Application of lactic acid in artemia breeding and simple, convenient and healthy artemia breeding method

Abstract

The invention firstly provides application of lactic acid in artemia culture. The lactic acid takes silicon dioxide or chitosan as a carrier, and is directly added into a culture water body; when the silicon dioxide is used as the carrier, the use concentration of the lactic acid in the water body is 180-300 micrometers; when the chitosan is used as the carrier, the use concentration of the lactic acid in the water body is 230-600 micrometers. The inventor accidentally finds that after the lactic acid is mixed with the silicon dioxide or the chitosan and then added into the culture water body, the sudden drop of the pH value of the water body cannot be caused, so that an acid-base regulator does not need to be added, and the incubation and growth of artemia cannot be influenced; and meanwhile, local high concentration of acid in the water body is realized by using the carrier, so that the antibacterial effect is remarkably improved, and the use amount of the acid is reduced. On the basis, the invention further provides an artemia breeding method adopting the lactic acid as the alternative antibody product. According to the artemia obtained through the breeding method, the hatching rate and the survival rate of the artemia are remarkably increased, the number of vibrios carried by the artemia is effectively reduced, expression of related genes such as growth and immunity of the artemia is up-regulated, synchronous development and growth of the artemia are promoted, and the unexpected technical effect is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of aquaculture, relates to a method for cultivating Artemia, and specifically relates to a method for cultivating Artemia by using lactic acid to reduce the hidden dangers of Artemia carrying opportunistic pathogens, thereby realizing simple, healthy, stable and sustainable cultivation of Artemia. Background Art

[0002] Artemia, commonly known as brine shrimp, brine shrimp, and brine shrimp, is a small, lower crustacean. Its small size, high nutritional value, strong environmental adaptability, rapid reproduction, and long-term storage make it an important biological feed for aquaculture's commercial animals. Market demand for Artemia has always been in short supply, and high-density, healthy Artemia farming offers significant application prospects and enormous economic benefits. Artemia's non-selective filter-feeding properties allow it to be used to enrich various nutrients and enhance its nutritional value. However, this can also easily lead to the accumulation of opportunistic pathogens, reducing the hatching and survival rates of Artemia, causing sudden collapse of high-density Artemia farming systems, and resulting in instability. Once Artemia carries opportunistic pathogens, they can infect the larvae of commercial aquaculture animals such as fish, shrimp, and crabs through the food chain, leading to the failure of seedling production. Therefore, effective control of opportunistic pathogens carried by Artemia (especially pathogenic Vibrio, such as Vibrio harveyi, Vibrio parahaemolyticus, Vibrio campbellii, etc.) is crucial to ensuring the high-density sustainable farming of Artemia, as well as the breeding of economic aquaculture animals and the healthy farming of aquatic animals.

[0003] Antibiotics are commonly used in aquaculture to control opportunistic pathogens carried by Artemia. However, antibiotic use is not only limited in effectiveness but also promotes the development of drug-resistant pathogens, which are then transmitted through the food chain, posing a significant threat to human health and the ecological environment. Currently, acidifiers (such as formic acid and acetic acid), probiotics (such as Bacillus marineus), and plant extracts (such as essential oils) are being used in Artemia aquaculture as alternatives to antibiotics. Acidifiers are a general term for organic acids that can be used as additives and are considered non-nutritional feed additives. However, the antibacterial effect of acids decreases with increasing pH, which is inconsistent with the neutral to slightly alkaline environment in which Artemia naturally lives. Studies have shown that the optimal pH for Artemia hatching is 7.5-8.5. A pH below 7 results in softened eggshells, embryonic deformities, and significantly reduced hatchability. Although adult Artemia have a wide pH range and can survive in pH ranges of 7.0-9.5, their optimal growth is between 7.5-8.5. Furthermore, sudden pH changes can trigger stress responses in Artemia (e.g., abnormal molting, decreased immunity, and susceptibility to infection). Immanuel et al. studied the effects of octanoic acid, acetic acid, propionic acid, and butyric acid on Artemia survival after artificial infection of sterile Artemia with Vibrio harveyi and Vibrio parahaemolyticus under experimental conditions. Adding octanoic acid (10 mM) to the culture water significantly reduced mortality by 16.30% for Artemia infected with Vibrio harveyi and 20.61% for Artemia infected with Vibrio parahaemolyticus (P < 0.05). Furthermore, incubation of Artemia nauplii infected with Vibrio parahaemolyticus in seawater supplemented with acetic acid (20 mM), propionic acid (20 mM), and butyric acid (20 mM) reduced mortality by 16.7%, 11.7%, and 10.0%, respectively, compared to the control group. However, despite the reduction in Artemia mortality, the high concentrations of these acids resulted in a low pH in the water, necessitating the addition of acid-base regulators to adjust the pH to 7 to meet Artemia's environmental needs. In actual aquaculture production, this significantly increases costs and operational steps, which is not conducive to the growth and industrial application of Artemia. As mentioned earlier, this study focused on artificially infected sterile Artemia, which carries a simpler bacterial flora than actual aquaculture systems.

[0004] Lactic acid is a carboxylic acid containing a hydroxyl group. It has a molecular weight of 90.08 (g / mol) and an acidity coefficient of 3.86, placing it between formic acid and acetic acid. It typically exists in liquid form and is readily soluble in water. The national food safety standard, "Food Additives Usage Standard GB2760-2014," stipulates that lactic acid can be added to infant formula as an acidity regulator. Currently, lactic acid is commonly used in fish and shrimp aquaculture as a feed additive. Direct application of lactic acid to aquaculture water poses a number of issues, including dissociation, irritation, and sensitivity to alkaline environments. Furthermore, high water hardness, alkalinity, and organic matter can neutralize the organic acid, weakening its antimicrobial activity. However, increasing lactic acid concentrations can cause a sudden drop in water pH, exceeding the tolerance range of Artemia, inhibiting hatching and growth, and further increasing costs and toxicity risks.

[0005] Due to the aforementioned technical problems, there are currently no reports on the use of lactic acid in Artemia farming. Summary of the Invention

[0006] In view of the problems of cumbersome steps and high costs in the prior art of Artemia cultivation using organic acids as alternative antibiotics, the present invention first provides the application of lactic acid in Artemia cultivation. The application achieves both the antibacterial effect of lactic acid and the stability of the pH value of the water body without adding acid-base regulators, overcoming the technical bias in the prior art that increasing the concentration of lactic acid leads to a sudden drop in the pH of the water body. On this basis, the present application also provides a method for cultivating Artemia using lactic acid as an alternative antibiotic. The Artemia obtained by the cultivation method not only significantly improves the hatching rate and survival rate of Artemia, and effectively reduces the number of Vibrio carried by Artemia, but also upregulates the expression of its growth and immunity related genes, promotes the synchronous development and growth of Artemia, and achieves unexpected technical effects.

[0007] The technical solution of the present invention:

[0008] The application of lactic acid in Artemia aquaculture, wherein the lactic acid is directly added to the aquaculture water body with silicon dioxide or chitosan as a carrier. Wherein, when the lactic acid is carried by silicon dioxide, the concentration of lactic acid in the water body is 180-300 μM; when the lactic acid is carried by chitosan, the concentration of lactic acid in the water body is 230-600 μM. The inventor unexpectedly discovered that after mixing lactic acid with the aforementioned silicon dioxide or chitosan and then adding it to the aquaculture water body, it will not cause a sudden drop in the pH of the water body, so there is no need to add an acid-base regulator and it will not affect the hatching and growth of Artemia; at the same time, the use of a carrier achieves a local high concentration of acid in the water body, which significantly improves the antibacterial effect and reduces the amount of acid used. Therefore, in the application described in the present application, the antibacterial effect of lactic acid and the stabilization of the pH value of the water body are achieved, overcoming the technical prejudice in the prior art that increasing the concentration of lactic acid to achieve the antibacterial effect, thereby causing a sudden drop in the pH value of the water body.

[0009] Preferably, when the lactic acid is carried by silica, the weight-to-volume ratio of SiO2 to lactic acid is (0.5-3):1 (g:ml), and the particle size of the silica is 10-40 μm; when the lactic acid is carried by chitosan, the weight-to-volume ratio of chitosan to lactic acid is (0.01-0.1):1 (g:ml), the degree of deacetylation of the chitosan is 75-88%, and the molecular weight is 100-180 kDa. Studies have shown that the silanol groups (Si-OH) on the surface of SiO2 can interact with the carboxylic acid groups (-COOH or -COO-) of organic acids through hydrogen bonds, and chitosan can react with the carboxylic acid groups (-COOH or -COO-) of organic acids through protonated amino groups (-NH3 + ) and the carboxylate ion of the organic acid (-COO - ) to form ion complexes; thereby controlling the release rate of organic acids, maintaining a high local concentration of acid, while prolonging the action time of the organic acids and achieving water pH stability. However, excessively high SiO2 concentrations or chitosan concentrations will affect the hatching rate of Artemia eggs and the survival rate of nauplii, while excessively low SiO2 concentrations or chitosan concentrations may not be able to achieve stable water pH.

[0010] Preferably, the density of Artemia ova in the aquaculture water body does not exceed 1000 / mL. The density range of Artemia ova in the aquaculture water body is large, which is not only suitable for experimental conditions, but also for large-scale actual aquaculture, and has important practical application value.

[0011] A simple and healthy method for cultivating Artemia comprises the following steps:

[0012] (1) According to the weight-to-volume ratio, the carrier SiO2 or chitosan is uniformly mixed with lactic acid to obtain a lactic acid product in the form of an emulsion or powder, wherein the weight-to-volume ratio of SiO2 to lactic acid is (0.5-3):1 (g:ml); the weight-to-volume ratio of chitosan to lactic acid is (0.01-0.1):1 (g:ml).

[0013] (2) Adding the lactic acid product described in step (1) to water used for hatching and breeding Artemia salina, so that the concentration of lactic acid in the breeding water reaches the required level, thereby obtaining lactic acid breeding water. When the lactic acid is carried by silica, the concentration of lactic acid in the water is 180-300 μM, and the particle size of the silica is 10-40 μm; when the lactic acid is carried by chitosan, the concentration of lactic acid in the water is 230-600 μM, and the chitosan has a degree of deacetylation of 75-88% and a molecular weight of 100-180 kDa.

[0014] This step slows down the release rate of organic acids through the interaction between the carrier and lactic acid, maintains a high local concentration of the acid (effective antibacterial effect), prolongs the action time and improves stability, avoids a sharp drop in the pH value of the water body, and keeps the pH of the water body stable within the range of 7.5-8.5 suitable for the survival of Artemia.

[0015] (3) Adding Artemia salina eggs to the lactic acid aquaculture water described in step (2), incubating at 28-30°C for 18-24 hours, and feeding a basic feed under continuous light conditions to obtain Artemia salina. The density of the Artemia salina eggs in the water is no more than 1000 eggs / mL; the continuous light conditions are 1000-2500 Lux; and the basic feed is chlorella, yeast powder, spirulina powder, rice bran, soybean meal, or artificial microparticle feed.

[0016] The Artemia ova adopted in this step is the commodity Artemia ova of actual cultivation, which is different from the sterile Artemia ova in the aforementioned research, so it itself often carries the conditional pathogens represented by pathogenic Vibrio. Through the aforementioned cultivation process, not only the quantity of the conditional pathogens carried by Artemia has been significantly reduced, but also the diversity of the carrying flora and the relative abundance of beneficial bacteria have been improved simultaneously. This not only improves the health of Artemia itself, reduces the death caused by Vibrio infection, but also optimizes the whole breeding microecological environment, reduces the risk of aquaculture animals becoming ill, reduces the use of drugs such as antibiotics, and meets the development trend of green and healthy breeding. The inventor believes, through analysis, that this is because lactic acid itself has antibacterial activity, and the sustained-release effect of the carrier and the local high concentration enable them to continue to effectively act on the Artemia living environment and Artemia body surface and body over a long period of time.

[0017] Moreover, the synchronized development and growth rate of the Artemia obtained by this step are significantly improved compared with those of ordinary breeding, producing unexpected technical effects. The inventors conducted transcriptomic sequencing analysis and qRT-PCR verification and believed that lactic acid, through the sustained release effect of the carrier system, regulated the growth of Artemia and the expression of immune-related genes, and comprehensively promoted the growth, development and survival rate of Artemia. This is the first discovery made by the inventors of this application, which provides a theoretical basis for further optimizing Artemia breeding technology; it not only shortens the breeding cycle, but also ensures the high-density, sustainable and healthy breeding of Artemia, which has important practical application value.

[0018] A method for the application of Artemia in aquaculture, wherein the Artemia are obtained using the aforementioned cultivation method. Compared with the prior art, Artemia obtained using the aforementioned cultivation method not only reduces the number of pathogenic bacteria carried, but also improves the diversity index of the carried bacterial community and the stability of the aquaculture microecological environment; therefore, the application of the Artemia in aquaculture reduces the risk of disease in farmed animals while reducing the use of antibiotics, which is in line with the development trend of green and healthy aquaculture. Moreover, the Artemia obtained using the aforementioned cultivation method not only significantly improves the hatching rate and survival rate, but also, when used as a biological bait for aquaculture, becomes a high-quality biological bait for aquaculture due to its developmental synchronization and the advantages of carrying a healthier bacterial community.

[0019] Beneficial effects of the present invention:

[0020] (1) This application first provides the application of lactic acid in Artemia aquaculture. This application achieves both the antibacterial effect of lactic acid and the stabilization of the pH value of the water body without adding an acid-base regulator, overcoming the technical problems in the prior art where increasing the lactic acid concentration causes a sudden drop in the pH value of the water body, while the reduced concentration is insufficient to produce an antibacterial effect.

[0021] (2) This application provides a method for cultivating Artemia using lactic acid as an alternative antibiotic. The method significantly improves the hatching and survival rates of Artemia and effectively reduces the number of Vibrio carried by Artemia. This provides more healthy Artemia larvae for aquaculture, ensures the production of seedlings for economic aquaculture animals, and improves the efficiency of aquaculture from the source.

[0022] (3) The cultivation method described in this application promotes the synchronization of Artemia growth and development by regulating the expression of genes related to growth and immunity, thereby enhancing Artemia immunity and ensuring the healthy and sustainable cultivation of Artemia. Compared with the existing technology that only utilizes the antibacterial properties of organic acids, the cultivation method achieves unexpected technical effects and provides a theoretical basis for further optimizing Artemia cultivation technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Attachment Figure 1 The effect of lactic acid concentration in aquaculture water (without carrier) on the hatching rate and survival rate of Artemia;

[0024] Attachment Figure 2 The emulsion prepared by using lactic acid as a carrier in Examples 1-3 of the present application;

[0025] Attachment Figure 3 The powder prepared from lactic acid using chitosan as a carrier in Examples 4-6 of the present application;

[0026] Attachment Figure 4is the relative abundance of bacterial genus levels carried by Artemia in the lactic acid group (LS) with SiO2 as a carrier, the lactic acid group (LC) with chitosan as a carrier, and the blank control group in Example 7;

[0027] Attachment Figure 5 The effect of the lactic acid group (LS) with SiO2 as the carrier and the lactic acid group (LC) with chitosan as the carrier on the number of Vibrio carried by Artemia in Example 7 (Day 7);

[0028] Attachment Figure 6 This is a comparison of the developmental synchronization of Artemia salina in the lactic acid group (LS) with SiO2 as a carrier and the blank control group after 24 hours of incubation in Example 9;

[0029] Attachment Figure 7 This is a KEGG enrichment analysis diagram of differentially expressed genes between the Artemia salina group (LS) and the control group using SiO2 as a carrier in Example 10;

[0030] Attachment Figure 8 This is a KEGG enrichment analysis diagram of differentially expressed genes between Artemia salina in the lactic acid group (LC) and the control group using chitosan as a carrier in Example 10;

[0031] Attachment Figure 9 The qRT-PCR results of differentially expressed genes of Artemia in Example 10 (A. lactic acid group (LS) with SiO2 as carrier and blank control group, B. lactic acid group (LC) with chitosan as carrier and blank control group). DETAILED DESCRIPTION

[0032] The present invention is further described below with reference to the following examples. The Bohai Bay Artemia eggs (hatching rate 80%), food-grade SiO2, chitosan, and lactic acid used in the examples were all purchased from commercial sources. The pH of the water used for Artemia hatching and culture was 8.23.

[0033] 1. Effects of lactic acid on the hatching and survival of Artemia in the absence of a carrier

[0034] In order to obtain the safe concentration of lactic acid on the hatching rate and survival rate of Artemia, the following experiment (experimental group and control group) was conducted.

[0035] The experimental groups were exposed to different concentrations of lactic acid (0, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, 1000 μM) in sterile seawater; the control group was not treated and Artemia was hatched and cultured. The specific operation was as follows: Artemia eggs were hatched in 5 mL of sterile seawater at a density of 30 eggs / mL for 24 hours, the aquaculture water temperature was maintained at 28-30 ℃, and the light was continuous (2000 Lux). Chlorella was fed twice a day at 7:00 and 19:00. The total number of Artemia eggs (H), the total number of Artemia hatched (h) and the total number of nauplii surviving for 48 hours (s) were recorded. Three replicates were set up for each group. For detailed results, see Figure 3 .

[0036] like Figure 1 As shown in the figure, when the lactic acid concentration was lower than 80 μM, there was no significant effect on the hatching rate of Artemia eggs (P>0.05), and there was no significant effect on the survival rate of Artemia nauplii (P>0.05). As the lactic acid concentration increased, the hatching rate of Artemia eggs and the survival rate of Artemia nauplii gradually decreased. The LC value of lactic acid on the hatching rate of Artemia eggs was calculated based on the number of Artemia eggs and nauplii deaths. 50 LC values for nauplii survival 50 values, which were 415.77 μM and 350.53 μM, respectively.

[0037] According to the Turubell safety concentration calculation formula:

[0038]

[0039] The safe concentration of lactic acid for Artemia was 74.75 μM.

[0040] Example 1: Hatching and Culture of Artemia (Lactic Acid-SiO2 as an Antibiotic Alternative)

[0041] A simple and healthy method for cultivating Artemia comprises the following steps:

[0042] (1) Preparation of lactic acid products: SiO2 and lactic acid are mixed evenly according to the weight-volume ratio to obtain a lactic acid product in the form of an emulsion (such as Figure 2 As shown); the weight volume ratio of SiO2 to lactic acid is 0.6:1 (g:ml).

[0043] (2) Preparing lactic acid aquaculture water: Add the lactic acid product described in step (1) to the water used for hatching and aquaculture of Artemia salina, so that the concentration of lactic acid in the aquaculture water reaches the required level, thereby obtaining lactic acid aquaculture water. The concentration of lactic acid in the water is 180 μM, and the particle size of the silica is 10 μm.

[0044] (3) Artemia hatching and culture: Artemia eggs are placed in the lactic acid culture water described in step (2), incubated at 28-30°C for a period of time, and fed with a basic feed under continuous light conditions to obtain Artemia. The incubation time is 18 hours, the density of the Artemia eggs in the water is 100 eggs / mL, the continuous light condition is 1000 Lux, and the basic feed is Chlorella vulgaris, which is fed twice a day at 7:00 and 19:00.

[0045] (4) Detection of Artemia hatching rate, survival rate and body length:

[0046] Hatching rate and survival rate of Artemia: Complete the hatching according to step (3) and record the total number of Artemia eggs (H), the total number of Artemia hatched (h), and the total number of Artemia nauplii surviving after 48 h (s). Set up three replicates for each group. The hatching rate is expressed as the percentage of the total number of Artemia hatched (h) to the total number of Artemia eggs (H), and the survival rate is expressed as the percentage of the total number of nauplii surviving (s) to the total number of Artemia eggs (H). The formula is as follows:

[0047]

[0048]

[0049] Note: h is the total number of Artemia hatched; H is the total number of Artemia eggs; s is the total number of nauplii surviving within 48 hours.

[0050] Body length detection: Since the Artemia hatched, 10 Artemia were randomly selected from each experimental group and the control group every day. The body length of Artemia was measured using a stereo microscope once a day. The body length was the distance from the front end of the Artemia head to the bottom end of the tail fork. The body length of Artemia was recorded and the average value was calculated.

[0051] Example 2: Hatching and Culture of Artemia (Lactic Acid-SiO2 as an Antibiotic Alternative)

[0052] Different from Example 1, a simple and healthy method for cultivating Artemia comprises the following steps:

[0053] (1) Preparation of lactic acid products: The weight-to-volume ratio of SiO2 to lactic acid is 1.5:1 (g:ml).

[0054] (2) Preparation of lactic acid aquaculture water: the concentration of lactic acid in the water is 250 μM, and the particle size of the silicon dioxide is 20 μm.

[0055] (3) Artemia hatching and breeding: the hatching time is 24 hours, the density of the Artemia eggs in the water body is 1000 / mL; the continuous illumination condition is 2000 Lux; the basic feed is spirulina powder, which is fed twice a day at 7:00 and 19:00.

[0056] (4) Detection of Artemia hatching rate, survival rate and body length: Same as Example 1.

[0057] Example 3: Hatching and Culture of Artemia (Lactic Acid-SiO2 as an Antibiotic Alternative)

[0058] Different from Example 1, a simple and healthy method for cultivating Artemia comprises the following steps:

[0059] (1) Preparation of lactic acid products: The weight-to-volume ratio of SiO2 to lactic acid is 2.5:1 (g:ml).

[0060] (2) Preparation of lactic acid aquaculture water: the concentration of lactic acid in the water is 300 μM, and the particle size of the silicon dioxide is 40 μm.

[0061] (3) Artemia hatching and breeding: the hatching time is 24 hours, the density of the Artemia eggs in the water body is 500 eggs / mL; the continuous illumination condition is 2500 Lux; the basic feed is yeast powder, which is fed twice a day at 7:00 and 19:00.

[0062] (4) Detection of Artemia hatching rate, survival rate and body length: Same as Example 1.

[0063] Comparative Example 1: Hatching and Culture of Artemia (Lactic Acid as an Antibiotic Alternative)

[0064] The difference from Example 2 is that no SiO2 is added.

[0065] Example 4: Hatching and Culture of Artemia (Lactic Acid-Chitosan as an Antibiotic Alternative)

[0066] A simple and healthy method for cultivating Artemia comprises the following steps:

[0067] (1) Preparation of lactic acid products: chitosan and lactic acid are mixed evenly according to the weight-volume ratio to obtain a lactic acid product in the form of a powder (such as Figure 3 As shown); the weight volume ratio of chitosan to lactic acid is 0.01:1 (g:ml).

[0068] (2) Preparing lactic acid aquaculture water: Add the lactic acid product described in step (1) to the water used for hatching and aquaculture of Artemia salina, so that the concentration of lactic acid in the aquaculture water reaches the required concentration, thereby obtaining lactic acid aquaculture water. The concentration of lactic acid in the water is 250 μM, and the chitosan has a degree of deacetylation of 75% and a molecular weight of 180 kDa.

[0069] (3) Artemia hatching and culture: Artemia eggs are placed in the lactic acid culture water described in step (2), incubated at 28-30°C for a period of time, and fed with a basic feed under continuous light conditions to obtain Artemia. The incubation time is 18 hours, the density of the Artemia eggs in the water is 100 eggs / mL, the continuous light condition is 1000 Lux, and the basic feed is Chlorella vulgaris, which is fed twice a day at 7:00 and 19:00.

[0070] (4) Detection of Artemia hatching rate, survival rate and body length: Same as Example 1.

[0071] Example 5: Hatching and Culture of Artemia (Lactic Acid-Chitosan as an Antibiotic Alternative)

[0072] Different from Example 4, a simple and healthy method for cultivating Artemia comprises the following steps:

[0073] (1) Preparation of lactic acid products: The weight-to-volume ratio of chitosan to lactic acid is 0.05:1 (g:ml).

[0074] (2) Preparation of lactic acid aquaculture water: the concentration of the lactic acid in the water is 500 μM, the deacetylation degree of the chitosan is 80%, and the molecular weight is 100 kDa.

[0075] (3) Artemia hatching and breeding: the hatching time is 24 hours, the density of the Artemia eggs in the water body is 1000 eggs / mL; the continuous illumination condition is 2000 Lux; the basic feed is spirulina powder, which is fed twice a day at 7:00 and 19:00.

[0076] (4) Detection of Artemia hatching rate, survival rate and body length: Same as Example 1.

[0077] Example 6: Hatching and Culture of Artemia (Lactic Acid-Chitosan as an Antibiotic Alternative)

[0078] Different from Example 4, a simple and healthy method for cultivating Artemia comprises the following steps:

[0079] (1) Preparation of lactic acid products: The weight-to-volume ratio of chitosan to lactic acid is 0.1:1 (g:ml).

[0080] (2) Preparation of lactic acid aquaculture water: The concentration of lactic acid in the water is 600 μM, the deacetylation degree of chitosan is 88%, and the molecular weight is 120 kDa.

[0081] (3) Artemia hatching and breeding: the hatching time is 24 hours, the density of the Artemia eggs in the water body is 500 eggs / mL; the continuous illumination condition is 2500 Lux; the basic feed is yeast powder, which is fed twice a day at 7:00 and 19:00.

[0082] (4) Detection of Artemia hatching rate, survival rate and body length: Same as Example 1.

[0083] Comparative Example 2: Hatching and Culture of Artemia (Lactic Acid as an Antibiotic Alternative)

[0084] The difference from Example 5 is that chitosan is not added.

[0085] Comparative Example 3: Hatching and Culture of Artemia (Blank Control Group)

[0086] The difference from Example 5 is that lactic acid, SiO2 and chitosan are not added.

[0087] Table 1. Hatching rate, survival rate and body length of Artemia cultured in Examples 1-6 and Comparative Examples 1-2

[0088]

[0089] As shown in Table 1, the blank control (no lactic acid and carrier added) was compared with Example 3. The pH of the aquaculture water was 8.23, the Artemia hatching rate and 48h survival rate were 81% and 78% respectively, and the average body length on the 7th day was 3.46 mm.

[0090] According to the Turubel safety concentration calculation formula, the safe concentration of lactic acid for Artemia hatching and survival was obtained (74.75 μM). In Examples 1-3, which used lactic acid-SiO2 as an antibiotic substitute, the initial pH of the aquaculture water was 8.23. After adding lactic acid-SiO2 at concentrations exceeding the safe concentration (180-300 μM), the final pH of the aquaculture water only decreased slightly, to between 7.58 and 7.86. The hatching rate of Artemia eggs was 80-82%, the 48-hour survival rate was 77-78%, and the average body length on the seventh day was 3.74-3.83 mm. In Comparative Example 1, which used only lactic acid as an antibiotic substitute, 250 μM lactic acid reduced the final pH of the aquaculture water to 6.86, significantly affecting the hatching rate of Artemia eggs and the survival rate of larvae. The hatching rate dropped significantly to 53%, the 48-hour survival rate was 48%, and the survival rate was even worse in the later stages. The hatching rate and survival rate of Artemia in Examples 1-3 were significantly improved compared with Comparative Example 1, and had no significant difference from Comparative Example 3. The body length of Artemia in Examples 1-3 was significantly promoted compared with Comparative Example 3.

[0091] In Example 4-6, which uses lactic acid-chitosan as an alternative to antibiotics, the initial pH of the aquaculture water was 8.23. After adding lactic acid-chitosan at a concentration far exceeding the safe use concentration (250-600 μM), the final pH of the aquaculture water only dropped slightly to between 7.69 and 7.94. The hatching rate of Artemia eggs was 77-79%, the survival rate was 71-74%, and the average body length on the 7th day was 3.77-3.99 mm. In Comparative Example 2, which only used lactic acid as an alternative to antibiotics, 500 μM lactic acid reduced the final pH of the aquaculture water to 6.72, affecting the hatching rate of Artemia eggs and the survival of Artemia larvae, resulting in a hatching rate of 50%, a 48h survival rate of 44%, and poor survival rate in the later stage. The hatching rate and survival rate of Artemia in Example 4-6 were significantly improved compared to Comparative Example 2, and slightly decreased compared to Comparative Example 3. The increase in Artemia body length in Example 4-6 was more significant than that in Comparative Example 3.

[0092] It can be seen that compared with the carrier-free system, (1) after adding 180-600 μM lactic acid-carrier to the aquaculture water of Examples 1-6 of the present application, the final pH is 7.58-7.94, and the pH of the aquaculture water is always stable, which is suitable for the growth and reproduction of Artemia; (2) the hatching rate and survival rate of Artemia obtained by the aquaculture method of Examples 1-6 of the present application are significantly improved (P < 0.05); (3) compared with the blank control system (no lactic acid and carrier addition), on the basis of not significantly affecting the hatching rate and survival rate of Artemia, not only the growth of Artemia is promoted (body length increases significantly), but also the safe use concentration of lactic acid is increased (i.e., the antibacterial effect is improved), achieving an unexpected technical effect. This shows that the lactic acid-SiO2 and lactic acid-chitosan are alternative antibiotic products. Under the premise of not adding acid-base regulators, they overcome the technical problem of increasing lactic acid concentration in the prior art, which causes the pH of the water to drop sharply and affects the hatching and survival of Artemia, and achieve the unexpected technical effect of promoting the growth and development of Artemia.

[0093] Example 7: Analysis of microbial flora carried by cultured Artemia

[0094] This example analyzes the bacterial flora carried by the cultured Artemia in Examples 1-6 based on 16S rRNA high-throughput sequencing, and the sequencing results are consistent with the actual results. The following uses Example 2 (LS group) and Example 5 (LC group) as examples, combined with the blank control group (Control, without lactic acid and carrier addition) for detailed description as follows:

[0095] (1) Sample preparation

[0096] Starting from the time Artemia hatched, samples were collected on days 1 (nauplii), 3 (pre-adult), and 7 (adult). From each group of three replicates, 200 Artemia were randomly sampled, mixed, rinsed repeatedly with ultrapure water, placed in 1.5 mL EP tubes, and quickly frozen in liquid nitrogen for 30 minutes before being stored at -80°C. After all samples were collected, 16S rRNA-based Artemia microbial flora analysis was performed by Shanghai Meiji Biological Co., Ltd.

[0097] (2) Data analysis

[0098] Paired-end reads generated by Illumina sequencing were spliced based on overlap, and sequence quality was controlled and filtered. After sample differentiation, OTU (Operational Taxonomic Unit) cluster analysis and species taxonomy analysis were performed. Bioinformatics statistical analysis was performed on OTUs at a 97% similarity level. Based on taxonomic information, statistical analysis of community structure was performed at various taxonomic levels.

[0099] Alpha diversity analysis was performed based on the high-throughput sequencing results of each Artemia sample group, and a statistical table of diversity indices was obtained (Table 2). The Chao, Ace, and Sob indices reflect the number of species in a sample, but do not consider the abundance of each species. The Shannon and Simpson indices reflect that the diversity of a sample is affected by species abundance and evenness. That is, under the condition of the same species abundance, the greater the evenness of each species in the sample, the greater the diversity.

[0100] (3) Verification of the effect of lactic acid carrier on the number of Vibrio carried by Artemia using the dilution plate method

[0101] On the 7th day (adult stage), 30 Artemia worms were taken from each of Example 2 (LS group), Example 5 (LC group) and the blank control group (Control, without adding lactic acid and carrier). The Artemia worms were repeatedly rinsed with ultrapure water in a clean bench, and then 1 mL of physiological saline was added to the glass homogenizer for tissue homogenization. The homogenization was carried out until the Artemia tissue was no longer visible. The homogenized tissue fluid was diluted 10-fold and spread on a TCBS plate. The cells were cultured at 32°C for 2 days, and the morphological characteristics of the bacteria were observed and counted.

[0102] Lactic acid with SiO2 as carrier (LS group) and lactic acid with chitosan as carrier (LC group) were added to the water for Artemia culture. The relative abundance of the bacterial community carried by Artemia in the nauplii (day 1), quasi-imaginal (day 3) and adult (day 7) stages of each group was analyzed at the genus level. Figure 4 .like Figure 4 As shown in the figure, the top two dominant bacterial groups in relative abundance were counted, and it was found that: in the nauplii stage, the dominant bacterial groups carried by the Artemia in the control group were Exiguobacterium and Acinetobacter, with relative abundances of 52.2% and 12.5%, respectively; the Artemia in the LS group carried Acinetobacter and Marinobacter, with relative abundances of 14.4% and 7.6%, respectively; and the Artemia in the LC group carried Psychrobacter and Exiguobacterium, with relative abundances of 52.6% and 37.3%, respectively. In the nauplii stage, Vibrio was not found as the dominant bacterial group in any of the three groups.

[0103] During the quasi-adult stage, the dominant bacterial communities in the control group were Psychrobacter and Exiguobacterium, with relative abundances of 79.43% and 6.4% respectively. The LS group carried Oceanobacter and Idiomarina, with relative abundances of 25.4% and 4.3% respectively. The LC group carried Pseudoalteromonas and Psychrobacter, with relative abundances of 15.2% and 13.1% respectively. Vibrio was not found as the dominant bacterial community in any of the three groups during the quasi-adult stage.

[0104] However, during the adult stage, both the blank control group and the LC group showed a high abundance of Vibrio, becoming the dominant bacterial community. The dominant bacterial communities in the blank control group were Vibrio and Psychrobacter, with relative abundances of 56.5% and 21.7%, respectively. The dominant bacterial communities in the LC group were Vibrio and Exiguobacterium, with relative abundances of 64.6% and 21.9%, respectively. In the LS group, the dominant bacterial communities were Oceanobacter and Exiguobacterium, with relative abundances of 8.9% and 5.1%, respectively. The relative abundance of Vibrio was only 0.28%. Studies have reported that Exiguobacterium, Psychrobacter, and Oceanobacterium are potential probiotics, and their increased abundance can help enhance the intestinal health of farmed animals, improving their immunity and disease resistance.

[0105] Table 2. Alpha diversity analysis of Artemia bacteria at different growth stages in LS, LC and blank control groups

[0106]

[0107] As shown in Table 2, the estimated Sobs (195, 102, 114), Chao (198.6, 106.4, 133.43), and Ace (197.5, 109.79, 126.43) indices of the Artemia microbiome in the LS group at each growth stage were significantly higher than those in the LC group and the blank control group, indicating that lactic acid, carried by SiO2, enriched the species diversity of the Artemia microbiome. The Shannon indices of Artemia in the LS group at each growth stage (2.47, 2.49, 2.58) were higher than those in the LC group and the blank control group, indicating a more uniform distribution of Artemia species in the LS group. Furthermore, the Simpson indices (0.19, 0.17, 0.13) were lower than those in the LC group and the blank control group, indicating fewer dominant species and higher microbial diversity. Compared with the LC and blank controls, the LS group exhibited a more stable and healthy microbial ecosystem.

[0108] Furthermore, the dilution plate method was used to verify the effect of lactic acid on the number of Vibrio carried by Artemia on the 7th day. Figure 5 .like Figure 5 The results showed that the number of Vibrio carried by Artemia in the LS group (1.8×10 2 CFU / tail) was significantly lower than that in the control group (P < 0.05), but the number of Vibrio carried by the LC group (3.27 × 10 5 CFU / tail) compared with the control group (4.03×10 5 There was no significant difference in the number of CFU / tail between the two groups (P>0.05). The results of the coating were consistent with the results of high-throughput sequencing. On day 7, the Artemia survival rate in the LS group (78 ± 5%) was significantly higher than that in the control group (68 ± 8%) and the LC group (71 ± 5%).

[0109] In summary, in this application, lactic acid with SiO2 as a carrier can significantly improve the hatching rate and survival rate of Artemia and effectively control the Vibrio carried by Artemia, and is superior to lactic acid with chitosan as a carrier.

[0110] Example 8: Anti-Vibrio infection experiment

[0111] According to the data of Examples 1-6 and Comparative Examples 1-2, the hatching rate, survival rate and body length of Artemia were significantly improved by using lactic acid-SiO2 and lactic acid-chitosan as alternative antibiotic products for Artemia cultivation. According to the results of the analysis of the microbial flora carried by cultured Artemia in Example 7, lactic acid-SiO2 significantly reduced the opportunistic pathogens carried by Artemia, and the carried flora was more diverse, with high distribution uniformity and diversity index, indicating that the cultured microecology was more stable and healthy.

[0112] Furthermore, Artemia challenge experiments were conducted using Vibrio campbellii BB120 (V. campbelli BB120) as the pathogen to verify the antibacterial effects of lactic acid-SiO2 and lactic acid-chitosan against pathogenic Vibrio. The following uses Example 2 (LS group) and Example 5 (LC group) as examples, where V. campbellii BB120 was added during Artemia hatching. The Vibrio group (only V. campbellii BB120) and the blank control group (no antibiotic replacement product or V. campbellii BB120) are described in detail below:

[0113] Three experimental groups were set up, namely Example 2 (lactic acid-SiO2+V. campbellii BB120), Example 5 (lactic acid-chitosan+V. campbellii BB120) and Vibrio group (only Vibrio campbellii BB120). When Artemia eggs were hatched, Vibrio campbellii BB120 was added to the aquaculture water of each experimental group to a concentration of 1×10 7 The cells were challenged with a final concentration of 10 cells / mL, and the blank control group was not treated. The total number of Artemia eggs (H), the total number of Artemia hatched (h), and the total number of Artemia nauplii surviving at 48 h (s) were recorded. Three replicates were set up for each group. The hatching rate was expressed as the percentage of the total number of Artemia hatched (h) to the total number of Artemia eggs (H), and the survival rate was expressed as the percentage of the total number of surviving Artemia nauplii (s) to the total number of Artemia eggs (H). The calculation formula was the same as in Example 1.

[0114] From Table 3, we can see that in 1×10 7 Under the challenge of Vibrio campbellii BB120 cell / mL, the hatching rate of Artemia eggs and the survival rate of nauplii added with lactic acid-SiO2 or lactic acid-chitosan were significantly higher than those in the Vibrio group (P < 0.05), indicating that lactic acid-carrier can significantly reduce the adverse effects of Vibrio on Artemia hatching and growth. In addition, the hatching rate and nauplii survival rate of Artemia in the LS group with lactic acid-SiO2 were not significantly different from those in the blank control group (P > 0.05), and the effect of lactic acid with chitosan as the carrier was second. The experimental results are consistent with those in Example 7.

[0115] Table 3. Effects of lactic acid carrier on the hatching rate and survival rate of Artemia salina under Vibrio campbellii challenge

[0116]

[0117] Note: Different letters indicate significant differences (P < 0.05), and the same letters indicate no significant differences (P > 0.05)

[0118] Example 9: Determination of Artemia Growth Rate and Developmental Synchronicity

[0119] According to the data of Examples 1-6 and Comparative Examples 1-2, the use of lactic acid-SiO2 and lactic acid-chitosan as antibiotic replacement products for Artemia culture has significantly increased the body length of Artemia. The inventors used Example 2 (LS group) and Example 5 (LC group) as examples and combined them with the blank control group to further study the effect on the growth rate of Artemia. The specific operation is as follows:

[0120] Ten Artemia were randomly selected from each experimental group and the control group. The body length of Artemia was measured using a stereo microscope once a day. The body length was the distance from the front end of the Artemia head to the bottom end of the tail fork. The body length of Artemia was recorded and the average value, average daily growth rate (ADG, Average Daily Growth Rate, mm / d) and specific growth rate (SGR, %) were calculated. The formulas for calculating the average daily growth rate and specific growth rate are as follows:

[0121]

[0122]

[0123] Note: ADG is the average daily growth rate, mm / d; SGR is the specific growth rate, %; L t is the average body length of Artemia in each group on the 7th day, mm; L0 is the average body length of Artemia in each group on the 1st day, mm; t is time, d.

[0124] As can be seen from Tables 1 and 4, lactic acid can increase the average daily growth rate and specific growth rate of Artemia. Among them, lactic acid with chitosan as a carrier has the best effect on promoting the growth of Artemia, with the average daily growth rate and specific growth rate of Artemia reaching 0.48 mm / d and 26.06%, respectively.

[0125] Table 4. Effects of lactic acid carrier on Artemia growth indicators

[0126]

[0127] The inventors took Example 2 (LS group, bottom figure) as an example and combined it with Comparative Example 1 (blank control group, top figure) to further study the synchronization of Artemia hatching and development. The Artemia eggs were photographed 24 hours after hatching. Figure 6 .like Figure 6 As shown, in Comparative Example 1 (blank control group, upper figure), after 24 hours of incubation, some Artemia eggs were still in the umbrella stage and had not fully hatched; while in the LS group (lower figure), Artemia eggs were fully hatched and in the active nauplii stage 1. This shows that lactic acid can accelerate the hatching and development of Artemia.

[0128] In conclusion, lactic acid can increase the hatching and development speed, average daily growth rate and specific growth rate of Artemia.

[0129] Example 10: Transcriptome Sequencing Analysis and qRT-PCR Verification of Artemia Cultured in Examples 1-6 and Comparative Examples 1-2

[0130] According to Examples 1-6 and 9, the use of lactic acid-chitosan or lactic acid-SiO2 as an alternative antibiotic product for Artemia cultivation significantly improved the body length and growth of Artemia, achieving unexpected technical effects. To get to the bottom of this, the inventors conducted transcriptome sequencing analysis on the Artemia cultured in Examples 1-6. The transcriptome sequencing results analyzed for the first time the molecular mechanism of lactic acid regulation of Artemia growth and immune protection at the gene level, revealing the physiological response characteristics of Artemia to lactic acid.

[0131] The following takes Example 2 (LS group) and Example 5 (LC group) as examples, and combines the blank control group (no antibiotic replacement product added) as an example to provide a detailed description as follows:

[0132] (1) Transcriptome Sequencing Analysis of Artemia Cultured in Examples 1-6 and Comparative Examples 1-2

[0133] Starting from the hatching of Artemia, each experimental group was sampled on the 7th day. 300 Artemia were randomly selected from the three replicates of each group, mixed and repeatedly rinsed with ultrapure water, placed in a 1.5 mL EP tube, and quickly frozen in liquid nitrogen for 30 minutes and then frozen in a -80 ℃ refrigerator for later use. After all sampling was completed, Shanghai Meiji Biological Co., Ltd. carried out Artemia transcriptome determination. 1) Functional annotation statistics: The obtained transcripts were compared in 6 databases, including NR, Swiss-Prot, Pfam, EggNOG, GO and KEGG; 2) Expression analysis: Based on the expression quantification results, inter-group differential gene analysis was performed to obtain differentially expressed genes between the two groups, and the DEGseq software was used for differential analysis; KEGG enrichment analysis was further performed on the differentially expressed genes, see for details. Figure 7-9 .

[0134] Attachment Figure 7 Figure 2 is the KEGG enrichment analysis of differentially expressed genes of Artemia in LS group and control group. Figure 7As shown, differentially expressed genes were annotated into 138 pathways between the LS group and the blank control group. Among the top 30 pathways with the highest enrichment, 25 were related to growth and metabolism. The top three pathways by enrichment were the glucagon signaling pathway, fatty acid degradation, and adipocytokine signaling pathway. Five immune-related signaling pathways were identified, with the top three pathways by enrichment being the PPAR signaling pathway, AMPK signaling pathway, and C-type lectin receptor signaling pathway. Specifically, the CPT1A and ACACA genes in the glucagon signaling pathway, fatty acid degradation, and adipocytokine signaling pathways were significantly upregulated in the LS group (P < 0.05). The significant upregulation of CPT1A and ACACA genes indicates that the dual activity of fatty acid synthesis and degradation allows Artemia to rapidly obtain energy in a short period of time while maintaining a long-term energy supply through fatty acid synthesis, which helps Artemia maintain growth and reproduction, thereby improving their survival rate. This dynamic balance of lipid metabolism plays a crucial role in the rapid growth and high energy requirements of Artemia during the hatching period.

[0135] Attachment Figure 8 Figure 2 is the KEGG enrichment analysis of differentially expressed genes of Artemia in LC group and control group. Figure 8 As shown, differentially expressed genes were annotated into 339 pathways between the LC and control groups. Among the top 30 pathways with the highest enrichment, 24 were related to growth metabolism. The top three pathways by enrichment were the insulin signaling pathway, the thyroid hormone signaling pathway, and carbohydrate digestion and absorption. Six immune-related signaling pathways were identified, with the top three pathways by enrichment being the PI3K-Akt signaling pathway, the mTOR signaling pathway, and the EMC receptor interaction pathway. In the LC group, the PI3KCA gene was significantly upregulated in the insulin signaling pathway, the thyroid hormone signaling pathway, and the carbohydrate digestion and absorption pathway (P < 0.05). TSC2 and mTOR genes were significantly upregulated in the insulin signaling pathway and the thyroid hormone signaling pathway (P < 0.05). The MGAM gene was significantly upregulated in the carbohydrate digestion and absorption pathway (P < 0.05). The upregulation of PI3KCA, mTOR, and MGAM increases the efficiency of carbohydrate digestion and absorption, enhances insulin signaling, further promotes glucose uptake and utilization, protein synthesis, and cell proliferation, enabling Artemia to utilize energy more efficiently, enhancing its energy metabolism and cell growth, and enabling Artemia to develop at the fastest rate and maintain survival rate under conditions of adequate nutrition and intensive farming.

[0136] Compared with the LS group, the LC group showed significantly more enriched KEGG pathways, consistent with the LC group's superior growth-promoting effect on experimental animals. This suggests that chitosan-based lactic acid triggers a broader biological response, activating more signaling and metabolic pathways (such as the insulin signaling pathway, carbohydrate digestion and absorption), further promoting cell proliferation, protein synthesis, and energy metabolism, thereby supporting Artemia growth. In contrast, the LS group was less effective in promoting growth and development.

[0137] (2) qRT-PCR verification

[0138] In order to verify the reliability of the transcriptome sequencing results, one gene with significant differential expression and closely related to metabolic or immune pathways was selected from each experimental group for qRT-PCR verification. The specific method was as follows: total RNA was extracted from Artemia tissue using the Trizol method, and the RNA was reverse transcribed into cDNA using the PrimeScript™ FAST RT reagent Kit with gDNA Eraser (TAKARA) kit. qRT-PCR was performed on a LightCyeler 96 (Roche, Germany) using the TB Green® Premix Ex Taq™ II FAST qPCR (TAKARA) kit. PCR primers were designed and synthesized by Qingdao Boshang Biotechnology Co., Ltd., using β-actin as the internal reference gene. The primer sequences are shown in Table 5. The relative quantification method (2 -ΔΔCT ) to determine the fold change of the target gene. T-test analysis was performed using SPSS 26.0 software, and P < 0.05 was considered statistically significant. Figure 9 .

[0139] Table 5. Primer sequences used for qRT-PCR

[0140]

[0141] Attachment Figure 9 The qRT-PCR results of differentially expressed genes in Artemia. Compared with the blank control group, the CPT1A gene in the LS group was significantly upregulated (P < 0.05) ( Figure 9 A); PI3KCA gene was significantly up-regulated in the LC group (P<0.05) ( Figure 9 B). This shows that the PCR verification results are consistent with the differential gene expression upregulation results of transcriptome sequencing analysis, confirming the reliability of the transcriptome sequencing analysis results of this application.

[0142] In summary, the present application first provides the application of lactic acid in Artemia cultivation. The application achieves both the antibacterial effect of lactic acid and the stability of the pH value of the water body without adding an acid-base regulator, overcoming the technical problems in the prior art that increasing the concentration of lactic acid leads to a sudden drop in the pH of the water body, the safety risk to Artemia, and the concentration reduction is not enough to produce an antibacterial effect. On this basis, the present application provides a method for cultivating Artemia using lactic acid as an alternative to antibiotics. The cultivation method not only significantly improves the hatching rate and survival rate of Artemia and effectively reduces the number of Vibrio carried by Artemia by regulating the expression of growth-related, immune-related genes, but also promotes the synchronization of Artemia growth and development, enhances the immunity of Artemia, and ensures the healthy and sustainable cultivation of Artemia. Compared with the prior art that only utilizes the antibacterial properties of organic acids, the cultivation method achieves unexpected technical effects and provides a theoretical basis for further optimizing Artemia cultivation technology.

Claims

1. The application of lactic acid in Artemia culture is characterized by: The lactic acid is directly added into the aquaculture water body with silicon dioxide or chitosan as a carrier.

2. The use according to claim 1, characterized in that: When the lactic acid uses silicon dioxide as a carrier, the concentration of the lactic acid in the water body is 180-300 μM; when the lactic acid uses chitosan as a carrier, the concentration of the lactic acid in the water body is 230-600 μM.

3. The use according to claim 2, characterized in that: When the lactic acid uses silicon dioxide as a carrier, the weight-to-volume ratio of SiO2 to lactic acid is (0.5-3):1, and the particle size of the silicon dioxide is 10-40 μm; when the lactic acid uses chitosan as a carrier, the weight-to-volume ratio of chitosan to lactic acid is (0.01-0.1):1, and the deacetylation degree of the chitosan is 75-88%, and the molecular weight is 100-180 kDa.

4. The use according to any one of claims 1 to 3, characterized in that: The density of Artemia eggs in the aquaculture water body is no more than 1000 eggs / mL.

5. A simple and healthy method for cultivating Artemia, characterized by: The following steps are involved: (1) The carrier SiO2 or chitosan is mixed evenly with lactic acid in a certain weight-to-volume ratio to obtain a lactic acid product in the form of an emulsion or powder; (2) adding the lactic acid product described in step (1) to the water used for hatching and breeding Artemia so that the concentration of lactic acid in the breeding water is as described in claim 2, thereby obtaining lactic acid breeding water; (3) Adding Artemia salina eggs into the lactic acid aquaculture water body described in step (2), incubating at 28-30°C for 18-24 hours, and feeding basic feed under continuous light conditions to obtain Artemia salina.

6. The Artemia culture method according to claim 4, wherein: In the lactic acid emulsion described in step (1), the weight-to-volume ratio of lactic acid to carrier is 0.5-3:1 (g:ml).

7. The Artemia culture method according to claim 4, wherein: In the lactic acid powder described in step (1), the weight-to-volume ratio of lactic acid to carrier is 1-10:1 (g:ml).

8. The Artemia culture method according to any one of claims 5 to 7, wherein: The density of the Artemia eggs in the water body in step (3) is no more than 1000 eggs / mL.

9. The Artemia culture method according to any one of claims 5 to 7, wherein: The continuous illumination condition in step (3) is 500-2500 Lux; the basic feed is chlorella, yeast powder, spirulina powder, rice bran, soybean meal, or artificial microparticle feed.

10. An application of Artemia in fish and shrimp farming, characterized by: The Artemia is obtained by the cultivation method according to any one of claims 5 to 9.

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