Method for efficiently extracting microalgae chlorophyll

Abstract

The invention belongs to the technical field of algae physiology, and particularly discloses a method for efficiently extracting microalgae chlorophyll, which comprises the following steps: a, taking an algae liquid sample, centrifuging, discarding supernatant, and adding 90% ethanol solution for resuspension; b, adding quartz sand into the resuspension, carrying out cell homogenization treatment under the condition of 60 Hz, repeating the cell homogenization treatment for 4 times, 45 seconds each time, and 5 seconds each time; and c, heating the sample in a constant-temperature water bath at 60 DEG C for 7 minutes, centrifuging, and collecting supernate. By optimizing the ethanol concentration, the cell homogenization time, the cell homogenization material and the hot bath time, the cell homogenization technology of combining an ethanol method with quartz sand broken algae cells is improved, the chlorophyll extraction efficiency is effectively improved, the stability is good, and large-scale application is easy. Compared with an acetone method, an ethanol method and an ethanol-steel ball method, the microalgae chlorophyll extraction method provided by the invention has obvious advantages.

Description

Technical Field

[0001] The present invention relates to the technical field of algae physiology, and in particular to a method for efficiently extracting microalgae chlorophyll. Background Art

[0002] The ethanol method is a classic chemical extraction method that uses high-concentration ethanol as a solvent to dissolve and extract chlorophyll through contact with microalgae cells. Ethanol effectively disrupts cell membranes, releasing chlorophyll into solution, enabling separation and extraction. Ethanol is less toxic and safer than acetone. The ethanol method offers advantages such as low cost, ease of use, and suitability for basic research.

[0003] However, ethanol extraction efficiency is low, not as good as acetone. Using only ethanol as a solvent without combining other techniques cannot completely destroy the cell wall, resulting in insufficient chlorophyll release and low extraction efficiency. The chlorophyll structure is easily destroyed and decomposed under high temperature or long extraction conditions, generating decomposition products such as dechlorophyll, which affects the purity and stability of the extract. The ethanol method is sensitive to extraction conditions and requires optimization steps, including ethanol concentration, sample handling method, hot water bath time, etc., to speed up the extraction process.

[0004] In summary, the ethanol method is limited in large-scale applications due to its low extraction efficiency, easy decomposition of chlorophyll, and high sensitivity to extraction conditions. Summary of the Invention

[0005] To solve the above problems, the present invention provides a method for efficiently extracting microalgae chlorophyll, which improves the overall performance of the ethanol method by optimizing extraction conditions and combining auxiliary technologies.

[0006] In order to achieve the above object, the specific technical solutions adopted by the present invention are as follows:

[0007] A method for efficiently extracting microalgae chlorophyll comprises the following steps:

[0008] a. Take an algae sample, centrifuge it, discard the supernatant, and resuspend it in 90% ethanol solution;

[0009] b. Add quartz sand to the resuspension and homogenize the cells at 60 Hz. Repeat the homogenization process four times, each for 45 seconds, with a 5-second interval.

[0010] c. Heat the sample in a 60°C water bath for 7 minutes, centrifuge, and collect the supernatant.

[0011] The invention optimizes ethanol concentration, cell homogenization time, cell homogenization material, and hot water bath time, and ultimately improves chlorophyll extraction efficiency by combining an improved ethanol method with a cell homogenization technology for crushing algal cells using quartz sand.

[0012] Furthermore, in step a, the volume ratio of the algae liquid sample to the 90% ethanol solution is 3:1.

[0013] Furthermore, in step a, the algae liquid sample is Chlorella algae liquid.

[0014] Furthermore, in step b, the amount of quartz sand added is 0.35 g / mL ethanol.

[0015] Furthermore, in step b, the cell homogenization treatment is to place the resuspension with quartz sand into a cell homogenizer (such as JXFSTPRP-24L) to break the cells by mechanical vibration. The quartz sand grinding and vibration crushing method is less damaging to chlorophyll than the commonly used ultrasonic crushing.

[0016] Furthermore, in steps a and c, the centrifugation condition is both 4000 r / min for 10 min.

[0017] Furthermore, in step c, the sample is diluted to the sampling volume in step a and then heated. The quantitative method for chlorophyll in the collected supernatant is to measure the OD of the supernatant. 665 ,OD 652 , substitute into the following formula (Ⅰ)-(Ⅱ) to calculate the content of chlorophyll a and chlorophyll b:

[0018] Chla (mg·L -1 )=16.82A 665 -9.28A 652 (Ⅰ);

[0019] Chlb (mg·L -1 )=36.92A 652 -16.54A 665 (Ⅱ).

[0020] The present invention has the following beneficial effects:

[0021] 1. The ethanol method of the present invention combined with the cell homogenization technology of quartz sand to break up algal cells greatly improves the chlorophyll extraction efficiency. Compared with the ethanol method alone and the ethanol-steel ball method, the extraction efficiency is increased by 1.28-1.45 times.

[0022] 2. The present invention sets algae solution with different concentration gradients and tests its OD 680 Then the chlorophyll content of each sample was determined and the microalgae OD 680 - Chlorophyll content standard curve, R 2 >0.99, indicating that the chlorophyll extraction method of the present invention has good stability.

[0023] 3. The extraction method of the present invention uses mild conditions (requires low temperatures and uses vibration grinding for cell disruption), is time-efficient, and chlorophyll is not easily degraded, ensuring the purity and stability of the extracted chlorophyll. Compared with the acetone method (which requires extraction in the dark for 24 hours in a refrigerator), the extraction method of the present invention only takes about 10 minutes, and the entire extraction process only takes about 30 minutes, significantly shortening the extraction time.

[0024] 4. The extraction method of the present invention uses conventional instruments such as an ultraviolet-visible spectrophotometer, a cell homogenizer, and a constant temperature water bath, which can meet the needs of large-scale extraction and industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : The optimization result of ethanol concentration among embodiment 1.

[0026] Figure 2 : Optimization results of cell homogenization time in Example 1.

[0027] Figure 3 : Optimization results of cell homogenate material in Example 1.

[0028] Figure 4 : Optimization result of hot water bath time among embodiment 1. DETAILED DESCRIPTION

[0029] The present invention is further described below with reference to the accompanying drawings and specific examples. The algae sample used in the following examples is Chlorella vulgaris, and the instrument used for the cell homogenization treatment involved in the extraction process is a JXFSTPRP-24L cell homogenizer from Shanghai Jingxin.

[0030] Example 1: Optimization of extraction conditions

[0031] (1) Optimization of ethanol concentration

[0032] Take 3 mL of algae solution and measure the OD using a UV-visible spectrophotometer. 680 0.5 (OD 680 The sample was then centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and 3 mL of 85%, 90%, and 95% ethanol solutions were added. The sample was then transferred to a 10 mL colorimetric tube, heated in a 60°C constant temperature water bath for 10 min, and centrifuged at 4000 rpm for 10 min. The supernatant was collected and the OD was measured. 665 ,OD 652 ,OD 470 Calculate the contents of chlorophyll a, chlorophyll b and carotenoids in the supernatant according to formulas (Ⅰ)-(Ⅲ):

[0033] Chla (mg·L -1)=16.82A 665 -9.28A 652 (Ⅰ);

[0034] Chlb (mg·L -1 )=36.92A 652 -16.54A 665 (Ⅱ);

[0035] Carotenoid (mg·L -1 )=(1000A 470 -1.91C a -95.15C b ) / 225 (Ⅲ).

[0036] The results are as follows Figure 1 As shown, 90% ethanol concentration has the best effect on extracting chlorophyll.

[0037] (2) Optimization of cell homogenization time

[0038] Take 3 mL of algae solution and measure the OD using a UV-visible spectrophotometer. 680 is 0.5. Subsequently, the sample was centrifuged at 4000r / min for 10min, the supernatant was discarded, and 1mL of 90% ethanol solution was added. The obtained algal solution was transferred to a 1.5mL centrifuge tube, 3 large steel balls were added, and the cells were homogenized at 60Hz (45s each time, 5s interval, repeated 2, 4 and 6 times). Subsequently, the sample was transferred to a 10mL colorimetric tube and diluted to 3mL, placed in a constant temperature water bath at 60℃ for 10min, and then centrifuged at 4000r / min for 10min, the supernatant was collected, and the OD was measured. 665 ,OD 652 ,OD 470 Calculate the contents of chlorophyll a, chlorophyll b and carotenoids according to formula (Ⅰ)-(Ⅲ). The results are as follows Figure 2 As shown in the figure, the best effect of chlorophyll extraction is 45s*4 times.

[0039] (3) Optimization of cell homogenate materials

[0040] Take 3 mL of algae solution and measure the OD using a UV-visible spectrophotometer. 680is 0.5. Subsequently, the sample was centrifuged at 4000r / min for 10min, the supernatant was discarded, and 1mL of 90% ethanol solution was added. The obtained algae solution was transferred to a 1.5mL centrifuge tube, 3 large steel balls, 3 small steel balls and 0.35g / mL quartz sand (equivalent to the mass of 3 large steel balls) were added, and the cells were homogenized at 60Hz (45s each time, 5s interval, repeated 4 times). Subsequently, the sample was transferred to a 10mL colorimetric tube, diluted to 3mL, placed in a constant temperature water bath at 60℃ and heated for 10min, then centrifuged at 4000r / min for 10min, the supernatant was collected, and the OD was measured. 665 ,OD 652 ,OD 470 Calculate the contents of chlorophyll a, chlorophyll b and carotenoids according to formula (Ⅰ)-(Ⅲ). The results are as follows Figure 3 As shown in the figure, 0.35 g / mL quartz sand has the best effect in extracting chlorophyll.

[0041] (4) Optimization of hot water bath time

[0042] Take 3 mL of algae solution and measure the OD using a UV-visible spectrophotometer. 680 The sample was then centrifuged at 4000 r / min for 10 min, the supernatant was discarded, and 1 mL of 90% ethanol solution was added. The resulting algal solution was transferred to a 1.5 mL centrifuge tube, 0.35 g / mL quartz sand was added, and the cells were homogenized at 60 Hz (45 s each time, 5 s interval, repeated 4 times). The sample was then transferred to a 10 mL colorimetric tube and diluted to 3 mL, placed in a 60 ° C constant temperature water bath and heated for 10, 7 and 4 min, and then centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the OD was measured. 665 ,OD 652 ,OD 470 Calculate the contents of chlorophyll a, chlorophyll b and carotenoids according to formula (Ⅰ)-(Ⅲ). The results are as follows Figure 4 As shown in the figure, the extraction time of chlorophyll for 7 minutes is the best.

[0043] Example 2: Improved ethanol extraction method and effect test

[0044] Based on the optimized conditions of Example 1, this example proposes the following method for extracting microalgae chlorophyll:

[0045] Take 3 mL of algae solution and measure the OD using a UV-visible spectrophotometer. 680. Subsequently, the sample was centrifuged at 4000r / min for 10min, the supernatant was discarded, and 1mL of 90% ethanol solution was added. The obtained algal liquid was transferred to a 1.5mL centrifuge tube, 0.35g / mL quartz sand was added, and the cells were homogenized at 60Hz (45s each time, 5s interval, repeated 4 times). Subsequently, the sample was transferred to a 10mL colorimetric tube, diluted to 3mL, placed in a constant temperature water bath at 60℃ and heated for 7min, and then centrifuged at 4000r / min for 10min, and the supernatant was collected.

[0046] The chlorophyll quantification method in the supernatant is: measuring the OD of the supernatant 665 ,OD 652 , substitute into the following formula (Ⅰ)-(Ⅱ) to calculate the content of chlorophyll a and chlorophyll b:

[0047] Chla (mg·L -1 )=16.82A 665 -9.28A 652 (Ⅰ);

[0048] Chlb (mg·L -1 )=36.92A 652 -16.54A 665 (Ⅱ).

[0049] The following is a test of the effectiveness of the extraction method:

[0050] (1) Stability test of the extraction method of this embodiment

[0051] In order to test the stability of the extraction method, chlorophyll was extracted and measured in algae solutions with different concentration gradients. 0.25, 0.5, 1, 2, and 3 mL of algae solution were taken, and the total volume was fixed to 3 mL with ultrapure water. The OD was measured using a UV-visible spectrophotometer. 680 Then, the chlorophyll extraction and OD 665 ,OD 652 Determine the chlorophyll content. 680 A standard curve was constructed with chlorophyll content as the horizontal axis and chlorophyll content as the vertical axis.

[0052] The results are shown in Table 1 below. The standard curves R for chlorophyll a and chlorophyll b were constructed. 2 >0.99, indicating that the chlorophyll extraction method of the present invention has good stability.

[0053] Table 1 Microalgae OD 680 - Chlorophyll content standard curve

[0054]

[0055] (2) Extraction efficiency test of the extraction method of this embodiment

[0056] Calculation method: Extraction efficiency Ea% = Chla / 840*100%, Eb% = Chlb / 840*100%; where 840 is OD 680 =0.5 when the dry weight of Chlorella cells, unit: mg·L -1 . Increase factor n=Chl(a,b) max / Chl(a,b) min .

[0057] Complete the chlorophyll extraction and OD 665 ,OD 652 The chlorophyll content was determined and substituted into the calculation formula of extraction efficiency and improvement factor, and the extraction efficiency of chlorophyll a and chlorophyll b was 0.27% and 0.32% respectively, which was significantly different from that of the ethanol method ( Figure 1 ) and ethanol-steel ball method ( Figure 2 ) compared with the control group, it increased by 1.28-1.45 times.

[0058] In summary, the extraction method of the present invention effectively overcomes the shortcomings of the existing ethanol extraction method, improves the chlorophyll extraction efficiency, shortens the extraction time, and is suitable for large-scale extraction needs.

[0059] This specific implementation is merely an explanation of the present invention and is not a limitation of the present invention. Any changes made by those skilled in the art after reading the specification of the present invention will be protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A method for efficiently extracting chlorophyll from microalgae, characterized in that: The following steps are involved: a. Take an algae sample, centrifuge it, discard the supernatant, and resuspend it in 90% ethanol solution; b. Add quartz sand to the resuspension and homogenize the cells at 60 Hz. Repeat the homogenization process four times, each for 45 seconds, with a 5-second interval. c. Heat the sample in a 60°C water bath for 7 minutes, centrifuge, and collect the supernatant.

2. The method for efficiently extracting microalgae chlorophyll according to claim 1, wherein: In step a, the volume ratio of the algae liquid sample to the 90% ethanol solution is 3:

1.

3. The method for efficiently extracting microalgae chlorophyll according to claim 1, wherein In step a, the algae liquid sample is Chlorella algae liquid.

4. The method for efficiently extracting microalgae chlorophyll according to claim 1, characterized in that: In step b, the addition amount of the quartz sand is 0.35 g / mL ethanol.

5. The method for efficiently extracting microalgae chlorophyll according to claim 1, characterized in that: In step b, the cell homogenization treatment is to put the resuspension with quartz sand into a cell homogenizer to break the cells through mechanical shaking.

6. The method for efficiently extracting microalgae chlorophyll according to claim 1, characterized in that: In steps a and c, the centrifugation condition is both 4000 r / min for 10 min.

7. The method for efficiently extracting microalgae chlorophyll according to claim 1, characterized in that: In step c, the sample is diluted to the sampling volume in step a and then heated. The chlorophyll in the collected supernatant is quantitatively determined by measuring the OD value of the supernatant. 665 ,OD 652 , substitute into the following formula (Ⅰ)-(Ⅱ) to calculate the content of chlorophyll a and chlorophyll b: <h2 style=";text-align:left;direction:ltr">Chla(mg·L<h2 style=";text-align:left;direction:ltr"> -1 <h2 style=";text-align:left;direction:ltr"> )=16.82A<h2 style=";text-align:left;direction:ltr"> 665 <h2 style=";text-align:left;direction:ltr"> -9.28A<h2 style=";text-align:left;direction:ltr"> 652 <h2 style=";text-align:left;direction:ltr"> (Ⅰ); Chlb(mg·L -1 )=36.92A 652 -16.54A 665 (Ⅱ)。

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