Rapid low-cost colorimetric detection method for nutrition activity of aquaculture tail water bait green algae

Abstract

The invention discloses a rapid low-cost colorimetric detection method for the nutrition activity of bait green algae in aquaculture tail water, and belongs to the technical field of aquaculture tail water detection. The method comprises the following steps: taking aquaculture tail water and pretreating to obtain a to-be-detected sample solution; sequentially adding a copper sulfate solution, a sodium hydroxide solution and a potassium sodium tartrate solution into the to-be-detected sample solution, uniformly mixing, and carrying out a water bath reaction; standing and precipitating after the reaction is finished, adding an ammonia water solution after complete precipitation to dissolve the precipitate, fully stirring, and observing the color of the solution; comparing the color of the solution with a standard colorimetric card, and judging the nutritional activity of the green algae; according to the invention, the oxidation-reduction reaction of reducing sugar and copper sulfate in green algae cells is utilized, the color depth of blue tetraamminecopper ions directly reflects the nutrition activity, and potassium sodium tartrate is matched to assist the stable reaction; core metabolites of green algae are directly associated, the reaction mechanism is clear and stable, and the accuracy and reliability of detection are guaranteed from the source.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture tail water detection, and particularly relates to a rapid and low-cost colorimetric detection method for the nutritional activity of green algae as bait in aquaculture tail water. Background Art

[0002] In the field of detecting the nutritional activity of green algae in aquaculture tail water bait, existing technologies have many limitations and are unable to meet the industry's actual needs for fast, accurate, and low-cost testing. From the perspective of traditional detection methods, microscopic observation and blood cell counting methods can only observe the morphology and quantity of green algae, and cannot deeply evaluate the nutritional activity of green algae, which is crucial to the growth and health of farmed organisms. This makes it difficult for breeders to accurately grasp the quality of green algae, and lacks key basis when adjusting breeding strategies, which makes it easy to fall into decision-making dilemmas. Some existing colorimetric detection technologies also have many problems. Their operating procedures are cumbersome, involving complex reagent ratios and special operating links, and require high professional skills from operators, which invisibly increases the threshold for technology application. In addition, the above methods rely on special colorimetric reagents, which have high procurement costs, further limiting their promotion and use.

[0003] Patent CN 112608974A discloses a rapid and low-cost colorimetric method for detecting the nutritional activity of green algae in aquaculture tailwater bait. This method utilizes the glucose-feeding properties of green algae to optimize the combination of glucose oxidation colorimetry and iodine titration for glucose measurement. The detection conditions are limited to a relatively high alkalinity, preventing the algae from surviving in the alkaline environment and causing them to settle. The supernatant is then used for color development, avoiding the influence of the green algae's inherent color on glucose color development. This method effectively reflects the growth status of the bait algae through color development, allowing for rapid comparison of the cell density of the fresh algae solution, thereby determining the nutritional activity of the green algae. This method relies on the indirect reaction of green algae feeding on externally added glucose, and infers the activity through the residual glucose amount. It has a weak correlation with the green algae's own metabolism and is easily interfered by external factors such as the feeding environment. In order to address the problem of complex composition of aquatic tail water (including residual bait, suspended matter and other impurities), this patent relies on a high alkaline environment separation system, which can easily destroy the stability of the reaction. In addition, this method requires tedious steps such as glucose cultivation and iodine titration, which is costly, relies on professional skills and takes 3-4 hours, making it difficult to adapt to the needs of aquaculture sites.

[0004] The composition of aquaculture tailwater is extremely complex, containing a large amount of impurities such as residual bait, biological metabolites, organic matter, and suspended solids, which can easily interfere with the detection process, resulting in a significant reduction in the accuracy and reliability of the test results. In summary, existing technologies have obvious deficiencies in detection principles, operating procedures, cost control, anti-interference capabilities, and application scenarios, and are unable to adapt to the needs of the rapid development of the aquaculture industry. An innovative detection method is urgently needed to break through the bottleneck of existing technologies, solve the practical problems in the detection of the nutritional activity of green algae in aquaculture tailwater bait, and provide strong support for the high-quality development of the industry. Summary of the Invention

[0005] In response to the problems of low detection accuracy, high detection cost and complex detection methods in the existing technology, the present invention provides a rapid and low-cost colorimetric detection method for the nutritional activity of green algae in aquaculture tail water bait. The method is fast, accurate and has low detection cost. The present invention is achieved through the following technical solutions: The present invention provides a rapid and low-cost colorimetric detection method for the nutritional activity of green algae in aquaculture tail water bait, comprising the following steps: (1) Take aquaculture tail water and pre-treat it to obtain the sample solution to be tested; (2) Add copper sulfate solution, sodium hydroxide solution and potassium sodium tartrate solution to the sample solution in sequence, mix well and then react in a water bath; after the reaction is completed, let it stand and precipitate. After the precipitation is complete, add excess ammonia solution to dissolve the precipitate, stir thoroughly and observe the color of the solution; (3) Compare the solution color with the standard colorimetric card to determine the nutritional activity of green algae; The preparation method of the standard colorimetric card is as follows: preparing green algae solutions with different cell concentrations, and using the above-mentioned steps (1) and (2) to photograph and record the colors of the green algae solutions with different cell concentrations after reaction to obtain a standard colorimetric card.

[0006] Furthermore, the aquaculture tail water is sampled 2-3 hours after the cultured organisms are fed, at the pond outlet, in an area where the water flow is relatively stable and there are no obvious vortices.

[0007] Furthermore, the pretreatment method is as follows: the aquaculture tail water is centrifuged at 3500r / min for 12 minutes, the supernatant is removed after the centrifugation, the green algae precipitate is resuspended with a phosphate buffer solution, vortexed at 2000r / min for 1.5 minutes, and centrifuged at 2200r / min for 6 minutes for separation, repeated 2-3 times, resuspended with a phosphate buffer solution again, and vortexed at 2000r / min for 1 minute to obtain a sample solution to be tested.

[0008] Furthermore, the concentration of the copper sulfate solution is 0.5-0.7 mol / L; the mass percentage concentration of the sodium hydroxide solution is 8-12%, the mass percentage concentration of the potassium sodium tartrate solution is 2%; and the concentration of the ammonia water is 4-7%.

[0009] Furthermore, the volume ratio of the sample solution to be tested to the copper sulfate solution, sodium hydroxide solution, and potassium sodium tartrate solution is 6:2:1:0.5.

[0010] Furthermore, the water bath reaction conditions are 35-45° C. for 15-25 min.

[0011] Furthermore, the green algae is Chlorella, Platymonas or Dunaliella.

[0012] While the species of fish farms vary, the vast majority utilize a single bait algae feeding system. Because the quantity and concentration of the bait algae must be sufficient to satisfy the feeding needs of the fish, these selected bait algae become the dominant species in the aquaculture water, far exceeding other algae in both concentration and abundance. The remaining algae are mostly foreign algae that enter the pond with filtered aquaculture water. Due to the frequent (and short) water changes, these foreign algae are unlikely to reproduce rapidly in a short period of time. Therefore, aside from the bait algae, their presence is negligible.

[0013] In the present invention, reducing sugar (core metabolite, whose content directly reflects the nutritional activity of green algae) in green algae cells is used as a reducing agent to react with Cu in copper sulfate under alkaline conditions (provided by sodium hydroxide). 2+ Reactions occur, Cu 2+ Reduced to Cu + , reducing sugars are oxidized. Potassium sodium tartrate acts as a coordination agent and reacts with Cu 2+ Form a stable coordination compound to avoid Cu 2+ Under alkaline conditions, copper hydroxide precipitation is directly generated to interfere with the reaction, helping to maintain the stability of the reaction system and ensure that the redox reaction proceeds in an orderly manner. 2+ Reduced to Cu + After that, a precipitate will form under alkaline conditions. After adding ammonia water, Cu + It reacts with ammonia and oxygen to form blue tetraammine copper ions ([Cu(NH4)4] 2+ ), the color depth is positively correlated with the content of reducing sugars in the green algae cells (i.e. nutritional activity): the higher the reducing sugar content (the stronger the nutritional activity of the green algae), the more [Cu(NH4)4] 2+ The more the amount, the darker the blue of the solution, and vice versa. The nutritional activity of green algae can be intuitively judged by color comparison.

[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention innovatively uses reducing sugars (core metabolites) in green algae cells as the direct detection object. The reducing sugars in the green algae cells undergo an oxidation-reduction reaction with copper sulfate to generate cuprous oxide precipitates. Potassium sodium tartrate is used to assist in stabilizing the reaction. After adding ammonia water to dissolve, the depth of the blue tetraammine copper ion color directly reflects the nutritional activity. The "precipitation-dissolution" two-step reaction is adopted. Compared with the traditional colorimetric method, which is easily interfered by tail water impurities, the single color development reaction, the fluorescent probe method, which relies on expensive equipment and is easily affected by background fluorescence, and the biosensor method, which is complex and has high equipment maintenance costs, the detection principle of this patent is more directly related to the core metabolites of green algae. The reaction mechanism is clear and stable, and is not affected by interference factors such as impurities, thus fundamentally ensuring the accuracy and reliability of the detection.

[0015] (2) Change the current situation of complex detection technology, reliance on special reagents and expensive equipment, and develop a simple, easy-to-use and low-cost detection process, which will shorten the time from sampling to obtaining results to within 1-2 hours, greatly improve the detection efficiency, truly meet the rapid detection needs of aquaculture sites, reduce technical barriers and application costs, and adapt to various types of farms.

[0016] (3) The present invention uses common and inexpensive chemical reagents such as copper sulfate, sodium hydroxide, and potassium sodium tartrate, and uses conventional instruments such as centrifuges and water baths to significantly reduce the cost of testing.

[0017] (4) Fill the gap in rapid and accurate on-site testing, formulate standardized operating procedures and result judgment specifications, solve the problem that existing technologies are difficult to quantify and lack unified standards, and promote the large-scale application of testing technology in breeding sites. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The pictures of fresh Chlorella vulgaris with different concentrations from high to low are shown in Example 1; the concentration gradient is 0, 1×10 0 cell / mL, 1×10 1 cell / mL, 1×10 2 cell / mL, 1×10 3 cell / mL, 1×10 4 cell / mL, 1×10 5 cell / mL, 1×10 6 cell / mL, 1×10 7 cell / mL, 1×10 8 cell / mL; Figure 2 The standard colorimetric card of fresh Chlorella of different concentrations in Example 1; Figure 3 The pictures of fresh Platymonas with different concentrations from high to low were taken in Example 2; the concentration gradient was 1×101 cell / mL, 1×10 2 cell / mL, 1×10 3 cell / mL, 1×10 4 cell / mL, 1×10 5 cell / mL, 1×10 6 cell / mL, 1×10 7 cell / mL; Figure 4 The standard colorimetric card of fresh Platymonas with different concentrations in Example 2; Figure 5 The pictures of fresh algae with different concentrations from high to low were taken in Example 3; the concentration gradient was 1×10 1 cell / mL, 1×10 2 cell / mL, 1×10 3 cell / mL, 1×10 4 cell / mL, 1×10 5 cell / mL, 1×10 6 cell / mL, 1×10 7 cell / mL; Figure 6 This is the standard colorimetric card of fresh Dunaliella dulcis at different concentrations in Example 3. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0020] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. The reagents and raw materials used in the present invention can be purchased through conventional channels. Unless otherwise specified, the reagents and raw materials used in the present invention are used in accordance with conventional methods in the art or in accordance with the product instructions.

[0021] The present invention will now be further described with reference to the accompanying drawings and specific implementation methods.

[0022] Example 1 Establishment of a standard color chart for the nutritional activity of fresh Chlorella: (1) Chlorella was cultured under laboratory conditions (BG-11 medium, light intensity 2500-3000 lux, LED light source, light-dark cycle 12h:12h, temperature controlled at 22-25°C), and the concentrations of Chlorella cells were 0 and 1×10 0cell / mL, 1×10 1 cell / mL, 1×10 2 cell / mL, 1×10 3 cell / mL, 1×10 4 cell / mL, 1×10 5 cell / mL, 1×10 6 cell / mL, 1×10 7 cell / mL, 1×10 8 cell / mL standard solution; (2) Pretreatment: Take 50 mL of the above standard solution and place it in a centrifuge tube. Set the centrifuge speed to 3500 r / min and the centrifugation time to 12 min. After centrifugation, remove the supernatant and add 25 mL of phosphate buffer solution (0.08 mol / L, pH = 7.8) to the precipitate. Place the centrifuge tube in a vortex oscillator (model: Vortex-Genius 3) and shake for 1.5 min (2000 r / min). Place it in the centrifuge again and centrifuge (2200 r / min, 6 min). After the centrifugation is completed, remove the supernatant and add phosphate buffer solution (0.08 mol / L, pH = 7.8) to the precipitate to make up to 50 mL as the test solution. (3) Take 6 mL of the above-mentioned test solution of different concentrations and place it in a 25 mL stoppered conical flask, add 2 mL of 0.6 mol / L copper sulfate solution, 1 mL of 10 wt% sodium hydroxide solution and 0.5 mL of 2 wt% potassium sodium tartrate solution, mix well and place in a 40 ° C constant temperature water bath to react for 18 minutes; after the reaction is completed, let it stand and precipitate. After the precipitation is complete, add 5 mL of 5 wt% ammonia solution to dissolve the precipitate, stir thoroughly, and use a high-resolution camera to capture the color of the solution after the reaction. The pictures of Chlorella with different cell concentrations are as follows. Figure 1 As shown, the concentration gradient is 0, 1×10 0 cell / mL, 1×10 1 cell / mL, 1×10 2 cell / mL, 1×10 3 cell / mL, 1×10 4 cell / mL, 1×10 5 cell / mL, 1×10 6 cell / mL, 1×10 7 cell / mL, 1×10 8 cell / mL, and prepare the corresponding standard colorimetric card such as Figure 2 shown.

[0023] Example 2 (1) Fresh Platymonas were cultured under laboratory conditions (BG-11 culture medium, light intensity 2500-3000 lux, LED light source, light-dark cycle 12h:12h, temperature controlled at 22-25°C), and the concentration of Chlorella vulgaris cells was 1×10 0 cell / mL, 1×10 1 cell / mL, 1×10 2 cell / mL, 1×10 3 cell / mL, 1×10 4 cell / mL, 1×10 5 cell / mL, 1×10 6 cell / mL, 1×10 7 cell / mL standard solution; (2) Pretreatment: Take 50 mL of the above standard solution and place it in a centrifuge tube. Set the centrifuge speed to 3500 r / min and the centrifugation time to 12 min. After centrifugation, remove the supernatant and add 25 mL of phosphate buffer solution (0.08 mol / L, pH = 7.8) to the precipitate. Place the centrifuge tube in a vortex oscillator (model: Vortex-Genius 3) and shake for 1.5 min (2000 r / min). Place it in the centrifuge again and centrifuge (2200 r / min, 6 min). After the centrifugation is completed, remove the supernatant and add phosphate buffer solution (0.08 mol / L, pH = 7.8) to the precipitate to make up to 50 mL as the test solution. (3) Take 6 mL of the above-mentioned test solution of different concentrations and place it in a 25 mL stoppered conical flask, add 2 mL of 0.6 mol / L copper sulfate solution, 1 mL of 10 wt% sodium hydroxide solution and 0.5 mL of 2 wt% potassium sodium tartrate solution, mix well and place in a 40 ° C constant temperature water bath to react for 18 minutes; after the reaction is completed, let it stand and precipitate. After the precipitation is complete, add 5 mL of 5% ammonia solution to dissolve the precipitate, stir thoroughly, and use a high-resolution camera to capture the color of the solution after the reaction. The pictures of Platymonas with different cell concentrations are shown in the figure below. Figure 3 As shown, the concentration gradient is 1×10 0 cell / mL, 1×10 1 cell / mL, 1×10 2 cell / mL, 1×10 3 cell / mL, 1×10 4 cell / mL, 1×10 5 cell / mL, 1×10 6 cell / mL, 1×10 7 cell / mL, and prepare the corresponding standard colorimetric card such as Figure 4 shown.

[0024] Example 3 (1) Fresh Dunaliella was cultured under laboratory conditions (BG-11 culture medium, light intensity 2500-3000 lux, LED light source for illumination, light-dark cycle of 12h:12h, temperature controlled at 22-25℃), and the concentration of Chlorella vulgaris cells was prepared to be 1×10 0 cell / mL, 1×10 1 cell / mL, 1×10 2 cell / mL, 1×10 3 cell / mL, 1×10 4 cell / mL, 1×10 5 cell / mL, 1×10 6 cell / mL, 1×10 7 cell / mL standard solution; (2) Pretreatment: Take 50 mL of the above standard solution and place it in a centrifuge tube. Set the centrifuge speed to 3500 r / min and the centrifugation time to 12 min. After centrifugation, remove the supernatant and add 25 mL of phosphate buffer solution (0.08 mol / L, pH = 7.8) to the precipitate. Place the centrifuge tube in a vortex oscillator (model: Vortex-Genius 3) and shake for 1.5 min (2000 r / min). Place it in the centrifuge again and centrifuge (2200 r / min, 6 min). After the centrifugation is completed, remove the supernatant and add phosphate buffer solution (0.08 mol / L, pH = 7.8) to the precipitate to make up to 50 mL as the test solution. (3) Take 6 mL of the above-mentioned test solution of different concentrations and place it in a 25 mL stoppered conical flask, add 2 mL of 0.6 mol / L copper sulfate solution, 1 mL of 10 wt% sodium hydroxide solution and 0.5 mL of 2 wt% potassium sodium tartrate solution, mix them evenly and place them in a 40 °C constant temperature water bath to react for 18 minutes; after the reaction is completed, let it stand and precipitate. After the precipitation is complete, add 5 mL of 5 wt% ammonia solution to dissolve the precipitate, stir it thoroughly, and use a high-resolution camera to capture the color of the solution after the reaction. The pictures of Dunaliella with different cell concentrations are shown in the figure. Figure 5 As shown, the concentration gradient is 1×10 0 cell / mL, 1×10 1 cell / mL, 1×10 2 cell / mL, 1×10 3 cell / mL, 1×10 4 cell / mL, 1×10 5 cell / mL, 1×10 6 cell / mL, 1×10 7cell / mL, and prepare the corresponding standard colorimetric card such as Figure 6 shown.

[0025] Example 4 (1) At the outlet of the Oriental Aquaculture Pond (taking grouper culture as an example), in order to accurately evaluate the nutritional activity of the green algae (chlorella) in the tail water, a sterilized 500 mL brown glass sampling bottle was used to collect aquaculture tail water sample 20-30 cm below the water surface at 9-10 am (water quality stable period); (2) First, pour 50 mL of tail water into a sterile centrifuge tube, place it in a refrigerated centrifuge, centrifuge at 3500 r / min for 12 min, discard the supernatant, add 25 mL of 0.08 mol / L phosphate buffer solution (pH = 7.8) to the precipitate, oscillate on a vortex oscillator at 2000 r / min for 1.5 min to resuspend it, then centrifuge again at 2200 r / min for 6 min, discard the supernatant, repeat this resuspension-centrifugation operation twice, and finally add phosphate buffer solution to resuspend it to 50 mL to prepare the sample solution to be tested; (3) Before the colorimetric reaction, prepare 0.6 mol / L copper sulfate solution, 10% sodium hydroxide solution and 2% potassium sodium tartrate solution; take 6 mL of the sample solution to be tested and place it in a 25 mL stoppered test tube, add 2 mL of copper sulfate solution, 1 mL of sodium hydroxide solution and 0.5 mL of potassium sodium tartrate solution in sequence, cover the test tube stopper tightly, invert and mix thoroughly, place in a 40°C constant temperature water bath to react for 18 minutes, and take out and shake every 3-4 minutes; (4) After the reaction is completed, the precipitate was allowed to settle for 10 minutes. After the precipitation was complete, 5 mL of 5 wt% ammonia solution was added to dissolve the precipitate. The mixture was stirred thoroughly. Under natural light, the color of the supernatant in the test tube was compared with a colorimetric card prepared in advance using standard samples of Chlorella with different nutrient activity gradients. The nutrient activity of Chlorella in the culture pond was 1×10 5 cell / mL.

Claims

1. A rapid and low-cost colorimetric detection method for the nutritional activity of green algae in aquaculture tail water bait, characterized in that: The following steps are involved: (1) Take aquaculture tail water and pre-treat it to obtain the sample solution to be tested; (2) Add copper sulfate solution, sodium hydroxide solution and potassium sodium tartrate solution to the sample solution in sequence, mix well and then react in a water bath; after the reaction is completed, let it stand and precipitate. After the precipitation is complete, add excess ammonia solution to dissolve the precipitate, stir thoroughly and observe the color of the solution; (3) Compare the solution color with the standard colorimetric card to determine the nutritional activity of green algae; The preparation method of the standard colorimetric card is as follows: preparing green algae solutions with different cell concentrations, and using the above-mentioned steps (1) and (2) to photograph and record the colors of the green algae solutions with different cell concentrations after reaction to obtain a standard colorimetric card.

2. The rapid and low-cost colorimetric detection method for the nutritional activity of green algae in aquaculture tail water bait according to claim 1, characterized in that: The aquaculture tail water is sampled 2-3 hours after the cultured organisms are fed, at the pond outlet, where the water flow is relatively stable and there is no obvious vortex.

3. The rapid and low-cost colorimetric detection method for the nutritional activity of green algae in aquaculture tail water bait according to claim 1 is characterized in that: The pretreatment method is as follows: the aquaculture tail water is centrifuged at 3500 r / min for 12 minutes, the supernatant is removed after the centrifugation, the green algae precipitate is resuspended with a phosphate buffer solution, vortexed at 2000 r / min for 1.5 minutes, and centrifuged at 2200 r / min for 6 minutes for separation, repeated 2-3 times, and resuspended with a phosphate buffer solution again, vortexed at 2000 r / min for 1 minute to obtain a sample solution to be tested.

4. The rapid and low-cost colorimetric detection method for the nutritional activity of green algae in aquaculture tail water bait according to claim 1, characterized in that: The concentration of the copper sulfate solution is 0.5-0.7 mol / L; the mass percentage concentration of the sodium hydroxide solution is 8-12%, the mass percentage concentration of the potassium sodium tartrate solution is 2%; and the concentration of the ammonia water is 4-7%.

5. The rapid and low-cost colorimetric detection method for the nutritional activity of green algae in aquaculture tail water bait according to claim 1 is characterized in that: The volume ratio of the sample solution to be tested to the copper sulfate solution, sodium hydroxide solution, and potassium sodium tartrate solution is 6:2:1:0.

5.

6. The rapid and low-cost colorimetric detection method for the nutritional activity of green algae in aquaculture tail water bait according to claim 1, characterized in that: The water bath reaction conditions are 35-45° C. for 15-25 min.

7. The rapid and low-cost colorimetric detection method for the nutritional activity of green algae in aquaculture tail water bait according to claim 1, characterized in that: The green algae is Chlorella, Platyphylla or Dunaliella.

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