A strain of Bacillus subtilis HASX5 producing chlorogenic acid and mangiferin and its application

Abstract

A strain of Bacillus subtilis HASX5 producing chlorogenic acid and mangiferin and its application, which relates to the field of microorganisms, is to solve the problem that the existing extraction of mangiferin and chlorogenic acid requires the consumption of a large amount of plant resources. Bacillus subtilis HASX5 has been deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, the deposit date is July 10, 2024, and the deposit number is CCTCC NO: M 20241528. The strain can ferment to produce chlorogenic acid and mangiferin, and also has good antioxidant activity. The present invention is used for in vitro antioxidant and production of Acanthopanax secondary metabolites.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a Bacillus subtilis HASX5 producing chlorogenic acid and mangiferin and applications thereof. Background Art

[0002] Mangiferin exists in many plants, such as mango and Anemarrhena asphodeloides, and has multiple active effects such as hypoglycemic, anti-tumor, liver protection, anti-oxidation, antibacterial, analgesic and antipyretic, etc. Chlorogenic acid is a pure natural product with high medicinal and economic value, recognized as "plant gold", and exists in many plants such as honeysuckle and Acanthopanax senticosus, and has antibacterial, antiviral, anti-inflammatory, liver protection, hypoglycemic and lipid-lowering, immunomodulatory, neuroprotective, antihypertensive, anti-oxidant, anti-aging, anti-tumor and other biological activities, and is widely used in medicine, food and cosmetics.

[0003] At present, the production of mangiferin and chlorogenic acid is mainly extracted and separated from plant raw materials, but it consumes a lot of plant resources. Although the chemical synthesis method can produce chlorogenic acid raw materials, by-products will be produced during the synthesis process, and the chemical synthesis method is not an environmentally friendly production method. Summary of the invention

[0004] The present invention aims to solve the problem that the existing method for extracting mangiferin and chlorogenic acid needs to consume a large amount of plant resources, and provides a Bacillus subtilis HASX5 producing chlorogenic acid and mangiferin and application thereof.

[0005] The present invention provides a Bacillus subtilis HASX5, which has been deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, the deposit date is July 10, 2024, and the deposit number is CCTCCNO: M 20241528.

[0006] The colony of the Bacillus subtilis HASX5 of the present invention is white, with a rough surface and a gray matrix, and the bacterial body is rod-shaped and has flagella.

[0007] The Bacillus subtilis HASX5 of the present invention is an obligate aerobic bacterium, which is positive in Gram staining, starch hydrolysis test, gelatin liquefaction test, catalase test, oil hydrolysis test, biofilm formation, VP test, indole test, nitrate reduction test and motility test.

[0008] The 16S rDNA sequence comparison analysis of the Bacillus subtilis HASX5 of the present invention shows that the similarity with Bacillus subtilis (MT491101.1) reaches 100%. Combining the bacterial morphological characteristics and physiological and biochemical identification results, it is determined that the endophytic bacterium HASX5 of the present invention is Bacillus subtilis.

[0009] The present invention also provides the use of Bacillus subtilis HASX5 in fermentation to produce chlorogenic acid and mangiferin.

[0010] The present invention also provides the use of Bacillus subtilis HASX5 in in vitro anti-oxidation.

[0011] Beneficial effects of the present invention:

[0012] The present invention separates an endophytic bacterium HASX5 from Acanthopanax senticosus, which is identified as Bacillus subtilis. After secondary metabolite analysis of its fermentation broth, it is found that the strain can ferment to produce chlorogenic acid and mangiferin. The strain also has good antioxidant activity.

[0013] The production of chlorogenic acid and mangiferin by microbial fermentation can avoid the consumption of a large amount of plant resources and is suitable for industrial production. The present invention provides a new method for producing secondary metabolites of Acanthopanax senticosus by microbial fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a colony photo of the endophytic bacterium HASX5;

[0015] Figure 2 is the phylogenetic tree of the endophytic bacterial HASX5;

[0016] Figure 3 The scavenging ability of the fermentation broth of endophytic bacteria HASX5 on DPPH free radicals;

[0017] Figure 4 The scavenging ability of the fermentation broth of endophytic bacteria HASX5 on •OH free radicals;

[0018] Figure 5 The results of HPLC analysis of chlorogenic acid in the fermentation broth of endophytic bacteria HASX5;

[0019] Figure 6 The results of HPLC analysis of mangiferin in the fermentation broth of endophytic bacteria HASX5;

[0020] Figure 7 For crystal A 1 H NMR spectroscopy;

[0021] Figure 8 For crystal B1 H NMR spectrum. DETAILED DESCRIPTION

[0022] The embodiments of the present invention are described in detail below. The following embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0023] Embodiment 1:

[0024] The Bacillus subtilis HASX5 of this example has been deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, the deposit date is July 10, 2024, and the deposit number is CCTCC NO: M20241528.

[0025] The separation method of Bacillus subtilis HASX5 in this embodiment is:

[0026] The stems of healthy Araliaceae plant Acanthopanax seuticosus (Rupr. et Maxim. ) Harms (collected from Qing'an County, Heilongjiang Province) were disinfected on the surface. The disinfection procedure was as follows: the stems were soaked in 75% alcohol for 30 s, then washed with sterile water, rinsed in 7% sodium hypochlorite for 10 min, washed with sterile water, then soaked in 75% alcohol for 30 s, and finally washed three times with sterile water.

[0027] Use a sterile scalpel to remove the outer skin of the sterilized stem and cut it in the middle. Place the cut surface in NA medium and culture it at 37℃ for 7 days. Remove the last sterile water wash in the sterilization procedure and spread it in the NA medium. Place the sterilized uncut experimental material on the NA medium, roll it once, take it out, and culture it with the endophytic bacteria isolation medium as a control.

[0028] Preparation of NA medium: Take 3g beef extract and 10g peptone and add them into boiling water in small amounts several times. After they are completely dissolved, add 20g agar and 5g sodium chloride, stir, and make up to 1000mL after dissolution.

[0029] The endophytic bacteria HASX5 were isolated from the stems of Acanthopanax senticosus. No bacteria grew in the negative control plate and the negative control liquid culture medium, and this was repeated many times, thus proving that the isolated bacteria were endophytic bacteria of Acanthopanax senticosus.

[0030] Example 2: Identification of endophytic bacteria HASX5

[0031] 1. Morphological characteristics of endophytic bacteria HASX5

[0032] The colony of endophytic bacteria HASX5 is white, with a rough surface and a gray matrix. The bacteria are rod-shaped and have flagella. Figure 1 shown.

[0033] 2. Physiological and biochemical characteristics of endophytic bacteria HASX5

[0034] The endophytic bacterium HASX5 was an obligate aerobe, with positive results in Gram staining, starch hydrolysis test, gelatin liquefaction test, catalase test, lipid hydrolysis test, biofilm formation, VP test, indole test, nitrate reduction test, and motility test.

[0035] 3. Molecular identification of endophytic bacterial HASX5

[0036] PCR amplification was performed using universal primers for bacterial 16S rDNA genes, and the product was tested by agarose gel electrophoresis. As a result, an amplified fragment was obtained at 1486 bp. Shanghai Shenggong Biotechnology Co., Ltd. was commissioned to sequence the endophytic bacterium HASX5. The 16S rDNA sequence of the endophytic bacterium HASX5 is shown in SEQ ID NO: 1 in the sequence list. The sequencing results were submitted to the NCBI database, analyzed and aligned using BLAST, and then the phylogenetic tree of the endophytic bacterium HASX5 was constructed using MEGA 7 software. The results of the phylogenetic tree construction are shown in Figure 2 The sequence of endophytic bacteria HASX5 has a 100% similarity with Bacillus subtilis (MT491101.1). Combining the bacterial morphological characteristics and physiological and biochemical identification results, it was determined that the endophytic bacteria HASX5 was Bacillus subtilis.

[0037] Example 3: Analysis of the antioxidant activity of endophytic bacteria HASX5

[0038] Activation of endophytic bacteria HASX5: Take endophytic bacteria HASX5, inoculate it on the surface of NA culture medium under sterile conditions, and culture it at 37°C for 3 days for later use.

[0039] Preparation of fermentation broth of endophytic bacteria HASX5: Under sterile conditions, an appropriate amount of activated endophytic bacteria HASX5 was selected and inoculated into 50 mL NB medium, and cultured in a shaking incubator at 37 °C and 120 r / min for 3 days to make 1×10 7CFU / mL seed liquid; the seed liquid was transferred to 3L sterilized NB medium at an inoculum volume of 20% (v / v), and cultured in a shaking incubator at 37°C and 120 r / min for 5 days. The fermentation broth was collected, concentrated under reduced pressure, extracted with ethyl acetate (3×100mL), and concentrated at low temperature to an extract, dried, and the fermentation broth powder of the endophytic bacteria HASX5 was obtained for later use.

[0040] 1. Determination of DPPH free radical scavenging ability

[0041] Dissolve an appropriate amount of DPPH in methanol and dilute to 100 mL to prepare a solution with a mass concentration of 0.15 mmol•L -1 DPPH solution; take an appropriate amount of Vc and dissolve it in methanol and make up to 10mL to prepare 200μg•mL -1 of reference solution;

[0042] The fermentation broth powder of the endophytic bacteria HASX5 was dissolved in an appropriate amount of methanol to prepare a solution with a concentration of 200 μg•mL -1 of the test sample solution.

[0043] Dilute the reference solution and the test solution in sequence to a concentration gradient of 100 μg•mL -1 , 50μg•mL -1 , 25μg•mL -1 , 12.5μg•mL -1 、6.25μg•mL -1 , accurately pipette 2 mL of each solution into a test tube, and use methanol as a blank control group. Add 0.15 mmol•L -1 4 mL of DPPH solution was placed in the dark for 30 min, and the absorbance of each sample was measured at 517 nm (n=3). The DPPH free radical scavenging rate was calculated as follows:

[0044]

[0045] In the formula, A i Indicates the absorbance value when the test product and DPPH solution are added simultaneously; A j A0 represents the absorbance value when the test sample and blank control are added at the same time; A1 represents the absorbance value when the blank control and DPPH solution are added at the same time.

[0046] The scavenging ability of endophytic bacteria HASX5 fermentation broth on DPPH free radicals Figure 3 shown.

[0047] from Figure 3It can be seen that the scavenging rate of DPPH free radicals is related to the concentration of endophytic bacteria HASX5 fermentation liquid, and the concentration of endophytic bacteria HASX5 fermentation liquid of Acanthopanax senticosus is positively correlated with the scavenging ability of DPPH free radicals. The experimental results show that the fermentation liquid of endophytic bacteria HASX5 of Acanthopanax senticosus has a good ability to scavenge DPPH free radicals.

[0048] 2. Determination of OH radical scavenging ability

[0049] Accurately pipette 2 mL of each test solution of different concentrations into a test tube, and add 2 mL of 6 mmol•L -1 FeSO4, 2mL 6mmol•L -1 of H2O2, shake and mix, let stand for 10 min, then add 2mL 6mmol•L -1 The salicylic acid-ethanol solution was placed at 37°C for 30 min, and the absorbance of each sample was measured at 510 nm (n=3). The •OH radical scavenging rate was calculated as follows:

[0050]

[0051] In the formula, A1 represents the absorbance value of the added sample; A2 represents the absorbance value when distilled water replaces H2O2 to eliminate the influence of the sample's own color; A0 represents the absorbance value when anhydrous ethanol replaces the sample as a blank control.

[0052] The scavenging ability of the fermentation broth of endophytic bacteria HASX5 on •OH free radicals is as follows Figure 4 shown.

[0053] from Figure 4 It can be seen that the scavenging ability of the fermentation liquid of endophytic bacteria HASX5 on •OH free radicals is related to its fermentation liquid concentration, and the concentration of the fermentation liquid of endophytic bacteria HASX5 in Acanthopanax senticosus is positively correlated with the scavenging ability of •OH free radicals. The experimental results show that the fermentation liquid of endophytic bacteria HASX5 in Acanthopanax senticosus has good antioxidant activity.

[0054] Example 4: HPLC analysis of fermentation broth of endophytic bacteria HASX5

[0055] Activation of endophytic bacteria HASX5: Take endophytic bacteria HASX5, inoculate it on the surface of NA culture medium under sterile conditions, and culture it at 37°C for 3 days for later use.

[0056] Preparation of fermentation broth of endophytic bacteria HASX5: Under sterile conditions, an appropriate amount of activated endophytic bacteria HASX5 was selected and inoculated into 50 mL NB medium, and cultured in a shaking incubator at 37 °C and 120 r / min for 3 days to make 1×10 7CFU / mL of seed liquid; transfer the seed liquid to 3L sterilized NB medium at a 20% (v / v) inoculation rate, culture at 37°C, 120 r / min shaking for 5 days, collect the fermentation liquid and set aside.

[0057] 1. Preparation of test solution

[0058] Take 300 mL of the fermentation broth of endophytic bacteria HASX5, concentrate it under reduced pressure at 50 °C, extract the concentrated fermentation broth with ethyl acetate three times (3×100 mL), combine the extracts, concentrate to dryness at 50 °C, dissolve it in 1 mL of methanol, and use it as the test solution for later use.

[0059] 2. Preparation of reference solution

[0060] Accurately weigh 1 mg each of chlorogenic acid and mangiferin reference substances, place them in a 1 mL centrifuge tube, add 1 mL of chromatographic methanol to dissolve, and prepare reference substance solutions for later use.

[0061] 10 g of Acanthopanax senticosus stem powder was dissolved in 100 mL of ethyl acetate solution, extracted by ultrasonic wave for 1 hour, filtered, and the solution was concentrated to 10 mL of Acanthopanax senticosus solution under low temperature environment, which was used as a reference substance.

[0062] Take 300 mL of NB culture medium and concentrate it under reduced pressure at 50°C. Extract the concentrated NB culture medium with ethyl acetate for 3 times (3×100 mL). Combine the extracts, concentrate to dryness at 50°C, dissolve them in 1 mL of methanol and use them as blank control solution for later use.

[0063] 3. HPLC conditions

[0064] Chromatographic column: Venusil XBP-C18 column (4.6 mm × 250 mm, 5 μm, USA); mobile phase: methanol: water (45:55) 0.2% phosphoric acid; flow rate: 0.8 mL•min -1 ; Column temperature: 25℃; Detection wavelength: 260 nm; Injection volume: 10 μL.

[0065] The fermentation broth of endophytic bacteria HASX5 was analyzed by HPLC. Figure 5 , Figure 6 shown. Figure 5 The HPLC analysis results of chlorogenic acid in the fermentation broth of endophytic bacteria HASX5 are shown in Figure 1. Figure 5 A is the test sample, B is the chlorogenic acid reference substance, C is the Acanthopanax senticosus herbal medicine, and D is the NB blank control. Figure 5 It can be seen that the fermentation broth of the endophytic bacteria HASX5 of Acanthopanax senticosus has the same chromatographic peak at the same retention time as the chlorogenic acid reference substance and Acanthopanax senticosus reference medicinal material, and the blank control (NB) has no interference, indicating that the fermentation broth of the bacteria may contain chlorogenic acid. Figure 6The HPLC analysis results of mangiferin in the fermentation broth of endophytic bacteria HASX5 are shown in Figure 1. Figure 6 A is the test sample, B is the reference substance of mangiferin, C is the herbal medicine of Acanthopanax senticosus, and D is the NB blank control. Figure 6 It can be seen that the fermentation broth of the endophytic bacteria HASX5 of Acanthopanax senticosus has the same chromatographic peak at the same retention time as the mangiferin reference substance and Acanthopanax senticosus reference medicinal material, and there is no interference in the blank control (NB), indicating that the fermentation broth of the bacteria may contain mangiferin.

[0066] Example 5: Separation of secondary metabolites from fermentation broth of endophytic bacteria HASX5

[0067] According to the HPLC analysis results, mangiferin and chlorogenic acid may be contained in the fermentation broth of endophytic bacteria HASX5, so a separation process was designed for the above two secondary metabolites. The specific method is as follows:

[0068] Take 7L of the fermentation liquid of endophytic bacteria HASX5 and slowly pass it through D 101 After the macroporous resin column, ethanol gradient elution (30%, 50%, 70%, 90%) was used, and the ethanol elution fractions were collected respectively. The 30% ethanol elution fraction was concentrated and the solvent was recovered, and then the polyamide resin column was separated, and gradient elution was performed with 30%, 50%, 70%, and 90% ethanol. The elution fractions were collected, the 30% ethanol elution fraction was taken, the solvent was recovered to dryness, dissolved in water, extracted with ethyl acetate, the ethyl acetate extraction fraction was collected, the solvent was recovered to dryness, dissolved in methanol, and refrigerated in a 4°C refrigerator for 24 hours to precipitate crystals. After repeated refrigeration treatment twice, white crystals A were obtained.

[0069] Take another D 101 The 50% ethanol elution portion of the macroporous resin column was recovered from the solvent until dry, dissolved in water, extracted with water-saturated n-butanol, the water-saturated n-butanol extraction portion was collected, the solvent was recovered until dry, dissolved in water, sampled and separated on a polyamide resin column, and gradient eluted with 30%, 50%, 70%, and 90% ethanol, the 50% ethanol elution portion was collected, the solvent was recovered, dissolved with a small amount of 50% ethanol, and refrigerated at 4°C for 24 hours to obtain light yellow crystals B.

[0070] White crystal A is easily soluble in polar solvents such as ethanol and acetone, but difficult to dissolve in low-polarity solvents such as chloroform. 1 H-NMR analysis, the results are shown in Figure 7 .

[0071] 1 H-NMR (400 MHz, MeOD) δ: 7.56 (1H, d, J = 15.9 Hz, H-7′), 7.05 (1H, d, J=2.1 Hz, H-2′), 6.96 (1H, dd, J = 8.2, 2.1 Hz, H-6′), 6.77 (1H, d, J = 8.2 Hz, H-5′), 6.26 (1H, d, J = 15.9 Hz, H-8′), 5.33 (1H, td, J = 8.8, 4.1 Hz, H-3), 4.16 (1H, q, J =3.9 Hz, H-5), 3.72 (1H, dd, J = 8.4, 3.1 Hz, H-4), 2.13-1.99 (2H, m, H-2, H-6).

[0072] The above NMR data are basically consistent with the relevant data of chlorogenic acid reported in the literature, and have the same Rf value through co-thin layer analysis with three solvent systems of chlorogenic acid reference substances, so the white crystal A is identified as chlorogenic acid.

[0073] Light yellow crystal B is slightly soluble in methanol, ethanol, and water, soluble in hot dilute methanol and dilute ethanol, and insoluble in non-polar solvents. 1 H-NMR analysis, the results are shown in Figure 8 .

[0074] 1 H-NMR (400 MHz, MeOD) δ: 7.45 (s, 1H, 8-H), 6.83 (s, 1H, 5-H), 6.37 (s, 1H, 4-H), 4.20 (d, 1H, J = 8.7 Hz, 1′-H).

[0075] The above NMR data are basically consistent with the relevant data of mangiferin reported in the literature, and through co-thin layer analysis with three solvent systems of mangiferin reference substance, they have the same Rf value, so the yellow crystal B is identified as mangiferin.

Claims

1. A strain of Bacillus subtilis HASX5 producing chlorogenic acid and mangiferin, characterized in that: The Bacillus subtilis HASX5 has been deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan City. The deposit date is July 10, 2024, and the deposit number is CCTCC NO: M 20241528.

2. Use of the Bacillus subtilis HASX5 as claimed in claim 1 in fermentation to produce chlorogenic acid and mangiferin.

3. Use of Bacillus subtilis HASX5 as claimed in claim 1 in in vitro anti-oxidation.

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