CN116555096A - Paramycolatopsis Diels strain and application thereof - Google Patents

Paramycolatopsis Diels strain and application thereof Download PDF

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CN116555096A
CN116555096A CN202310441608.7A CN202310441608A CN116555096A CN 116555096 A CN116555096 A CN 116555096A CN 202310441608 A CN202310441608 A CN 202310441608A CN 116555096 A CN116555096 A CN 116555096A
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ginsenoside
strain
parabacteroides
dirachta
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刘洪涛
胡海明
齐子涵
张志刚
朱天翔
郑军平
杨化冰
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Wuhan Sishengyuan Biotechnology Co.,Ltd.
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Abstract

The application discloses a parabacteroides dirachta strain and application thereof. The strain is named Parabacteroides distasonis QZH 1201 and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2022813 in 2022, 6 and 7. The strain can rapidly convert ginsenoside Re into ginsenoside Rg2 and protopanaxatriol PPT. After the ginsenoside Re and the strain are cultured for 48 hours at 37 ℃, the ginsenoside Re and the strain are converted into ginsenoside Rg2 and protopanaxatriol PPT, the conversion efficiency is high, and a novel production way is provided for obtaining rare saponins.

Description

Paramycolatopsis Diels strain and application thereof
Technical Field
The application relates to the technical field of parabacteroides dirachta, in particular to a parabacteroides dirachta strain and application thereof.
Background
The parabacteroides dieldrin (Parabacteroides distasonis) belongs to the phylum bacteroides, is a gram-negative bacterium, has wide cholic acid conversion function, and can improve lipid metabolism disorder, repair intestinal wall integrity and activate intestinal gluconeogenesis, thereby regulating appetite, promoting liver glycogen synthesis and improving host glycometabolism disorder. The method mainly activates different signal paths by generating succinic acid and secondary cholic acid, plays a role in overall regulation of multiple targets, and is a potential novel anti-metabolic syndrome probiotic. The product development, processing and utilization of next generation probiotics represented by parabacteroides dieldrin will become a new research hotspot.
Disclosure of Invention
The inventor of the application has long-term practical exploration, and has separated a strain of Paralopecuroides dirachta from human intestinal tracts and preserved in China center for type culture collection. The embodiment of the application also discovers that the strain can utilize ginsenoside to realize conversion of ginsenoside Re into rare ginsenoside Rg2 and protopanaxatriol PPT, overcomes the defects of low content, high cost, environmental pollution and the like in the traditional acquisition mode of the rare ginsenoside, and has the advantages of simple conversion process, various action substrates, abundant conversion products, higher practical application value and the like. Therefore, the embodiment of the application at least discloses the following technical scheme:
(1) A Paralopecuroides dirachta strain is named Parabacteroides distasonis QZH 1201 and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2022813 and the preservation address of 2022, 6 and 7 days: chinese armed chinese.
(2) A microbial inoculum for fermenting ginsenoside comprises the bacterial powder of the Paramycolatopsis Diels strain of (1) and a strain protecting agent.
(3) An immobilized preparation for converting ginsenoside Re, said immobilized preparation comprisingThe immobilizing preparation contains not less than 10 5 cfu/g of the bacterial powder of the parabacteroides dirachta strain (1) and an immobilization auxiliary agent.
(4) A method of fermenting ginsenoside, comprising:
inoculating the parabacteroides dirachta strain in the step (1) into a culture medium containing ginsenoside for fermentation culture; or alternatively
And (3) adding the microbial inoculum of the step (1) into a culture medium containing ginsenoside for fermentation culture.
(5) A method for converting ginsenoside Re, which comprises the steps of contacting and reacting ginsenoside Re with the immobilized preparation of (3).
(6) The use of the parabacteroides dirachta strain as defined in (1) in ginsenoside fermentation and conversion.
Compared with the prior art, the application has at least one of the following beneficial effects:
the Parabacteroides distasonis QZH 1201 strain provided by the example can rapidly convert ginsenoside Re into ginsenoside Rg2 and protopanaxatriol PPT. After the ginsenoside Re is cultured for 48 hours at 37 ℃ with the strain, the ginsenoside Re is converted into ginsenoside Rg2 and protopanaxatriol PPT, the conversion efficiency is high, and a novel production way is provided for obtaining rare saponins.
Drawings
Fig. 1 is a colony morphology diagram of Parabacteroides distasonis QZH strain 1201 provided in the examples of the present application, and the right image is a 4-fold enlarged view of the left image.
Fig. 2 is a gram-stained microscopic morphology of Parabacteroides distasonis QZH 1201 strain provided in the examples of the present application, with a left magnification of 1000×, and a right magnification of 3000×.
Fig. 3 is a phylogenetic tree diagram of Parabacteroides distasonis QZH 1201 strain provided in the examples of the present application.
Fig. 4 is a thin-layer chromatogram of ginsenoside Re, rg1, rg2, rh1, F1, and protopanaxatriol provided in the examples of the present application.
Fig. 5 is a high performance liquid chromatogram of ginsenoside Re, rg1, rg2, rh1, F1, and protopanaxatriol standard provided in the examples of the present application.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be further described in detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application. Reagents not specifically and individually described in this application are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.
It should be noted that, the terms "first," "second," and the like in the description and the claims of the present invention and the above drawings are used for distinguishing similar objects, and are not necessarily used for describing a particular sequence or order, nor do they substantially limit the technical features that follow. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
For a better understanding of the present invention, and not to limit its scope, all numbers expressing quantities, percentages, and other values used in the present application are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. Each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Isolation, purification and identification of strains
10% (w/v) human fecal suspension was prepared: 3 healthy volunteers were recruited, required that the volunteers were free of digestive system disease, and did not receive antibiotic treatment for the last 3 months. Weighing fresh human feces, adding sterile PBS (phosphate buffer solution) vortex into an ultra-clean bench, centrifuging, and taking supernatant to obtain human feces bacterial suspension.
Isolation of strains: a dilution was performed with sterile PBS, 100. Mu.L of each of the dilutions was pipetted onto a GAM solid culture plate and incubated at 37℃for 24 hours in a constant temperature incubator.
Purification of the strain: and (3) picking single colonies with different color forms on the GAM solid culture medium in an ultra-clean workbench, respectively inoculating the single colonies into the GAM liquid culture medium for enrichment, and then carrying out flat streaking again, and gradually separating and purifying until the single bacterial strains are obtained after purification. Then, the single bacteria and ginsenoside Re are subjected to anaerobic co-culture for 7d at 37 ℃ to verify whether the strain has Re metabolic capability. The parabacteroides dirachta (Parabacteroides distasonis) in this example is initially numbered as F5.
Microscopic morphological identification of strain F5: the bacterial colony is observed to be like a circle, a milky white or an off-white shape, the middle process is thin around, the diameter is 1.4-2 mm, the surface is smooth and glossy, semitransparent and the edge is irregular after the bacterial colony is stained with a small amount of bacterial liquid and is subjected to three-area lineation on a GAM solid culture medium and cultured for 24 hours at 37 ℃.
Gram staining microscopic morphological identification: the bacteria after gram staining were morphologically observed with an optical microscope, and as can be seen from fig. 2, the bacteria were red, gram-negative bacteria, and rod-like or sphere-like.
Bacterial 16S rDNA sequencing identification: the DNA of the strain is extracted and sequenced by using an extraction kit, the sequencing result is submitted to an NCBI database for homology comparison, and a phylogenetic evolutionary tree is drawn, as shown in figure 3, from the phylogenetic tree, the highest similarity of the strain F5 and the bacteria of the genus Porphyromonadaceae, parabacteroides of the family rhodomonas can be primarily judged, so the strain is named as Paramethod jejunum dieldrin Parabacteroides distasonis QZH 1201 and is preserved in China Center for Type Culture Collection (CCTCC) at 6 months and 7 days 2022, and the preservation number is CCTCC NO: M2022813.
Application of strain
The research shows that the strain can ferment ginsenoside. For this reason, the embodiment of the application also provides a method for fermenting ginsenoside, which comprises the following steps: inoculating the parabacteroides dirachta Parabacteroides distasonis QZH 1201 provided in the embodiment into a culture medium containing ginsenoside for fermentation culture; or adding the microbial inoculum provided in the example into a culture medium containing ginsenoside for fermentation culture.
Based on the above, the embodiment of the application also provides a microbial inoculum for fermenting ginsenoside, which comprises the bacterial powder of the parabacteroides dirtiens Parabacteroides distasonis QZH 1201 provided by the embodiment and the bacterial strain protective agent. In some embodiments, the number of the Paralopecis jejuni Parabacteroides distasonis QZH 1201 in the microbial inoculum is not less than 10 5 cfu/g, preferably not less than 10 6 cfu/g, or not less than 10 7 cfu/g, or not less than 10 8 cfu/g。
In some embodiments, the strain protectant is independently selected from at least one of fructo-oligosaccharides (or FOS), short-chain fructo-oligosaccharides, inulin, isomalt-oligosaccharides, pectin, xylo-oligosaccharides (or XOS), chitosan-oligosaccharides (or COS), beta-glucans, modified gum arabic and resistant starches, polydextrose, D-tagatose, acacia fiber, carob, oat, and citrus fiber. In some embodiments, the strain protectant further comprises a vitamin selected from at least one of vitamin B6, magnesium, dimethylglycine (vitamin B16), and vitamin C.
In some examples, GAM liquid medium containing 0.5mg/mL ginsenoside Re was prepared, and Parabacteroides distasonis QZH 1201 colonies on a plate as shown in FIG. 2 were inoculated at an inoculum size of 2%, placed in a constant temperature shaker at 200rpm/min for 48h, and sampled every 12 h; drying n-butanol extract at each time point with air pump, re-dissolving with liquid phase methanol, filtering with 0.22 μm microporous membrane (organic phase), and performing thin layer chromatography and HPLC analysis.
Wherein, the thin layer chromatography uses chloroform, ethyl acetate, methanol, distilled water=15:40:22:10 mixed solution as developing agent.
Wherein, the HPLC analysis conditions include: YMC-PackODS-AM (250 mm. Times.4.6 mm,5 μm) column; the mobile phase A is acetonitrile, and the mobile phase B is water; gradient elution was 0min,20% a;8min,30% A;12min,40% A;15min,65% A;20min,100% A;25min,100% A;30min,20% A. The elution flow rate is 0.6mL/min, and the sample injection amount is 20 mu L; the detection wavelength is 203nm; the column temperature was 30 ℃. Establishment of a standard curve: the ginsenoside Re, the ginsenoside Rg2, the ginsenoside Rh1, the protopanaxatriol reference substances 0.0017g, 0.0028g, 0.0021g, 0.0030g, 0.0025g and 0.0027g are respectively and accurately weighed, placed in a 2mL measuring flask, and the mixed reference substance stock solution is obtained by metering the volume to the scale with methanol. The mixed reference substance stock solutions are respectively sucked and diluted by 1.33, 2, 4, 8, 16 and 32 times in sequence, 7 concentration gradient mixed reference substance solutions are obtained, and 20 mu L of mixed reference substance solutions are respectively injected. And (3) carrying out linear regression on the mass concentration (X) by using the peak area (Y), substituting the peak area of the sample to be detected into a regression equation, and calculating to obtain the content of the active ingredient in the sample to be detected.
Fig. 4 shows thin layer chromatograms of ginsenoside Re, rg1, rg2, rh1, F1 and protopanaxatriol (PPT) (columns 1 and 2), and 0h, 12h, 24h, 36h and 48h broth samples. As can be seen from fig. 4, the ginsenoside Re content in the fermentation broth gradually decreased and the ginsenoside Rg2 content gradually increased as the fermentation time progressed.
In order to further accurately analyze the ginsenoside content in each fermentation broth sample, fig. 5 shows HPLC chromatograms of ginsenoside Re, rg1, rg2, rh1, F1 and protopanaxatriol (PPT) standards, and it can be seen that the chromatographic conditions can well achieve separation. And calculating the ginsenoside content in each fermentation broth according to a standard curve fitted by the HPLC detection spectrum, and calculating the conversion rate of ginsenoside Re to Rg1, rg2, rh1, F1 and PPT respectively, wherein the conversion rate is the mole amount of the generated product divided by the mole amount of added ginsenoside Re. As a result, when fermentation was carried out using GAM liquid medium of 0.5mg/mL ginsenoside Re, after 48 hours of culture at Parabacteroides distasonis 37 ℃the conversion rates of ginsenoside Rg2 and protopanaxatriol PPT were 88.9% and 16.4%, respectively. Thus, parabacteroides distasonis QZH 1201 provided by the embodiment of the application can effectively convert ginsenoside Re into ginsenoside Rg2 and protopanaxatriol PPT.
Immobilization and application of bacterial strain
For this reason, the embodiment of the application also provides an immobilization preparation and an immobilization method of the parabacteroides dirfar strain Parabacteroides distasonis QZH 1201. And also provides the application of the immobilized preparation in the conversion of ginsenoside Re.
Some embodiments provide an immobilized preparation of converted ginsenoside Re, comprising not less than 10 5 cfu/g of Paralopecuroides dirachta strain Parabacteroides distasonis QZH 1201 and immobilization aid. Preferably, the number of the Paecilomyces dieldahl strain Parabacteroides distasonis QZH 1201 is not less than 10 6 cfu/g, or not less than 10 7 cfu/g, or not less than 10 8 cfu/g。
In some embodiments, the immobilization aid is selected from at least one of lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol, gelatin, glucose, anhydrous lactose, oxidized dextran containing polyaldehyde groups, corn sweetener, gum acacia, tragacanth or sodium alginate, carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, sodium benzoate, esters of sorbic acid and p-hydroxybenzoic acid, and sericin.
In some embodiments, the enriched suspension of the cultured Paramycolatopsis Diels strain Parabacteroides distasonis QZH 1201 is concentrated by centrifugation to give a bacterial count of 1X 10 8 CFU/mL of the bacterial concentrate; dissolving sericin by using ultrapure water to obtain a sericin aqueous solution with the mass percentage concentration of 2%, and uniformly mixing the sericin aqueous solution with the bacteria concentrated solution to obtain liquid A; dripping the obtained liquid A into carboxymethyl cellulose and oxidized dextran containing polyaldehyde groups, fully and uniformly mixing, and crosslinking for 8 hours at 36 ℃ to obtain hydrogel containing lactobacillus; dissolving cellulose powder in water to obtain 1% swelling cellulose solution, adding into hydrogel, mixing, and stirring at 36 deg.CLinking for 5h; and (3) slowly dripping the obtained crosslinked solution into a calcium chloride solution with the mass percentage concentration of 3% by a constant flow pump, reacting for 3 hours at the temperature of 34 ℃, filtering, washing with deionized water, and thus obtaining immobilized lactobacillus, and storing in a refrigerator at the temperature of 4 ℃. Wherein the volume ratio of the lactobacillus suspension to the sericin to the carboxymethyl cellulose to the oxidized dextran containing the polyaldehyde group is 2:4:3:4.
In some embodiments, the immobilized paramecium dieldae strain Parabacteroides distasonis QZH 1201 is inoculated with 2% inoculum size to GAM liquid medium containing 0.5mg/mL ginsenoside Re, cultured for 48h, and after centrifugation and sampling, the same HPLC method is used to detect the conversion of ginsenoside Rg2 and protopanaxatriol to ginsenoside Rg2 and protopanaxatriol PPT at 86.6% and 28.7%, respectively.
The embodiment of the application applies the parabacteroides dieldrin to the field of ginsenoside Re conversion for the first time, and two rare ginsenosides (ginsenoside Rg2 and protopanaxatriol PPT) can be obtained from the aspect of metabolic products, so that the parabacteroides dieldrin has a larger application value in the aspect of converting the ginsenosides Re into the rare ginsenosides. The microbial transformation has the advantages of low cost, easy operation and the like, is suitable for large-scale industrial production, and has wide application prospect in the aspect of utilizing the parabacteroides dirachta to transform the ginsenoside Re.
The foregoing is merely a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions easily contemplated by those skilled in the art within the technical scope of the present application should be covered by the scope of the present application.

Claims (9)

1. A Paralopecuroides dirachta strain, named Parabacteroides distasonis QZH 1201, is preserved in China Center for Type Culture Collection (CCTCC) No. M2022813 at the 6 th month and 7 th year of 2022.
2. A bacterial agent for fermenting ginsenoside, comprising the parabacteroides dirachta according to claim 1 and a strain protectant.
3. The microbial inoculum according to claim 2, wherein the number of Paralopecuroides dirachta according to claim 1 in the microbial inoculum is not less than 10 5 cfu/g。
4. An immobilized preparation for converting ginsenoside Re, characterized in that the immobilized preparation comprises not less than 10 5 cfu/g of the parabacteroides dieldrin strain as defined in claim 1, and an immobilization aid.
5. A method of fermenting ginsenoside, comprising:
inoculating the paramamopsis di-bacillus strain of claim 1 into a culture medium containing ginsenoside for fermentation culture; or alternatively
The microbial inoculum according to claim 2 or 3 is added into a culture medium containing ginsenoside for fermentation culture.
6. The method of claim 4, wherein the medium is PDB or GAM broth.
7. The method according to claim 4, wherein the content of ginsenoside in the culture medium is not less than 0.5 μg/mL, preferably 0.5 μg/mL to 20.5mg/mL.
8. A method for converting ginsenoside Re, wherein ginsenoside Re is contacted and reacted with the immobilized preparation of claim 4.
9. The use of the parabacteroides dirachta strain of claim 1 in ginsenoside fermentation and conversion.
CN202310441608.7A 2023-04-23 2023-04-23 Paramycolatopsis Diels strain and application thereof Pending CN116555096A (en)

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