Composite probiotic preparation for breeding macrobrachium rosenbergii as well as preparation method and application of composite probiotic preparation

Abstract

The invention discloses a compound probiotic preparation for breeding macrobrachium rosenbergii as well as a preparation method and application of the compound probiotic preparation, and relates to the technical field of microorganisms, the compound probiotic preparation comprises the following active strains: Lactococcus gasseri, Bacillus subtilis and Nitrobacter winogradski, and the compound probiotic preparation comprises the following active strains: Lactococcus gasseri, Bacillus subtilis and Nitrobacter winogradski, and the compound probiotic preparation comprises the following active strains: Lactococcus gasseri, Bacillus subtilis and Nitrobacter winogradski. Wherein the preservation number of the lactococcus format is CGMCC (China General Microbiological Culture Collection Center) No.31024. According to the physiological requirements of different development stages of the Z1-Z4 stage and the Z5-P1 stage of larvae, 2: 1 and 2: 1: 1 bacterial strains are adopted for staged accurate addition respectively, meanwhile, artificial seawater of a specific formula is matched, baits such as fairy shrimps and egg custard are quantitatively fed according to the development stage, key water quality indexes such as dissolved oxygen, ammonia nitrogen and nitrite in a water body are strictly regulated and controlled, and the quality of the larvae is improved. A green seedling raising technical system integrating probiotic micro-ecological regulation and control, precise nutrition supply and water quality management is formed, and finally the effects of improving the survival rate of larvae, reducing the metamorphosis rate, promoting growth and development and replacing antibiotics for use are achieved.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a compound probiotic preparation for the breeding of giant freshwater prawns, its preparation method, and its application. Background Technology

[0002] As a globally important freshwater economic shrimp species, the cultivation of giant freshwater prawns (Macrobrachium rosenbergii) seedlings is a crucial link in the aquaculture industry chain, directly affecting the yield and profitability of subsequent adult shrimp farming. In intensive seedling cultivation, high larval density and large feeding volumes easily lead to the accumulation of harmful substances such as ammonia nitrogen and nitrite in the cultivation water due to uneaten feed and excrement. This poses severe environmental stress to the still-developing immune systems of giant freshwater prawn larvae and often induces bacterial diseases such as vibriosis, ultimately resulting in low larval survival rates and inconsistent seedling quality—a bottleneck in the industry. To address these challenges, probiotic technology, as a potential green solution, has been introduced into the aquaculture seedling cultivation field.

[0003] A thorough analysis of existing technical solutions reveals areas for optimization in addressing the unique microecological needs of giant freshwater prawns during their larval stage. Giant freshwater prawn larvae exhibit variations in digestive physiology, feeding habits, immune capacity, and sensitivity to the aquatic environment at different developmental stages. For instance, in the early stages (Z1-Z4), when larvae are first feeding, the intestinal flora colonization is at a critical window, requiring easily accessible probiotics that can quickly occupy ecological niches. In the later stages (Z5-P1), with increased feeding and metabolic waste, the pressure of ammonia nitrogen conversion in the water increases. Therefore, there remains a need in this field for a more precise, tailored probiotic solution that synergistically regulates both intestinal health and the external aquatic environment, precisely matching the developmental patterns of giant freshwater prawn larvae. Summary of the Invention

[0004] In view of the aforementioned existing problems, the present invention is proposed.

[0005] Therefore, this invention provides a compound probiotic preparation for raising giant freshwater prawns, which solves the problem that existing technologies cannot simultaneously meet the dynamic needs of intestinal microecological colonization and ammonia nitrogen transformation in water bodies at different developmental stages of larvae.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: In a first aspect, the present invention provides a compound probiotic preparation for raising giant freshwater prawns, comprising, wherein the active bacterial strains of the compound probiotic preparation include Lactococcus garvieae, Bacillus subtilis, and Nitrifying Bacillus winogradskyi; wherein the Lactococcus garvieae has the preservation number CGMCC No. 31024.

[0007] As a preferred embodiment of the compound probiotic preparation for raising giant freshwater prawns according to the present invention, the compound probiotic preparation adopts different strain compositions and ratios according to different developmental stages of giant freshwater prawn larvae: During the Z1 to Z4 stages of giant freshwater prawn larvae, the compound probiotic preparation is composed of Lactococcus griseus and Bacillus subtilis, with a strain ratio of 2:1. During the Z5 to P1 stages of giant freshwater prawn larvae, the compound probiotic preparation consists of Lactococcus griseus, Bacillus subtilis, and Nitrifying Bacillus vesiculosus, with a strain ratio of 2:1:1.

[0008] Furthermore, its phased and precise application strategy is deeply aligned with the physiological and ecological needs of giant freshwater prawn larvae (specifically "Shufeng No. 1") at different developmental stages from Z1 to P1. During Z1 to Z4 (early zoea larvae), the larvae deplete their yolk sac and begin exogenous feeding, placing intestinal flora colonization in a critical window. At this time, the formulation consists of Lactococcus gravidarum and Bacillus subtilis in a 2:1 ratio, aiming to preferentially promote the early colonization and dominance of beneficial bacteria in the larval intestine, inhibit pathogens, and aid digestion. Entering Z5 to P1 (mid-to-late zoea larvae to postlarvae), the larvae's food intake increases dramatically, and the accumulation of metabolic waste leads to a significant increase in ammonia nitrogen conversion pressure in the water. Therefore, Nitrifying Bacillus vesiculosus is added to the existing bacterial strains, forming a 2:1:1 ratio. This stage of the program balances intestinal health maintenance and water environment purification, particularly by converting toxic ammonia nitrogen and nitrite into non-toxic nitrate through nitrification, providing stable water quality assurance for successful metamorphosis of the larvae.

[0009] As a preferred embodiment of the compound probiotic preparation for raising giant freshwater prawns according to the present invention, the compound probiotic preparation needs to be diluted with sterile physiological saline and activated at room temperature for 10-20 minutes before use.

[0010] Furthermore, the high-concentration compound probiotic suspension (or powder) is diluted appropriately with sterile physiological saline to achieve the target application concentration. After dilution, it needs to be allowed to stand at room temperature (usually 25-30℃) for 10 to 20 minutes to activate it. This process aims to revive the probiotic cells that are dormant or stored, restore their metabolic activity, and thus enable them to immediately adapt to the environment and rapidly proliferate, colonize, and exert their beneficial functions (such as degrading organic matter, inhibiting pathogens, and converting ammonia nitrogen) after being introduced into the seedling water. This effectively avoids problems such as insufficient bacterial activity and delayed effects that may result from direct application.

[0011] In a second aspect, the present invention provides a method for preparing a compound probiotic preparation for raising giant freshwater prawns, comprising: (1) Preparing the water body for raising seedlings: preparing artificial seawater, wherein the artificial seawater is formulated as follows: each ton of water contains 11000g of sodium chloride, 3700g of magnesium sulfate, 420g of calcium chloride, 190g of potassium chloride, 20g of sodium bicarbonate, 20g of potassium bromide, 120g of boric acid, 6g of EDTA and 1g of strong chlorine, and aerated for 24-48 hours after dissolution for later use; (2) Addition of compound probiotics: According to the developmental stage of giant freshwater prawn larvae, add the compound probiotic preparation with the corresponding ratio described in claim 2 to the seedling water; (3) Feeding: From Z2 stage, feed brine nauplii, from Z5 stage, add egg custard, and from Z11 stage, supplement with shrimp chips; (4) Water quality control: Maintain dissolved oxygen content in the water body at ≥5 mg / L throughout the process, control ammonia nitrogen concentration at ≤0.5 mg / L, and nitrite concentration at ≤0.1 mg / L.

[0012] This invention provides a method for preparing a compound probiotic preparation for raising giant freshwater prawns, comprising the following steps: the compound probiotic addition scheme in step (2) is as follows: During periods Z1 to Z4, the bacteria were added every two days to achieve final concentrations of 1×10^5 CFU / mL and 5×10^4 CFU / mL in the culture water, respectively. During periods Z5 to Z9, the bacteria were added every two days to achieve final concentrations of 2×10^5 CFU / mL, 1×10^5 CFU / mL, and 1×10^5 CFU / mL in the aquaculture water, respectively. During the Z10 to P1 phases, add once a day, with the final concentration of each strain being the same as that during the Z5 to Z9 phases.

[0013] Furthermore, dynamic and precise regulation was implemented based on the physiological and ecological needs of the "Shufeng No. 1" giant freshwater prawn larvae at different developmental stages. During stages Z1 to Z4 (early zoea larvae), the initial exogenous feeding of the larvae and the colonization of the intestinal flora are crucial. Therefore, a bacterial agent composed of *Lactococcus gravidarum* (final concentration 1×10^5 CFU / mL) and *Bacillus subtilis* (final concentration 5×10^4 CFU / mL) in a 2:1 ratio was added every two days to preferentially promote the early colonization and occupancy of beneficial bacteria in the larval intestine. Entering stages Z5 to Z9 (mid-to-late zoea larvae), with increased feeding and metabolic waste, the pressure on water purification became more pronounced. Therefore, while maintaining the frequency of the first two bacterial additions, *Nitrifying Bacillus vesiculosus* (final concentration 1×10^5 CFU / mL) was added, forming a 2:1:1 bacterial agent ratio to synergistically achieve intestinal health maintenance and efficient conversion of ammonia nitrogen and nitrite in the water. From Z10 to P1 stages (late zoea larvae to postlarvae), the larvae are in a critical period of metamorphosis and are extremely sensitive to environmental fluctuations. Therefore, the frequency of addition was increased to once a day, while the final concentration of each strain remained constant. Through continuous and intensive microecological regulation, a stable internal and external environment was provided to ensure successful metamorphosis of the larvae. This scheme, through the phased optimization of strain combination, concentration, and frequency, achieved precise dual regulation of both the "internal gut" and the "external water" throughout the entire seedling cultivation process.

[0014] This invention provides a method for preparing a compound probiotic preparation for raising giant freshwater prawns, including, in step (3), the total daily feeding amount of the brine shrimp is calculated according to the formula "total feeding amount (number) = number of larvae (tails) × daily addition amount per tail for this developmental stage (number / tail)", wherein the daily addition amount of brine shrimp per tail for each developmental stage is: Z2 stage 1-2, Z3-Z4 stage 2-3, Z5-Z6 stage 3-5, Z7-Z10 stage 5-8, Z11-Z12 stage 8-12.

[0015] Furthermore, it is crucial to ensure that the food supply matches the developmental needs of the larvae. The total daily feeding amount for Artemia is strictly calculated according to the formula: "Total feeding amount (individuals) = Number of larvae (tails) × Daily feeding amount per tail for this developmental stage (individuals / tail)". The daily feeding amount per tail for each developmental stage is dynamically adjusted based on the increase in larval mouth size and feeding ability: Z2 stage (1-2 larvae / tail) is the adaptation stage for initial feeding; Z3-Z4 stages (2-3 larvae / tail) see a moderate increase as activity increases; Z5-Z6 stages (3-5 larvae / tail) see a significant increase in feeding due to mouth size expansion; Z7-Z10 stages (5-8 larvae / tail) meet the high nutritional needs of rapid growth; and Z11-Z12 stages (8-12 larvae / tail) reach the peak feeding period to accumulate energy for metamorphosis. This quantitative standard effectively avoids growth inhibition caused by insufficient feeding or water quality deterioration caused by overfeeding, and is a crucial management step in achieving high survival rates and uniformity.

[0016] This invention provides a method for preparing a compound probiotic preparation for raising giant freshwater prawns, comprising, in step (3), the proportion of the amount of egg custard added to the total feed is: 20%-40% for Z5-Z6 stage, 40%-60% for Z7-Z10 stage, and 50%-75% for Z11-Z12 stage; and the egg custard is prepared by steaming for 3-5 minutes and then passing it through a 60-80 mesh sieve.

[0017] Furthermore, as an important supplementary feed for larvae in the mid-to-late stages of development, the proportion of egg custard added gradually increases with each developmental stage, and the preparation method is strictly regulated. Regarding the addition ratio: it begins to be added in stages Z5-Z6, accounting for 20%-40% of the total feed, guiding larvae to adapt to artificial feed; in stages Z7-Z10, it increases to 40%-60%, becoming an important source of nutrition; in stages Z11-Z12, it further increases to 50%-75%, preparing for metamorphosis and transition to artificial feed. Regarding the preparation method: fresh eggs are selected, thoroughly stirred, and then steamed under normal pressure for 3-5 minutes to ensure the egg custard solidifies, matures, and is effectively sterilized. It is then immediately passed through a 60-80 mesh sieve to produce fine particles of suitable size, easily ingested by larvae. This process ensures nutrition and hygiene while also aligning with the changes in mouth size at different developmental stages of larvae, significantly improving feed utilization.

[0018] This invention provides a method for preparing a compound probiotic preparation for raising giant freshwater prawns, including, in step (3), the feeding frequency of the feed is: 3-4 times a day for Z1-Z4 period, and 3 times a day from Z5 period onwards, with the feeding time and feeding amount ratios being 30% at 6:00 am, 20% at 11:00 am, and 50% at 5:00 pm.

[0019] Furthermore, feeding management emphasizes reasonable frequency and scientific time allocation to match the larvae's diurnal feeding rhythm. During stages Z1-Z4, the larvae have a high metabolic rate but small stomach capacity, so high-frequency feeding (3-4 times daily) ensures a continuous supply of nutrients. From stage Z5 onwards, the larvae's digestive system becomes more developed, and feeding is adjusted to 3 times daily, with precise time and quantity allocation based on their activity patterns: 30% of the daily ration is fed at 6:00 AM to replenish energy after a night's consumption; 20% is fed at 11:00 AM to maintain daytime activity needs; and the remaining 50% is fed at 5:00 PM to provide sufficient energy reserves for slow digestion at night. This feeding strategy effectively reduces uneaten feed contamination and promotes synchronous growth of the larvae.

[0020] This invention provides a method for preparing a compound probiotic preparation for raising giant freshwater prawns, comprising: the giant freshwater prawn larvae being the "Shufeng No. 1" strain, and the stocking density of the larvae being 100,000 larvae / cubic meter.

[0021] Furthermore, this technology is applicable to the cultivation of larvae of the nationally approved new species of giant freshwater prawn, "Shufeng No. 1." This strain possesses excellent traits such as rapid growth and strong resistance to adverse conditions, providing an ideal genetic basis for the successful implementation of this technology. In large-scale seedling cultivation, the stocking density of larvae is strictly controlled at 100,000 larvae per cubic meter. This density represents the optimal solution for balancing unit water output and ecological carrying capacity, ensuring both overall population yield and providing sufficient living space and dissolved oxygen for each larva. This effectively avoids stress, cannibalism, and rapid water quality deterioration caused by excessive density, laying the foundation for creating a stable micro-ecosystem for probiotics to exert their optimal efficacy.

[0022] Thirdly, the present invention provides the application of the compound probiotic preparation for raising giant freshwater prawns in improving the survival rate of giant freshwater prawn larvae, reducing the rate of metamorphosis and deformity, promoting growth and development, purifying the water quality of the seedling raising water body, and / or replacing antibiotics for green and healthy seedling raising.

[0023] The beneficial effects of this invention are as follows: Based on the physiological needs of different developmental stages of larvae (Z1-Z4 and Z5-P1), strain ratios of 2:1 and 2:1:1 are used for precise addition in stages. At the same time, artificial seawater with a specific formula is used, and feed such as Artemia and egg custard is given in quantitative amounts according to the developmental stage. Key water quality indicators such as dissolved oxygen, ammonia nitrogen, and nitrite are strictly controlled. This forms a green seedling cultivation technology system that integrates probiotic microecological regulation, precise nutrient supply, and water quality management. Ultimately, it can improve the survival rate of larvae, reduce the rate of abnormal deformities, promote growth and development, and replace the use of antibiotics. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 Images of giant freshwater prawn larvae at different developmental stages.

[0026] Figure 2 The ammonia nitrogen content in the water used for laboratory culture of giant freshwater prawn larvae.

[0027] Figure 3 The nitrite content in the water used for culturing juvenile giant freshwater prawns in the laboratory was measured.

[0028] Figure 4 The ammonia nitrogen content in the water used for raising juvenile giant freshwater prawns in cement ponds.

[0029] Figure 5The nitrite content in the water used for raising juvenile giant freshwater prawns in cement ponds. Detailed Implementation

[0030] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0031] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0032] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0033] Reference Figures 1-5 This is one embodiment of the present invention, which provides a compound probiotic preparation for the breeding of giant freshwater prawns, its preparation method and application, including the following steps: Example 1: Laboratory-scale probiotic breeding experiment of giant freshwater prawn larvae "Shufeng No. 1" 1. Experimental materials Seedling containers: 6 round 100L water buckets (3 for the experimental group and 3 for the control group), each equipped with a miniature oxygenation pump and a temperature control device.

[0034] Larvae source: Z1 stage larvae of the nationally approved new variety of giant freshwater prawn "Shufeng No. 1", selected individuals with consistent vitality, and 5000 larvae were released into each group.

[0035] Aquaculture water: Prepare artificial seawater according to the invented formula, specifically by adding 11,000 g of sodium chloride, 3,700 g of magnesium sulfate, 420 g of calcium chloride, 190 g of potassium chloride, 20 g of sodium bicarbonate, 20 g of potassium bromide, 120 g of boric acid, 6 g of EDTA, and 1 g of strong chlorine per ton of water. After fully dissolving, aerate for 24 hours for later use. The initial salinity is 14‰.

[0036] Compound probiotic preparation: Lactococcus faecium (CGMCC No. 31024, concentration 10) cultured according to the invented method. 9 CFU / mL), Bacillus subtilis (concentration 10) 10 CFU / mL), Nitrifying Bacillus vesiculosus (concentration 10) 8 (CFU / mL), diluted with sterile saline before use.

[0037] Feed: Artemia eggs (hatching rate ≥85%), fresh eggs (for making egg custard), shrimp chips.

[0038] 2. Test Methods The experimental group was given compound probiotics according to the protocol in Table 1: Lactococcus lactis + Bacillus subtilis were added every 2 days during periods Z1 to Z4, with a final concentration of 1×10⁻⁶. 5 5×10 4 CFU / mL (2:1 ratio); add the three-strain compound preparation every 2 days during Z5-Z9, with a final concentration of 2×10⁻⁶. 5 1×10 5 1×10 5 CFU / mL (ratio 2:1:1); during Z10-P1 phases, the three-strain compound preparation was added once daily, with the final concentration the same as during Z5-Z9 phases. The control group received no probiotics, but all other conditions were the same.

[0039] Feeding: Follow Table 2 and the egg custard addition plan. Feed brine nauplii starting from stage Z2. Add egg custard (steamed for 3-5 minutes and then passed through a 60-mesh sieve) starting from stage Z5. Add shrimp chips in stage Z11. The amount of each feeding is calculated as "total feeding amount = number of nauplii × amount added in this stage ÷ number of feedings per day", and ensure that the food is consumed within 1 hour.

[0040] Water quality control: The aeration pump is kept running throughout the process to ensure dissolved oxygen ≥5 mg / L; the water is changed every 2-3 days, with 1 / 3 to 1 / 2 of the water being changed; ammonia nitrogen (≤0.5 mg / L) and salinity are monitored daily (maintained at 13-15‰ during Z1-Z11, and gradually desalinated to fresh water during P1).

[0041] Monitoring indicators: Record the number of larval deaths daily and calculate the survival rate at each stage; record the metamorphosis time from Z1 to P1 and calculate the metamorphosis deformity rate; regularly test the concentration of ammonia nitrogen and nitrite in the water.

[0042] 3. Experimental Results The cumulative survival rate and hatching rate of larvae in the Z1-P1 stage of the experimental group reached over 80% and 60%, respectively, which were significantly higher than those in the control group (around 70% and 50%). The rate of abnormal deformities was only 2.1% to 2.5%, compared to 8.7% in the control group.

[0043] In the experimental group, the concentrations of ammonia nitrogen and nitrite in the water remained consistently below 0.6 mg / L and 0.05 mg / L, respectively; in the control group, ammonia nitrogen rose to 1.0 mg / L and nitrite rose to 0.1 mg / L in the later stages. Figure 2 and Figure 3 ).

[0044] The experimental group of larvae developed from Z1 to P1 in only 25 days, which is 3 days shorter than the control group (28 days). The uniformity of the size of the larvae reached 91%, which is significantly better than the control group (82%).

[0045] Example 2: Large-scale application of "Shufeng No. 1" probiotics in the breeding of giant freshwater prawns. 1. Seedling raising conditions Seedling nursery area: 50 m 3 Two cement seedling beds (one for the experimental group and one for the control group) were equipped with high-power aerators, circulating water systems, and temperature control equipment.

[0046] Larval release: 500,000 "Shufeng No. 1" Z1 larvae were released per pond, with a larval density of 100,000 larvae / m³. 3 .

[0047] Artificial seawater: Prepared on a large scale according to the invented formula for 100m³ 3 The salinity is 14‰. After aeration for 48 hours, the larvae are injected into the seedling pond. The pond is disinfected with strong chlorine before release, and the larvae are released after the residual chlorine disappears.

[0048] Compound probiotic preparation: Lactococcus gravidarum (CGMCC No. 31024), Bacillus subtilis and Nitrifying Bacillus vesiculosus were cultured in batches. After centrifugation under appropriate conditions, the bacterial cells were collected and initially resuspended in sterile physiological saline to prepare a bacterial suspension (concentration 10). 9 -10 11 (CFU / mL); Before use, dilute with sterile saline to a suitable concentration according to the water volume of the seedling pond and the target final concentration. Activate at room temperature for 10-20 minutes, stir thoroughly and evenly, and then sprinkle evenly throughout the pond to ensure that the probiotics are evenly distributed in the water and improve the colonization efficiency.

[0049] 2. Seedling raising operations Probiotic addition: Follow the final concentrations set in Table 1 for each stage and 50mg / L. 3 The water volume of the seedling pond, combined with the concentration of the compound probiotic suspension (10... 9 -10 11The dosage was precisely calculated using CFU / mL. The specific addition protocol for the experimental group was as follows: For periods Z1-Z4, addition was given every 2 days, with each addition consisting of 500 mL of *Lactococcus gravidarum* suspension and 250 mL of *Bacillus subtilis* suspension (strain ratio 2:1); for periods Z5-Z9, addition was given every 2 days, with each addition consisting of 1000 mL of *Lactococcus gravidarum* suspension, 500 mL of *Bacillus subtilis* suspension, and 500 mL of *Nitrobacterium velutipes* suspension (strain ratio 2:1:1); for periods Z10-P1, addition was given daily, with the same dosage of the three strains as for periods Z5-Z9. All bacterial suspensions were diluted with sterile saline and activated at room temperature for 10-20 minutes before use, thoroughly stirred, and then evenly sprinkled throughout the tank. The control group received no probiotics, but all other procedures were the same as the experimental group.

[0050] Feeding: Artemia are fed according to the formula "daily feeding amount = number of larvae × amount added during this period", and fed after batch hatching; egg custard is prepared according to the proportion of total feeding amount, and after passing through an 80-mesh sieve, it is sprinkled throughout the pond. Z11 stage is fed with shrimp chips.

[0051] Water quality management: Maintain dissolved oxygen ≥5mg / L throughout the process; maintain salinity at 13~15‰ during Z1~Z12 periods; desalinate to fresh water by 1‰ daily during P1 period; change water every 3 days, replacing half of the water volume; supplement with probiotics after water change.

[0052] Routine monitoring: Check the vitality and mortality of juveniles every morning and evening, and test water quality indicators (ammonia nitrogen, dissolved oxygen, salinity, etc.) three times a week.

[0053] 3. Application Effects The survival rate of seedlings in the experimental group reached 80.5%, with a seedling output of 300,000; the survival rate of the control group was only 70%, with a seedling output of 249,000. The seedling output of the experimental group was significantly increased.

[0054] No disease outbreaks occurred in the experimental group throughout the entire process, and the rate of larval deformities was only 2.3%; in the control group, vibriosis broke out in the middle stage, with a deformity rate of 9.2%, requiring the administration of antibiotics for control.

[0055] The seedling period for the experimental group was 23 days, while that for the control group was 28 days, meaning the experimental group had 5 days less seedling time than the control group.

[0056] The experimental group required no chemical water conditioner during seedling cultivation, maintained stable water quality, and consistently kept ammonia nitrogen and nitrite levels below 0.4 mg / L and 0.05 mg / L, respectively. Seedling cultivation costs were significantly lower than the control group, achieving green, efficient, and large-scale seedling cultivation.

[0057] This invention provides a compound probiotic preparation for aquaculture. The main bacterial strains in the preparation are Lactococcus gravidarum, Bacillus subtilis and Nitrifying Bacillus vesiculosus. Among them, Lactococcus gravidarum is a typical probiotic isolated and cultured by our team from the intestine of Macrobrachium rosenbergii, and its strain number is CGMCC No.31024.

[0058] Lactococcus faecalis and Clostridium butyricum have antibacterial, acid-producing, and enzyme-producing effects, while Nitrosobacterium vesiculosum has the function of degrading nitrite.

[0059] Lactococcus faecium: LB medium, anaerobic culture at 28℃ for 24-36 h, centrifuged at 6000 rpm for 10 min, collected, resuspended in physiological saline, and quantified by plate counting (10⁻⁶). 9 -10 10 (CFU / mL).

[0060] Bacillus subtilis: LB medium, 28°C, aerobic shaking (180-200 rpm) for 18-24 h, centrifuged at 8000 rpm for 8 min, collected, resuspended, and plate counted (10⁻⁶). 10 -10 11 (CFU / mL), if spores are required, extend the culture time to 36 hours.

[0061] Nitrifying Bacillus vesiculosus: Use a dedicated autotrophic medium for nitrifying bacteria. Incubate at 28°C with aerobic shaking (150 rpm) for 7-10 days. Centrifuge at 4000 rpm for 15 minutes (to prevent cell rupture). Resuspend and count on plates or use turbidimetric assay for quantification (10⁻⁶). 8 -10 9 (CFU / mL).

[0062] The objective of this invention is achieved through the following technical solution: The giant freshwater prawn larvae cultivated in this invention are the nationally approved new variety "Shufeng No. 1" which was innovatively cultivated by our team. This variety is characterized by rapid growth and strong resistance to adverse conditions.

[0063] The aquaculture water used in this invention is artificial seawater prepared by our team, with the following formula: sodium chloride (NaCl): 11,000 g / ton water, magnesium sulfate (MgSO4): 3,700 g / ton water, calcium chloride (CaCl): 420 g / ton water, potassium chloride (KCl): 190 g / ton water, sodium bicarbonate (NaHCO3): 20 g / ton water, potassium bromide (KBr): 20 g / ton water, boric acid (BH2O): 120 g / ton water, ETDA: 6 g / ton, and strong chlorine: 1 g / ton.

[0064] The method of adding the compound probiotic preparation is shown in Table 1. During the Z1 to Z4 stages of the development of giant freshwater prawn larvae, the compound probiotic preparation (Lactococcus gestrinus and Bacillus subtilis) was added every two days, with final concentrations in the culture water of 1×10⁵ and 5×10⁴ cfu / ml, respectively (addition ratio of 2:1).

[0065] During the Z5-Z9 stage of giant freshwater prawn larval development, a compound probiotic preparation (Lactococcus gravidarum, Bacillus subtilis and Nitrifying Bacillus vesiculosus) was added every two days, with final concentrations in the culture water of 2×10⁵, 1×10⁵ and 1×10⁵ cfu / ml (addition ratio of 2:1:1).

[0066] During the Z10 to P1 stage of giant freshwater prawn larval development, a compound probiotic preparation was added every other day. The final concentrations of the compound probiotic preparation in the culture water were 2×10⁵, 1×10⁵ and 1×10⁵ cfu / ml (the addition ratio was 2:1:1).

[0067] Table 1. Methods of Adding Compound Probiotics

[0068] The feeding amount for brine shrimp is calculated based on the number of larvae: Total feeding amount (larvae / time) = Number of larvae (tails) × Amount added during this period (larvae / tail) ÷ Number of feedings per day. Feeding frequency and time: Z1-Z4 stages: 3-4 times a day, around 7:00, 11:00, 15:00, and 19:00, with an interval of 3-4 hours. From Z5 stage onwards: 3 times a day, at 6:00 in the morning (30%), 11:00 at noon (20%), and 17:00 in the afternoon (50%).

[0069] The amount of Artemia added is shown in Table 2.

[0070] Table 2. Amount of Artemia var. mongolica added

[0071] Adding egg custard: Z1 stage: Relying on yolk nutrition, no feeding is given; from Z2 stage onwards, the main food is Artemia nauplii. Z5–Z6 stages: The larvae's mouth diameter increases and their digestive capacity improves, so egg custard artificial feed is added, accounting for 20%–40% of the total feed, gradually increasing to 50%–75% as they develop. Before and after metamorphosis (Z11–Z12 stages): The proportion of cake can be further increased, while shrimp chips are added to prepare for acclimatization and transition to a new diet (Table 3).

[0072] Table 3. Amount of steamed egg custard added

[0073] During the cultivation of giant freshwater prawns, ammonia nitrogen should be controlled at ≤0.5 mg / L and nitrite at ≤0.1 mg / L, regulated by regular water changes (every 2-3 days, replacing 1 / 3-1 / 2 of the water) and probiotics. Dissolved oxygen should be maintained at ≥5 mg / L, with aeration equipment running continuously to avoid hypoxia stress. Salinity should be adjusted in stages: maintained at 13-15‰ during Z1-Z12 stages, and gradually reduced to freshwater during P1 stage. Daily checks should be conducted to monitor larval mortality, promptly counting and investigating the causes. If mortality suddenly increases, the water exchange rate should be increased immediately, and water quality indicators should be tested to ensure a stable cultivation environment and improve larval survival rates.

[0074] In summary, this invention, based on the physiological needs of different developmental stages of larvae (Z1-Z4 and Z5-P1), employs a 2:1 and 2:1:1 ratio of bacterial strains for precise, staged addition. This is combined with artificial seawater of a specific formula, quantitative feeding of Artemia and egg custard according to developmental stages, and strict control of key water quality indicators such as dissolved oxygen, ammonia nitrogen, and nitrite. This forms a green seedling cultivation technology system integrating probiotic microecological regulation, precise nutrient supply, and water quality management, ultimately achieving the effects of improving larval survival rate, reducing deformity rate, promoting growth and development, and replacing antibiotic use.

[0075] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention. Preservation information: Name of depositary institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Deposit date: June 20, 2024 Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Accession number: CGMCC No. 31024 Classification and nomenclature: Lactococcus garvieae.

Claims

1. A compound probiotic preparation for the breeding of giant freshwater prawns, characterized in that: include, The active bacterial strains in the compound probiotic preparation include Lactococcus garvieae, Bacillus subtilis, and Nitrobacter winogradskyi; wherein the preservation number of Lactococcus garvieae is CGMCC No. 31024.

2. The compound probiotic preparation for raising giant freshwater prawns as described in claim 1, characterized in that: The compound probiotic preparation uses different strains and ratios according to different developmental stages of giant freshwater prawn larvae: During the Z1 to Z4 stages of giant freshwater prawn larvae, the compound probiotic preparation is composed of Lactococcus griseus and Bacillus subtilis, with a strain ratio of 2:

1. During the Z5 to P1 stages of giant freshwater prawn larvae, the compound probiotic preparation consists of Lactococcus griseus, Bacillus subtilis, and Nitrifying Bacillus vesiculosus, with a strain ratio of 2:1:

1.

3. The compound probiotic preparation for raising giant freshwater prawns as described in claim 2, characterized in that: The compound probiotic preparation needs to be diluted with sterile saline before use and activated at room temperature for 10-20 minutes.

4. A method for preparing a compound probiotic preparation for raising giant freshwater prawns, based on the compound probiotic preparation for raising giant freshwater prawns according to any one of claims 1 to 3, characterized in that: include, (1) Preparation of seedling water: Prepare artificial seawater. The formula of artificial seawater is as follows: per ton of water, there are 11,000g of sodium chloride, 3,700g of magnesium sulfate, 420g of calcium chloride, 190g of potassium chloride, 20g of sodium bicarbonate, 20g of potassium bromide, 120g of boric acid, 6g of EDTA and 1g of strong chlorine. After dissolving, aerate for 24-48 hours for later use. (2) Addition of compound probiotics: According to the developmental stage of giant freshwater prawn larvae, add the compound probiotic preparation with the corresponding ratio described in claim 2 to the seedling water; (3) Feeding: From Z2 stage, feed brine nauplii, from Z5 stage, add egg custard, and from Z11 stage, supplement with shrimp chips; (4) Water quality control: Maintain dissolved oxygen content in the water body at ≥5 mg / L throughout the process, control ammonia nitrogen concentration at ≤0.5 mg / L, and nitrite concentration at ≤0.1 mg / L.

5. The method for preparing the compound probiotic preparation for raising giant freshwater prawns as described in claim 4, characterized in that: The addition scheme for the compound probiotics mentioned in step (2) is as follows: During periods Z1 to Z4, the bacteria were added every two days to achieve final concentrations of 1×10^5 CFU / mL and 5×10^4 CFU / mL in the culture water, respectively. During periods Z5 to Z9, the bacteria were added every two days to achieve final concentrations of 2×10^5 CFU / mL, 1×10^5 CFU / mL, and 1×10^5 CFU / mL in the aquaculture water, respectively. During the Z10 to P1 phases, add once a day, with the final concentration of each strain being the same as that during the Z5 to Z9 phases.

6. The method for preparing the compound probiotic preparation for raising giant freshwater prawns as described in claim 4, characterized in that: In step (3), the total daily feeding amount of the brine shrimp is calculated according to the formula "total feeding amount (shrimp) = number of larvae (tails) × daily addition amount per tail for this developmental stage (shrimp shrimp / tail)". The daily addition amount per tail for each developmental stage is as follows: Z2 stage 1-2 shrimp, Z3-Z4 stage 2-3 shrimp, Z5-Z6 stage 3-5 shrimp, Z7-Z10 stage 5-8 shrimp, Z11-Z12 stage 8-12 shrimp.

7. The method for preparing the compound probiotic preparation for raising giant freshwater prawns as described in claim 4, characterized in that: In step (3), the proportion of the amount of egg custard added to the total amount of feed is as follows: 20%-40% in Z5-Z6, 40%-60% in Z7-Z10, and 50%-75% in Z11-Z12; and the egg custard is prepared by steaming for 3-5 minutes and then passing it through a 60-80 mesh sieve.

8. The method for preparing the compound probiotic preparation for raising giant freshwater prawns as described in claim 4, characterized in that: In step (3), the feeding frequency of the bait is: 3-4 times a day for Z1-Z4 period, and 3 times a day from Z5 period onwards. The feeding time and feeding amount ratio are 30% at 6:00 am, 20% at 11:00 am and 50% at 5:00 pm respectively.

9. The method for preparing the compound probiotic preparation for raising giant freshwater prawns as described in claim 4, characterized in that, The juvenile giant freshwater prawns were of the "Shufeng No. 1" strain, and the stocking density of the juveniles was 100,000 prawns per cubic meter.

10. The application of the compound probiotic preparation for raising giant freshwater prawns according to any one of claims 1-3 in improving the survival rate of giant freshwater prawn larvae, reducing the rate of metamorphosis and deformity, promoting growth and development, purifying the water quality of the raising water body, and / or replacing antibiotics for green and healthy raising.

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