阿兹威毕赤酵母、贝莱斯芽胞杆菌Mixed strains: Pichia Kudriazwei, Bacillus velezia
HPLC检测条件如下:The HPLC detection conditions are as follows:
设备型号:Agilent 1200RRLC-G6410BDevice model: Agilent 1200RRLC-G6410B
色谱柱:Zorbax C18 column(150mm×2.1mm,3.5μm particle size);HPLC条件:流动相水(A)-乙腈(B);梯度洗脱:0~6min:10%~15%B,6~50min:15%~25%B,50~60min:25%~47.5%B;流速:0.2mL/min;温度:室温;检测波长:210nm;进样量1.0μL。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条件:ESI-负离子模式,电喷雾离子源,4KV的离子喷射电压,-15V毛细管电压,300℃毛细管温度,扫描次数:500,总离子色谱图:100~1000m/z。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.
本发明首次在发现两种菌株对去芹糖桔梗皂苷具有不同的转化途径,如下,本发明为去芹糖桔梗皂苷D的微生物生产提供基础。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.
在本发明中,去芹糖桔梗皂苷转化率计算方法如下:In the present invention, the calculation method for the conversion rate of de-celery platycodin is as follows:
去芹糖桔梗皂苷转化率%=(Y-X)/Y×100%Conversion rate of celeryside platycodin saponins%=(Y-X)/Y×100%
式中:Y-发酵前含量(mg/mL)In the formula: Y- content before fermentation (mg/mL)
X-发酵后含量(mg/mL)X-content after fermentation (mg/mL)
利用SPSS 17.0软件对试验结果进行处理,采用Duncan法进行多重比较,试验结果用平均值±标准差来表示。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.
在本发明中,库德里阿兹威毕赤酵母30℃发酵结果,如下详细说明:In the present invention, the 30°C fermentation results of Pichia kudriazwei are described in detail as follows:
在本发明中,库德里阿兹毕威赤酵母对于去芹糖桔梗皂苷存在(PE→PD
3→PD)、(dPE→dPD
3→dPD)和(PD
3→PD)和(dPD
3→dPD)转化途径。
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.
表2库德里阿兹威毕赤酵母30℃下发酵桔梗过程中皂苷含量的变化Table 2 Changes of saponins content during the fermentation of Platycodon grandiflorum by Pichia kudriazwei at 30℃
图1和表2为库德里阿兹威毕赤酵母30℃条件下发酵桔梗过程中皂苷含量的变化,由图1可知,库德里阿兹威毕赤酵母在30℃条件下发酵桔梗过程中,桔梗发酵液中dPE、PE、dPD
3呈下降趋势,PD
3呈先上升后下降趋势,dPD、PD含量呈上升趋势;其中dPE、PE在0~24h内含量未发生显著性变化,在24h后迅速下降,dPE在48h后、PE在60h后未检出,转化率达100%,可能已经完成全部转化,这可能是因为发酵前期桔梗提取液中含有充足的桔梗多糖作为碳源,供库德里阿兹威毕赤酵母生长利用,在发酵后期多糖消耗殆尽,开始产生生物转化酶水解桔梗皂苷所致。dPD
3在0~48h呈缓慢下降趋势,在48~60h内迅速下降,72h后仍存在剩余,含量为0.0023mg/mL,PD
3在0~36h内含量未发生显著性变化,在36~48h内含量显著性上升(P<0.01),这是由于PE的分解转化所致,在48h后含量逐渐下降,转化产生PD,在72h后仍存在剩余,含量为0.0229mg/mL,这表明对PD
3的转化仍存在转化空间,需要进一步对菌株的产酶条件进行研究;dPD、PD在发酵结束时含量都得到了极显著的提高(P<0.01),dPD在72h后含量为0.054mg/mL,比初始的0.0462mg/mL提高了16.88%、PD在72h后含量为0.2445mg/mL,比初始的0.2246mg/mL提高了8.86%。
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.
表3库德里阿兹威毕赤酵母37℃下发酵桔梗过程中皂苷含量的变化Table 3 Changes of saponins content during the fermentation of Platycodon grandiflorum by Pichia Kudriazwei at 37℃
图2和表3示出库德里阿兹威毕赤酵母37℃条件下发酵桔梗过程中皂苷含量的变化,由图2可知,库德里阿兹威毕赤酵母在37℃发酵桔梗过程中,发酵 液内桔梗皂苷含量的变化趋势和30℃发酵条件相类似,但dPE、PE含量在36h后才开始下降,对比30℃发酵条件推迟了12h,此外dPD
3和PD
3含量的变化也不明显,这表明库德里阿兹威毕赤酵母在37℃发酵条件下的发酵途径效率不高,这可能是因为所产生的转化酶在37℃条件下活性不强的原因;与30℃条件下发酵相比,30℃更适合库德里阿兹威毕赤酵母对桔梗皂苷的转化。
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.
表4贝莱斯芽孢杆菌37℃下发酵桔梗过程中皂苷含量的变化Table 4 Changes of saponins content during the fermentation of Platycodon grandiflorum by Bacillus Velez at 37℃
图3和表4示出贝莱斯芽孢杆菌37℃条件下发酵桔梗过程中皂苷含量的变化,由图3可知,贝莱斯芽孢杆菌在37℃条件下发酵桔梗过程中,桔梗发酵液中,dPE、PE呈下降趋势,dPD
3、PD
3含量无明显变化,dPD、PD含量呈上升趋势;其中dPE、PE在24h内含量迅速下降,dPE在24h后未检出,可能已经转化完全,结合dPE、PE含量变化情况分析,推测贝莱斯芽孢杆菌的桔梗皂苷转化酶可能是在发酵初期产生,不会受到桔梗中碳源的影响;dPD、PD含量在发酵后期含量分别为0.0434mg/mL、0.2847mg/mL,对比发酵前的0.0418mg/mL、0.2560mg/mL分别提高了3.83%和11.21%。
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.
表5贝莱斯芽孢杆菌30℃下发酵桔梗过程中皂苷含量的变化Table 5 Changes of saponins content during the fermentation of Platycodon grandiflorum by Bacillus Velez at 30℃
注:同列肩标字母相同表示差异不显著(P>0.05),大写字母不同表示差异极显著(P<0.01),小写字母不同表示差异显著(P<0.05),下表同。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.
图4和表5示出贝莱斯芽孢杆菌30℃条件下发酵桔梗过程中皂苷含量的变化,由图4可知,贝莱斯芽孢杆菌在30℃条件下发酵桔梗过程中,桔梗发酵液中dPE、PE在8~12h内含量迅速下降,dPE在12h后未检出,可能已经转化完全;dPD、PD在发酵后期含量有所下降,与37℃条件下发酵相比,37℃更适合库德里阿兹威毕赤酵母对桔梗皂苷的转化。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.
此外,在贝莱斯芽孢杆菌30℃、37℃发酵过程中,观察到了一种色谱峰面积逐渐增加,图示出贝莱斯芽孢杆菌发酵桔梗过程中皂苷的变化,如图5所示,其在dPD和PD
3之间,并且随着未知峰的峰面积增加,dPD
3附近的其它峰面积逐渐减少,推测其可能是其它皂苷含量的变化,贝莱斯芽孢杆菌对桔梗皂苷的生物转化可能还存在其它的转化机制,需要进一步试验研究。
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.
表6混合菌株30℃下发酵桔梗过程中皂苷含量的变化Table 6 Changes of saponin content during the fermentation of Platycodon grandiflorum by mixed strains at 30℃
图6和表6示出混合菌株30℃条件下发酵桔梗过程中皂苷含量的变化,由图6可知,混合菌株在37℃条件下发酵桔梗过程中,桔梗发酵液中dPE、PE呈下降趋势,dPD
3含量在发酵前中期呈波动性变化,在发酵后期呈上升趋势,PD
3含量呈上升趋势,dPD含量呈上升趋势,PD含量呈先下降后上升趋势;其中dPE、PE在24~36h内迅速下降,发酵结束时仍存在剩余;dPD
3在24h后开始上升,PD
3在12h后开始上升,并且未出现下降情况,结合库德里阿兹威毕 赤酵母37℃的发酵结果分析,库德里阿兹威毕赤酵母可能在混合菌株37℃发酵过程中受到了一定程度的抑制,导致dPD
3、PD
3含量无法下降;dPD、PD含量在发酵后期含量分别为0.0562mg/mL、0.3213mg/mL,对比发酵前的0.0458mg/mL、0.2960mg/mL分别提高了22.71%和8.55%,因此37℃条件可用于混合菌株发酵转化桔梗皂苷。
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.
表7混合菌株37℃下发酵桔梗过程中皂苷含量的变化Table 7 Changes of saponin content during the fermentation of Platycodon grandiflorum by mixed strains at 37℃
图7和表7示出混合菌株发酵桔梗过程中皂苷含量的变化,由图7可知,混合菌株在30℃条件下发酵桔梗过程中,桔梗发酵液中dPD含量呈波动性变化、PD含量几乎不变,dPE、PE呈下降趋势,dPD
3含量呈波动性变化,PD
3含量出现了先上升后下降的变化,其中dPE、PE在4h内含量无显著性变化(P>0.05),dPE在4h后迅速下降,24h后未检出,可能已经转化完全,PE在4~8h内缓慢下降,在12~24h内迅速下降,dPE、PE前期含量的变化表明贝莱斯芽孢杆菌参与了发酵前期的皂苷转化;dPD
3含量在8~12h出现上升,但无显著性差异(P>0.05)、PD
3含量在12~36h内极显著性上升(P<0.01),随后在36~48h缓慢下降(P<0.01),这表明库德里阿兹威毕赤酵母参与了发酵中后期的皂苷转化;与30℃条件下相比,37℃更适合混合菌株的发酵。
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.
本发明首次利用不同转化机制的两种菌株,以混合发酵的方式转化桔梗皂苷,有效提高了桔梗皂苷D和去芹糖桔梗皂苷D的产率。为桔梗皂苷D、去芹糖桔梗皂苷D的微生物生产和库德里阿兹威毕赤酵母、贝莱斯芽孢杆菌对桔梗的混合发酵提供理论基础。根据本发明,单一菌株的转化途径有限,无法完全利用皂苷资源,显著优于传统的单一菌株转化。利用菌株发酵特性对桔梗皂苷 进行生物转化,不仅可以实现对桔梗皂苷安全、环保的生产,还可以充分利用桔梗资源,使其内在含有的生物活性低的桔梗皂苷转化为生物活性高的皂苷,达到变废为宝的效果。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.
本发明利用库德里阿兹威毕赤酵母、贝莱斯芽孢杆菌对桔梗皂苷水提液进行发酵,分别监测了单独菌株和混合菌株在30℃、37℃条件下对桔梗皂苷的生物转化情况。库德里阿兹威毕赤酵母在30℃条件下对桔梗皂苷的发酵优于37℃,dPD提高了16.88%、PD提高了8.86%;贝莱斯芽孢杆菌37℃条件下对桔梗皂苷的发酵优于30℃,dPD提高了3.83%、PD提高了11.21%;两种菌株混合发酵37℃条件下对桔梗皂苷的发酵优于30℃,dPD提高了22.71%、PD提高了8.55%,混合发酵模式可以作为桔梗皂苷转化的一种途径。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.
在本发明中,阿兹威毕赤酵母的转化机制为(PE→PD
3→PD)、(dPE→dPD
3→dPD),其中对dPD
3和PD
3的转化速度显著高于PE、dPE;贝莱斯芽胞杆菌(Bacillus velezensis)的转化机制为(PE→PD)、(dPE→dPD),转化速度快,从桔梗皂苷资源充分利用的角度考虑,可以尝试进行混合菌株转化。
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.
根据本发明,桔梗最适转化资源筛选:十二份不同地区的桔梗中水分、总灰分、醇浸出物及桔梗皂苷D均达到了药典规定的要求;粗蛋白含量在4.38%~20.13%之间;粗纤维含量在3.80%~12.70%之间;多糖含量在18.86%~51.00%%之间;总黄酮含量在0.22%~0.54%之间;总酚含量在0.64%~0.96%之间;各地区桔梗中dPD、PD及总皂苷的含量差异较大,其中安徽大别山桔梗总皂苷含量最高,值为11.88%,其次为山东淄博、吉林长白山,分别为11.25%、11.00%;浙江桔梗桔梗皂苷D含量最高,值为0.8212%,其次为吉林长白山桔梗,值为0.6280%;去芹糖桔梗皂苷D含量最高的为浙江桔梗,值为0.1196%,其次为广东和吉林桔梗,值分别为0.0793%、0.0777%;根据多糖、总皂苷及六种皂苷含量综合考虑,选择吉林长白山桔梗作为生物转化材料。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.
根据本发明,库德里阿兹威毕赤酵母在30℃条件下对桔梗皂苷的发酵优于37℃,dPD提高了16.88%、PD提高了8.86%;贝莱斯芽孢杆菌37℃条件下优于30℃,dPD提高了3.83%、PD提高了11.21%;两种菌株混合发酵37℃条件下优于30℃,dPD提高了22.71%、PD提高了8.55%,确定混合发酵模式可以作为桔梗皂苷转化的一种途径。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.
本发明采用如下实验材料进行试验得到,贝莱斯芽胞杆菌和库德里阿兹威 毕赤酵母菌株由本实验室筛选,桔梗片购自河北全泰药业有限公司。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.
本发明中使用的实验试剂购于天津科密欧化学试剂有限公司,包括,亚硝酸钠、铁氰化钾、三氯乙酸、三氯化铁、氢氧化钠、磷酸氢二钠、磷酸二氢钠、乙醇、硝酸铝、福林酚、碳酸钠,水杨酸,双氧水,硫酸亚铁等;Tris-HCl缓冲液、邻苯三酚、抗坏血酸购自北京索莱宝科技有限公司;芦丁标准品、DPPH购于上海源叶生物科技有限公司。没食子酸标准品购于上海麦克林生化科技有限公司。总抗氧化能力试剂盒购于南京建成生物工程研究所。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.
总抗氧化能力的测定Determination of total antioxidant capacity
按照表8配制试剂二(R2)和试剂三(R3)应用液,精密量取0.1mL待测样品溶液,按照表9步骤配置测定管和对照管,用浓度为0.5mg/mL,1mg/mL,2mg/mLVC溶液做阳性对照。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.
表8试剂组成及配制Table 8 Reagent composition and preparation
表9总抗氧化能力测定操作步骤表Table 9 Operation steps for determination of total antioxidant capacity
注:a为待测样品的量取体积,mL。Note: a is the volume of the sample to be tested, mL.
配置后混匀,静置10min。以蒸馏水为空白,在520nm下测定各管吸光度值。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.
总抗氧化能力(U/mL)的计算公式:The calculation formula of total antioxidant capacity (U/mL):
ODU-测定管的吸光度值;ODU-measure the absorbance value of the tube;
ODC-对照管的吸光度值;ODC- the absorbance value of the control tube;
V-测定时反应液的总体积(mL);V- the total volume of the reaction solution (mL) during measurement;
V0-样品取样量(mL);V0-sample sampling volume (mL);
M-样品的稀释倍数。M-the dilution factor of the sample.
DPPH·清除率的测定DPPH·Determination of clearance rate
精密称取5mg DPPH,用无水乙醇溶解,并定容在100mL容量瓶中,用三蒸水将样品溶液稀释2倍,4倍,6倍,8倍,配置成50mg/mL,25mg/mL,12.5mg/mL,8.33mg/mL,6.25mg/mL的不同浓度的样品溶液,分别取不同浓度的样品溶液2mL,并加入2mLDPPH溶液,用无水乙醇做对照,空白组为无水乙醇,蒸馏水各2mL,摇匀,在室温下避光静置30min,用Vc做阳性对照,进行3次平行试验,在517nm波长下测定吸光度。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.
计算公式:Calculation formula:
DPPH·清除率=[1-(Ai-Aj)/A0]×100%,DPPH·Clearance rate=[1-(Ai-Aj)/A0]×100%,
A0为未加清除剂的吸光值(加入无水乙醇)A0 is the absorbance value without scavenger (add absolute ethanol)
Ai为加入清除剂的吸光值Ai is the absorbance value of adding scavenger
Aj为样品溶液+无水乙醇的吸光度Aj is the absorbance of sample solution + absolute ethanol
在水杨酸体系中羟基自由基清除率的测定Determination of Scavenging Rate of Hydroxyl Radical in Salicylic Acid System
按照上述方法稀释样品溶液,分别取不同浓度的样品溶液0.5mL于试管中,按顺序加入,8mmol/L的硫酸亚铁溶液0.6mL,20mmol/L的双氧水0.5mL和3mmol/L的水杨酸(无水乙醇溶解)2mL,混匀后放在水浴锅中,37℃反应30分钟,流水冷却。用无水乙醇作对照组,去离子水代替水杨酸作空白组,用Vc作阳性对照做三次平行实验。在510nm波长下测定吸光度。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.
按下式计算清除率:Calculate the clearance rate as follows:
清除率(%)=[1-(Ai-Aj)/A0]×100%,Clearance rate (%)=[1-(Ai-Aj)/A0]×100%,
A0为未加清除剂的吸光值(加入无水乙醇)A0 is the absorbance value without scavenger (add absolute ethanol)
Ai为加入清除剂的吸光值Ai is the absorbance value of adding scavenger
Aj为空白组吸光值Aj is the absorbance value of the blank group
超氧阴离子清除率的测定Determination of superoxide anion scavenging rate
按上述方法配置不同浓度样品溶液,分别取不同浓度的样品溶液0.1mL,加入0.05mol/LTris-HCl缓冲液(pH=8.2)2.8mL,0.1mL 3mmol/L的邻苯三酚,用蒸馏水代替样品溶液作为对照组,空白组为2.8mL Tris-HCl缓冲液,0.1m L蒸馏水,0.1mL 0.01mol/L的HCl,用Vc作阳性对照,进行三次平行实验。开始反应30s后,在320nm波长下测定其吸光度,每隔30s记录一次吸光度值,反应5min。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.
计算公式:Calculation formula:
氧化速率=(最后一次记录值-第一次记录值)/5Oxidation rate = (last recorded value-first recorded value)/5
清除率(%)=(A0-A1)/A0×100Clearance rate (%) = (A0-A1)/A0×100
式中:A0为未加清除剂的吸光值(加入蒸馏水)In the formula: A0 is the absorbance value without scavenger (adding distilled water)
A1为加入清除剂的吸光值。A1 is the absorbance value of adding scavenger.
还原力的测定Determination of reducing power
按上述方法配置不同浓度的样品溶液,分别取不同浓度的样品液1mL于试管中,向其中加入pH=6.6的磷酸缓冲液和0.3%的铁氰化钾溶液各2.5mL,混匀后放在水浴锅中,55℃恒温温育20min,之后取出,迅速冷却,加入2.5mL10%的三氯乙酸溶液,3000rpm,离心10min,取上清液2.5mL,加入2mL蒸馏水,0.5mL质量分数0.1%的FeCl 3溶液,充分混合,静置10min后,在波长700nm下测定吸光度值,吸光度值越大,代表还原力越强。蒸馏水组作为空白组,Vc为阳性对照,进行3次平行试验。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.
计算公式:Calculation formula:
还原力=样品吸光度一空白对照组吸光度Reducing power = absorbance of sample-absorbance of blank control group
总抗氧化能力Total antioxidant capacity
表10发酵对桔梗提取液总抗氧化能力的影响Table 10 The effect of fermentation on the total antioxidant capacity of Platycodon grandiflorum extract
注:同行肩标字母相同表示差异不显著(P>0.05),字母不同表示差异显著(P<0.05)。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).
如上表10所示,50mg/mL的未发酵组,库德里阿兹威毕赤酵母发酵组总抗氧化能力和1mg/mL抗坏血酸相当,而50mg/mL贝莱斯芽胞杆菌发酵组总抗氧化能力强于1mg/mL的抗坏血酸,可见微生物发酵提高了桔梗提取液的总抗氧化能力。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·的清除能力DPPH·Removal ability
表11发酵对桔梗提取液DPPH清除能力的影响Table 11 The effect of fermentation on the DPPH scavenging ability of Platycodon grandiflorum extract
注:同列肩标字母相同表示差异不显著(P>0.05),字母不同表示差异显著(P<0.05)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)
如表11所示,发酵组与未发酵组对DPPH·的清除能力均随着浓度的不断增加而增大,具有浓度依赖性,当浓度为50mg/mL时,各液体清除能力达到最大,其中贝莱斯芽胞杆菌发酵组清除率最高,高达90.73%,库德里阿兹威毕赤酵母发酵组为85.16%,未发酵组为80.23%。在相同的浓度下,各液体对DPPH·清除能力的强弱依次为:贝莱斯芽胞杆菌发酵组>库德里阿兹威毕赤酵母发酵组>未发酵组,组间差异显著,可见发酵显著提高了桔梗提取液对DPPH·的清除能力。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·.
水杨酸羟基清除能力Salicylic acid hydroxyl scavenging ability
表12发酵对桔梗提取液羟基清除能力的影响Table 12 The effect of fermentation on the hydroxyl scavenging ability of Platycodon grandiflorum extract
注:同列肩标字母相同表示差异不显著(P>0.05),字母不同表示差异显著(P<0.05)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)
从上表12可见:发酵组与未发酵组均具有一定的清除羟自由基的能力,且随着浓度的增加,清除羟基的能力也随之增大。当浓度为50mg/mL时,既溶液浓度达到最大时,各液体羟基清除能力也达到最高,其中贝莱斯芽胞杆菌发酵组清除率最大,为70.28%,库德里阿兹威毕赤酵母发酵组为61.54%,未发酵组为57.30%。在相同浓度下,羟基清除能力的强弱依次为:贝莱斯芽胞杆菌发酵组>库德里阿兹威毕赤酵母发酵组>未发酵组。库德里阿兹威毕赤酵母发酵组与未发酵组相比,组间差异不显著,贝莱斯芽胞杆菌组与未发酵组及库德里阿兹威毕赤酵母组相比,组间差异显著。可见贝莱斯芽胞杆菌菌株发酵后显著提高了桔梗提取液清除羟自由基的能力。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.
超氧负离子清除能力Superoxide anion scavenging ability
表13发酵对桔梗提取液超氧负离子清除能力的影响Table 13 The effect of fermentation on the scavenging ability of Platycodon grandiflorum extract on superoxide anions
注:同列肩标字母相同表示差异不显著(P>0.05),字母不同表示差异显著(P<0.05)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)
从表13可见:发酵组与未发酵组其超氧负离子清除能力均随浓度的增加而增大。与浓度呈正相关,在相同浓度下各液体超氧负离子的清除能力强弱依次为:贝莱斯芽胞杆菌发酵组>库德里阿兹威毕赤酵母发酵组>未发酵组。在浓度为50mg/mL时,贝莱斯芽胞杆菌发酵组清除率最大,为63.28%,库德里阿兹威毕赤酵母发酵组为54.68,未发酵组为47.74%。发酵组与未发酵组相比, 组间差异显著,可见,微生物发酵后桔梗提取液的超氧负离子清除能力也会有显著提高。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.
还原力Reducing power
表14发酵对桔梗提取液还原力的影响Table 14 The influence of fermentation on the reducing power of Platycodon grandiflorum extract
注:同列肩标字母相同表示差异不显著(P>0.05),字母不同表示差异显著(P<0.05)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)
如表14所示:发酵组与未发酵组其还原力均随浓度的增加而增大,在相同浓度下,各液体还原力强弱依次为:贝莱斯芽胞杆菌发酵组>库德里阿兹威毕赤酵母发酵组>未发酵组。贝莱斯芽胞杆菌组与库德里阿兹威毕赤酵母发酵组相比,组间差异不显著,但与未发酵组相比,组间差异显著,可见,微生物发酵后桔梗提取液的还原力也会有显著提高。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.
通过本发明,贝莱斯芽胞杆菌和库德里阿兹威毕赤酵母菌株发酵桔梗提取液后,总黄酮、总酚含量均显著提高,王行等通过测定蓝莓酒发酵过程中酚类等物质,发现蓝莓酒在发酵过程中,总酚、总黄酮含量呈先增加后减少的趋势;但显著高于初始含量。陈彩云利用纳豆菌对槐米进行发酵,提高了芦丁和槲皮素含量。但本实验各液体中总黄酮、总酚不论是发酵或未发酵,含量均较低,查阅文献发现桔梗水提黄酮含量会少于醇提,黄酮更易溶于醇类试剂,故含量会较低。钱骅等也表明:抗氧化活性物质如酚类、黄酮类等多存在于醇提物中。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.
在利用微生物发酵提高体外抗氧化能力方面,杨学娟利用纳豆芽孢杆菌对豆粕进行固体发酵,证明了豆粕提取物发酵后不但在体外具有更高的清除DPPH自由基和超氧负离子的活性、还原力及抑制脂质过氧化的能力,而且在体内也具有更高的抗氧化能力。由此,我们可以推断发酵后的提取液,可能是因为提高或生成了多种具有抗氧化活性的物质的含量,从而使抗氧化能力得到 显著的提高。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.
本发明将贝莱斯芽胞杆菌和库德里阿兹威毕赤酵母接种到桔梗提取液中,进行微生物发酵,并与未发酵的提取液做对比,研究微生物发酵对桔梗提取液体外抗氧化能力的影响。结果如下: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.微生物发酵后,桔梗提取液中的总黄酮、总酚含量提高显著,50mg/mL桔梗提取液和库德里阿兹威毕赤酵母发酵组的总抗氧化能力相当于1mg/mL的抗坏血酸。而50mg/mL贝莱斯芽胞杆菌发酵液总抗氧化能力强于1mg/mL的抗坏血酸溶液。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.通过考察DPPH·,羟基自由基,超氧负离子的清除能力及还原力来研究其体外抗氧化能力,发现发酵组与未发酵组的清除自由基的能力与还原力均随溶液浓度的增大而增大,且呈正相关。其中2个发酵组均比未发酵组的抗氧化能力高,贝莱斯芽胞杆菌发酵后,更是显著提高了桔梗提取液的体外抗氧化能力。说明微生物发酵会显著提高桔梗提取液的体外抗氧化能力。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.
通过本发明,可以低成、保存条件不苛刻,在短时间内大量应用于生产。本领域的技术人员容易理解,以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。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)
- 一种库德里阿兹威毕赤酵母菌株,其特征在于,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏日期为2019年6月17日,保藏编号为CGMCC No.17939。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.
- 一种采用权利要求1所述的库德里阿兹威毕赤酵母菌株制备去芹糖桔梗皂苷D的方法,包括以下步骤:A method for preparing de-celerylated Platycodin D by using the Pichia Kudriazwei Pichia strain according to claim 1, comprising the following steps:培养步骤:所述阿兹威毕赤酵母的培养选用YPD液体培养基、酵母浸出粉胨葡萄糖琼脂培养基;Cultivation steps: the culture of the Pichia azweed yeast selects YPD liquid medium and yeast extract peptone glucose agar medium;种子培养步骤:菌落接种于20mL的液体培养基中,置于30℃,110r/min条件下震荡培养14h~18h,作为种子培养液;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;转化步骤:2%接种量加入0.1mg/mL皂苷标准品溶液中,在30℃下转化24h~72h;Transformation steps: add 2% inoculum to 0.1mg/mL saponin standard solution, transform at 30℃ for 24h~72h;所述菌落包括所述库德里阿兹威毕赤酵母菌株的菌落。The colony includes the colony of the Pichia kudriazwe strain.
- 根据权利要求2所述的方法,其特征在于,所述菌落包括所述库德里阿兹威毕赤酵母菌株的菌落。The method of claim 2, wherein the colony comprises a colony of the Pichia kudriazwe strain.
- 根据权利要求3所述的制备方法,其特征在于,所述菌株为库德里阿兹威毕赤酵母菌株和贝莱斯芽孢杆菌,所述库德里阿兹威毕赤酵母菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏日期为2019年6月17日,保藏编号为CGMCC No.17939;所述贝莱斯芽孢杆菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏日期为2019年6月17日,保藏编号为CGMCC No.17938。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.
- 根据权利要求3所述的制备方法,其特征在于,所述发酵的温度控制在30~37℃。The preparation method according to claim 3, wherein the temperature of the fermentation is controlled at 30-37°C.
- 根据权利要求4所述的制备方法,其特征在于,所述混合发酵的发酵底物包括桔梗水提液。The preparation method according to claim 4, wherein the fermentation substrate of the mixed fermentation comprises an aqueous extract of Platycodon grandiflorum.
- 根据权利要求6所述的制备方法,其特征在于,所述库德里阿兹威毕赤酵母菌株、贝莱斯芽胞杆菌两菌株种子培养液以2%的接种量接种到桔梗水提液中。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%.
- 权利要求2~7任意一项所述的制备方法制备得到的去芹糖桔梗皂苷D在制备抑制肿瘤药物、抑制类风湿关节炎药物、抗肥胖类药物、护肾类药物、护 肝类药物、调节免疫活性类药物、抗糖尿病类药物、镇痛类药物或酶抑制类药物中的应用。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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