Method for rapidly extracting industrial microorganism lycopene
By using mild alkaline alkaline sodium carbonate solution and enzymatic lysis technology, combined with low toxic extractants, the problem of using toxic solvents and high-cost equipment in the existing lycopene extraction methods is solved, and an efficient, environmentally friendly and economical lycopene extraction effect is achieved.
Patent Information
- Application Number
- CN202510137477.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-07
- Publication Date
- 2025-05-16
AI Technical Summary
The existing lycopene extraction methods have problems such as the use of toxic solvents, high equipment costs, complex operation and environmental pollution, making it difficult to achieve large-scale industrial production.
The alkaline mild sodium carbonate solution is used for saponification, combined with the use of enzymatic lysis and low toxic extractants, and efficient extraction of lycopene is achieved by adjusting the temperature, time and extractant ratio.
It significantly improves the extraction efficiency of lycopene, reduces the amount of solvent used, reduces production costs, and improves production efficiency. The solvent content of the extract is low and the environmental pollution is less.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microbial fermentation product extraction, in particular to a method for rapidly extracting industrial microbial lycopene. Background Art
[0002] Lycopene is a type of carotenoid and also a fat-soluble natural pigment. It is widely found in tomatoes, watermelons, papayas, grapes, plums and guavas. It was first extracted and separated from tomatoes, so it is called lycopene. Lycopene is the most effective antioxidant active substance known so far. Its antioxidant capacity is 2-3.2 times that of β-carotene and 100 times that of vitamin E. Lycopene has a strong ability to scavenge free radicals and destroy singlet oxygen. It can relieve aging, improve the body's immunity, and prevent the occurrence of prostate cancer, breast cancer and digestive tract (including colon, rectum and stomach) cancer. The excellent performance of lycopene has attracted widespread attention at home and abroad, and the potential value of lycopene has not been fully developed. However, the human body cannot synthesize this substance by itself and can only extract it from other substances, so the extraction of lycopene has become an important issue.
[0003] The main methods for extracting lycopene include organic solvent extraction, microwave-assisted extraction, ultrasonic-assisted extraction, supercritical fluid extraction, etc. The organic solvent extraction process is relatively simple, but the use of acetone makes the extractant toxic, there will be toxic solvent residues in the product and the production cost is high; the microwave-assisted extraction method is limited to a small amount of extraction experiments and does not have the ability for large-scale industrial production; because the microwave-assisted extraction method uses microwave radiation, it may cause certain heat-sensitive substances to degrade during the extraction process, and the microwave radiation leaked from the microwave equipment will cause chronic damage to the human body. The equipment is expensive and requires professional operators to operate and maintain it.
[0004] Organic solvent extraction technology is a relatively widely used extraction technology for extracting lycopene in industry. This technology is simple to use and convenient for industrial production. Under different extractants, the proportion of organic reagents can be adjusted according to the different solubility of lycopene, making the extraction effect more efficient and convenient. At the same time, the enzyme digestion method can be used to break the wall to better release lycopene, adjust the proportion of acetone in the extractant, use less or even no acetone, making industrial extraction safer and more economical; and the structure of the substance will not be destroyed during the rotary evaporation separation process, and no other impurities will be left in the extract after the separation. The new method for rapid extraction of lycopene from industrial microorganisms does not have too much organic solvent, which prevents some solvents from polluting the environment. Summary of the invention
[0005] In order to overcome the above-mentioned defects of the prior art, the present invention provides a method for rapidly extracting industrial microbial lycopene.
[0006] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions: The present invention provides a method for rapidly extracting industrial microbial lycopene, the method comprising the following steps: (1) Centrifuge the E. coli fermentation broth at 8000 rpm, discard the supernatant, and wash twice with water; (2) Add 0.03 mol / L Na2CO3 solution and saponify at 40°C for 30 min, then centrifuge and discard the supernatant; (3) Prepare the precipitate into a bacterial solution, adjust the pH to 3 to 8, add 25 mg / mL enzyme solution, incubate at 37°C for 6 h, and centrifuge to discard the supernatant; (4) Add the extractant, shake in the dark at room temperature for 1 min, centrifuge at 8000 rpm, and measure the absorbance of the supernatant containing lycopene at 472 nm.
[0007] Furthermore, the method according to claim 1 is characterized in that the ratio of the fermentation liquid in step (1) to the saponifying agent used subsequently is 1:2.
[0008] Furthermore, the method according to claim 1 is characterized in that the saponifying agent in step (2) is a 0.03 mol / L Na2CO3 solution.
[0009] Furthermore, the method according to claim 1 is characterized in that the volume of the bacterial liquid in step (3) is the same as that of the previous fermentation liquid.
[0010] Furthermore, the method according to claim 1 is characterized in that the enzymatic agent in step (3) is 25 mg / mL lysozyme solution, and the amount added is 100 μL of enzyme solution per 1 mL of fermentation liquid.
[0011] Furthermore, the method according to claim 1 is characterized in that the extractant in step (4) is a mixed solution of n-hexane:ethanol (1:2).
[0012] Furthermore, according to the method of claim 1, it is characterized in that the existing lycopene extraction method is to add an extractant (methanol:acetone=1:1) to the bacterial liquid, shake it in the dark for 5 minutes, and centrifuge it at 8000 rpm to obtain a supernatant containing lycopene.
[0013] Furthermore, according to the method of claim 1, it is characterized in that the lycopene standard curve determination method is to dilute the lycopene standard product with different concentration gradients of the extractant, measure the absorbance values at 472 nm respectively, and obtain the standard curve of lycopene after correction with n-hexane solution.
[0014] Furthermore, according to the method of claim 1, it is characterized in that the lycopene determination method is to take the supernatant of the last centrifugation, use the extractant as a blank control, determine the absorbance value x at 472 nm, and obtain the lycopene yield y (g / L) = (x+0.0261) / 0.1502 according to the standard curve.
[0015] Beneficial effects of the present invention: The present invention first uses a mild alkaline sodium carbonate solution for treatment, which can avoid the degradation of lycopene and preliminarily improve the extraction effect of lycopene; the presence of the stabilizer further avoids the decomposition of lycopene; the extraction method of the present invention can significantly improve the extraction efficiency of lycopene, making the release of lycopene more complete, and the extraction rate is greatly improved compared with the traditional method. By adjusting parameters such as pressure, temperature, and time, the lycopene yield is increased while achieving the purpose of low solvent content in lycopene extract and less environmental pollution in the production process. The entire extraction process is relatively simple, the requirements for equipment are not high, and it is easy to carry out industrial large-scale production, thereby effectively reducing production costs and improving production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 The lycopene content of the embodiment and the comparative example. DETAILED DESCRIPTION
[0017] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. The described embodiments are only part of the embodiments of the present invention, not all of the embodiments. The scope and core content of the present invention are determined according to the claims.
[0018] Embodiment 1:
[0019] Add 1 mL of fermentation broth to the centrifuge tube, centrifuge at 8000 rpm for 10 min, and discard the supernatant.
[0020] Add 1 mL of ddH2O to wash, centrifuge at 8000 rpm for 10 min, and discard the supernatant.
[0021] Add 30 mL of 0.03 mol / L Na2CO3 solution, saponify at 20°C for 30 min, centrifuge at 8000 rpm for 10 min, and discard the supernatant.
[0022] Add 1 mL of 0.5 mol / L hydrochloric acid, incubate in a 30°C water bath for 20 min, centrifuge at 8000 rpm and 10°C for 10 min, and discard the supernatant.
[0023] Add 10 mL of a 1:1 mixture of methanol and acetone, shake at room temperature in the dark for 5 min, and then centrifuge at 8000 rpm and 10°C for 10 min.
[0024] Take 1 mL of supernatant, add 2 mL of extractant and measure its absorbance at 472 nm. 472 It is 0.1066.
[0025] Embodiment 2:
[0026] Add 1 mL of fermentation broth to the centrifuge tube, centrifuge at 8000 rpm for 10 min, and discard the supernatant.
[0027] Add 1 mL ddH2O to wash, centrifuge at 8000 rpm for 10 min, and discard the supernatant.
[0028] Add 30 mL of 0.03 mol / L Na2CO3 solution, saponify at 40°C for 30 min, centrifuge at 8000 rpm for 10 min, and discard the supernatant.
[0029] Add 1 mL of 0.5 mol / L hydrochloric acid, incubate at 30°C for 20 min, centrifuge at 8000 rpm and 10°C for 10 min, and discard the supernatant.
[0030] Add 10 mL of a 1:1 mixture of methanol and acetone, shake at room temperature in the dark for 5 min, and then centrifuge at 8000 rpm and 10°C for 10 min.
[0031] Take 1 mL of supernatant, add 2 mL of extractant and measure its absorbance at 472 nm. 472 It is 0.1954.
[0032] Embodiment 3:
[0033] Add 1 mL of fermentation broth to the centrifuge tube, centrifuge at 8000 rpm for 10 min, and discard the supernatant.
[0034] Add 1 mL of ddH2O to wash, centrifuge at 8000 rpm for 10 min, and discard the supernatant.
[0035] Add 30 mL of 0.03 mol / L Na2CO3 solution and saponify at 40°C for 30 min in a water bath. After saponification, centrifuge at 8000 rpm at room temperature for 10 min and discard the supernatant.
[0036] Add 1 mL of 0.5 mol / L hydrochloric acid, incubate in a 30°C water bath for 20 min, centrifuge at 8000 rpm and 10°C for 10 min, and discard the supernatant.
[0037] Add 10 mL of hexane:acetone:ethanol (2:1:1) extractant, shake at room temperature in the dark for 5 min, and then centrifuge at 8000 rpm and 10°C for 10 min.
[0038] Take 1 mL of supernatant, add 2 mL of extractant and measure its absorbance at 472 nm. 472 It is 0.8013.
[0039] Embodiment 4:
[0040] Add 1 mL of fermentation broth to the centrifuge tube, centrifuge at 8000 rpm for 10 min, and discard the supernatant.
[0041] Add 1 mL of ddH2O to wash, centrifuge at 8000 rpm for 10 min, and discard the supernatant.
[0042] Add 30 mL of 0.03 mol / L Na2CO3 solution, saponify at 40°C for 30 min, centrifuge at 8000 rpm for 10 min, and discard the supernatant.
[0043] Add 1 mL of 0.5 mol / L hydrochloric acid, incubate in a 30°C water bath for 20 min, centrifuge at 8000 rpm and 10°C for 10 min, and discard the supernatant.
[0044] Add 10 mL of a mixed solution of hexane:acetone:ethanol (2:1:1), shake in the dark at 20°C for 5 min, and then centrifuge at 8000 rpm at 10°C for 10 min.
[0045] Take 1 mL of supernatant, add 2 mL of extractant and measure its absorbance at 472 nm. After extraction and centrifugation at 20°C, stratification occurs. 472 It is 0.5932, which is higher than the unlayered and lower layer solutions.
[0046] Embodiment 5:
[0047] Add 1 mL of fermentation broth to the centrifuge tube, centrifuge at 8000 rpm for 10 min, and discard the supernatant.
[0048] Add 1 mL of ddH2O to wash, centrifuge at 8000 rpm for 10 min, and discard the supernatant.
[0049] Add 30 mL of 0.03 mol / L Na2CO3 solution, saponify at 40°C for 30 min, centrifuge at 8000 rpm for 10 min, and discard the supernatant.
[0050] Add 2 mL of 0.5 mol / L hydrochloric acid, incubate in a 30°C water bath for 20 min, centrifuge at 8000 rpm and 10°C for 10 min, and discard the supernatant.
[0051] Add 10 mL of a mixed solution of hexane:acetone:ethanol (2:1:1), shake in the dark at 20°C for 5 min, and then centrifuge at 8000 rpm at 10°C for 10 min.
[0052] Take 1 mL of supernatant, add 2 mL of extractant and measure its absorbance at 472 nm. 472 It is 0.2627.
[0053] Embodiment 6:
[0054] Add 1 mL of fermentation broth to the centrifuge tube, centrifuge at 8000 rpm for 10 min, and discard the supernatant.
[0055] Add 1 mL of ddH2O to wash, centrifuge at 8000 rpm for 10 min, and discard the supernatant.
[0056] Add 30 mL of 0.03 mol / L Na2CO3 solution, saponify at 40°C for 30 min, centrifuge at 8000 rpm for 10 min, and discard the supernatant.
[0057] Add 2 mL of 0.5 mol / L hydrochloric acid, incubate in a 30°C water bath for 15 min, centrifuge at 8000 rpm and 10°C for 10 min, and discard the supernatant.
[0058] Add 10 mL of a mixed solution of hexane:acetone:ethanol (2:1:1), shake in the dark at 20°C for 5 min, and then centrifuge at 8000 rpm at 10°C for 10 min.
[0059] Take 1 mL of supernatant, add 2 mL of extractant and measure its absorbance at 472 nm. 472 It is 0.3341.
[0060] Embodiment 7:
[0061] Add 1 mL of fermentation broth to the centrifuge tube, centrifuge at 8000 rpm for 10 min, and discard the supernatant.
[0062] Add 1 mL of ddH2O to wash, centrifuge at 8000 rpm for 10 min, and discard the supernatant.
[0063] Add 2 mL of 0.03 mol / L Na2CO3 solution, saponify at 40°C for 30 min, centrifuge at 8000 rpm at room temperature for 10 min, and discard the supernatant.
[0064] Add 1 mL of ddH2O to prepare the bacterial solution, adjust the pH to 3 to 8, add 100 μL of 25 mg / mL enzyme solution, and perform enzymatic hydrolysis at 37°C for 6 h. Centrifuge at 8000 rpm at room temperature for 10 min and discard the supernatant.
[0065] Add 10 mL of a mixed solution of hexane:acetone:ethanol (2:1:1), shake in the dark at 20°C for 5 min, and then centrifuge at 8000 rpm at 10°C for 10 min.
[0066] Take 1 mL of supernatant, add 2 mL of extractant and measure its absorbance at 472 nm. 472 It is 0.3321.
[0067] Embodiment 8:
[0068] Add 1 mL of fermentation broth to the centrifuge tube, centrifuge at 8000 rpm for 10 min, and discard the supernatant.
[0069] Add 1 mL of ddH2O to wash, centrifuge at 8000 rpm for 10 min, and discard the supernatant.
[0070] Add 2 mL of 0.03 mol / L Na2CO3 solution, saponify at 40°C for 30 min, centrifuge at 8000 rpm at room temperature for 10 min, and discard the supernatant.
[0071] Add 1 mL of ddH2O to prepare the bacterial solution, adjust the pH to 3 to 8, add 100 μL of 25 mg / mL enzyme solution, and perform enzymatic hydrolysis at 37°C for 6 h. Centrifuge at 8000 rpm at room temperature for 10 min and discard the supernatant.
[0072] Add 2 mL of a mixed solution of n-hexane:ethanol (3:7), shake at room temperature in the dark for 5 min, and then centrifuge at 12000 rpm at room temperature for 5 min.
[0073] Take 1 mL of supernatant, add 2 mL of extractant and measure its absorbance at 472 nm. 472 It is 0.2154.
[0074] Comparative Example 1:
[0075] Add 1 mL of fermentation broth to each of the 7 centrifuge tubes, centrifuge at 8000 rpm for 10 min, and discard the supernatant.
[0076] Add 1 mL of ddH2O to wash, centrifuge at 8000 rpm for 10 min, and discard the supernatant.
[0077] Add 2 mL of 0.03 mol / L Na2CO3 solution, saponify at 40°C for 30 min, centrifuge at 8000 rpm at room temperature for 10 min, and discard the supernatant.
[0078] Add 1 mL of ddH2O to prepare the bacterial solution, adjust the pH to 3 to 8, add 100 μl of 25 mg / mL enzyme solution, hydrolyze at 37°C for 6 h, centrifuge at 8000 rpm at room temperature for 10 min, and discard the supernatant.
[0079] 2 mL of n-hexane:methanol (1:1), n-hexane:methanol (2:3), n-hexane:methanol (3:7), n-hexane:methanol (1:4), n-hexane:methanol (1:9), methanol, and n-hexane were added as extractants for extraction, shaken in the dark at room temperature for 5 min, and then centrifuged at 12000 rpm at room temperature for 5 min.
[0080] Take 1 mL of supernatant from each tube, add 2 mL of extractant and measure the absorbance at 472 nm.
[0081] As the ratio difference between n-hexane and methanol increases, the stratification phenomenon becomes less and less until it disappears, which is specifically manifested as the proportion of the upper layer decreases to nothing. During the final dilution, the stratified layer is basically insoluble in the extractant, making it impossible to measure A. 472 .
[0082] Comparative Example 2:
[0083] Add 200 mL of fermentation broth to each of 10 2 mL centrifuge tubes, centrifuge at 8000 rpm, and discard the supernatant.
[0084] Add 200 mL ddH2O for washing, centrifuge at 8000 rpm at room temperature, and discard the supernatant.
[0085] Add 2 mL of n-hexane:acetone:ethanol (2:1:1), 2 mL of n-hexane:ethanol (2:1), 2 mL of n-hexane:ethanol (1:1), 2 mL of n-hexane:ethanol (1:2), 2 mL of n-hexane:ethanol (1:4), 2 mL of n-hexane:ethanol (1:6), 2 mL of n-hexane:ethanol (1:8), 2 mL of n-hexane:ethanol (1:10), 2 mL of ethanol, and 2 mL of n-hexane respectively, observe and shake each group until complete extraction, and record the time.
[0086] After each group of samples was shaken and turned completely white, they were centrifuged at 12,000 rpm and room temperature for 5 min. 1 ml of supernatant was taken from each tube and the absorbance was measured at a wavelength of 472 nm using 2 ml of the extractant from each group.
[0087] The first group, n-hexane:acetone:ethanol (2:1:1), had the fastest extraction speed and turned white immediately after adding the extractant. The third group, n-hexane:ethanol (1:1), had the second fastest extraction speed and turned completely white after shaking for 40s after adding the extractant. The fourth, fifth and sixth groups turned completely white after shaking for 1 min. The second group, n-hexane:ethanol (2:1), turned completely white after adding the extractant for 80s. The extraction speeds of the seventh, eighth, ninth and tenth groups were relatively slow and all exceeded 5 min. The second group had the highest extraction volume, followed by the third, fourth and first groups, and the absorbance was all above 0.3500. The first group had the best extraction efficiency through calculation, but stratification occurred in one of the tubes. The third group had the second highest extraction efficiency, the fourth group had the third highest extraction efficiency, the fifth group had the fourth highest extraction efficiency, and the second and sixth groups had similar extraction efficiencies.
[0088] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.
Claims
1. A method for rapidly extracting industrial microbial lycopene, characterized in that: The method comprises the following steps: (1) Centrifuge the E. coli fermentation broth at 8000 rpm, discard the supernatant, and wash twice with water; (2) Add 0.03 mol / L Na2CO3 solution and saponify at 40°C for 30 min, then centrifuge and discard the supernatant; (3) Prepare the precipitate into a bacterial solution, adjust the pH to 3 to 8, add 25 mg / mL enzyme solution, incubate at 37°C for 6 h, and centrifuge to discard the supernatant; (4) Add the extractant, shake in the dark at room temperature for 1 min, centrifuge at 8000 rpm, and measure the absorbance of the supernatant containing lycopene at 472 nm.
2. The method according to claim 1, characterized in that The ratio of the fermentation liquid in step (1) to the saponifying agent used subsequently is 1:
2.
3. The method according to claim 1, characterized in that The saponifying agent in step (2) is a 0.03 mol / L Na2CO3 solution.
4. The method according to claim 1, characterized in that: The volume of the bacterial liquid in step (3) is the same as that of the previous fermentation liquid.
5. The method according to claim 1, characterized in that: The enzymatic agent in step (3) is 25 mg / mL lysozyme solution, and the amount added is 100 μL of enzyme solution per 1 mL of fermentation liquid.
6. The method according to claim 1, characterized in that The extractant in step (4) is a mixed solution of n-hexane:ethanol (1:2).
7. The method according to claim 1, characterized in that The existing lycopene extraction method is to add an extractant (methanol:acetone = 1:1) to the bacterial liquid, shake it in the dark for 5 minutes, and centrifuge it at 8000 rpm to obtain a supernatant containing lycopene.
8. The method according to claim 1, characterized in that The lycopene standard curve determination method is to dilute the lycopene standard with different concentration gradients of the extractant, measure the absorbance values at 472 nm respectively, and obtain the standard curve of lycopene after correction with n-hexane solution.
9. The method according to claim 1, characterized in that: The method for determining lycopene is to take the supernatant of the last centrifugation, use the extractant as a blank control, determine the absorbance value x at 472 nm, and according to the standard curve, obtain the lycopene yield y (g / L) = (x+0.0261) / 0.1502.