A method for efficiently purifying phospholipids from coenzyme Q10 fermentation powder

Abstract

The present invention belongs to the field of extraction of microbial phospholipids, and specifically discloses a method for efficiently purifying phospholipids from coenzyme Q10 fermentation powder, which comprises the following steps: 1) mother liquor culture, 2) seed tank culture, 3) fermenter culture, 4) centrifugation of fermentation broth, 5) high-pressure homogenization, 6) centrifugation of cell lysate, 7) vacuum freeze-drying, 8) subcritical extraction, 9) acetone extraction, 10) rotary evaporation; by adjusting the medium components and optimizing the culture process, the present invention can achieve high-density culture of Agrobacterium tumefaciens cells, obtain a large amount of cells, in the fermentation broth, the coenzyme Q10 content can reach 5.1 g / L of fermentation broth, and the phospholipid content can reach 13.5 g / L of fermentation broth. Moreover, the present method also discloses a separation and purification method for efficiently extracting high-purity phospholipids from coenzyme Q10 fermentation powder, turning waste into treasure, with good economic benefits, and the steps are simple and mild, with low requirements for equipment, and are environmentally friendly.

Description

Technical Field

[0001] The present invention belongs to the field of extraction of microbial phospholipids, and specifically discloses a method for efficiently purifying phospholipids from coenzyme Q10 fermentation powder. Background Art

[0002] Coenzyme Q10 is a lipid-soluble electron carrier between NADH dehydrogenase, succinate dehydrogenase and BC complex in the respiratory chain, an essential element for ATP generation, plays an important role in proton transfer and electron transfer in the mitochondrial respiratory chain of cells, is an activator of cell respiration and cell metabolism, and is also an important antioxidant and non-specific immune enhancer. Since coenzyme Q10 can enhance physical strength and energy, protect the cardiovascular system, reduce the side effects of lipid-lowering drugs, protect the skin and other functions, it also has important application values in aspects such as improving immunity, delaying aging, health care and beauty. At present, there are mainly three methods for preparing coenzyme Q10: extraction and separation method, chemical synthesis method, and biosynthesis method. The extraction and separation method mainly extracts from animal livers, myocardium, etc. and plant oil-containing seeds or tender buds and young leaves. The extraction method is a relatively traditional production method in modern bioengineering, with high production costs and difficult-to-obtain raw materials.

[0003] The chemical synthesis method of coenzyme Q10 can be divided into two types: total chemical synthesis method and semi-chemical synthesis method. The total chemical synthesis method refers to a synthesis method that does not use solanesol from tobacco and other raw materials and completely obtains the side chain of coenzyme Q10 through chemical methods. This method has inexpensive and easily available raw materials, high product regioselectivity and stereoselectivity, but the construction of the side chain requires multiple couplings, with a low overall yield and difficulty in industrialization. The semi-chemical synthesis method mainly includes the direct side chain introduction method and the side chain extension method. Both methods require natural solanesol as the key raw material to synthesize coenzyme Q10. Due to the extremely high price of high-quality and pure solanesol, the cost of chemically synthesizing coenzyme Q10 is high, and it also has disadvantages such as many synthesis steps, many by-products, and difficulty in separating chiral substances. Therefore, the chemical synthesis method is subject to certain limitations in industrial production.

[0004] Biosynthesis method The biosynthesis method is a technical process that utilizes the life activities of microbial cells, plant cells, or animal cells to obtain the substances required by people. Currently, the biosynthesis methods of coenzyme Q10 include two ways: plant cell culture and microbial cell fermentation. Plant cell culture mainly uses tobacco tissue as the material, through callus induction and suspension culture, to produce tobacco suspension culture cells with fast growth rate and high coenzyme Q10 content. In 1977, Japan first realized the industrial production of coenzyme Q10 by microbial fermentation method. The main strains applied to produce coenzyme Q10 include photosynthetic bacteria, Agrobacterium, Rhodopseudomonas, Schizosaccharomyces pombe, etc. This method has the advantages of cheap and abundant raw materials, relatively simple product separation process, and no chirality problem. Currently, there are also many reports on the production of coenzyme Q10 by fermentation method. For example, CN200910093966.3, a method for fermenting and producing coenzyme Q10, includes the following steps: first, Agrobacterium tumefaciens is inoculated at an inoculation amount of 5-15% for high-density aerobic fermentation for 60-84 hours. The fermentation medium components are glucose 12-20%, ammonium sulfate 0.5-1.5%, potassium dihydrogen phosphate 0.1-0.5%, magnesium sulfate 0.01-0.05%, corn steep liquor 0.5-2.5%, diammonium hydrogen phosphate 0.2-1%, pH 6-7.5, and the fermentation temperature is 28-31°C; then the obtained fermentation broth is separated to obtain separated bacteria; finally, the separated bacteria are resuspended and then refluxed and extracted to obtain coenzyme Q10. Its process is simple, the cell concentration in the fermentation broth is low, and the production cost is low, which is suitable for large-scale production. Also, CN202011030403.2, a fermentation method of coenzyme Q10, controls the ammonium ion concentration in cycles during the method, adds lysine salt to the fermentation medium and replenishes lysine salt again during the fermentation process, and adds sodium sulfate once during the fermentation process. This fermentation control process significantly enhances the growth and metabolic ability of coenzyme Q10-producing bacteria, significantly increases the cell mass, the fermentation unit of coenzyme Q10 increases rapidly, the titer of coenzyme Q10 at the end of fermentation reaches more than 4000 mg / L, the fermentation cycle is significantly extended, the bacteria are easy to filter and collect, and the batch production of coenzyme Q10 increases significantly, significantly reducing the fermentation cost of coenzyme Q10. However, in the above methods, for other active ingredients in the bacterial powder, such as phospholipids, they are discarded in the form of bacterial residues or destroyed during the saponification process, resulting in the waste of phospholipids.

[0005] Phospholipids play a significant role in activating cells, maintaining metabolism, basal metabolism, and the balanced secretion of hormones, enhancing the body's immunity and regenerative capacity. Additionally, phospholipids also have the effects of promoting fat metabolism, preventing fatty liver, reducing serum cholesterol, improving blood circulation, and preventing cardiovascular diseases. In the coenzyme Q10 fermented bacteria powder, the phospholipid content is above 10%, which is more than three times the coenzyme Q10 content. However, both coenzyme Q10 and phospholipid molecules have long carbon chain structures and functional groups that can form hydrogen bonds, making it technically difficult to efficiently separate phospholipids from coenzyme Q10 fermented bacteria powder. And there are few reports on the phospholipid extraction technology from coenzyme Q10 fermented bacteria powder in the existing technology. Summary of the Invention

[0006] To solve the above problems, the present invention discloses a method for efficiently purifying phospholipids from coenzyme Q10 fermented powder.

[0007] The technical solution of the present invention is as follows:

[0008] A method for efficiently purifying phospholipids from coenzyme Q10 fermented powder, comprising the following steps:

[0009] 1) Mother liquor culture: Dilute Agrobacterium tumefaciens to 1 - 5x10 3 cells / mL and inoculate 0.1 - 1 mL onto the slant medium. Place the slant medium in an incubator at a temperature of 30 - 34 °C and a relative humidity of 45% - 55% for 2 - 6 days. Then pick 1 colony and inoculate it into the mother flask medium. Place the mother flask medium in a shaker at 30 - 34 °C, a relative humidity of 45% - 55%, and a rotation speed of 220 - 350 rpm for 24 - 48 hours to obtain the mother liquor strain;

[0010] 2) Seed tank culture: Inoculate the mother liquor strain into the seed tank medium in the seed tank at an inoculation amount of 1 - 3‰. Culture at 30 - 35 °C, a tank pressure of 0.03 - 0.05 MPa, an aeration ratio of 0.3 - 0.6, and a stirring speed of 200 - 250 rpm for 24 - 48 hours to obtain a primary seed liquid with a residual glucose amount of 0.5 - 1.0 g / L, uniform cell shape, and good asepsis;

[0011] 3) Fermentation tank culture: Inoculate the seed liquid into the fermentation medium in the fermentation tank at an inoculation amount of 5 - 10%. Culture at 35 - 38 °C, a tank pressure of 0.03 - 0.06 MPa, an aeration ratio of 0.6 - 0.8, and a stirring speed of 110 - 140 rpm for 24 - 36 hours;

[0012] 4) Fermentation broth centrifugation: Centrifuge the fermentation broth at 8000 - 15000 rpm at low temperature for 10 - 20 minutes, and take the precipitate to obtain the cells;

[0013] 5) High-pressure homogenization: After dissolving the bacterial cell precipitate in the bacterial cell lysate according to the solid-liquid ratio of 1:10 - 20 by mass, add it to a high-pressure homogenizer for cell disruption, and collect the cell disruption liquid;

[0014] 6) Centrifugation of cell disruption liquid: Centrifuge the cell disruption liquid at 12000 - 15000 rpm at low temperature, discard the precipitate, and take the supernatant;

[0015] 7) Vacuum freeze-drying: Perform vacuum freeze-drying on the supernatant with a vacuum degree less than 133 Pa and a temperature below -50 °C to obtain dry fermentation powder;

[0016] 8) Subcritical extraction: Put the dry fermentation powder into the extraction tank. After evacuating the extraction tank to a vacuum state, add the extractant for subcritical extraction. After the extraction is completed, pour the subcritical fluid containing fat-soluble components obtained from the extraction from the extraction tank into the solvent removal tank, and perform heating and reduced-pressure evaporation. After the solvent is completely removed, obtain the crude extract;

[0017] 9) Acetone extraction: Add the crude extract to an acetone solution with a mass fraction of 50 - 60%, cool and crystallize at 4 - 10 °C for 4 - 8 h, and then filter through a 0.45 μm filter to obtain the filter cake;

[0018] 10) Rotary evaporation: Rinse the filter cake with deionized water and then perform rotary evaporation to remove water and organic solvents to obtain phospholipids.

[0019] Furthermore, in the above method for efficiently purifying phospholipids from coenzyme Q10 fermentation powder, the composition of the slant medium in step 1) is as follows: peptone 8 g / L, yeast extract 5 g / L, sodium chloride 3 g / L, agar 18 g / L, pH value 7.2, atrazine 0.05 g / L, pH value 7.4.

[0020] Furthermore, in the above method for efficiently purifying phospholipids from coenzyme Q10 fermentation powder, the composition of the seed tank medium in step 2) is as follows: sucrose 35 g / L, corn steep liquor 15 g / L, yeast extract 10 g / L, NaCl 3 g / L, atrazine 0.03 g / L, pH value 7.4.

[0021] Furthermore, in the above method for efficiently purifying phospholipids from coenzyme Q10 fermentation powder, the composition of the fermentation medium in step 3) is as follows: sucrose 45 g / L, corn steep liquor 30 g / L, yeast extract 10 g / L, NaCl 3 g / L, sodium dihydrogen phosphate 0.5 g / L, disodium hydrogen phosphate 0.5 g / L, nicotinamide adenine dinucleotide 0.02 g / L.

[0022] Furthermore, in the above method for efficiently purifying phospholipids from coenzyme Q10 fermentation powder, in step 5) high-pressure homogenization, the working pressure is 1200 - 1500 Bar, and the homogenization is performed 3 - 6 times.

[0023] Further, in the above method for efficiently purifying phospholipids from coenzyme Q10 fermentation powder, the composition of the cell lysate in step 5) is as follows: 50 mmol / L Tris-Cl pH 6.8, 100 mmol / L DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol.

[0024] Further, in the above method for efficiently purifying phospholipids from coenzyme Q10 fermentation powder, in the subcritical extraction in step 8), the vacuum state refers to a pressure less than 133 Pa, and the extractant is ethanol.

[0025] Further, the phospholipids prepared by the above method.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention discloses a method for efficiently purifying phospholipids from coenzyme Q10 fermentation powder. By adding a trace amount of atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) during the mother liquor culture and seed tank culture processes, it is found that it can effectively inhibit the growth of miscellaneous bacteria, improve the fermentation density of Agrobacterium tumefaciens, obtain a higher cell density and a larger amount of coenzyme Q10 and phospholipids. Further, by adding a high concentration of corn steep liquor to the seed medium and fermentation medium, the fermentation density can be further increased; further, through process optimization of the cell disruption of the fermentation broth, high-purity fermentation powder is obtained through cell disruption and freeze-drying. On this basis, the fermentation powder is subjected to subcritical extraction and combined with acetone extraction to separate coenzyme Q10 and phospholipids as much as possible to obtain high-purity phospholipids. The present invention can turn waste into treasure, has good economic benefits, and the separation steps are simple and mild, with low requirements for equipment, and is environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is a process flow schematic diagram of the present invention;

[0029] Figure 2 is a comparison of the wet cell weight obtained per liter of fermentation broth in the examples and comparative examples;

[0030] Figure 3 is a comparison of coenzyme Q10 and phospholipids obtained per liter of fermentation broth in the examples and comparative examples;

[0031] Figure 4 is a comparison of the phospholipid harvest amounts in Example 2 and Comparative Examples 4 and 5;

[0032] Figure 5 is a comparison of the purities of the phospholipids in Example 2 and Comparative Examples 4 and 5. DETAILED DESCRIPTION OF THE INVENTION

[0033] As Figure 1 shown, a method for efficiently purifying phospholipids from coenzyme Q10 fermentation powder includes the following steps:

[0034] 1) Mother liquor culture: Dilute Agrobacterium tumefaciens to 1 - 5×10 3 cells / mL, then inoculate 0.1 - 1 mL onto a slant medium. Place the slant medium in an incubator at a temperature of 30 - 34°C and a relative humidity of 45% - 55% for 2 - 6 days. Then pick 1 colony and inoculate it into a mother flask medium. Place the mother flask medium in a shaker at 30 - 34°C, a relative humidity of 45% - 55%, and a rotation speed of 220 - 350 rpm for 24 - 48 hours to obtain a mother liquor strain;

[0035] 2) Seed tank culture: Inoculate the mother liquor strain into the seed tank medium in the seed tank at an inoculation amount of 1 - 3‰. Culture at 30 - 35°C, a tank pressure of 0.03 - 0.05 MPa, an aeration ratio of 0.3 - 0.6, and a stirring speed of 200 - 250 rpm for 24 - 48 hours to obtain a first - stage seed liquid with a residual glucose amount of 0.5 - 1.0 g / L, uniform cell shape, and good asepsis;

[0036] 3) Fermentation tank culture: Inoculate the seed liquid into the fermentation medium in the fermentation tank at an inoculation amount of 5 - 10%. Culture at 35 - 38°C, a tank pressure of 0.03 - 0.06 MPa, an aeration ratio of 0.6 - 0.8, and a stirring speed of 110 - 140 rpm for 24 - 36 hours;

[0037] 4) Fermentation broth centrifugation: Centrifuge the fermentation broth at 8000 - 15000 rpm at low temperature for 10 - 20 minutes, and take the precipitate to obtain the cells;

[0038] 5) High - pressure homogenization: Add the cell precipitate to the cell lysate for dissolution according to a solid - to - liquid ratio of 1:10 - 20 by mass, then add it to a high - pressure homogenizer for cell disruption, and collect the cell disruption liquid;

[0039] 6) Centrifugation of cell disruption liquid: Centrifuge the cell disruption liquid at 12000 - 15000 rpm at low temperature, discard the precipitate, and take the supernatant;

[0040] 7) Vacuum freeze - drying: Perform vacuum freeze - drying on the supernatant, with a vacuum degree less than 133 Pa and a temperature below - 50°C to obtain the dried fermentation powder;

[0041] 8) Sub - critical extraction: Put the dried fermentation powder into an extraction tank. After evacuating the extraction tank to a vacuum state, add an extractant for sub - critical extraction. After the extraction is completed, pour the sub - critical fluid containing fat - soluble components obtained from the extraction from the extraction tank into a stripping tank for heating and reduced - pressure evaporation. After the solvent is completely removed, obtain a crude extract;

[0042] 9) Acetone extraction: Add the crude extract to an acetone solution with a mass fraction of 50 - 60%, cool and crystallize at 4 - 10°C for 4 - 8 h, then filter through a 0.45 μm filter to obtain a filter cake; the mass ratio of the crude extract to the acetone solution is 1:5 - 10

[0043] 10) Rotary evaporation: Rinse the filter cake with deionized water and then rotary evaporate the water and organic solvents to obtain phospholipids;

[0044] The composition of the slant medium in step 1) is as follows: peptone 8 g / L, yeast extract 5 g / L, sodium chloride 3 g / L, agar 18 g / L, pH 7.2, atrazine 0.05 g / L, pH 7.4;

[0045] The composition of the seed tank medium in step 2) is as follows: sucrose 35 g / L, corn steep liquor 15 g / L, yeast extract 10 g / L, NaCl 3 g / L, atrazine 0.03 g / L, pH 7.4;

[0046] The composition of the fermentation medium in step 3) is as follows: sucrose 45 g / L, corn steep liquor 30 g / L, yeast extract 10 g / L, NaCl 3 g / L, sodium dihydrogen phosphate 0.5 g / L, disodium hydrogen phosphate 0.5 g / L, nicotinamide adenine dinucleotide 0.02 g / L;

[0047] In step 5) high-pressure homogenization, the working pressure is 1200 - 1500 Bar, and the homogenization is carried out 3 - 6 times;

[0048] The cell lysate in step 5) is composed of: 50 mmol / L Tris-Cl pH 6.8, 100 mmol / L DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol.

[0049] In step 8) subcritical extraction, the vacuum state refers to a pressure less than 133 Pa, and the extractant is ethanol.

[0050] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0051] The reagents or instruments used in the embodiments of the present invention that are not specified by the manufacturer can all be obtained as conventional reagent products through commercial purchase.

[0052] Example 1

[0053] As Figure 1A method for efficiently purifying phospholipids from coenzyme Q10 fermentation powder, comprising the following steps:

[0054] 1) Mother liquor culture: Dilute Agrobacterium tumefaciens to 1x10 3 cells / mL, then inoculate 0.1 mL onto the slant medium. Place the slant medium in an incubator at a temperature of 30 °C and a relative humidity of 45% for 2 days. Then pick 1 colony and inoculate it into the mother flask medium. Place the mother flask medium in a shaker at 30 °C, a relative humidity of 45%, and a rotation speed of 220 rpm for 24 h to obtain the mother liquor strain;

[0055] 2) Seed tank culture: Inoculate the mother liquor strain into the seed tank medium in the seed tank at an inoculation amount of 1‰. Culture at 30 °C, a tank pressure of 0.03 MPa, an aeration ratio of 0.3, and a stirring speed of 200 rpm for 24 h to obtain a primary seed liquid with a residual glucose amount of 0.5 - 1.0 g / L, uniform cell shape, and good sterility;

[0056] 3) Fermentation tank culture: Inoculate the seed liquid into the fermentation medium in the fermentation tank at an inoculation amount of 5%. Culture at 35 °C, a tank pressure of 0.03 MPa, an aeration ratio of 0.6, and a stirring speed of 110 rpm for 24 h;

[0057] 4) Fermentation broth centrifugation: Centrifuge the fermentation broth at 8000 rpm at low temperature for 20 min, and take the precipitate to obtain the cells;

[0058] 5) High-pressure homogenization: Add the cell precipitate to the cell lysate for dissolution according to a solid-liquid ratio of 1:10 by mass, then add it to a high-pressure homogenizer for cell disruption, and collect the cell disruption liquid;

[0059] 6) Centrifugation of cell disruption liquid: Centrifuge the cell disruption liquid at 12000 rpm at low temperature, discard the precipitate, and take the supernatant;

[0060] 7) Vacuum freeze-drying: Perform vacuum freeze-drying on the supernatant with a vacuum degree less than 133 Pa and a temperature below -50 °C to obtain the dried fermentation powder;

[0061] 8) Subcritical extraction: Put the dried fermentation powder into the extraction tank. After evacuating the extraction tank to a vacuum state, add the extractant for subcritical extraction. After the extraction is completed, pour the subcritical fluid containing fat-soluble components obtained from the extraction from the extraction tank into the stripping tank for heating and reduced-pressure evaporation. After the solvent is completely removed, obtain the crude extract;

[0062] 9) Acetone extraction: Add the crude extract to a 50% by mass acetone solution, cool and crystallize at 4 °C for 4 h, and then filter through a 0.45 μm filter to obtain the filter cake. Preferably, the mass ratio of the crude extract to the acetone solution is 1:5;

[0063] 10) Rotary evaporation: After rinsing the filter cake with deionized water, rotary evaporate to remove water and organic solvents to obtain phospholipids;

[0064] The composition of the slant medium in step 1) is as follows: peptone 8 g / L, yeast extract 5 g / L, sodium chloride 3 g / L, agar 18 g / L, pH 7.2, atrazine 0.05 g / L, pH 7.4;

[0065] The composition of the seed tank medium in step 2) is as follows: sucrose 35 g / L, corn steep liquor 15 g / L, yeast extract 10 g / L, NaCl 3 g / L, atrazine 0.03 g / L, pH 7.4;

[0066] The composition of the fermentation medium in step 3) is as follows: sucrose 45 g / L, corn steep liquor 30 g / L, yeast extract 10 g / L, NaCl 3 g / L, sodium dihydrogen phosphate 0.5 g / L, disodium hydrogen phosphate 0.5 g / L, nicotinamide adenine dinucleotide 0.02 g / L;

[0067] In step 5) high-pressure homogenization, the working pressure is 1200 Bar and homogenize 3 times;

[0068] The cell lysate in step 5) has the following composition: 50 mmol / L Tris-Cl pH 6.8, 100 mmol / L DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol.

[0069] In step 8) subcritical extraction, the vacuum state means the pressure is less than 133 Pa, and the extractant is ethanol.

[0070] Example 2

[0071] Such as Figure 1 A method for efficiently purifying phospholipids from coenzyme Q10 fermentation powder as shown, comprising the following steps:

[0072] 1) Mother liquor culture: Dilute Agrobacterium tumefaciens to 2x10 3 cells / mL and inoculate 0.5 mL onto the slant medium. Place the slant medium in an incubator at 32 °C and a relative humidity of 50% for 4 days. Then pick 1 colony and inoculate it into the mother flask medium. Place the mother flask medium in a shaker at 32 °C, a relative humidity of 50%, and a rotation speed of 300 rpm for 36 h to obtain the mother liquor strain;

[0073] 2) Seed tank culture: Inoculate the mother liquor strain into the seed tank medium in the seed tank at an inoculation amount of 2‰. Culture at 33 °C, a tank pressure of 0.04 MPa, an aeration ratio of 0.5, and a stirring speed of 220 rpm for 36 h to obtain a primary seed liquor with a glucose residue of 0.5 - 1.0 g / L, uniform cell shape, and good sterility;

[0074] 3) Fermentation tank culture: Inoculate the seed liquid into the fermentation medium in the fermentation tank at an inoculation amount of 8%, and culture at 36 °C, tank pressure 0.05 MPa, aeration ratio 0.7, stirring speed 130 rpm for 32 h;

[0075] 4) Centrifugation of fermentation broth: Centrifuge the fermentation broth at 12000 rpm at low temperature for 15 min, and take the precipitate to obtain the thallus;

[0076] 5) High-pressure homogenization: After dissolving the thallus precipitate in the thallus lysate according to the solid-liquid ratio of 1:15 by mass, add it to a high-pressure homogenizer for thallus disruption, and collect the thallus disruption liquid;

[0077] 6) Centrifugation of thallus disruption liquid: Centrifuge the thallus disruption liquid at 13500 rpm at low temperature, discard the precipitate, and take the supernatant;

[0078] 7) Vacuum freeze-drying: Perform vacuum freeze-drying on the supernatant, with a vacuum degree less than 133 Pa and a temperature lower than -50 °C to obtain the dried fermentation powder;

[0079] 8) Subcritical extraction: Put the dried fermentation powder into the extraction tank. After evacuating the extraction tank to a vacuum state, add the extractant for subcritical extraction. After the extraction is completed, pour the subcritical fluid containing fat-soluble components obtained by extraction from the extraction tank into the stripping tank, and perform heating and reduced-pressure evaporation. After the solvent is completely removed, obtain the crude extract;

[0080] 9) Acetone extraction: Add the crude extract to an acetone solution with a mass fraction of 55%, cool and crystallize at 8 °C for 6 h, and then filter with 0.45 μm to obtain the filter cake; the mass ratio of the crude extract to the acetone solution is 1:8;

[0081] 10) Rotary evaporation: Rinse the filter cake with deionized water and then rotary evaporate the water and organic solvents to obtain phospholipids;

[0082] The composition of the slant medium in step 1) is as follows: peptone 8 g / L, yeast extract 5 g / L, sodium chloride 3 g / L, agar 18 g / L, pH value 7.2, atrazine 0.05 g / L, pH value 7.4;

[0083] The composition of the seed tank medium in step 2) is as follows: sucrose 35 g / L, corn steep liquor 15 g / L, yeast extract 10 g / L, NaCl 3 g / L, atrazine 0.03 g / L, pH value 7.4;

[0084] The composition of the fermentation medium in step 3) is as follows: sucrose 45 g / L, corn steep liquor 30 g / L, yeast extract 10 g / L, NaCl 3 g / L, sodium dihydrogen phosphate 0.5 g / L, disodium hydrogen phosphate 0.5 g / L, nicotinamide adenine dinucleotide 0.02 g / L;

[0085] In step 5) of high-pressure homogenization, the working pressure is 1400 Bar, and the homogenization is performed 4 times;

[0086] The cell lysate in step 5) is composed of: 50 mmol / L Tris-Cl pH 6.8, 100 mmol / L DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol.

[0087] In step 8) of subcritical extraction, the vacuum state refers to a pressure less than 133 Pa, and the extractant is ethanol.

[0088] Example 3

[0089] As Figure 1 shown, a method for efficiently purifying phospholipids from coenzyme Q10 fermentation powder includes the following steps:

[0090] 1) Mother liquor culture: Dilute Agrobacterium tumefaciens to 5x10 3 cells / mL, then inoculate 1 mL onto the slant medium. Place the slant medium in an incubator at a temperature of 34°C and a relative humidity of 55% for 6 days. Then pick 1 colony and inoculate it into the mother flask medium. Place the mother flask medium in a shaker at 34°C, a relative humidity of 55%, and a rotation speed of 350 rpm for 48 h to obtain the mother liquor strain;

[0091] 2) Seed tank culture: Inoculate the mother liquor strain into the seed tank medium in the seed tank at an inoculation amount of 1-3‰. Culture at 35°C, a tank pressure of 0.05 MPa, an aeration ratio of 0.6, and a stirring speed of 250 rpm for 48 h to obtain a first-stage seed liquid with a residual glucose amount of 1.0 g / L, uniform cell shape, and good sterility;

[0092] 3) Fermentation tank culture: Inoculate the seed liquid into the fermentation medium in the fermentation tank at an inoculation amount of 10%. Culture at 38°C, a tank pressure of 0.06 MPa, an aeration ratio of 0.8, and a stirring speed of 140 rpm for 36 h;

[0093] 4) Fermentation broth centrifugation: Centrifuge the fermentation broth at 15000 rpm at low temperature for 20 min, and take the precipitate to obtain the cells;

[0094] 5) High-pressure homogenization: Add the cell precipitate to the cell lysate for dissolution according to the solid-liquid ratio of 1:20 by mass, then add it to a high-pressure homogenizer for cell disruption, and collect the cell disruption liquid;

[0095] 6) Centrifugation of cell disruption liquid: Centrifuge the cell disruption liquid at 15000 rpm at low temperature, discard the precipitate, and take the supernatant;

[0096] 7) Vacuum freeze-drying: The supernatant was subjected to vacuum freeze-drying with a vacuum degree less than 133 Pa and a temperature below -50 °C to obtain dry fermentation powder;

[0097] 8) Subcritical extraction: The dry fermentation powder was put into an extraction tank. After the inside of the extraction tank was evacuated to a vacuum state, an extraction agent was added for subcritical extraction. After the extraction was completed, the subcritical fluid containing fat-soluble components obtained from the extraction was poured from the extraction tank into a solvent removal tank for heating and reduced-pressure evaporation. After the solvent was completely removed, a crude extract was obtained;

[0098] 9) Acetone extraction: The crude extract was added to an acetone solution with a mass fraction of 60%. It was cooled and crystallized at 10 °C for 8 h, and then filtered through a 0.45 μm filter to obtain a filter cake; the mass ratio of the crude extract to the acetone solution was 1:10;

[0099] 10) Rotary evaporation: The filter cake was rinsed with deionized water and then rotary evaporated to remove water and organic solvents to obtain phospholipids;

[0100] The composition of the slant medium in step 1) is as follows: peptone 8 g / L, yeast extract 5 g / L, sodium chloride 3 g / L, agar 18 g / L, pH value 7.2, atrazine 0.05 g / L, pH value 7.4;

[0101] The composition of the seed tank medium in step 2) is as follows: sucrose 35 g / L, corn steep liquor 15 g / L, yeast extract 10 g / L, NaCl 3 g / L, atrazine 0.03 g / L, pH value 7.4;

[0102] The composition of the fermentation medium in step 3) is as follows: sucrose 45 g / L, corn steep liquor 30 g / L, yeast extract 10 g / L, NaCl 3 g / L, sodium dihydrogen phosphate 0.5 g / L, disodium hydrogen phosphate 0.5 g / L, nicotinamide adenine dinucleotide 0.02 g / L;

[0103] In step 5) high-pressure homogenization, the working pressure is 1500 Bar and homogenization is carried out 6 times;

[0104] The cell lysate in step 5) has the following composition: 50 mmol / L Tris-Cl pH 6.8, 100 mmol / L DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol.

[0105] In step 8) subcritical extraction, the vacuum state refers to a pressure less than 133 Pa, and the extraction agent is ethanol.

[0106] Comparative Example 1

[0107] A method for efficiently purifying phospholipids from coenzyme Q10 fermentation powder. In 1) mother liquor culture and 2) seed tank culture, atrazine 0.05 g / L is not contained, and the others are the same as in Example 2

[0108] Comparative Example 2

[0109] A method for efficiently purifying phospholipids from coenzyme Q10 fermentation powder, using peptone instead of corn steep liquor in the seed tank medium and fermentation medium, and the rest is the same as in Example 2

[0110] Comparative Example 3

[0111] A method for efficiently purifying phospholipids from coenzyme Q10 fermentation powder, 1) The mother liquor culture and 2) The seed tank culture do not contain atrazine at 0.05 g / L, using peptone instead of corn steep liquor in the seed tank medium and fermentation medium, and the rest is the same as in Example 2

[0112] Test Example 1

[0113] Fermentation test

[0114] This test example compares the fermentation processes of Examples 1-3 and Comparative Examples 1-3, comparing the wet bacterial cell mass (the supernatant was discarded after centrifugation at 15000 rpm), coenzyme Q10 content, and phospholipid content obtained per liter of the final fermentation broth. The results are shown in Table 1 and Figure 2-3 。

[0115] Table 1 Wet bacterial cell production, coenzyme Q10, and phospholipid content of different fermentation processes

[0116] Wet cell mass (g) Coenzyme Q10 (g) Phospholipid content (g) Example 1 354 4.8 12.3 Example 2 365 5.0 12.6 Example 3 375 5.1 14.5 Comparative Example 1 245 3.1 6.7 Comparative Example 2 235 2.8 5.9 Comparative Example 3 212 2.9 5.4

[0117] From Table 1 and Figure 2-3 the test data, it can be seen that by adding a trace amount of atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) during the mother liquor culture and seed tank culture processes, it was found that it could effectively inhibit the growth of miscellaneous bacteria, increase the fermentation density of Agrobacterium tumefaciens, obtain a higher cell density and a larger amount of coenzyme Q10 and phospholipids. Further, by adding a high concentration of corn steep liquor to the seed medium and fermentation medium, the fermentation density can be further increased, and the combined action of the two can exert a more significant yield increase effect than the simple addition of single factors

[0118] Comparative Example 4

[0119] A method for efficiently purifying phospholipids from coenzyme Q10 fermentation powder, without using 5) high-pressure homogenization, but using ultrasonic disruption method, and the rest is the same as in Example 2

[0120] Comparative Example 5

[0121] A method for efficiently purifying phospholipids from coenzyme Q10 fermentation powder, without using 8) subcritical extraction, but using ethanol extraction under normal pressure, and the rest is the same as in Example 2

[0122] Test Example 2

[0123] Phospholipid separation test

[0124] This test example compares the phospholipid purification processes of Example 2 and Comparative Examples 4 and 5, and finally compares the yield and purity (HPLC) of the phospholipids obtained after separation and purification per liter of fermentation broth.

[0125] In the determination of the phospholipid purity, the HPLC method is used

[0126] The stationary phase is Hypersil Si-5μ, and the mobile phase is n-hexane - isopropanol - water = 6:8:1 (V / V / V).

[0127] The results are shown in Table 2 and Figure 4 and 5 .

[0128] Table 2 Yield and purity of phospholipids

[0129] Phospholipid yield (g) Purity (%) Example 2 12.6 99.31 Comparative Example 4 8.6 96.23 Comparative Example 5 8.4 85.17

[0130] From the test data in Table 2 and Figure 4-5 It can be seen that high-pressure homogenization and subcritical extraction in the separation process have a greater impact on the final yield and purity of phospholipids. High-pressure homogenization is more suitable for obtaining phospholipids from Agrobacterium tumefaciens cells compared to ultrasound, and the use of subcritical extraction can greatly improve the obtained purity of phospholipids.

[0131] The limited number of preferred embodiments of the invention are described in relatively specific and detailed terms, but this should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the invention, several modifications and improvements can still be made, and these all fall within the protection scope of the invention.

Claims

1. A method for efficiently purifying phospholipids from coenzyme Q10 fermentation powder, characterized in that, It includes the following steps: 1) Mother liquor culture: Dilute Agrobacterium tumefaciens to 1 - 5x10 3 CFU / mL, then inoculate 0.1 - 1 mL onto the slant medium. Place the slant medium in an incubator at a temperature of 30 - 34°C and a relative humidity of 45% - 55% for 2 - 6 days. Then pick 1 colony and inoculate it into the mother flask medium. Place the mother flask medium in a shaker at 30 - 34°C, a relative humidity of 45% - 55%, and a rotation speed of 220 - 350 rpm for 24 - 48 hours to obtain the mother liquor strain; 2) Seed tank culture: Inoculate the mother liquor strain into the seed tank medium in the seed tank at an inoculation amount of 1-3‰, culture at 30-35°C, tank pressure of 0.03-0.05 MPa, aeration ratio of 0.3-0.6, stirring speed of 200-250 rpm for 24-48 h to obtain a primary seed liquid with a residual glucose amount of 0.5-1.0 g / L, uniform cell shape, and good sterility; 3) Fermentation tank culture: Inoculate the seed liquid into the fermentation medium in the fermentation tank at an inoculation amount of 5-10%, culture at 35-38°C, tank pressure of 0.03-0.06 MPa, aeration ratio of 0.6-0.8, stirring speed of 110-140 rpm for 24-36 h; 4) Fermentation broth centrifugation: Centrifuge the fermentation broth at 8000-15000 rpm at low temperature for 10-20 min, and collect the precipitate to obtain cells; 5) High-pressure homogenization: After dissolving the cell precipitate in the cell lysate according to the solid-liquid ratio of 1:10-20 by mass, add it to a high-pressure homogenizer for cell disruption, and collect the cell disruption liquid; 6) Centrifugation of cell disruption liquid: Centrifuge the cell disruption liquid at 12000-15000 rpm at low temperature, discard the precipitate, and take the supernatant; 7) Vacuum freeze-drying: Perform vacuum freeze-drying on the supernatant, with a vacuum degree less than 133 Pa and a temperature below -50°C to obtain dry fermentation powder; 8) Subcritical extraction: Put the dry fermentation powder into the extraction tank, evacuate the extraction tank to a vacuum state, add an extractant for subcritical extraction. After the extraction is completed, pour the subcritical fluid containing liposoluble components obtained from the extraction from the extraction tank into the stripping tank, and perform heating and reduced-pressure evaporation. After the solvent is completely removed, obtain the crude extract; 9) Acetone extraction: Add the crude extract to an acetone solution with a mass fraction of 50-60%, cool and crystallize at 4-10°C for 4-8 h, and then filter through a 0.45 μm filter to obtain a filter cake; 10) Rotary evaporation: Rinse the filter cake with deionized water and then rotary evaporate the water and organic solvents to obtain phospholipids; The composition of the slant medium in step 1) is as follows: peptone 8 g / L, yeast extract 5 g / L, sodium chloride 3 g / L, agar 18 g / L, pH value 7.2, atrazine 0.05 g / L, pH value 7.4; The composition of the seed tank medium in step 2) is as follows: sucrose 35 g / L, corn steep liquor 15 g / L, yeast extract 10 g / L, NaCl 3 g / L, atrazine 0.03 g / L, pH value 7.

4.

2. The method for highly efficiently purifying phospholipids from coenzyme Q10 fermentation powder according to claim 1, characterized in that, The composition of the fermentation medium in step 3) is as follows: sucrose 45 g / L, corn steep liquor 30 g / L, yeast extract 10 g / L, NaCl 3 g / L, sodium dihydrogen phosphate 0.5 g / L, disodium hydrogen phosphate 0.5 g / L, nicotinamide adenine dinucleotide 0.02 g / L.

3. A method for efficiently purifying phospholipids from coenzyme Q10 fermentation powder according to claim 1, characterized in that, In step 5) of high-pressure homogenization, the working pressure is 1200-1500 Bar, and homogenization is performed 3-6 times.

4. A method for highly purifying phospholipids from coenzyme Q10 fermentation powder according to claim 1, characterized in that, The composition of the cell lysate in step 5) is as follows: 50 mmol / L Tris-Cl pH 6.8, 100 mmol / L DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol.

5. A method for highly purifying phospholipids from coenzyme Q10 fermentation powder according to claim 1, characterized in that, In the subcritical extraction in step 8), the vacuum state means a pressure less than 133 Pa, and the extractant is ethanol.

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