Bacillus capable of promoting magnolia officinalis to generate magnolol and honokiol and application thereof

Abstract

The invention belongs to the field of microorganisms, and discloses bacillus velezensis capable of promoting mangnolia officinalis to generate magnolol and honokiol and application of the bacillus velezensis. The strain is separated from dried magnolia officinalis after spontaneous perspiration, and the preservation number of the strain is GDMCC NO: 65701. Tests prove that the strain can promote the generation of magnolol and honokiol in the mangnolia officinalis sweating process, and can be used for preparing traditional Chinese medicinal materials or traditional Chinese medicinal ingredients.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms and relates to Bacillus velez which can promote magnolia bark to produce magnolol and honokiol and application thereof. Background Art

[0002] Magnolia officinalis Rehd et Wils belongs to the Magnoliaceae family and the genus Magnolia. It is a traditional Chinese medicinal material with the effects of promoting gastrointestinal function, drying dampness and eliminating phlegm, dispelling wind and relieving pain, and promoting blood circulation and removing blood stasis. Magnolia officinalis is rich in a variety of chemical components, among which the content of lignan compounds is particularly prominent, mainly represented by magnolol and honokiol. Magnolia officinalis (5',5-diallyl-2,2'-biphenyldiphenol) is a bisphenol compound, which is an isomer of honokiol (3',5-di-2-propenyl-1,1'-biphenyl-2,4'-diphenol). The different positions of the phenolic hydroxyl groups of the two lead to slight differences in their physical properties. Due to their similar molecular structures, the two compounds show a high degree of consistency in chemical properties and therefore have similar pharmacodynamic characteristics. Modern pharmacological studies have shown that magnolol and honokiol exhibit a variety of pharmacological activities such as intestinal protection, anticancer and antitumor effects, antibacterial effects, and neuroprotective effects.

[0003] In my country, Chinese herbal medicines must be processed through specific processing techniques before they become commercial medicinal materials. In traditional processing techniques, "sweating" is a unique method of treating dry materials and is an important factor affecting the quality of some medicines. The so-called "sweating" is a process in which the materials are piled up, the heat increases, and the moisture inside the medicinal materials diffuses outward. When the fresh medicinal materials are dehydrated to a certain extent, the "sweating" of Magnolia officinalis ends. Modern research shows that "sweating" has an important influence on the properties, chemical composition and efficacy of medicinal materials. Compared with Magnolia officinalis that has not been "sweated", the content of magnolol and honokiol in "sweating" Magnolia officinalis is significantly increased. During the "sweating" process, the temperature and humidity of the medicinal materials are constantly changing, which will inevitably lead to changes in the microbial community, and changes in their primary metabolism, secondary metabolism and enzyme activity. These changes will directly affect the quality of Magnolia officinalis. Therefore, it is speculated that the change in the microbial community is the cause of the change in the content of active substances in Magnolia officinalis, but the specific strains have not yet been determined. Summary of the invention

[0004] The present invention isolates a Bacillus HP3 from dry Magnolia officinalis. Microorganisms on the surface of Magnolia officinalis are separated by TSA culture medium, and HP3 is screened by culturing at 37°C for 24 hours. The pure culture of the strain is identified at the morphological and molecular biological levels to be Bacillus Velez. The strain can significantly promote the production of magnolol and honokiol in the "sweating" process of Magnolia officinalis.

[0005] The invention provides a Velez bacillus capable of promoting magnolia bark to produce magnolol and honokiol, and the preservation number of the bacillus is GDMCC NO:65701.

[0006] The present invention thus provides a method for culturing the Bacillus Velezii, which is cultured in TSB medium at a temperature of 25-35° C. and with shaking at 150-210 rmin.

[0007] Preferably, the culture temperature is 30°C and the shaking culture is 160-200 rmin.

[0008] The invention provides the use of the Bacillus Velezii in producing magnolol and / or honokiol by taking magnolia bark as a raw material.

[0009] The present invention also provides a method for producing magnolol and / or honokiol by using magnolia bark as raw material, which comprises inoculating the bacterial liquid of Bacillus velezensis into the magnolia bark raw material to produce magnolol and / or honokiol.

[0010] The raw material of Magnolia officinalis is prepared as follows: the fresh raw material of Magnolia officinalis is cut and placed in deionized water for steaming until the bark becomes soft and the inner epidermis becomes brown-yellow, taken out and soaked in alcohol, and then soaked in deionized water, and then the bacterial liquid is inoculated on the inner surface of the Magnolia officinalis sample, spread evenly, covered with plastic film for accumulation, and sweating is induced to produce magnolol and / or honokiol.

[0011] Preferably, the sweating is sweating at 35-39°C for 2-5 days.

[0012] Specifically, the specific operation of preparing the raw material of Magnolia officinalis is as follows: cut the fresh raw material of Magnolia officinalis into strips of about 10 cm × 3 cm in length and width, weigh it, and steam the sample in deionized water for 5 min before "sweating" (steam until the bark becomes soft and the inner skin becomes brown-yellow), take it out and soak it in 75% alcohol for 5 s, and then soak it in deionized water for 5 s, and repeat this process three times. 8 CFU / mLHP3 bacterial solution was inoculated on the inner surface of the pre-treated Magnolia officinalis sample, spread evenly, covered with plastic film, and sweated at 37°C for 3 days.

[0013] The method further includes taking out the magnolia bark sample after sweating and drying it, specifically putting it into an oven for drying, and the drying temperature is 55-65°C, for example 60°C.

[0014] The strain HP3 of the present invention was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC for short) on December 31, 2024. The address of the depository unit is: 5th Floor, Dayuan Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province; the deposit number is GDMCC NO.65701, and the classification name is: Bacillus Velezii (Bacillus velezensis ). BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the colony morphology of strain HP3 cultured on plates.

[0016] Figure 2 A phylogenetic tree was constructed by selecting closely related model species based on sequence similarity.

[0017] Figure 3 The contents of magnolia bark and honokiol produced by sweating fresh magnolia bark after inoculation with strain HP3 are shown. DETAILED DESCRIPTION

[0018] The present invention is described below through specific implementation modes in order to better understand the present invention, but it does not constitute a limitation of the present invention.

[0019] Example 1. Isolation and identification of HP3 strain 1. Isolation and purification of strains The fresh Magnolia bark samples were collected in Sichuan Province in June 2023. After the samples were collected, they were placed in a -80℃ refrigerator for standby use; the dry Magnolia bark samples after sweating were from a traditional Chinese medicine factory in Sichuan. Weigh 50.0g of dry Magnolia bark sample and add it to a conical flask, then add 500mL of liquid culture medium and shake thoroughly to make a dry Magnolia bark suspension. The components of the liquid culture medium involved are (g / L): sodium butyrate 6.2, sodium formate 1.4, yeast powder 1.0, peptone 1.0, mineral element solution 50ml, trace element solution 1ml, B vitamin solution 5ml, sodium bicarbonate solution 70ml. Adjust the pH to 7.4±0.2 before sterilization, and sterilize at 121℃ for 15min. After sterilization, add vitamin solution and sodium bicarbonate solution in proportion. Among them, Mineral element solution mother liquor (g / L): 10.0 potassium dihydrogen phosphate, 6.6 magnesium chloride hexahydrate, 8.0 sodium chloride, 8.0 ammonium chloride, 1.0 calcium chloride.

[0020] Trace element solution mother liquor (g / L): 0.1 zinc sulfate heptahydrate, 0.03 manganese chloride tetrahydrate, 0.3 boric acid, 0.2 cobalt chloride hexahydrate, 0.01 calcium chloride dihydrate, 0.02 nickel chloride hexahydrate, 0.03 sodium molybdate dihydrate, 1.5 ferrous chloride tetrahydrate.

[0021] B vitamin solution stock solution (g / L): niacin 0.02, cobalamin 0.02, thiamine hydrochloride 0.01, p-aminobenzoic acid 0.01, pyridoxine hydrochloride 0.05, calcium pantothenate 0.005, sterilized by 0.22um filter membrane.

[0022] 5% (w / v) sodium bicarbonate solution: 5.0 g sodium bicarbonate, add distilled water to make up to 100 mL, and filter through a 0.22 um filter membrane for sterilization.

[0023] Take 1 mL of the dried magnolia bark suspension for gradient dilution and coating. According to the 10-fold gradient dilution method, add sterile 0.85% (w / v) physiological saline and mix thoroughly by pipetting. Dilute to 10 in a sterile centrifuge tube. -4 , 200 μL was applied to the surface of soybean casein agar (TSA) medium. Then, the medium was cultured at 37°C. After 24 hours of culture, the growth of the colonies on the plate was analyzed morphologically, and single colonies of different morphologies on the plate (about 30 colonies in total) were picked for separation and purification. The three-zone line was purified three times to obtain the purified culture HP3 strain. Figure 1 As shown, the isolated strain colonies are white, round, slightly convex, with relatively neat colony edges and slightly diffused in some places.

[0024] 2. Identification and phylogenetic analysis of strains The purified HP3 strain single colony was inoculated into TSB liquid medium and cultured overnight on a shaking table. The bacterial broth was cultured using the Tiangen Bacterial Genome Kit according to the instructions for bacterial genome extraction. The whole genome DNA was used as the PCR template, 2×Taq PCR Mix product, 25μL PCR reaction system, and universal primers 27F (SEQ ID NO.2: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (SEQ ID NO.3: 5'-TACGGCTACCTTGTTACGACTT-3') were used to amplify the 16S rRNA gene sequence. The PCR amplification program was 94℃ pre-denaturation for 3min, 30 cycles including 94℃ denaturation for 30s, 55℃ annealing for 30s, and 72℃ extension for 1min. Finally, 72 degrees extension for 10min. The PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0025] Then, the HP3 16S rRNA gene sequence (SEQ ID NO.1) obtained by sequencing was used for NCBI Blast search (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The comparison results showed that HP3 and Bacillus velezinoffii ( Bacillus velezensis ) had the highest similarity of 99.71%. In order to further clarify the taxonomic status of HP3, strains with close genetic relationship with HP3 16S rRNA sequence were selected to construct phylogenetic tree and perform phylogenetic analysis. Figure 2 As shown, HP3 is most closely related to Bacillus velezinsis ( Figure 2 ).

[0026]

[0027] Combining morphological and molecular biological characteristics, HP3 was preliminarily identified as Bacillus velez. The strain was submitted to the Guangdong Microbiological Culture Collection Center for preservation on December 31, 2024, with the collection number: GDMCC NO:65701.

[0028] Example 2. Analysis of the ability of HP3 strain to promote the production of magnolol and honokiol by Magnolia officinalis A single HP3 colony was selected and placed in TSB medium and cultured in a shaking incubator at 30°C and 180 rmin to prepare 1×10 8 CFU / mL bacterial solution, put it in a 4℃ refrigerator for later use. Cut the fresh Magnolia officinalis raw material into strips of about 10 cm × 3 cm in length and width, weigh it, and steam the sample in deionized water for 5 min before "sweating" (steam until the bark becomes soft and the inner skin turns brown), take it out and soak it in 75% alcohol for 5 s, then soak it in deionized water for 5 s, and repeat this process three times. 8 CFU / mLHP3 bacterial solution was inoculated on the inner surface of the pre-treated magnolia bark sample, spread evenly, covered with plastic film, and sweated at 37°C for 3 days. The magnolia bark sample that had been "sweated" was taken out and placed in an oven for drying at 60°C.

[0029] Preparation of sample solution: Grind the sample through a No. 3 sieve with 50 mesh, accurately weigh 0.2 g, place it in a stoppered conical bottle, accurately add 25 mL of methanol, shake well, seal, soak, ultrasonic-assisted extraction for 30 min, filter, accurately measure 2 mL of the filtrate, place it in a 10 mL volumetric flask, add methanol to the scale, mix and shake well, use a 0.22 μm filter membrane to treat the solution to obtain the sample solution. Preparation of standard solution: Take magnolol reference substance and honokiol reference substance, accurately weigh them, add methanol to make solutions containing 42 μg of magnolol and 24 μg of honokiol per 1 mL respectively.

[0030] HPLC determination: Accurately measure 2, 5, 10, 15, 20, and 25 µL of the reference solution and inject them respectively, and determine the peak areas of magnolia and honokiol respectively. The injection volume (µL) is used as the horizontal axis, and the peak area (mV) is used as the vertical axis to draw the standard curve. The injection volume of the sample is 10 µL, and the chromatographic conditions are as follows: the chromatographic column is a Thermo C18 column (250 mm×4.6 mm, 5 μm); methanol-water (78:22) is used as the mobile phase; the flow rate is 1 mL / min, the detection wavelength is 294 nm, and the column temperature is 35°C. Quantify magnolia and honokiol according to the standard curve. Figure 3After inoculation with HP3, the fresh Magnolia officinalis was sweated, and the content of honokiol increased by 137.41% and the content of magnolol increased by 81.74%. Therefore, HP3 can effectively increase the content of magnolol and honokiol in the sweating process of Magnolia officinalis.

Claims

1. A strain of Bacillus velezine that can promote Magnolia officinalis to produce magnolol and honokiol, characterized in that: Its deposit number is GDMCC NO:65701.

2. The method for culturing Bacillus Velezii according to claim 1, wherein The cells were cultured in TSB medium at 25-35°C and with shaking at 150-210 rmin.

3. The culture method according to claim 2, characterized in that The culture temperature was 30℃ and the shaking speed was 160-200 rmin.

4. The application of Bacillus Velezii as claimed in claim 1 in producing magnolol and / or honokiol with magnolia bark as raw material.

5. A method for producing magnolol and / or honokiol using magnolia bark as raw material, characterized in that: The bacterial liquid of the Bacillus according to claim 1 is inoculated into a raw material of Magnolia bark to produce magnolol and / or honokiol.

6. The method according to claim 5, characterized in that The raw material of Magnolia officinalis is prepared as follows: the fresh raw material of Magnolia officinalis is cut and placed in deionized water for steaming until the bark becomes soft and the inner epidermis becomes brown-yellow, taken out and soaked in alcohol, and then soaked in deionized water, and then inoculated with the bacterial solution on the inner surface of the Magnolia officinalis sample, spread evenly, covered with plastic film for accumulation, and sweating to produce magnolol and / or honokiol.

7. The method according to claim 6, characterized in that The sweating is sweating at 35-39°C for 2-5 days.

8. The method according to claim 6, characterized in that The specific operation of preparing Magnolia officinalis raw material is as follows: cut the fresh Magnolia officinalis raw material into strips of about 10 cm × 3 cm in length and width, weigh it, and steam the sample in deionized water for 5 min before "sweating" (steam until the bark becomes soft and the inner skin turns brown), take it out and soak it in 75% alcohol for 5 s, then soak it in deionized water for 5 s, and repeat this process three times. 8 CFU / mLHP3 bacterial solution was inoculated on the inner surface of the pre-treated Magnolia officinalis sample, spread evenly, covered with plastic film, and sweated at 37°C for 3 days.

9. The method according to claim 6, characterized in that The method further comprises taking out and drying the Magnolia bark sample after the sweating is completed.

10. The method according to claim 9, characterized in that Specifically, the mixture is placed in an oven for drying at a temperature of 55-65°C, for example, 60°C.

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