CN108997153B - Method for extracting multiple high-value substances from photosynthetic bacteria step by step - Google Patents
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- CN108997153B CN108997153B CN201810826047.1A CN201810826047A CN108997153B CN 108997153 B CN108997153 B CN 108997153B CN 201810826047 A CN201810826047 A CN 201810826047A CN 108997153 B CN108997153 B CN 108997153B
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- C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
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- C07C403/24—Derivatives of cyclohexane or of a cyclohexene or of cyclohexadiene, having a side-chain containing an acyclic unsaturated part of at least four carbon atoms, this part being directly attached to the cyclohexane or cyclohexene or cyclohexadiene rings, e.g. vitamin A, beta-carotene, beta-ionone having side-chains substituted by six-membered non-aromatic rings, e.g. beta-carotene
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- C07D487/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains four or more hetero rings
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- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention discloses a method for extracting various high-value substances from photosynthetic bacteria step by step. The invention adopts a step-by-step extraction method to determine the optimal extraction sequence for the high-value substances of coenzyme Q10, carotenoid, bacteriochlorin, 5-ALA and protein in the photosynthetic bacteria after the high-value substances are examined according to the factors of market price, difficulty and easiness in extraction, whether thallus is destructive and the like, extracts the high-value substances in the photosynthetic bacteria to the maximum extent, can synchronously extract other substances while ensuring the yield of the preferentially extracted substances, and finally uses the residual thallus to purify the protein, thereby achieving the maximum extent of utilization of thallus resources. The adopted technology has low energy consumption and cost and mild condition, can effectively ensure the components of the extract and can obtain high-quality extract and other functional components.
Description
Technical Field
The invention belongs to the technical field of biological processing, and particularly relates to a method for extracting multiple high-value substances from photosynthetic bacteria step by step.
Background
Photosynthetic bacteria contain various abundant nutrients, of which coenzyme Q10, carotenoid, bacteriochlorin, 5-ALA and single-cell protein are the most prominent and typical ones, and are called high-value substances. Some of the high-value substances can be used as raw materials in the industries of planting, medicine and cosmetics, some have the effects of delaying senility, inhibiting tumors, resisting oxidation and the like, and some can be used as plant additives and animal feed additives, so that the high-value substances have high economic value and social benefit. The existing research already introduces methods for extracting different high-value substances in photosynthetic bacteria, wherein the research on carotenoids is more, mainly ultrasonic-assisted extraction (UAE), pressurized liquid-phase extraction, Pulsed Electric Field (PEF) extraction and enzyme-assisted extraction (UNK), while the research on methods for extracting substances such as coenzyme Q10 and 5-ALA is less, the existing extraction method of coenzyme Q10 mainly adopts freeze thawing + extraction method, ultrasonic + extraction method, enzymolysis-assisted ultrasonic extraction method, alkaline saponification method and the like, and only a few researches show that a small amount of 5-ALA can be extracted from photosynthetic bacteria. So far, the existing research focuses on the single extraction of a certain high-value substance in photosynthetic bacteria, only one substance can be extracted at a time, only partial value of the photosynthetic bacteria can be exerted, and no researcher continuously and simultaneously extracts a plurality of high-value substances in the photosynthetic bacteria, so that a large amount of other resources in thalli are wasted. Therefore, if one method is available, various high-value substances in the photosynthetic bacteria can be extracted at one time, the thallus resources can be utilized to the maximum extent, and greater economic benefit and social value are realized.
Disclosure of Invention
The invention aims to provide a method for extracting multiple high-value substances from photosynthetic bacteria step by step.
The invention provides a method for extracting coenzyme Q10, carotenoid, bacteriochlorin, 5-ALA and protein from photosynthetic bacteria step by step, which comprises the following steps:
1) centrifuging the photosynthetic bacteria liquid, removing supernatant, collecting thallus precipitate, and breaking cell wall;
2) extracting coenzyme Q10 from the system with the broken cell walls in the step 1), and collecting the obtained supernatant to obtain coenzyme Q10;
3) collecting the lower layer bacterial liquid of the system obtained by extraction in the step 2), extracting carotenoid and bacteriochlorin, and collecting the obtained supernatant to obtain carotenoid and bacteriochlorin;
4) collecting the lower layer bacterial liquid of the system obtained by extraction in the step 3), and extracting 5-ALA;
5) collecting the lower layer bacterial liquid of the system obtained by extraction in the step 4), and extracting the protein.
In the step 1) of the method, the centrifugal speed is 6000-; specifically 9000 r/min; the time is 5 to 15 minutes; specifically 10 minutes;
in the step of breaking cell walls, the breaking method comprises the following steps: mixing the thallus precipitate with a solvent, and placing the mixture in an ultrasonic cell crusher for ultrasonic treatment; the ultrasonic cell pulverizer can be JY 92-IIDN;
specifically, the solvent is acetone;
the dosage of the solvent is 70-73 ml/g DCW, specifically 71.74ml/g DCW;
in the ultrasonic step, the ultrasonic power is 300-600W; in particular 400W; the ultrasonic temperature is 10-25 ℃; the ultrasound is carried out for 2s every time at intervals of 1 s; the ultrasonic time is 5-20 min.
In the step 2), the extraction method is oscillation in a shaking table;
specifically, the temperature of the shaking table is 20-40 ℃, specifically 25 ℃; the oscillation time is 20-40 min; the rotating speed is 85-135 r/min; in particular 120 r/min.
In the step 3), the extraction method is to stand in an extraction solvent;
specifically, the extraction solvent is acetone and methanol; in the used extraction solvent, the volume ratio of acetone to methanol is 6-8: 2, specifically 7: 2; the volume ratio of the lower layer bacterial liquid to the extraction solvent is 1-3: 1; specifically 2: 1; the extraction temperature is 20-40 deg.C; the standing time is 1-6 hours.
In the step 4), the extraction method comprises the following steps:
adding a 5-ALA extraction solvent into the lower layer bacterial liquid of the system obtained by the extraction in the step 3), uniformly mixing, heating in a water bath, cooling to room temperature, centrifuging, and collecting the supernatant to finish the extraction of the 5-ALA.
Specifically, the 5-ALA extraction solvent consists of a sodium acetate buffer solution and acetylacetone;
the concentration of the sodium acetate buffer solution is 1-2 mol/L; the pH value is 4-5; in particular 4.6;
the volume ratio of the sodium acetate buffer solution to the acetylacetone is 0.8-1.5: 0.25 to 1; specifically, 1: 0.25 mL; the volume ratio of the bacterial liquid to the sodium acetate buffer solution is 9-11: 1; specifically, 10: 1;
in the step of heating in the water bath, the temperature is 90-100 ℃; the time is 20-40 min; specifically 30 min;
in the centrifugation step, the centrifugation rotating speed is 6000-; specifically 9000 r/min; the time is 3-6 minutes; specifically 5 minutes.
The absorbance of 5-ALA can be measured using a modified Ehrlich reagent. The improved Ehrlich reagent can be prepared according to the following method: in a 50mL measuring cylinder, 30mL of glacial acetic acid, 1g of p-dimethylaminobenzaldehyde and 8mL of 70% perchloric acid were sequentially added, and after dissolution, the mixture was diluted to 50mL with glacial acetic acid and mixed well. The mixture is contained in a brown bottle and stored in a refrigerator to be prepared and used immediately.
In the step 5), the extraction method is a salting-out method. The method is a conventional method for extracting protein.
Specifically, the salting-out method comprises the following steps: adding a Tris-HCL buffer solution with the pH value of 8.0 and the concentration of 10mM into the lower layer bacterial liquid of the system extracted in the step 4), adding 10ml of buffer solution into each g of wet thalli to suspend cells, and repeatedly centrifuging for 2-3 times; adding 1% Triton-X100 and 0.1mol/l NaCl into the thallus, stirring at room temperature in the dark for 2 hours, sealing the mouth with a preservative film, and standing in a refrigerator at 4 ℃ overnight to solubilize membrane protein; adding the overnight solubilized membrane protein solution into a centrifuge tube for balancing, centrifuging at 9000r/min for 30min, and collecting supernatant; adding 60% ammonium sulfate solution into the collected supernatant, standing in a refrigerator at 4 deg.C for 1min, centrifuging the standing solution at 9000r/min for 30min, collecting supernatant aerosol, and dissolving in 10mM Tris-HCl buffer solution (pH8.0) to obtain crude protein extract;
and then placing the obtained crude protein extract in a dialysis bag, sealing, placing the dialysis bag in a big beaker filled with 10mM Tris-HCL buffer solution with the pH value of 8.0 for overnight dialysis, and obtaining the purified protein after 2-3 times of dialysis. The absorbance of the protein was measured at 280nm using an ultraviolet spectrophotometer, and the protein concentration was calculated using an empirical formula.
In addition, the invention also claims a method for extracting coenzyme Q10, carotenoid and bacteriochlorin from photosynthetic bacteria step by step, and the method comprises the steps 1) to 3) of the method.
Aiming at the characteristics of rich varieties and high content of high-value substances in photosynthetic bacteria, comprehensive extraction is carried out on the basis of integrating the respective extraction methods of each substance, and coenzyme Q10, carotenoid, bacteriochlorin, 5-ALA and protein are extracted successively by the same batch of bacteria, so that the high-value substances in the photosynthetic bacteria are extracted and utilized to the maximum extent, and the recycling effect of the high-value substances is better exerted.
The invention has the following beneficial effects:
1. the invention adopts a step-by-step extraction method to determine the optimal extraction sequence for the high-value substances of coenzyme Q10, carotenoid, bacteriochlorin, 5-ALA and protein in the photosynthetic bacteria after the high-value substances are examined according to the factors of market price, difficulty and easiness in extraction, whether thallus is destructive and the like, extracts the high-value substances in the photosynthetic bacteria to the maximum extent, can synchronously extract other substances while ensuring the yield of the preferentially extracted substances, and finally uses the residual thallus to purify the protein, thereby achieving the maximum extent of utilization of thallus resources. The adopted technology has low energy consumption and cost and mild condition, can effectively ensure the components of the extract and can obtain high-quality extract and other functional components.
2. The method has the advantages of simple equipment, safe and simple operation, strong operation repeatability, and contribution to popularization and application in industrial production and greater economic benefit. The obtained coenzyme Q10 can be used for processing and manufacturing medical health products, the carotenoid and the bacteriochlorin can be used as additives of edible pigments, the 5-ALA can be used as a green herbicide and a photodynamic agent, the protein can be used as raw materials of health products and feed additives, a foam extinguishing agent and the like, and the method has remarkable economic and social benefits.
Drawings
FIG. 1 is a technical scheme of the process.
FIG. 2 is a graph showing the content of each high-value substance obtained by the method and the effect of the extraction of each substance alone.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified. The modified Ehrlich reagents used in the following examples were prepared as follows: in a 50mL measuring cylinder, 30mL of glacial acetic acid, 1g of p-dimethylaminobenzaldehyde and 8mL of 70% perchloric acid were sequentially added, and after dissolution, the mixture was diluted to 50mL with glacial acetic acid and mixed well. The mixture is contained in a brown bottle and stored in a refrigerator to be prepared and used immediately.
Example 1 extraction of coenzyme Q10, carotenoids, bacteriochlorins, 5-ALA and proteins from photosynthetic bacteria in stages
1) Centrifuging the photosynthetic bacteria liquid for 10 minutes at 9000r/min, discarding supernatant, washing with deionized water for 2-3 times, collecting thallus precipitate, adding acetone (71.74ml/g DCW), and crushing cells with an ultrasonic cell crusher under the following crushing conditions: the ultrasonic power is 400W, the ultrasonic temperature is 10 ℃, the ultrasonic time is 5min, and the working time is 2s and 1 s;
2) extraction of coenzyme Q10:
oscillating for 40min at 25 ℃ in a shaking table, and rotating at 120 r/min;
taking the supernatant, and detecting the content of coenzyme Q10 by a colorimetric method;
detecting carotenoid absorbance at 473nm wavelength and bacteriochlorin absorbance at 771nm wavelength by using an ultraviolet spectrophotometer; detecting the content of 5-ALA by using an improved Ehrlich reagent to obtain the coenzyme Q10 with the content of 84.4mg/g, the concentration of carotenoid of 1.132mg/L, the concentration of bacteriochlorin of 0.997mg/L and the content of 5-ALA of 2.145 mg/g;
3) extraction of carotenoids and bacteriochlorins:
collecting the lower layer bacterial liquid of the system extracted in the step 2), and supplementing acetone and methanol to ensure that the acetone: methanol 7:2(v: v) and left to stand at 40 ℃ for 1 hour; the volume ratio of the lower layer bacterial liquid to the extraction solvent is 2: 1;
collecting supernatant, detecting carotenoid absorbance at 473nm wavelength and bacteriochlorin absorbance at 771nm wavelength with ultraviolet spectrophotometer to obtain carotenoid concentration of 3.704mg/L, bacteriochlorin concentration of 1.176mg/L and 5-ALA content of 2.224 mg/g;
4) extraction of 5-ALA: washing the lower layer bacterial liquid of the system extracted in the step 3) by using deionized water, adding sodium acetate buffer solution (1mol/L, pH 4.6), and preparing the bacterial liquid: sodium acetate buffer 10:1(v: v) and acetylacetone, bacterial solution: mixing acetylacetone 40:1(v: v), heating in 100 deg.C water bath for 30min, cooling to room temperature, centrifuging at 9000r/min for 5min, collecting supernatant, and detecting 5-ALA absorbance with improved Ehrlich reagent; the content of 5-ALA is 4.409 mg/g;
5) protein extraction:
adding a Tris-HCL buffer solution with the pH value of 8.0 and the concentration of 10mM, suspending cells according to the proportion of adding 10ml of the buffer solution into each g of wet thalli, and repeatedly centrifuging for 2-3 times; adding 1% Triton-X100 and 0.1mol/l NaCl into the thallus, stirring at room temperature in the dark for 2 hours, sealing the mouth with a preservative film, and standing in a refrigerator at 4 ℃ overnight to solubilize membrane protein; the overnight solubilized membrane protein solution was added to the centrifuge tube and equilibrated, centrifuged at 9000r/min for 30min and the supernatant collected. Adding 60% ammonium sulfate solution into the collected supernatant, standing in a refrigerator at 4 deg.C for 1min, centrifuging the standing solution at 9000r/min for 30min, collecting supernatant aerosol, and dissolving in 10mM Tris-HCl buffer solution (pH8.0) to obtain crude protein extract;
then placing the obtained crude protein extract into a dialysis bag, sealing, placing the dialysis bag into a big beaker containing 10mM Tris-HCL buffer solution with the pH value of 8.0 for overnight dialysis, and obtaining purified protein after 2-3 times of dialysis;
the absorbance of the protein was measured at 280nm with an ultraviolet spectrophotometer, and the protein concentration was calculated using an empirical formula to obtain a protein content of 335.868 mg/g.
FIG. 2 is a graph showing the content of each high-value substance obtained by the method and the effect of the extraction of each substance alone. As can be seen from the figure, the experimental method can realize the effective extraction of 97.4% of coenzyme Q10 in photosynthetic bacteria, and the extraction effects of the carotenoid and the chlorophyll are improved by more than 100% compared with the single extraction effect, the extraction effect of the 5-ALA only accounts for 34.4% of the single extraction effect, and the extraction effect of the protein accounts for 75.0% of the single extraction effect.
Claims (14)
1. A method for extracting coenzyme Q10, carotenoid, bacteriochlorin, 5-ALA and protein from photosynthetic bacteria in a step-by-step manner comprises the following steps:
1) centrifuging the photosynthetic bacteria liquid, removing supernatant, collecting thallus precipitate, and breaking cell wall;
2) extracting coenzyme Q10 from the system with the broken cell walls in the step 1), and collecting the obtained supernatant to obtain coenzyme Q10;
3) collecting the lower layer bacterial liquid of the system obtained by extraction in the step 2), extracting carotenoid and bacteriochlorin, and collecting the obtained supernatant to obtain carotenoid and bacteriochlorin;
4) collecting the lower layer bacterial liquid of the system obtained by extraction in the step 3), and extracting 5-ALA;
5) collecting the lower layer bacterial liquid of the system obtained by extraction in the step 4), and extracting the protein.
2. The method of claim 1, wherein: in the step 1), the centrifugal speed is 6000-10000 r/min; the time is 5 to 15 minutes;
in the step of breaking cell walls, the breaking method comprises the following steps: and mixing the thallus precipitate with a solvent, and putting the mixture into an ultrasonic cell crusher for ultrasonic treatment.
3. The method of claim 2, wherein: the ultrasonic cell pulverizer is Ningbo Xinzhi ultrasonic cell pulverizer JY 92-IIDN.
4. The method of claim 3, wherein: the solvent is acetone;
the dosage of the solvent is 70-73 ml/g DCW;
in the ultrasonic step, the ultrasonic power is 300-600W; the ultrasonic temperature is 10-25 ℃; the ultrasound is carried out for 2s every time at intervals of 1 s; the ultrasonic time is 5-20 min.
5. The method of claim 1, wherein: in the step 2), the extraction method is shaking in a shaking table.
6. The method of claim 5, wherein: the temperature of the shaking table is 20-40 ℃; the oscillation time is 20-40 min; the rotation speed is 85-135 r/min.
7. The method of claim 1, wherein: in the step 3), the extraction method is to stand in an extraction solvent.
8. The method of claim 7, wherein: in the step 3), the used extraction solvents are acetone and methanol; in the used extraction solvent, the volume ratio of acetone to methanol is 6-8: 2; the volume ratio of the lower layer bacterial liquid to the extraction solvent is 1-3: 1; the extraction temperature is 20-40 deg.C; the standing time is 1-6 hours.
9. The method of claim 8, wherein: in the step 3), the volume ratio of acetone to methanol in the extraction solvent is 7: 2; the volume ratio of the lower layer bacterial liquid to the extraction solvent is 2: 1.
10. The method of claim 1, wherein: in the step 4), the extraction method comprises the following steps:
adding a 5-ALA extraction solvent into the lower layer bacterial liquid of the system obtained by the extraction in the step 3), uniformly mixing, heating in a water bath, cooling to room temperature, centrifuging, and collecting the supernatant to finish the extraction of the 5-ALA.
11. The method of claim 10, wherein: the 5-ALA extraction solvent consists of sodium acetate buffer solution and acetylacetone.
12. The method of claim 11, wherein: the concentration of the sodium acetate buffer solution is 1-2 mol/L; the pH value is 4-5;
the volume ratio of the sodium acetate buffer solution to the acetylacetone is 0.8-1.5: 0.25 to 1; the volume ratio of the bacterial liquid to the sodium acetate buffer solution is 9-11: 1;
in the step of heating in the water bath, the temperature is 90-100 ℃; the time is 20-40 min;
in the centrifugation step, the centrifugation rotating speed is 6000-; the time is 3-6 minutes.
13. The method according to any one of claims 1-12, wherein: in the step 5), the extraction method is a salting-out method.
14. A method for stepwise extraction of coenzyme Q10, carotenoids and bacteriochlorins from photosynthetic bacteria, comprising steps 1) to 3) of the method of any one of claims 1-13.
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