Determination of chlorophyll content in kelp

Chlorophyll is a product of natural photosynthesis in the natural world. It is non-toxic and edible, so it is widely used in the coloring of foods, cosmetics, and drugs. In addition, it also plays an important role in biochemical research. Therefore, the demand for chlorophyll is very large. The exit is about 1000t. At present, people focus their efforts on the production of chlorophyll or on the silkworms with high chlorophyll content, but this is affected by the seasons, and some leaves contain toxins that are difficult to separate, and the chlorophyll content of terrestrial plants is about It is 0.3%-0.5%. In order to expand the production of chlorophyll and discover new raw materials for production, we have turned our attention to the ocean.
As we all know, seaweed and land-based plants are also used by photosynthesis to convert light energy into chemical energy and maintain its own growth. Since the available light energy in water is much less than on land, it is necessary to complete energy conversion. The amount of chlorophyll involved in photosynthesis must be increased. Therefore, in general, the content of chlorophyll and other supplementary pigments in marine plants is higher than that of terrestrial plants, and marine plants grow fast and have large yields. Therefore, the study of the use of marine plants to extract chlorophyll has led to the comprehensive development of marine resources and natural pigments. Extraction has a very significant and far-reaching significance. For this purpose, we used kelp as a raw material for the study of chlorophyll extraction.
1.Materials and methods 1.1 Materials: Zhejiang Province Wenling kelp farm provides dried kelp 1.2 instrument 721 spectrophotometer, top chlorophyll detector, pH meter, electric constant temperature water bath, electronic scale, rack balance, miniature plant test Sample grinder.
1.3 Reagent petroleum ether, 95% ethanol, 95% acetone, sodium hydroxide, copper sulfate, acetone and ethanol mixture (prepared according to the volume ratio of 95:5)
1.4 Processes Seaweed → Pulverization → Washing with water → Paste-like kelp → Copper sulfate treatment → Washing and drying → Extraction → Purification → Concentration → Saponification → Drying → Product 1.5 Determination method 1.5.1 Determination of absorbance (OD): 721 Spectrophotometer determination, according to absorbance values ​​and changes were analyzed, according to absorbance values ​​to determine and select the optimal conditions.
1.5.2 Product Quality Analysis: Determined according to Food Additives Manual P314-315.
1.5.3 pH: Determination with a pH meter.
1.5.4 crude product rate: X%=W2/W1×100
Weight of W1-membrane after mechanical dehydration after rehydration, and the weight of the dried water on the filter paper (g)
W2- refers to the weight of the final crude copper chlorophyllin (g)
2. Results and discussion 2.1. Pretreatment of raw materials 2.1.1 Pulverized pigments are contained in cells or in some cellular tissues filled in the cell cavities, and crushing destroys the cell wall tissues of raw materials to a certain extent, removing the main obstacle to the diffusion of pigments into solvents. This accelerates the process of seepage of the solvent and dissolution and diffusion of the pigment.
2.1.2 Cold water washing can remove sediment and impurities, and can sink the mannitol attached to the surface layer of kelp during the washing process. Therefore, mannitol can be extracted from the washing solution.
2.1.3 Hot Water Soaking After washing with cold water, there are some substances that are not suitable for pigment extraction, such as alginate, which are soluble in warm water and are soaked in warm water at 40-50°C to remove these substances. The ratio of kelp to warm water is 1:4. Soaking 10-15 and n each time is appropriate.
2.1.4 Copper sulfate treatment The ton of chlorophyll molecules is replaced by Cu. The resulting green salt is more stable. It has strong lightfastness, is not easily replaced by proteins, and can reduce the effects of lutein and carotenoids on extraction and purification. The pigment is distributed in the cytoplasm of the plant tissue. It takes a certain amount of time for the copper salt to penetrate. Therefore, different treatment times have different effects on pigment extraction. The experiment was performed by soaking kelp with a 5% CuS instrument solution.
2.1.5 Washing. After the dehydration of the copper sulfate is completed, the mannitol copper precipitate and copper sulfate are washed first. Dry seaweed absorbs a large amount of water after the above operation, and the presence of water will affect the extraction rate and the leaching rate. Therefore, mechanical dehydration is first performed, and then the filter paper is used to reduce the effect of water on the leaching.
2.2 Extraction 2.2.1 Determination of the maximum absorption peak γ After 13 hours of kelp treatment, an acetone-ethanol mixture was added at a feed-to-liquid ratio of 1:50. Incubate in a thermostatic bath at 25°C for 2 hours, filter the resulting extract and set to volume. Scan with a 721 spectrophotometer in the wavelength range of 380 to 680 nm. Take the average of the three measurements and plot as shown in Figure 1.

Figure 1 Relationship between wavelength and OD

Figure 1 Relationship between wavelength and OD Figure 1 shows that at the two wavelengths of 410nm and 660nm, the chlorophyll copper salt has two maximum absorption peaks. Green is a mixture of purple and red. 410nm is in the violet region (400-435nm). Since there is no impurity in the leaching process, the content of impurities in the extract is high, such as protein, lutein, carotenoids, etc., and the maximum absorption peak of these impurities is close to 410nm, so the experiment should be based on the OD of only 660nm.
2.2.2 Determination of copper sulfate processing time

Table 1 Effect of Copper Sulfate Treatment Time on Leaching
Table 1 Effect of copper sulfate treatment time on leaching The results show that the treatment time of copper sulfate is 13h.
2.2.3 Selection of extraction solvent

Table 2 Effect of different solvents on yield
Table 2 shows the effect of different solvents on the yield. It can be seen from Table 2 that 95% acetone extraction has the best effect, but when acetone is used for leaching, when acetone is concentrated to a paste product, acetone remains in the product as a food additive. Is not allowed. The extraction with acetone ethanol mixture, its extraction rate and acetone extraction is not much difference, while the concentration recovery, the residual ethanol in the product has little effect on the human body, so choose to use acetone ethanol mixture extraction.
2.2.4 Selection of extraction time

Table 3 Relationship between extraction time and yield
Table 3 Relation between extraction time and yield Visible extraction time is 2h.
2.2.5 Determination of extraction temperature

Table 4 Effect of extraction temperature
Table 4 shows the influence of extraction temperature. The results show that 60°C is appropriate. Increasing the temperature can increase the leaching rate of the pigment and increase the yield, but the high temperature affects the stability of the pigment.
In summary, the optimal conditions for extraction are: raw materials are treated with 5% CuSO4 for 13 h, and extracted with 50 times acetone-ethanol mixture at 60° C. for 2 h.
2.3 Concentration, saponification, and drying The extract obtained above was placed in a separatory funnel, and one-third volume of petroleum ether was added to vortex and n was allowed to stand for stratification. The lower aqueous phase was a chlorophyll copper salt solution, and the upper oil was added. Phase is lutein, folic acid and other impurities. Take off the liquid, adjust the pH = 11 with 5% Na0H, install the flask, turn on the condenser, heat the water bath on the reflux for 1h for saponification, then recover the acetone, concentrate the product, as the amount of acetone decreases, the temperature can be appropriately increased Recovery of acetone, the general temperature does not exceed 70 °C, until it can not recover acetone. The concentrate was transferred to a baking tray and placed in a dry oven at a constant temperature of 0°C to obtain a black-green, block-like product that was ground into a powder. The crude sodium copper chlorophyllin was obtained and protected from light. The average yield measured was 5.03%.
3. Product performance test 3.1 The product is a dark green powder, easy to absorb moisture, soluble in water, the aqueous solution is green and transparent, slightly soluble in ethanol and acetone, there are precipitation of calcium ions in the presence of precipitation.
3.2 Take the sample for the flame color test. When the concentration of the sample solution is high, it can be clearly observed that the flame starts to appear green and yellow.
3.3 pH value was formulated with distilled water to make a 1% solution and the pH value was 9.70.
3.4 Absorption Peak Take a sample of 0.1g into 100ml aqueous solution and scan in the wavelength range of 380nm-660nm. The maximum absorption peak of this solution is 405 and 630nm. The ratio of extinction value E405/E630 = 0.686/0.178 = 3.85, which meets the requirements of the standard.

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