Method for rapidly detecting content of xanthophyll in microalgae

Abstract

The invention discloses a mild and efficient method for rapidly detecting the content of lutein in microalgae, and belongs to the technical field of microalgae pigment detection.The detection method comprises the steps that microalgae liquid is centrifugally concentrated, repeatedly frozen and thawed and then subjected to ultrasonication; adding an organic solvent into the obtained mixture, extracting in a dark place, and concentrating; adding an alkaline solution for saponification and then cooling; adding an organic solvent and a sodium sulfate aqueous solution, fully mixing, and standing in a dark place for layering; taking supernate, detecting absorbance, and calculating the content of xanthophyll. According to the detection method, the sample treatment process is mild, the loss of the xanthophyll in the treatment process is reduced to the maximum extent, the accuracy of the detection result is improved, meanwhile, operation is easy, the detection cost is low, consumed time is short, and mass detection of the xanthophyll content in the microalgae and dynamic monitoring of the xanthophyll content in the microalgae growth process can be efficiently achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of microalgae pigment detection, and particularly relates to a method for quickly detecting lutein content in microalgae. Background Art

[0002] Lutein is a fat-soluble carotenoid that is mainly found in the leaves, flowers and fruits of plants. It is also distributed in human serum, liver and other tissues, with the highest density in the retinal macula. Lutein is an important plant pigment that protects the retina of the eyes. It can protect the eyes from light damage, relieve visual fatigue, increase macular pigment density, and prevent macular degeneration. It is of great significance to human health.

[0003] The human body cannot synthesize lutein on its own, so it needs to be supplemented through exogenous intake; chemically synthesized lutein has no biological function, and extraction from plants such as marigold is currently the main source of lutein. However, the planting cycle of marigold is long, the land occupation and labor costs are high, and the extraction efficiency is low, which makes it difficult to meet the growing market demand; microalgae have a short growth cycle, and the use of reactor cultivation can effectively break through the occupation of arable land and the influence of natural climate, making it suitable for industrial production management and quality control, and is an important source of lutein production in the future.

[0004] The production of lutein using microalgae requires staged growth regulation to increase the lutein content in the microalgae. The all-trans structure of natural lutein is easily destroyed under the influence of high temperature and other factors. Therefore, the development of a mild and efficient method for rapid detection of lutein content in microalgae is of great significance for the dynamic monitoring of lutein content during the growth of microalgae. Summary of the invention

[0005] The purpose of the present invention is to provide a mild and efficient method for rapid detection of lutein content in microalgae, so as to realize dynamic monitoring of lutein content during the growth of microalgae.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is: S1: The algae solution was heated to 4000 g The mixture was centrifuged and concentrated at a speed of 5000rpm for 5 minutes to obtain microalgae mud.

[0007] S2: After the algae mud obtained by centrifugation in step S1 is completely frozen in liquid nitrogen or at -80°C, it is taken out and thawed in a 25°C water bath, and the above freezing and thawing process is repeated 2 to 5 times.

[0008] S3: The algae solution after freeze-thawing in step S2 was subjected to ultrasonic disruption, with the ultrasonic power set to 200-400 W, the ultrasonic time to 5-10 seconds, the ultrasonic interval to 5-10 seconds, and the total ultrasonic disruption time to 5-10 minutes. During the ultrasonic disruption process, an ice bath was used to control the temperature of the algae solution below 10°C.

[0009] S4: Add 60%~100% (v / v) methanol aqueous solution, ethanol aqueous solution or acetone aqueous solution to the broken algae liquid obtained by ultrasonic disruption in step S3, with the volume ratio of organic solvent to sample being 2:1~5:1. Repeat the extraction 3~5 times in the dark, then combine the extracts and concentrate.

[0010] S5: Add 40% (v / v) potassium hydroxide aqueous solution to the concentrated solution obtained by extraction in step S4, saponify at 40°C in the dark for 20 to 60 minutes, and immediately cool to below 25°C with cold water after the saponification is completed.

[0011] S6: Add n-hexane and 10% (v / v) sodium sulfate aqueous solution to the algae solution after saponification in step S5, mix thoroughly, and then stand in the dark to separate the layers.

[0012] S7: Take the supernatant in step S6, dilute it with n-hexane, detect the absorbance A at 474 nm with a spectrophotometer, and calculate the lutein content Y (g / kg) in the microalgae. The calculation formula is: Where: V is the volume of the supernatant (mL), K is the sample dilution factor, 236 is the absorbance of a 1 g / L lutein standard n-hexane solution at a wavelength of 474 nm, M is the mass of the sample (g), and c is the water content of the sample.

[0013] Preferably, in step S2, the microalgae mud is frozen and thawed 3 to 4 times.

[0014] Preferably, in the step S3, the ultrasonic power is 300-400 W.

[0015] Preferably, in the step S3, during the ultrasonic fragmentation process, the ultrasonic time is 10 seconds, the ultrasonic interval is 10 seconds, and the total time is 8 to 10 minutes.

[0016] Preferably, in step S4, the organic solvent is a 60-80% (v / v) methanol aqueous solution or an acetone aqueous solution.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The sample processing process of the present invention is gentle and the temperature is controlled throughout the process, which minimizes the damage to the lutein in the microalgae during the processing and improves the accuracy of the detection result; at the same time, by optimizing the pretreatment method, simplifying the operation process, shortening the sample processing time, and improving the detection efficiency, the dynamic monitoring of the lutein content during the growth of microalgae can be efficiently realized. Specific implementation method: In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with embodiments; those skilled in the art should understand that the embodiments described below are part of the embodiments of the present invention, which are only to help understand the present invention and should not be regarded as limiting the present invention; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0019] Example 1 After 14 days of culture, the Chlorella algae solution was heated at 4000 g Centrifuge for 5 minutes, collect the algae mud, and determine the water content of the algae mud to be 80%; weigh 2 g of algae mud and freeze it in liquid nitrogen, take it out and thaw it in a 25℃ water bath, and repeat the above freeze-thaw process 3 times; ultrasonically break the frozen and thawed algae liquid, set the ultrasonic power to 300 W, the ultrasonic time to 10 seconds, the ultrasonic interval to 10 seconds, and the total ultrasonic break time to 10 minutes; add 80% (v / v) methanol aqueous solution to the broken algae liquid, the volume ratio of organic solvent to sample is 3:1, and extract it 3 times in the dark, then combine the extracts and concentrate; add 40% (v / v) potassium hydroxide aqueous solution to the concentrate, saponify it in the dark at 40℃ for 60 minutes, and immediately cool it to below 25℃ with cold water after the saponification is completed; add 50 mL of n-hexane and shake to mix, then add 10 mL of 10% (v / v) sodium sulfate aqueous solution, mix thoroughly, stand in the dark for 60 minutes, and separate the layers, take 2 mL of the supernatant to 50 mL volumetric flask, dilute to the mark with n-hexane solvent and shake well; use a spectrophotometer to measure the absorbance of the three samples in parallel at a wavelength of 474 nm. The average absorbance A is 0.475. According to the formula The lutein content in the microalgae was calculated to be 6.29 g / kg.

[0020] Example 2 After 14 days of culture, the Chlorella algae solution was heated at 4000 gCentrifuge for 5 minutes, collect the algae mud, and determine the water content of the algae mud to be 80%; weigh 2 g of algae mud and freeze it in liquid nitrogen, take it out and thaw it in a 25℃ water bath, and repeat the above freeze-thaw process 3 times; ultrasonically break the frozen and thawed algae liquid, set the ultrasonic power to 400 W, the ultrasonic time to 10 seconds, the ultrasonic interval to 10 seconds, and the total ultrasonic break time to 8 minutes; add 60% (v / v) methanol aqueous solution to the broken algae liquid, the volume ratio of organic solvent to sample is 3:1, and extract it 3 times in the dark, then combine the extracts and concentrate; add 40% (v / v) potassium hydroxide aqueous solution to the concentrate, saponify it in the dark at 40℃ for 60 minutes, and immediately cool it to below 25℃ with cold water after the saponification is completed; add 50 mL of n-hexane and shake to mix, then add 10 mL of 10% (v / v) sodium sulfate aqueous solution, mix thoroughly, stand in the dark for 30 minutes to separate, take 2 mL of the supernatant to 50 mL volumetric flask, dilute to the mark with n-hexane solvent and shake well; use a spectrophotometer to measure the absorbance of the three samples in parallel at a wavelength of 474 nm. The average absorbance A is 0.472. According to the formula The lutein content in the microalgae was calculated to be 6.25 g / kg.

[0021] Example 3 After 14 days of culture, the Chlorella algae solution was heated at 4000 g Centrifuge for 5 minutes, collect the algae mud, and determine the water content of the algae mud to be 80%; weigh 2 g of algae mud and freeze it in liquid nitrogen, then take it out and thaw it in a 25℃ water bath, and repeat the above freeze-thaw process 3 times; ultrasonically break the frozen and thawed algae liquid, set the ultrasonic power to 400 W, the ultrasonic time to 10 seconds, the ultrasonic interval to 10 seconds, and the total ultrasonic break time to 8 minutes; add 60% (v / v) acetone aqueous solution to the broken algae liquid, the volume ratio of organic solvent to sample is 3:1, and extract it 3 times in the dark, then combine the extracts and concentrate; add 40% (v / v) potassium hydroxide aqueous solution to the concentrate, saponify it in the dark at 40℃ for 60 minutes, and immediately cool it to below 25℃ with cold water after the saponification is completed; add 50 mL of n-hexane and shake to mix, then add 10 mL of 10% (v / v) sodium sulfate aqueous solution, mix thoroughly, stand in the dark for 30 minutes to separate the layers, take 2 mL of the supernatant to 50 mL volumetric flask, dilute to the mark with n-hexane solvent and shake well; use a spectrophotometer to measure the absorbance of the three samples in parallel at a wavelength of 474 nm. The average absorbance A is 0.477. According to the formula The lutein content in the microalgae was calculated to be 6.32 g / kg.

[0022] Example 4 After 14 days of culture, the Chlorella algae solution was heated at 4000 gCentrifuge for 5 minutes, collect the algae mud, and determine the water content of the algae mud to be 80%; weigh 2 g of algae mud and freeze it in liquid nitrogen, then take it out and melt it in a 25℃ water bath, and repeat the above freeze-thaw process 5 times; ultrasonically break the frozen and thawed algae liquid, set the ultrasonic power to 400 W, the ultrasonic time to 10 seconds, the ultrasonic interval to 10 seconds, and the total ultrasonic break time to 10 minutes; add 80% (v / v) acetone aqueous solution to the broken algae liquid, the volume ratio of organic solvent to sample is 5:1, and extract it three times in the dark, then combine the extracts and concentrate; add 40% (v / v) potassium hydroxide aqueous solution to the concentrate, saponify it at 40℃ in the dark for 60 minutes, and immediately cool it to below 25℃ with cold water after the saponification is completed; add 50 mL of n-hexane and shake to mix, then add 10 mL of 10% (v / v) sodium sulfate aqueous solution, mix thoroughly, stand in the dark for 60 minutes, and separate the layers, take 2 mL of the supernatant to 50 mL volumetric flask, dilute to the mark with n-hexane solvent and shake well; use a spectrophotometer to measure the absorbance of the three samples in parallel at a wavelength of 474 nm. The average absorbance A is 0.481. According to the formula The lutein content in the microalgae was calculated to be 6.37 g / kg.

[0023] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the ultrasonic crushing process is not included, and other conditions remain unchanged. The specific steps are as follows.

[0024] After 14 days of culture, the Chlorella algae solution was heated at 4000 g Centrifuge for 5 minutes, collect the algae mud, and determine the water content of the algae mud to be 80%; weigh 2 g of algae mud and freeze it in liquid nitrogen, take it out and thaw it in a 25℃ water bath, and repeat the above freeze-thaw process 3 times; add 80% (v / v) methanol aqueous solution to the frozen and thawed algae solution, the volume ratio of organic solvent to sample is 3:1, and extract it 3 times in the dark, then combine the extracts and concentrate; add 40% (v / v) potassium hydroxide aqueous solution to the concentrate, saponify it in the dark at 40℃ for 60 minutes, and immediately cool it to below 25℃ with cold water after saponification; add 50 mL of n-hexane and shake to mix, then add 10 mL of 10% (v / v) sodium sulfate aqueous solution, mix thoroughly, stand in the dark for 60 minutes to separate, take 2 mL of the supernatant to a 50 mL volumetric flask, dilute to the scale with n-hexane solvent and shake well; use a spectrophotometer to measure the absorbance of the three samples in parallel at a wavelength of 474 nm, and the average absorbance A is 0.206. According to the formula The lutein content in the microalgae was calculated to be 2.73 g / kg.

[0025] Comparative Example 2 The difference between Comparative Example 1 and Example 1 is that the freeze-thaw process is not included, and other conditions remain unchanged. The specific steps are as follows.

[0026] After 14 days of culture, the Chlorella algae solution was heated at 4000 g Centrifuge for 5 minutes, collect the algae mud, and determine the water content of the algae mud to be 80%; weigh 2 g of algae mud for ultrasonic crushing, set the ultrasonic power to 300 W, the ultrasonic time for 10 seconds, the ultrasonic interval for 10 seconds, and the total ultrasonic crushing time for 10 minutes; add 80% (v / v) methanol aqueous solution to the broken algae liquid, the volume ratio of organic solvent to sample is 3:1, and the extraction is repeated 3 times in the dark, and the extract is combined and concentrated; add 40% (v / v) potassium hydroxide aqueous solution to the concentrate, saponify at 40°C in the dark for 60 minutes, and immediately cool to below 25°C with cold water after the saponification is completed; add 50 mL of n-hexane and shake to mix, then add 10 mL of 10% (v / v) sodium sulfate aqueous solution, mix thoroughly, stand in the dark for 60 minutes to separate, take 2 mL of the supernatant into a 50 mL volumetric flask, dilute to the scale with n-hexane solvent and shake well; use a spectrophotometer at a wavelength of 474 The absorbance of three samples was measured in parallel at nm, and the average absorbance A was 0.417. According to the formula The lutein content in the microalgae was calculated to be 5.52 g / kg.

[0027] Comparative Example 3 The difference between Comparative Example 1 and Example 1 is that light protection is not performed throughout the process, and other conditions remain unchanged. The specific steps are as follows.

[0028] After 14 days of culture, the Chlorella algae solution was heated at 4000 g Centrifuge for 5 minutes, collect the algae mud, and determine the water content of the algae mud to be 80%; weigh 2 g of algae mud and freeze it in liquid nitrogen, then take it out and thaw it in a 25℃ water bath, and repeat the above freeze-thaw process 3 times; ultrasonically break the frozen and thawed algae liquid, set the ultrasonic power to 300 W, the ultrasonic time to 10 seconds, the ultrasonic interval to 10 seconds, and the total ultrasonic break time to 10 minutes; add 80% (v / v) methanol aqueous solution to the broken algae liquid, the volume ratio of organic solvent to sample is 3:1, repeat the extraction 3 times and combine the extracts to concentrate; add 40% (v / v) potassium hydroxide aqueous solution to the concentrate, saponify at 40℃ for 60 minutes, and immediately cool it to below 25℃ with cold water after the saponification is completed; add 50 mL of n-hexane and shake to mix, then add 10 mL of 10% (v / v) sodium sulfate aqueous solution, mix thoroughly and let stand for 60 minutes to separate, take 2 mL of the supernatant to 50 mL volumetric flask, dilute to the mark with n-hexane solvent and shake well; use a spectrophotometer to measure the absorbance of the three samples in parallel at a wavelength of 474 nm. The average absorbance A is 0.442. According to the formula The lutein content in the microalgae was calculated to be 5.85 g / kg.

[0029] Although the implementation scheme of the present invention has been described above in conjunction with the embodiments, it should be noted that the described embodiments are only a part of the specific implementation methods of the present invention and are not intended to limit the scope of protection of the present invention. Any equivalent replacements, improvements, etc. made within the scope of the technical concept and technical method of the present invention belong to the protection scope and disclosure scope of the present invention.

Claims

1. A rapid detection method for lutein content in microalgae, It is characterized in that The detection method comprises: (1) centrifuging and concentrating the microalgae liquid, and then repeatedly freezing and thawing and then ultrasonically crushing it; (2) adding an organic solvent to the obtained mixture, extracting in the dark, and then concentrating; (3) Add alkaline solution for saponification and then cool; (4) Add an organic solvent and an aqueous sodium sulfate solution, mix thoroughly, and then stand in a dark place to separate into layers; (5) Take the supernatant and measure the absorbance using a spectrophotometer to calculate the lutein content.

2. The method for rapid detection of lutein content in microalgae according to claim 1, It is characterized in that The concentration method in step (1) is centrifugal collection, and the centrifugal speed is 4000 g , the centrifugation time is 5 minutes.

3. The rapid detection method for lutein content in microalgae according to claim 1, It is characterized in that The freeze-thaw step in step (1) is to freeze the sample in liquid nitrogen or at -80°C, then take it out and thaw it in a 25°C water bath. This freeze-thaw process is repeated 2 to 5 times.

4. The rapid detection method for lutein content in microalgae according to claim 1, It is characterized in that In the ultrasonic disruption step in step (1), the temperature of the algae solution is controlled to be below 10° C. by an ice bath, the ultrasonic power is 200-400 W, the ultrasonic time is 5-10 seconds, the ultrasonic interval is 5-10 seconds, and the total ultrasonic disruption time is 5-10 minutes.

5. The rapid detection method for lutein content in microalgae according to claim 1, It is characterized in that In step (2), the organic solvent is a 60% to 100% (v / v) methanol aqueous solution, an ethanol aqueous solution or an acetone aqueous solution, the volume ratio of the organic solvent to the sample is 2:1 to 5:1, and the extraction is repeated 3 to 5 times.

6. The method for rapid detection of lutein content in microalgae according to claim 1, It is characterized in that In step (3), the alkaline solution is a 40% (v / v) potassium hydroxide aqueous solution, the treatment temperature is 40° C., the treatment time is 20 to 60 minutes, and after the saponification is completed, the temperature is immediately cooled to below 25° C. with cold water.

7. The method for rapid detection of lutein content in microalgae according to claim 1, It is characterized in that In the step (4), the organic solvent is n-hexane, and the sodium sulfate content in the sodium sulfate aqueous solution is 10%.

8. The method for rapid detection of lutein content in microalgae according to claim 1, It is characterized in that In the step (5), the absorbance measurement wavelength is 474 nm, and the supernatant is diluted with n-hexane to an absorbance of 0.2-0.8.