A Bacillus bacterium capable of promoting the production of magnolol and honokiol in Magnolia officinalis and its application

Abstract

The present invention belongs to the field of microorganisms, and discloses Bacillus velezensis that can promote the production of magnolol and honokiol in Magnolia officinalis Rehd. et Wils., and its application. This strain was isolated from dried Magnolia officinalis Rehd. et Wils. after sweating, and its preservation number is GDMCC NO: 65701. Experiments have proved that this strain can promote the production of magnolol and honokiol during the sweating process of Magnolia officinalis Rehd. et Wils., and can be used for preparing Chinese medicinal materials or preparing Chinese medicine components.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms and relates to Bacillus velezensis that can promote the production of magnolol and honokiol in Magnolia officinalis and its application. Background Art

[0002] Magnolia officinalis Rehd et Wils belongs to the Magnoliaceae family and Magnolia genus, and is a traditional Chinese medicinal material in China, with the effects of promoting digestion, drying dampness and resolving phlegm, expelling wind and relieving pain, activating blood circulation and removing stasis, etc. Magnolia officinalis is rich in various chemical components. Among them, the content of lignan compounds is particularly prominent, mainly represented by magnolol and honokiol. Magnolol (5',5-diallyl-2,2'-biphenyldiol) is a bisphenol compound and is an isomer of honokiol (3',5-di-2-propenyl-1,1'-biphenyl-2,4'-diol). The different positions of their phenolic hydroxyl groups result in slight differences in their physical properties. Due to their similar molecular structures, these two compounds show high consistency in chemical properties and thus have similar pharmacodynamic characteristics. Modern pharmacological studies have shown that magnolol and honokiol exhibit various pharmacological activities such as intestinal protection, anti-cancer and anti-tumor effects, antibacterial effects, and neuroprotective effects.

[0003] In China, Chinese herbal medicines need to be processed by specific processing techniques before becoming commercial medicinal materials. In traditional processing techniques, "sweating" is a unique method for treating dried materials and is an important factor affecting the quality of some medicines. The so-called "sweating" is a process in which materials are piled up, the heat increases, and the internal moisture of the medicinal materials diffuses outward. When the fresh medicinal materials are dehydrated to a certain extent, the "sweating" of Magnolia officinalis ends. Modern research has shown that "sweating" has an important impact on the properties, chemical components and efficacy of medicinal materials. Compared with non-"sweated" Magnolia officinalis, the contents of magnolol and honokiol in "sweated" Magnolia officinalis are significantly increased. During the "sweating" process, the temperature and humidity of the medicinal materials change continuously, which will inevitably lead to changes in the microbial community and the changes in its primary metabolism, secondary metabolism and enzyme activities. These changes will directly affect the quality of Magnolia officinalis. Therefore, it is speculated that the change in the microbial community is the reason for the change in the content of active substances in Magnolia officinalis, but the specific acting strains have not been clarified. Summary of the Invention

[0004] A Bacillus sp. HP3 was isolated from dried Magnolia officinalis. The surface microorganisms of Magnolia officinalis were isolated by TSA medium, and HP3 was screened by culturing at 37°C for 24 h. Identification of the pure culture of this strain at the morphological and molecular biological levels showed that the strain was Bacillus velezensis. This strain can significantly promote the production of magnolol and honokiol during the "sweating" process of Magnolia officinalis.

[0005] The present invention provides a Bacillus velezensis strain that can promote the production of magnolol and honokiol in Magnolia officinalis, and its preservation number is GDMCC NO: 65701.

[0006] The present invention thus provides a culture method of the said Bacillus velezensis, which is cultured in TSB medium, the culture temperature is 25 - 35 °C, and it is cultured with shaking at 150 - 210 r / min.

[0007] Preferably, the culture temperature is 30 °C, and it is cultured with shaking at 160 - 200 r / min.

[0008] The present invention provides the application of the said Bacillus velezensis in the production of magnolol and / or honokiol using Magnolia officinalis as a raw material.

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

[0010] Among them, the Magnolia officinalis raw material is prepared as follows: The fresh Magnolia officinalis raw material is cut and placed in deionized water and steamed until the bark becomes soft and the inner epidermis turns brownish-yellow. After taking it out and soaking it in alcohol, then soaking it in deionized water, and then inoculating the said bacterial liquid on the inner surface of the Magnolia officinalis sample, smearing it evenly, covering it with a plastic film and piling it up to sweat to produce magnolol and / or honokiol.

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

[0012] Specifically, the specific operation of preparing the Magnolia officinalis raw material is: Cut the fresh Magnolia officinalis raw material into strips with a length and width of about 10 cm × 3 cm, weigh it, and before "sweating", place the sample in deionized water and steam for 5 min (steam until the bark becomes soft and the inner epidermis turns brownish-yellow), take it out and soak it in 75% alcohol for 5 s, and then soak it in deionized water for 5 s. This process is repeated three times. Inoculate 1×10 8 CFU / mL HP3 bacterial liquid on the inner surface of the pretreated Magnolia officinalis sample, smear it evenly, cover it with a plastic film and pile it up, and sweat at 37 °C for 3 days.

[0013] It further includes taking out the Magnolia officinalis sample after sweating is completed and drying it. Specifically, it is put into an oven for drying treatment, and the drying temperature is 55 - 65 °C, for example, 60 °C.

[0014] The strain HP3 of the present invention was preserved in the Guangdong Provincial Culture Collection of Microorganisms (abbreviated as GDMCC) on December 31, 2024. The address of the preservation unit: 5th Floor, Experimental Building, No. 100 Yard, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province; The preservation number is GDMCC NO.65701, and the taxonomic name: Bacillus velezensis (Bacillus velezensis ). Description of the Drawings

[0015] Figure 1 It is the colony morphology of strain HP3 in plate culture.

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

[0017] Figure 3 It shows the contents of magnolol and honokiol produced by sweating fresh Magnolia officinalis after inoculating strain HP3. Detailed Implementation Modes

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

[0019] Example 1. Isolation and Identification of Strain HP3

[0020] 1. Isolation and Purification of the Strain

[0021] The fresh Magnolia officinalis sample was collected in June 2023 in Sichuan Province. After collection, the sample was placed in a -80°C refrigerator for later use; the dried Magnolia officinalis sample after sweating was from a traditional Chinese medicine factory in Sichuan. Weigh 50.0 g of the dried Magnolia officinalis sample and add it to a conical flask, then add 500 mL of liquid medium and shake well to make a dried Magnolia officinalis suspension. The composition of the liquid medium involved (g / L) is: sodium butyrate 6.2, sodium formate 1.4, yeast powder 1.0, peptone 1.0, mineral element solution 50 ml, trace element solution 1 ml, B vitamin solution 5 ml, sodium bicarbonate solution 70 ml. Adjust the pH to 7.4 ± 0.2 before sterilization and sterilize at 121°C for 15 min. After sterilization, add the vitamin solution and sodium bicarbonate solution in proportion. Among them,

[0022] Mineral element solution stock solution (g / L): potassium dihydrogen phosphate 10.0, magnesium chloride hexahydrate 6.6, sodium chloride 8.0, ammonium chloride 8.0, calcium chloride 1.0.

[0023] Trace element solution stock solution (g / L): zinc sulfate heptahydrate 0.1, manganese chloride tetrahydrate 0.03, boric acid 0.3, cobalt chloride hexahydrate 0.2, calcium chloride dihydrate 0.01, nickel chloride hexahydrate 0.02, sodium molybdate dihydrate 0.03, ferrous chloride tetrahydrate 1.5.

[0024] B vitamin solution stock solution (g / L): nicotinic acid 0.02, cobalamin 0.02, thiamine hydrochloride 0.01, p-aminobenzoic acid 0.01, pyridoxol hydrochloride 0.05, calcium pantothenate 0.005, filtered and sterilized with a 0.22 μm filter membrane.

[0025] 5% (w / v) sodium bicarbonate solution: 5.0 g of sodium bicarbonate, add distilled water to make up to 100 mL, and filter and sterilize with a 0.22-μm filter membrane.

[0026] Take 1 mL of the dried Magnolia officinalis suspension for gradient dilution and coating. According to the 10-fold gradient dilution method, add sterile 0.85% (w / v) normal saline and mix well by pipetting. Dilute it to 10 -4 , and pipette 200 μL to coat on the surface of tryptic soy agar (TSA) medium. Then, incubate the medium at 37°C. After 24 h of incubation, perform morphological analysis on the growth of colonies on the plate, pick different morphological single colonies on the plate (about 30 colonies in total) for isolation and purification, and purify them 3 times by three-zone streaking to obtain the purified HP3 strain. As Figure 1 shown, the colonies of the isolated strain are white, round, slightly convex, with relatively regular colony edges and slightly spreading in some places.

[0027] 2. Identification and phylogenetic analysis of the strain

[0028] Inoculate a single colony of the purified HP3 strain into TSB liquid medium and incubate it overnight on a shaker. The cultured bacterial solution is used to extract bacterial genomes using the Tiangen Bacterial Genome Kit according to the instructions. Using the whole-genome DNA as the PCR template, a 25-μL PCR reaction system is used with the 2×Taq PCR Mix product, and the universal primers 27F (SEQ ID NO.2: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (SEQ ID NO.3: 5'-TACGGCTACCTTGTTACGACTT-3') are used to amplify the 16S rRNA gene sequence. The PCR amplification program is pre-denaturation at 94°C for 3 min, 30 cycles including denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min. Finally, extend at 72°C for 10 min. The PCR product is sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0029] Then, use the HP3 16S rRNA gene sequence (SEQ ID NO.1) obtained by sequencing for NCBI Blast search (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The alignment results show that HP3 has the highest similarity with Bacillus velezensis ( Bacillus velezensis ), which is 99.71%. To further clarify the taxonomic status of HP3, select strains with a close phylogenetic relationship to the HP3 16S rRNA sequence to construct a phylogenetic tree for phylogenetic analysis. As Figure 2As shown, HP3 has the closest genetic relationship with Bacillus velezensis ( Figure 2 ).

[0030]

[0031] Combined with morphological and molecular biological characteristics, HP3 was preliminarily identified as Bacillus velezensis. This strain was submitted to the Guangdong Provincial Microbial Culture Collection Center for preservation on December 31, 2024, and the preservation number is: GDMCC NO: 65701.

[0032] Example 2. Analysis of the ability of HP3 strain to promote the production of magnolol and honokiol in Magnolia officinalis

[0033] Pick a single colony of HP3 into TSB medium, place it in a shaker incubator at 30 °C and 180 r / min to culture, and prepare a bacterial solution of 1×10 8 CFU / mL, and put it in a refrigerator at 4 °C for refrigerated standby. Cut the fresh Magnolia officinalis raw material into strips about 10 cm × 3 cm in length and width, weigh it, and steam the sample in deionized water for 5 min (steam until the bark becomes soft and the inner epidermis turns brownish-yellow) before "sweating". After taking it out, soak it in 75% alcohol for 5 s, and then soak it in deionized water for 5 s. This process is repeated three times. Inoculate 1×10 8 CFU / mL HP3 bacterial solution on the inner surface of the pre-treated Magnolia officinalis sample, smear it evenly, cover it with plastic film and stack it, and "sweat" at 37 °C for 3 days. Take out the "sweated" Magnolia officinalis sample and put it in an oven for drying treatment, and the drying temperature is 60 °C.

[0034] Preparation of sample solution: Grind the sample through a No. 3 sieve of 50 mesh, accurately weigh 0.2 g, place it in a stoppered conical flask, accurately add 25 mL of methanol, shake well, stopper tightly, soak, extract with ultrasonic assistance for 30 min, filter, accurately measure 2 mL of the subsequent filtrate, place it in a 10 mL volumetric flask, add methanol to the scale, mix well, and treat the solution with a 0.22 μm filter membrane to obtain the sample solution. Preparation of standard solution: Take magnolol reference substance and honokiol reference substance, accurately weigh them, and prepare solutions containing 42 μg of magnolol and 24 μg of honokiol per 1 mL of methanol respectively.

[0035] HPLC determination: Accurately measure 2, 5, 10, 15, 20, 25 μL of the reference solution respectively for injection, and measure the peak areas of magnolol and honokiol respectively. Take the injection volume (μL) as the abscissa and the peak area (mV) as the ordinate to plot the standard curve. The injection volume of the sample is 10 μL, and the chromatographic conditions are: the chromatographic column is a Thermo C18 column (250 mm×4.6 mm, 5 μm); the mobile phase is methanol-water (78∶22); the flow rate is 1 mL / min, the detection wavelength is 294 nm, and the column temperature is 35 °C. Quantify magnolol and honokiol according to the standard curve. As Figure 3, after inoculating with HP3, the fresh Magnolia officinalis was sweat-treated. After sweating, the content of honokiol increased by 137.41%, and the content of magnolol increased by 81.74%. Therefore, HP3 can effectively increase the contents of magnolol and honokiol during the sweating process of Magnolia officinalis.

Claims

1. A strain of Bacillus velezensis 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 culture is carried out in TSB medium at a temperature of 25-35°C and a shaking culture at 150-210 r / min.

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

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 Bacillus velezensis according to claim 1 is inoculated into a raw material of Magnolia officinalis 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 materials is: Cut the fresh Magnolia officinalis raw material into strips of about 10cm×3cm in length and width, weigh it, and cook the sample in deionized water until the bark becomes soft and the inner skin turns brown before "sweating". Take it out and soak it in 75% alcohol for 5s, then soak it in deionized water for 5s. Repeat this process three times to obtain the pre-treated Magnolia officinalis sample. Then 1×10 8 The bacterial solution of Bacillus Velezii described in claim 1 was inoculated on the inner surface of the pre-treated Magnolia officinalis sample, spread evenly, covered with a 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, it is placed in an oven for drying at a drying temperature of 55-65°C.

11. The method according to claim 9, characterized in that The drying temperature is 60°C.

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