Bacterial strain and method for producing pyrroloquinoline quinone

Abstract

The invention discloses a bacterial strain and a method for producing pyrroloquinoline quinone. The strain is pseudomonas putida N1-1 which is preserved in the China General Microbiological Culture Collection Center on January 2, 2025, the preservation number is CGMCC NO.33266, and the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No.1 yard, West Beichen Road, Chaoyang District, Beijing. The strain is separated from myxomycete. The bacterial strain is inoculated into a fermentation culture medium, standing fermentation culture is carried out for 7 days at room temperature under the dark condition, a fermentation product is obtained and filtered, fermentation filtrate containing pyrroloquinoline quinine is obtained, and the yield of pyrroloquinoline quinine reaches 12.21 mg / L.

Description

Technical Field

[0001] The invention belongs to the technical field of fermentation, and particularly relates to a strain and a method for producing pyrroloquinoline quinone. Background Art

[0002] Tai Sui is the folk name for Meat-like Ganodermalucidum (MGL). Modern research shows that Tai Sui is a slime mold, a protoplast organism between organisms and fungi, and a "large rare slime mold complex". Studies have shown that Tai Sui has multiple functions such as anti-oxidation, anti-inflammatory, and immunity enhancement, and has important application prospects. Pyrroloquinolinequinone (PQQ) is a bacterial dehydrogenase coenzyme. PQQ is a vitamin that has the functions of improving human metabolism, regulating the level of free radicals in the body, promoting plant growth, protecting liver damage, promoting the synthesis of nerve growth factor, and improving bacteria's tolerance to extreme conditions such as toxicity and radiation. In addition, in 2022, my country approved the disodium salt of pyrroloquinoline quinone produced by synthetic methods as a new food raw material. It is managed under GRAS in the United States and is allowed to be used as a food raw material for energy drinks, sports drinks, electrolyte drinks, etc.; the European Union and Canada also allow it to be used as a dietary supplement or natural health food production. Although Tai Sui contains more PQQ in more conventional breast milk, fruits, and fermented foods, the content is extremely small. At present, the production method of PQQ is mainly based on chemical synthesis, which has problems such as high cost, complicated steps and environmental pollution. Microbial fermentation not only improves efficiency but also reduces costs, which is the main direction of PQQ industrial production in the future.

[0003] It is reported that the PQQ production of wild PQQ-producing bacteria is not high, about 2-3 mg / L, and the sources of isolation are mostly soil, chemical wastewater, endophytic bacteria of Polygonum cuspidatum and myxobacteria. This patent is the first to isolate PQQ-producing bacteria from Gastrodia elata. The culture medium for culturing this bacteria is different from the reported culture mediums such as grains and traditional Chinese medicine. Instead, it uses a culture medium that meets the "water" requirement, which is colorless and tasteless and can increase PQQ production. The culture medium needs to meet the oral safety requirements, and the Gastrodia elata water is used in various products. Summary of the invention

[0004] The object of the present invention is to provide a strain and a method for producing pyrroloquinoline quinone.

[0005] To achieve the above object, the present invention adopts the following technical solutions: The first aspect of the present invention provides a strain for producing pyrroloquinoline quinone, wherein the strain is Pseudomonas putida N1-1, which has been deposited in the General Microbiological Center of China Microbiological Culture Collection Committee on January 2, 2025, with a deposit number of CGMCCNO.33266, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Furthermore, the strain is isolated from Gastrodia elata.

[0006] The second aspect of the present invention provides the use of the above-mentioned Pseudomonas putida N1-1 in the production of pyrroloquinoline quinone.

[0007] The third aspect of the present invention provides the use of the above-mentioned Pseudomonas putida N1-1 in the preparation of a product containing pyrroloquinoline quinone as an active ingredient.

[0008] The fourth aspect of the present invention provides a method for producing pyrroloquinoline quinone, which is to inoculate the spores and / or the above-mentioned Pseudomonas putida N1-1 into a fermentation medium, and ferment and culture for 3 to 7 days at room temperature and in the dark to obtain a fermentation product, and filter to obtain a fermentation filtrate containing pyrroloquinoline quinone; the formula of the fermentation medium is: Monosaccharides and / or oligosaccharides 0-50g / L, inorganic ions 0-10g / L, and the rest is natural drinking water; Furthermore, the monosaccharides include but are not limited to glucose, galactose, ribose, and deoxyribose; the oligosaccharides include but are not limited to fructooligosaccharides, galacto-oligosaccharides, and xylooligosaccharides; the inorganic ion salts include but are not limited to ammonium ions, magnesium ions, iron ions, chloride ions, manganese ions, potassium ions, sodium ions, hydrogen ions, phosphate ions, and sulfate ions; Furthermore, the method is as follows: inoculating the Tai Sui strain into a fermentation medium, fermenting and culturing for 3 to 7 days at room temperature and in the dark, obtaining a fermentation product, filtering, and obtaining a fermentation filtrate containing pyrroloquinoline quinone; the formula of the fermentation medium is: drinking natural water; Furthermore, the method can also be: inoculating Pseudomonas putida N1-1 into a fermentation medium, fermenting and culturing at room temperature and in the dark for 3 to 7 days, obtaining a fermentation product, filtering, and obtaining a fermentation filtrate containing pyrroloquinoline quinone; the formula of the fermentation medium is: 7.5 g / L glucose, 1 g / L ammonium sulfate, 1 g / L magnesium sulfate, 1.4 g / L potassium dihydrogen phosphate, 3 g / L disodium hydrogen phosphate, and the rest is drinking natural water.

[0009] The fifth aspect of the present invention provides a product containing pyrroloquinoline quinone as an active ingredient, wherein the product is prepared by the above method.

[0010] The sixth aspect of the present invention provides the use of the above-mentioned product containing pyrroloquinoline quinone as an active ingredient in the fields of food, health products, cosmetics, medicine, and agriculture.

[0011] The significant advantages of the present invention are: The Tai Sui water containing pyrroloquinoline quinone can be used for patients to assist chemotherapy, reduce blood lipids, and other products. It has the functions of improving human metabolism, regulating the level of free radicals in the body, promoting plant growth, protecting liver damage, promoting nerve growth factor synthesis, and improving bacteria's tolerance to extreme conditions such as toxicity and radiation. However, in practical applications, due to the low content of pyrroloquinoline quinone, and laboratory tests have found that refrigeration and heating can greatly reduce the content of pyrroloquinoline quinone. In order to achieve the therapeutic effect, it is necessary to take 1000 to 2000 mL of Tai Sui water orally every day, and low temperature and high oral dosage are a challenge for patients to take. The present invention provides a strain and method for producing pyrroloquinoline quinone, which is different from the culture medium using grains, traditional Chinese medicine, etc. as raw materials reported, and drinking natural water containing inorganic ions, monosaccharides and or oligosaccharides is used as a fermentation medium, and Tai Sui and / or strains separated from Tai Sui are inoculated in the fermentation medium, and fermented and cultured at room temperature and in dark, and the yield of pyrroloquinoline quinone can reach 12.21 mg / L. The present invention increases the pyrroloquinoline quinone active ingredient of Tai Sui water without changing the color and taste of Tai Sui water, providing a new method for the production and application of Tai Sui water. With the improvement of people's quality of life and the increase in demand for food nutrition and health care, the application of Tai Sui water rich in pyrroloquinoline quinone will be a good direction. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 : Colony morphology of strain N1-1.

[0013] Figure 2 : Microscopic morphology of strain N1-1.

[0014] Figure 3 : Phylogenetic tree of strain N1-1.

[0015] Figure 4 : HPLC chromatogram of pyrroloquinoline quinone.

[0016] Figure 5 : Schematic diagram of the fermentation culture of Example 1.

[0017] Figure 6 : Schematic diagram of the fermentation culture of Example 3. DETAILED DESCRIPTION

[0018] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the present invention equally.

[0019] In the present invention, the method for determining the content of pyrroloquinoline quinone is a high performance liquid chromatography method: chromatographic column: Agilent Poroshell 120 EC-C18 (4.6 mm×150 mm, 5 μm); mobile phase: methanol (A)-0.1% phosphoric acid aqueous solution (B); elution conditions: 0-3 min, 5% A, 95% B, isocratic elution; 3-3.5 min, 5%→50% A, 95%→50% B, gradient elution; 3.5-8 min, 50% A, 50% B, isocratic elution; flow rate: 1 mL / min; column temperature: 30° C.; fluorescence detector wavelength: excitation wavelength 331 nm, emission wavelength 451 nm.

[0020] In the present invention, the tested Gastrodia elata has an appearance like compacted cotton wool, with the epidermis and deep tissues tightly connected, and a light pink color.

[0021] In the present invention, the drinking natural water is produced by Nongfu Spring Co., Ltd., and the food production license number is QS330106010554.

[0022] In the present invention, the NA medium formula is: peptone 10g / L, beef extract powder 3g / L, sodium chloride 5g / L, agar 15g / L, and the rest is pure water; pH value is 7.3±0.2. MRS medium formula is: peptone 10g / L, beef extract powder 10g / L, yeast extract powder 5g / L, glucose 20g / L, sodium acetate 5g / L, diammonium citrate 2g / L, Tween 80 1g / L, dipotassium hydrogen phosphate 2g / L, magnesium sulfate heptahydrate 0.2g / L, manganese sulfate tetrahydrate 0.05g / L, agar 15g / L, and the rest is pure water; pH value is 6.2±0.2. PDA medium formula is: peeled potato 200g / L, glucose 20g / L, agar 15g / L, and the rest is pure water; pH value is 5.6±0.2. Rose Bengal medium formula: peptone 5g / L, glucose 10g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, rose bengal sodium 0.0133g / L, agar 14g / L, the rest is pure water; pH value 6.0±0.2. Malt extract medium formula: malt extract powder 30g / L, agar 15g / L, the rest is pure water; pH value 5.6±0.2. Sabouraud medium formula: peptone 10g / L, glucose 40g / L, agar 20g / L, the rest is pure water; pH value 5.6±0.2. NA liquid medium formula: peptone 10g / L, beef extract powder 3g / L, sodium chloride 5g / L, the rest is pure water; pH value 7.3±0.2.

[0023] Embodiment 1: According to the requirements of aseptic operation, the test Gastrodia elata was inoculated into drinking natural water at an inoculum rate of 2 wt %, and the water was allowed to ferment at room temperature in the dark for 7 days to obtain a fermentation product; the fermentation product was filtered using a 0.45 μm filter membrane to obtain a fermentation filtrate.

[0024] The content of pyrroloquinoline quinone in the fermentation filtrate was measured by high performance liquid chromatography and was found to be 3.18 ng / mL.

[0025] Embodiment 2: After surface disinfection, the test taro was cut into small pieces with a sterilized scalpel. The cut samples were placed in a sterilized mortar, ground with a grinding rod, and then transferred to a triangular flask filled with sterile saline and sterile glass beads, and shaken on a shaker at room temperature for 20 minutes; then, according to the requirements of sterile operation, 1 mL of supernatant was aspirated, diluted, and spread on NA medium, MRS medium, PDA medium, rose red medium, malt extract medium, and Sabouraud medium, and cultured upside down in an incubator for 2 to 5 days (the culture temperature of NA medium is 37°C, and the culture temperature of MRS medium, PDA medium, rose red medium, malt extract medium and Sabouraud medium is 28°C), and a single colony was picked for further streaking purification through sterile operation. After multiple streaking purifications, a pure cultured strain was obtained, which was numbered N1-1. The genomic DNA of strain N1-1 was extracted according to the instructions of the bacterial genomic DNA extraction kit and used as a template for PCR amplification reaction. The 16S rRNA amplification primers were: upstream primer 5'-AGAGTTTGATCCTGGCTCAG-3', downstream primer 5'-ACGGCTACCTTGTTACGACTT-3'; the target fragment after PCR amplification was recovered and purified after gel excision and sent to a professional biotechnology company for sequencing; after sequencing, the obtained sequence was compared with the relevant sequence in the GenBank database, and then the sequence was subjected to multiple sequence matching analysis with the 16S rRNA sequence with higher homology in GenBank, and the phylogenetic tree was drawn using MEGA software and the Neighborjoining method.

[0026] On NA medium, strain N1-1 has vigorous colony growth, its surface is smooth and round, the colony is milky white and opaque, the colony surface is slightly moist, the colony edge is very regular, and there is a bulge in the center of the colony. Microscopic examination results of strain N1-1 show that the bacterial body is short rod-shaped. The 16S rRNA sequence of strain N1-1 is shown in SEQ ID NO.1. Comparison and analysis of it with the existing bacterial 16S rRNA in GenBank showed that strain N1-1 has the highest homology with Pseudomonas putida (GenBank accession numbers: PQ781503.1, KR045590.1, AB680362.1, OQ592394.1 and HQ270550.1, etc.). The phylogenetic tree was constructed from the 16S rRNA sequence of strain N1-1, and the results showed that the bacteria clustered with Pseudomonas putida.

[0027] Based on the morphological characteristics of the strain and the results of 16S rRNA sequence analysis, strain N1-1 was identified as Pseudomonas putida and recorded as Pseudomonas putida N1-1. Pseudomonas putida N1-1 was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on January 2, 2025, with the deposit number CGMCCNO.33266, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0028] Embodiment 3: According to the aseptic operation requirements, Pseudomonas putida N1-1 was inoculated into NA liquid culture medium and cultured at 30°C under static conditions until the bacterial concentration reached 11×10 7 CFU / mL, and obtain seed liquid; then, the obtained seed liquid is inoculated into the fermentation medium at an inoculation rate of 5vol%, and the fermentation culture is statically cultured at room temperature and in the dark for 7 days to obtain the fermentation product; the obtained fermentation product is filtered with a 0.45μm filter membrane to obtain the fermentation filtrate. Among them, the fermentation medium formula is: glucose 7.5g / L, ammonium sulfate 1g / L, magnesium sulfate 1g / L, potassium dihydrogen phosphate 1.4g / L, disodium hydrogen phosphate 3g / L, and the rest is drinking natural water; pH value 6.7.

[0029] The content of pyrroloquinoline quinone in the fermentation filtrate was measured by high performance liquid chromatography and was found to be 12.21 mg / L.

[0030] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A strain for producing pyrroloquinoline quinone, characterized in that: The strain is Pseudomonas putida ( Pseudomonas putida ) N1-1, was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 2, 2025, with the deposit number CGMCC NO.33266, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The strain according to claim 1, characterized in that: The strain is isolated from Gastrodia elata.

3. Use of the strain as claimed in claim 1 in producing pyrroloquinoline quinone.

4. Use of the strain according to claim 1 in preparing a product containing pyrroloquinoline quinone as an active ingredient.

5. A method for producing pyrroloquinoline quinone, characterized in that: Inoculate the strain described in Tai Sui and / or claim 1 into a fermentation medium, and culture it at room temperature in the dark for 3 to 7 days to obtain a fermented product, filter it, and obtain a fermentation filtrate containing pyrroloquinoline quinone; the formula of the fermentation medium is: monosaccharide and / or oligosaccharide 0 to 50 g / L, inorganic ion 0 to 10 g / L, and the rest is drinking natural water.

6. The method according to claim 5, characterized in that: The monosaccharides include but are not limited to glucose, galactose, ribose, and deoxyribose; the oligosaccharides include but are not limited to fructooligosaccharides, galacto-oligosaccharides, and xylooligosaccharides; the inorganic ion salts include but are not limited to ammonium ions, magnesium ions, iron ions, chloride ions, manganese ions, potassium ions, sodium ions, hydrogen ions, phosphate ions, and sulfate ions.

7. The method according to claim 6, characterized in that: The Tai Sui strain is inoculated into a fermentation medium, and the fermentation culture is statically carried out at room temperature and in the dark for 3 to 7 days to obtain a fermentation product, which is filtered to obtain a fermentation filtrate containing pyrroloquinoline quinone; the formula of the fermentation medium is: drinking natural water.

8. The method according to claim 6, characterized in that: The strain described in claim 1 is inoculated into a fermentation medium, and the fermentation culture is statically carried out at room temperature and in the dark for 3 to 7 days to obtain a fermentation product, which is filtered to obtain a fermentation filtrate containing pyrroloquinoline quinone; the formula of the fermentation medium is: 7.5 g / L glucose, 1 g / L ammonium sulfate, 1 g / L magnesium sulfate, 1.4 g / L potassium dihydrogen phosphate, 3 g / L disodium hydrogen phosphate, and the rest is drinking natural water.

9. A product containing pyrroloquinoline quinone as an active ingredient, characterized in that: The product is prepared by the method according to any one of claims 4 to 8.

10. Application of the product according to claim 9 in the fields of food, health products, cosmetics, medicine and agriculture.

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