阅读说明：本技术 饵料绿藻营养活性的比色检测方法 (Colorimetric detection method for bait green algae nutrition activity ) 是由 赵文 王宇 魏杰 张湾 潘立峰 于 2021-01-12 设计创作，主要内容包括：本发明公开一种饵料绿藻营养活性的比色检测方法,是利用绿藻摄食葡萄糖的特性,将葡萄糖氧化显色与碘量法测葡萄糖方法优化结合并将检测条件限定为较高的碱性,使藻类在碱性环境中无法存活而沉降,进而取上清液进行显色,避免绿藻本身具有颜色对葡萄糖显色的影响,可有效地通过显色来反映饵料藻的生长状况,进而迅速比对出鲜活藻液的细胞密度,从而确定绿藻的营养活性,无需养殖经验及血细胞计数仪等设备,具有操作简单、检测效率高等优点。(The invention discloses a colorimetric detection method for the nutritional activity of bait green algae, which utilizes the characteristic that green algae ingest glucose, optimally combines glucose oxidation color development with a method for measuring glucose by an iodometric method, limits the detection condition to be higher alkalinity, ensures that algae cannot survive and settle in an alkaline environment, further takes supernatant for color development, avoids the influence of the color of the green algae on the glucose color development, can effectively reflect the growth condition of the bait algae through color development, and further quickly compares the cell density of fresh and live algae liquid, thereby determining the nutritional activity of the green algae, does not need equipment such as culture experience, a blood cell counter and the like, and has the advantages of simple operation, high detection efficiency and the like.)

1. A colorimetric detection method for the nutritional activity of bait green algae is characterized by comprising the following steps:

a. adding anhydrous glucose into a bait green alga solution, shaking uniformly, sealing, standing at 20-25 ℃, and performing illumination culture for 2h to obtain a culture solution, wherein the dosage ratio of the alga solution to the anhydrous glucose is 150 ml: 1.5 g;

b. taking 25ml of culture solution, adding 5ml of iodine standard solution with the concentration of 0.05mol/L and 2ml of sodium hydroxide aqueous solution with the concentration of 2mol/L, shaking uniformly, and sealing and performing dark treatment for 10-15 min;

c. adding 1ml of hydrochloric acid with the concentration of 6mol/L, 25ml of sodium thiosulfate aqueous solution with the concentration of 0.1mol/L and 1ml of starch indicator with the mass fraction of 0.01 into the liquid, and shaking up; then 2ml of 2mol/L sodium hydroxide aqueous solution and 3ml of 0.5g/L indigo carmine aqueous solution are added and the mixture is kept stand for two hours;

d. comparing the color of the clear liquid with a color comparison card to determine the nutritional activity of the bait green alga; and c, culturing fresh and live algae solutions corresponding to different concentration gradients, and preparing according to colors corresponding to the concentration gradients after the operation according to the steps a-c.

Technical Field

The invention relates to a method for detecting the nutritional activity of bait green algae, in particular to a colorimetric detection method for the nutritional activity of the bait green algae.

Background

The green algae is the largest one of algae, and has a wide variety and extremely wide distribution, about 90% of the green algae are grown in fresh water, and only about 10% of the green algae live in seawater. The green algae has wide application, and floating species such as Chlorella, Platymonas, Dunaliella, etc. are important baits for marine economic animal larvae. Experiments show that when the bait green algae is in the exponential growth phase with strong cell activity, the nutritional ingredients such as total fat content and unsaturated fatty acid content are higher than the death phase, so if the bait green algae which is already in the death phase is fed, the growth of the organisms is influenced because the cultured organisms do not eat and the nutritional activity is low, and the residual bait amount is increased to cause water quality deterioration and economic loss. However, in the current bait green alga management for aquaculture industry, the number of algae is often measured by observing the living body state through a microscope, visually observing through experience, counting by a blood cell counting plate, and the like, and the nutritional activity of green algae cannot be accurately analyzed.

Disclosure of Invention

The invention aims to solve the technical problems in the prior art and provides a colorimetric detection method for the nutritional activity of the bait green alga.

The technical solution of the invention is as follows: a colorimetric detection method for the nutritional activity of bait green algae is characterized by comprising the following steps:

a. adding anhydrous glucose into a bait green alga solution, shaking uniformly, sealing, standing at 20-25 ℃, and performing illumination culture for 2h to obtain a culture solution, wherein the dosage ratio of the alga solution to the anhydrous glucose is 150 ml: 1.5 g;

b. taking 25ml of culture solution, adding 5ml of iodine standard solution with the concentration of 0.05mol/L and 2ml of sodium hydroxide aqueous solution with the concentration of 2mol/L, shaking uniformly, and sealing and performing dark treatment for 10-15 min;

c. adding 1ml of hydrochloric acid with the concentration of 6mol/L, 25ml of sodium thiosulfate aqueous solution with the concentration of 0.1mol/L and 1ml of starch indicator with the mass fraction of 0.01 into the liquid, and shaking up; then 2ml of 2mol/L sodium hydroxide aqueous solution and 3ml of 0.5g/L indigo carmine aqueous solution are added and the mixture is kept stand for two hours;

d. comparing the color of the clear liquid with a color comparison card to determine the nutritional activity of the bait green alga; and c, culturing fresh and live algae solutions corresponding to different concentration gradients, and preparing according to colors corresponding to the concentration gradients after the operation according to the steps a-c.

The method optimally combines the glucose oxidation color development and the glucose measurement method by an iodometric method by utilizing the characteristic that green algae ingest glucose, limits the detection condition to be higher alkalinity, ensures that the algae cannot survive and settle in an alkaline environment, further takes the supernatant for color development, avoids the influence of the color of the green algae on the glucose color development, can effectively reflect the growth condition of the bait algae through color development, and further quickly compares the cell density of the fresh and live algae liquid, thereby determining the nutritional activity of the green algae, does not need equipment such as culture experience, a blood cell counter and the like, and has the advantages of simple operation, high detection efficiency and the like.

Drawings

FIG. 1 is a picture of the present invention taken from high concentration to low concentration in step d of taking fresh chlorella as algae liquid.

Fig. 2 is a color chart produced by using the color of the picture shown in fig. 1.

FIG. 3 is a picture of the present invention taken from high to low concentrations in the step d of taking fresh Platymonas campestris as algae liquid.

Fig. 4 is a color chart made with the color of the picture shown in fig. 3.

FIG. 5 is a picture of the present invention taken from high concentration to low concentration in step d of taking fresh Dunaliella salina as the algae liquid.

Fig. 6 is a color chart made with the color of the picture shown in fig. 5.

Detailed Description

The invention relates to a colorimetric detection method for the nutritional activity of bait green algae, which is carried out according to the following steps:

a. adding 1.5g of anhydrous glucose into 150ml of tested bait chlorella solution, shaking uniformly, sealing with a sealing film, standing at 22 ℃ and culturing for 2h under illumination to obtain a culture solution, shaking the bottle for 1-2 times during the period without oxygen supply; the Chlorella liquid can be Chlorella pyrenoidosa (Chlorella pyrenoidosa) of Chlorella and Platymonas subcordiformis (Platymonas subcordiformis) of PlatymonasFetraselmis subcordiformis) Or Dunaliella salina of the genus Dunaliella (Dunaliella salina) (B)Dunaliella salina）；

b. Adding 5ml of iodine standard solution with concentration of 0.05mol/L and 2ml of sodium hydroxide aqueous solution with concentration of 2mol/L into 25ml of culture solution, shaking uniformly, sealing and performing dark treatment for 15 min;

c. adding 1ml of hydrochloric acid with the concentration of 6mol/L, 25ml of sodium thiosulfate aqueous solution with the concentration of 0.1mol/L and 1ml of starch indicator with the mass fraction of 0.01 into the liquid, and shaking up; then 2ml of 2mol/L sodium hydroxide aqueous solution and 3ml of 0.5g/L indigo carmine aqueous solution are added and the mixture is kept stand for two hours;

d. comparing the color of the clear liquid with a color comparison card to determine the nutritional activity of the bait green alga; and c, culturing fresh and live algae liquid corresponding to different concentration gradients, shooting by using a Sony A600 camera with pixels of 2000-4000 ten thousand after the operation according to the steps a-c, and drawing the color card from high concentration to low concentration according to the color corresponding to the picture.

The fresh green algae solution used for preparing the colorimetric card corresponds to the algae solution to be detected, and is Chlorella pyrenoidosa (Chlorella pyrenoidosa) of Chlorella and Platymonas subcordiformis (Platymonas subcordiformis) of Platymonas respectivelyFetraselmis subcordiformis) Or Dunaliella salina of the genus Dunaliella (Dunaliella salina) (B)Dunaliella salina) And the like.

The method has short operation time, and can ignore the influence of extracellular products and cell propagation generated by reactions such as photosynthesis of algae cells on experimental results. All the reagents are purchased from outsourcing and prepared into different concentrations according to the existing method.

FIG. 1 is a picture of 10 concentration-from-high-to-low pictures taken in step d of the invention using fresh chlorella as algae liquid. Concentration gradient of 0, 1 x 10⁰cell/ml、1*10¹cell/ml、1*10²cell/ml、1*10³cell/ml、1*10⁴cell/ml、1*10⁵cell/ml、1*10⁶cell/ml、1*10⁷cell/ml、1*10⁸cell/ml 。

Fig. 2 is a color chart produced by using the color of the picture shown in fig. 1.

FIG. 3 is a picture of 8 concentration pictures taken from high to low in step d of taking fresh Platymonas sobria as algae liquid.

Concentration gradient of 1 x 10⁰cell/ml、1*10¹cell/ml、1*10²cell/ml、1*10³cell/ml、1*10⁴cell/ml、1*10⁵cell/ml、1*10⁶cell/ml、1*10⁷cell/ml。

Fig. 4 is a color chart made with the color of the picture shown in fig. 3.

FIG. 5 is a picture of the present invention taken from 8 concentrations from high to low in the step d of taking fresh Dunaliella salina as the algae liquid.

Concentration gradient of 1 x 10⁰cell/ml、1*10¹cell/ml、1*10²cell/ml、1*10³cell/ml、1*10⁴cell/ml、1*10⁵cell/ml、1*10⁶cell/ml、1*10⁷cell/ml。

Fig. 6 is a color chart made with the color of the picture shown in fig. 5.