Method for realizing efficient nitrogen utilization and rapid growth of fishes
By adding the intestinal strain Exiguobacterium sp. strain WY(Y)3 of perciformes to fish feed, the intestinal flora of fish was improved, the problem of allometric growth was solved, nitrogen efficiency and rapid growth were achieved, and costs and individual differences were reduced.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANGHAI OCEAN UNIV
- Filing Date
- 2025-12-18
- Publication Date
- 2026-04-14
AI Technical Summary
Abnormal growth rates exist in fish farming, leading to increased farming costs and risks of injury, disability, and disease during grading. Existing exogenous activators are expensive and difficult to effectively promote fish growth.
The feed was made from heterotrophic nitrifying-aerobic denitrifying Bacillus sp. strain WY(Y)3 derived from the intestines of perciformes fish. By improving the intestinal flora structure, it promotes nitrogen utilization and rapid growth in fish.
Improve nitrogen utilization in fish, reduce growth aberrations, lower probiotic preparation costs, and achieve rapid fish growth while reducing individual differences.
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Figure CN121852249A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the technical field of exploring the differential systems of fish with different growth rates, specifically to a heterotrophic nitrifying-aerobic denitrifying Bacillus and a method for achieving efficient nitrogen utilization and rapid growth in fish using this strain. Background Technology
[0002] Biological growth is an extremely complex life phenomenon, with significant variations in growth among different species, populations, or individuals. This complexity stems from the interaction of multiple physiological, environmental, and genetic factors. During the growth process of different species, environmental conditions and genetic backgrounds influence their growth characteristics. Within the same species, different populations and individuals exhibit variations in growth rate and pattern due to genotype and environmental differences. These growth variations may involve multiple aspects such as body size, morphology, growth rate, lifespan, and reproductive capacity. Factors leading to intraspecific growth allergies in aquatic animals may include environmental factors, behavioral factors, social hierarchy behaviors, cannibalism, or genetic factors.
[0003] In aquaculture, the problem of abnormal growth rates in fish is becoming increasingly prominent. As fish from the same batch mature, size differences increase, necessitating regular grading to mitigate this impact. This not only increases the workload of aquaculture but also raises the probability of injury, disability, and death during grading, increasing the risk of disease across the entire tank and severely hindering the development of the entire industry.
[0004] Our team, through transcriptomic analysis of a species of perciformes fish, discovered that differentially expressed genes are mainly involved in processes such as muscle development, phosphagen synthesis and metabolism, carbohydrate synthesis and metabolism, myocardial contraction and disease, cytoskeleton, and ion binding. These genes are primarily enriched in signaling pathways related to myocardial contraction, calcium, gluconeogenesis / glycolysis, PPAR, thyroid hormones, purine metabolism, glucagon, galactose metabolism, and the pentose phosphate pathway. Significantly upregulated and enriched genes in larger individuals include ann, mid1, pur, fat, elo, acy, and dcK. The PPAR signaling and purine metabolism pathways involved may be closely related to the rapid growth of larger fish. Although this study elucidates the growth differences in this fish through genetic analysis, activating these pathways by adding exogenous activators to feed is unlikely to quickly promote fish growth and is technically costly, making it impractical.
[0005] The gastrointestinal tract of fish contains a vast array of microorganisms, forming a dynamic micro-ecosystem over a long evolutionary history. The gut microbiota plays a crucial role in the host's immunity, growth, and energy. Under normal circumstances, the gut microbiota maintains a dynamic equilibrium within the fish's intestines, thus preserving normal intestinal physiological functions. The growth and reproduction of the gut microbiota primarily depend on the fish's own nutrient supply or the decomposition of intestinal contents. Simultaneously, gut microbes secrete various digestive enzymes and synthesize a variety of nutrients, including vitamins. These products play a vital role in the digestion, absorption, growth, and development of fish. The fish gut microbiota plays a crucial role in the host's growth, immunity, and energy metabolism. Gut microorganisms can secrete various digestive enzymes beneficial to fish growth and synthesize a variety of nutrients required by fish. Fish growth is influenced by many factors, primarily including parental inheritance, food quality, environmental conditions, and the gut microbiota. Understanding the gut microbiota helps us explore the relationship between bacterial communities and host growth.
[0006] In recent decades, with the increase in intensive aquaculture globally, the incidence of disease outbreaks has risen. Antibiotics have long been the traditional disease control strategy in aquaculture, but their overuse easily leads to antibiotic resistance in pathogens, causing economic losses and threatening the environment and human health. Currently, my country prohibits the addition of antibiotic-based growth promoters to feed; therefore, probiotics have become the most promising feed additives for controlling or treating various diseases in fish and shellfish. Probiotics are microecological preparations made from the normal microbial flora or beneficial bacteria in animals. In water, probiotics can reduce the total number of bacteria or Vibrio bacteria; inside aquatic animals, they can promote the reproduction of beneficial microorganisms, regulate the flora structure, and promote growth. Currently, there is a lack of effective probiotic strains and related feeds that can improve aberrant growth in fish. Summary of the Invention
[0007] This invention addresses the phenomenon of aberrant growth in fish farming by focusing on gut microbiota. It identifies a probiotic strain and processes it into feed for fish, enabling them to grow rapidly and resolving the issue of increased farming costs due to growth differences.
[0008] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows:
[0009] In a first aspect, the present invention provides a probiotic derived from the intestinal flora of a perciform fish that can improve the nitrogen utilization rate and growth rate of the fish. It is a heterotrophic nitrifying-aerobic denitrifying Bacillus, specifically Exiguobacterium sp. strain WY(Y)3, classified as Exiguobacterium, and deposited at the China General Microbiological Culture Collection Center on December 8, 2025, with accession number CGMCC No. 36957.
[0010] Furthermore, the screening method for the heterotrophic nitrifying-aerobic denitrifying Bacillus is as follows:
[0011] S1. Screening of dominant gut microbiota: The test fish were divided into a fast-growing group and a slow-growing group according to their body weight. The gut samples of the two groups were subjected to full-length 16s amplicon sequencing to obtain the dominant microbiota with higher content in the fast-growing group than in the slow-growing group. The microbiota with the greatest difference was selected as the target microbiota.
[0012] S2. Preparation of dominant bacteria culture: Take the intestinal contents of the test fish, add sterile physiological saline to form an initial bacterial suspension, which is used to isolate, screen and purify fish-derived bacteria;
[0013] S3. Isolation and screening of target bacterial strains: The initial bacterial suspension was serially diluted and spread on LB agar plates and incubated at 32°C for 24 h; then single colonies with obvious morphological differences were picked and streaked on the plates, and the streaking was repeated 2-3 times until pure cultures were obtained.
[0014] S4. Identification of target bacterial species: Genomic DNA was extracted from the pure culture and then subjected to first-generation sequencing. The sequencing results were compared with the BLAST website sequence to screen for target probiotics in the dominant intestinal flora of the fast-growing group of fish. The identified bacterial species were then compared with metagenomic data to verify the target bacterial species.
[0015] Secondly, this invention provides a method for achieving efficient nitrogen utilization and rapid growth in fish. This method involves adding the aforementioned heterotrophic nitrifying-aerobic denitrifying Bacillus exiguobacterium sp. strain WY(Y)3 to a basal diet to prepare feed. Feeding fish with this feed can improve their nitrogen utilization rate, promote their growth, and alleviate growth abnormalities. The basal diet can be any commercially available or homemade conventional feed suitable for the corresponding fish species.
[0016] Furthermore, the addition method involves spraying an Exiguobacterium sp. strain WY(Y)3 bacterial suspension onto the surface of the basic feed.
[0017] Furthermore, the fish include fish from the orders Perciformes and Cypriniformes.
[0018] In one specific embodiment, the fish is a koi carp, and the corresponding Exiguobacterium sp. strain WY(Y)3 is added to the basal feed at a rate ≤1×10⁻⁶. 8 cfu / g, further preferably 1×10 6 cfu / g. When Exiguobacterium sp. strain WY(Y)3 is applied to koi, the addition amount is 1×10 6 Koi carp exhibited better growth indicators and nutritional performance when cfu / g was used.
[0019] Thirdly, the present invention provides the application of the above-mentioned heterotrophic nitrifying-aerobic denitrifying Bacillus or the method for achieving efficient nitrogen utilization and rapid growth in fish in promoting fish growth or in fish farming where there are abnormal growth rates.
[0020] The present invention has the following beneficial effects:
[0021] The probiotics added to the feed in this invention are extracted from the intestines of fast-growing fish, making them fish-derived probiotics. These probiotics more easily alter the gut microbiota of fish, promoting growth and improving aberrant growth. Compared to existing technologies, firstly, probiotics are easier to obtain than related genes; secondly, the feed prepared from dominant gut probiotics of fast-growing fish is less expensive than exogenous activators; and finally, this invention provides a more convenient and simple way to achieve efficient nitrogen utilization and rapid growth in fish. Attached Figure Description
[0022] Figure 1 Peak diagram of DNA sequence from the first-generation sequencing of the intestinal contents sample of the test fish in Example 1.
[0023] Figure 2 The relative abundance of gut microbiota at the genus level in the slow-growth group (S) and the fast-growth group (F) in Example 1.
[0024] Figure 3 Example 1: Difference analysis of the bacterial strains such as Exiguobacterium in the slow growth group (S) and the fast growth group (F).
[0025] Figure 4 Example 1: The results of first-generation sequencing of the pure culture of intestinal flora in Example 1 were obtained by BLAST alignment of the bacterial strains.
[0026] Figure 5 In Example 3, the results of screening out significantly upregulated metabolites from each experimental group (C1, C2, C3) and the control group (NC) and taking the intersection were obtained.
[0027] Figure 6 In Example 3, the metabolite abundance scores of each experimental group (C1, C2, C3) were significantly upregulated compared with the control group (NC). Detailed Implementation
[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, all materials involved in the following embodiments are commercially available conventional materials, and all experimental operations involved are conventional operations in the art unless otherwise specified.
[0029] Example 1: Screening and preparation of Exiguobacterium sp. strain WY(Y)3
[0030] 1. Sampling preparation
[0031] Juvenile fish from the same parent fish of the order Perciformes were fasted for 6 hours before sampling. The juveniles were divided into a fast-growing group (FG) and a slow-growing group (SG), with 9 fish randomly selected from each group for analysis. The FG group specifications were: body length, 11.44±0.61 cm; weight, 20.81±1.33 g. The SG group specifications were: body length, 7.13±0.31 cm; weight, 7.13±0.61 g. After sampling under aseptic conditions, the intestinal contents of three fish from each group were mixed in 2 mL sterile centrifuge tubes, immediately frozen in liquid nitrogen, and stored at −80 °C for intestinal flora analysis. Sampling was repeated three times for each group. Approximately 1 g of intestinal contents was then taken from each group, added to 9 mL of sterile physiological saline, and vortexed for 5 min to form an initial bacterial suspension (10⁻¹ concentration), used for isolating, screening, and purifying fish-derived bacteria.
[0032] 2. Screening of dominant gut microbiota in alligator fish
[0033] The intestinal contents sample stored in step 1 for determining the gut microbiota was subjected to full-length 16S amplicon sequencing. The results showed that ( Figure 1-3 In the FG group, the contents of unclassified bacteria of the family Hericium, Thermopyramus, Erythromyces, Paget's, Kovacea, Leptothermofonsia, Microbacterium, and Pantanalinema were all higher than those in the SG group, and there were significant differences in the presence of Microbacterium in the intestines of the two groups of experimental fish.
[0034] 3. Isolation of Microbacterial strains
[0035] The initial bacterial suspension (10⁻¹ concentration) obtained in step 1 was serially diluted 10-fold to 10⁻¹. ⁻6(Add 1 mL of each solution to 9 mL of physiological saline) to obtain different concentration gradients (10... ⁻2 ~10 ⁻6 Take 100 μL of bacterial suspension at each dilution (usually 10 μL is selected). -4 ~10 -6 The cultures were spread onto LB agar plates using a sterile spreader, rotating the plate 60° each time, three times. Each dilution was replicated three times. After standing at room temperature for 10 minutes, the plates were incubated upside down. The plates were then incubated at 32°C for 24 hours. Single colonies with significant morphological differences were picked from the spread plates and streaked in four zones using an inoculation loop. Single colonies formed in the third and fourth zones were picked and streaked 2-3 times until pure cultures were obtained. The colony morphology (size, color, edge, transparency, etc.) was observed.
[0036] 4. Identification of Microbacterial strains
[0037] DNA was extracted from the pure cultures of the rapid growth group using a bacterial genomic DNA extraction kit, and the obtained DNA was subjected to first-generation sequencing. The sequenced sequences were compared using a BLAST website to obtain the results, and probiotics (Exiguobacterium sp. strain WY(Y)3) were screened from the dominant intestinal flora of the fish in the rapid growth group. Figure 4 The identified bacterial strain was compared with the results obtained from metagenomics (differences in macroscopic gut microbiota), confirming the target bacterial strain as (Exiguobacterium sp. strain WY(Y)3). This strain was deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on December 8, 2025, with accession number CGMCC No. 36957.
[0038] Example 2: Test on the effect of adding screened Microbes to feed for koi
[0039] Exiguobacterium sp. strain WY(Y)3 bacterial suspension was evenly sprayed onto the surface of sterile feed (Tongwei feed, model Floating Feed 180) at a certain concentration, and four types of live bacteria were prepared with a dosage of 0 (NC control group), 1×10⁻⁶, and 1×10⁻⁶. 6 cfu / g (C1 group), 1×10 7 cfu / g (C2 group), 1×10 8 The fish were fed cfu / g (C3 group) of food. Each group had three replicates, with 15 juvenile koi carp in each replicate, for a total of 12 glass aquariums and 180 experimental fish. The experiment lasted for 56 days, after which the growth indicators and muscle nutrient composition of the koi carp were measured.
[0040] Growth indicators include survival rate, weight gain rate, specific growth rate, conditionality, visceral-to-body ratio, and liver-to-body ratio, calculated using the following formulas:
[0041] Survival rate (%) = 100 × (N0 - N) t ) / N0; where N0: the total number of fish at the start of the experiment. N t Number of fish that died at the end of the experiment.
[0042] Weight gain rate (%) = 100 × (W t -W0) / W0; where W0: average body weight (g) at the start of the experiment, W t Average body weight (g) at the end of the experiment.
[0043] Specific growth rate (% / d) = 100 × (lnW) t -lnW0) / t; where W0: average body weight (g) at the start of the experiment, W t : Average body weight (g) at the end of the experiment, t is the number of days in the experiment.
[0044] Condition factor (CF, g / cm³) 3 =100×W b / L 3 Among them, W b : Net weight of the fish (excluding internal organs, g); L: Body length (cm, usually from the snout to the end of the caudal peduncle).
[0045] Viscera weight index (VSI, %) = 100 × W v / W; where Wᵥ: weight of all internal organs (heart, liver, intestines, swim bladder, etc.) (g), W: total weight of the fish (g).
[0046] Hepatosomatic index (HSI, %) = 100 × (W) h / W b ); where W h Liver weight (g), W b Fish net weight (g).
[0047] The results of the growth index tests are shown in Table 1. It can be seen that the addition concentration of 1×10 6At cfu / g (C1 group), the weight gain rate (WGR%) was significantly higher than that of the control group (186.26±1.07 vs. 181.65±3.77); the liver-to-body ratio (HSI%) of C1 group was significantly lower than that of the medium-to-high concentration groups (C1: 0.83±0.06 vs. C2: 0.98±0.11, C3: 1.01±0.10), while there were no significant differences in survival rate (SR%) and specific growth rate (SGR%) among the groups. (Note: 1×10⁻⁶) 6 The cfu / g addition rate of koi carp showed higher weight gain and growth rate than other experimental groups. Overall, the addition of Exiguobacterium sp. strain WY(Y)3 to the feed effectively promoted the growth of koi carp, and there was little individual variation in growth among the koi carp in the group, with no obvious abnormal growth rate issues.
[0048] Table 1. Effects of different levels of Exiguobacterium sp. strain WY(Y)3 in feed on the growth of koi carp.
[0049]
[0050] In the table: a represents a specific mean group or category, b represents another mean group or category that is different from a but may not be significantly different from some other means, and ab indicates that the two groups of data are not significantly different at the 5% significance level.
[0051] The results of the muscle nutrient composition test are shown in Table 2. In the table, different treatment groups (NC, C1, C2, C3) are represented as follows: a represents a specific mean group or category, b represents another mean group or category that is different from a but may not be significantly different from some other means, and ab represents that the two groups of data are not significantly different at the 5% significance level.
[0052] The fish exhibited differential characteristics in terms of moisture, ash, crude fat, and crude protein content. Moisture content fluctuated little among the groups (78.22%-80.62%), with a slightly higher level in group C2 (80.62±2.23%), but the difference was not statistically significant (p>0.05), possibly related to external factors such as environmental humidity or feed moisture content. Ash content was highest in group C1 (3.46±0.43%) and lowest in group C3 (3.07±0.32%), but the difference between groups was also not statistically significant (p>0.05), suggesting that its influence on feed mineral addition or fish metabolism regulation was limited. Crude fat content remained relatively stable among the groups (3.07±0.19-3.18±0.28, p>0.05), indicating that the experimental treatment had a weak regulatory effect on fat deposition and energy distribution. Notably, the crude protein content in group C1 was significantly higher than in other groups (18.08±0.15b vs. NC: 17.43±0.42a, C2: 16.80±0.16a, C3: 16.93±0.05a, p<0.05). This result may be attributed to the promoting effect of *Exiguobacterium* in the feed of group C1 on protein digestion and absorption, for example, by enhancing intestinal enzyme activity or improving amino acid utilization. In summary, the advantage of group C1 in crude protein accumulation suggests it as a potential optimization direction for efficient protein utilization, while the stability of other nutrients (moisture, ash, crude fat) reflects the buffering capacity of the fish's metabolic system against experimental intervention. Overall, feeding koi with *Exiguobacterium* sp. strain WY(Y)3 did not adversely affect their nutritional composition. The weight gain rate in Table 1 and the crude protein content in Table 2 together demonstrate that group C1 has high nitrogen utilization.
[0053] Table 2. Effects of different levels of Exiguobacterium sp. strain WY(Y)3 in feed on the nutritional composition of koi muscle.
[0054]
[0055] Example 3: Study on the mechanism by which microbacteria screened through omics analysis promote the growth of koi carp
[0056] Experimental Methods: 180 healthy, deformed, and relatively uniform juvenile koi were selected and randomly divided into 4 groups, with 3 replicates per group and 15 koi per replicate. Each replicate of koi was placed in 12 cement ponds measuring 2 m × 3 m × 1.1 m. Feeding was initiated, with the 4 groups fed Exiguobacterium sp. strain WY(Y)3) at a dosage of 0 (NC control group), 1 × 10⁻⁶, and 1 × 10⁻⁶, respectively. 6 cfu / g (C1 group), 1×10 7cfu / g (C2 group), 1×10 8 Feed containing cfu / g (C3 group) was used. After the experiment, five koi were selected from each cement pond, anesthetized, and their intestines were dissected with a scalpel. The intestinal contents were placed in 10 mL cryovials, rapidly frozen in liquid nitrogen, and then stored at -80°C. The samples were sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for metabolomics sequencing.
[0057] We compared the sequencing results of each experimental group with the control group to screen for significantly upregulated metabolites and took the intersection. The results showed that the following metabolic pathways related to promoting fish growth were significantly upregulated: tryptophan metabolism, biosynthesis of valine, leucine and isoleucine, D-amino acid metabolism, phenylalanine metabolism, alanine, aspartic acid and glutamate metabolism. Figure 5 ).
[0058] Analysis of metabolites related to amino acid metabolic pathways in each group showed that the abundance of all four metabolites was higher in group C1 than in the control group. Indole-3-acetic acid, indole-3-acetamide, and 2-isopropylmalic acid were the most abundant in group C1. Figure 6 ).
[0059] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. Any changes made by those skilled in the art after reading the specification of the present invention, as long as they are within the scope of the claims of the present invention, will be protected by patent law.
Claims
1. A heterotrophic nitrifying-aerobic denitrifying Bacillus, characterized in that, The strain is *Exiguobacterium* sp. strain WY(Y)3, classified as *Exiguobacterium*, and deposited at the China General Microbiological Culture Collection Center on December 8, 2025, with accession number CGMCC No. 36957.
2. The heterotrophic nitrifying-aerobic denitrifying Bacillus according to claim 1, characterized in that, The screening method is as follows: S1. Screening of dominant gut microbiota: A species of fish in the order Perciformes was divided into a fast-growing group and a slow-growing group according to its body weight. Intestinal samples from both groups were subjected to full-length 16s amplicon sequencing to obtain the dominant microbiota with higher content in the fast-growing group than in the slow-growing group. The microbiota with the greatest difference was selected as the target microbiota. S2. Sampling preparation for dominant bacteria culture: Take the intestinal contents of the test fish, add sterile physiological saline to form an initial bacterial suspension, which is used to isolate, screen and purify fish-derived bacteria; S3. Target bacterial strain isolation and screening: The initial bacterial suspension was serially diluted and spread on LB agar plates and incubated at 32°C for 24 h; then single colonies with obvious morphological differences were picked and streaked on the plates, and the streaking was repeated 2-3 times until pure cultures were obtained; S4. Identification of target bacterial species: Genomic DNA was extracted from the pure culture and then subjected to first-generation sequencing. The sequencing results were compared with the BLAST website sequence to screen for target probiotics in the gut microbiota of the test fish in the fast-growing group. The identified bacterial species were then compared with metagenomic data to verify the target bacterial species.
3. A method for achieving efficient nitrogen utilization and rapid growth in fish, characterized in that, Fish are fed with a feed made by adding the heterotrophic nitrifying-aerobic denitrifying Bacillus as described in claim 1 to a basic feed.
4. The method according to claim 3, characterized in that, The addition method involves spraying an Exiguobacterium sp. strain WY(Y)3 bacterial suspension onto the surface of the basic feed.
5. The method according to claim 3, characterized in that, The fish mentioned include fish from the orders Perciformes and Cypriniformes.
6. The method according to claim 5, characterized in that, When the fish species is koi, the amount of Exiguobacterium sp. strain WY(Y)3 added to the basal feed should be ≤1×10⁻⁶. 8 cfu / g.
7. The method according to claim 6, characterized in that, The *Exiguobacterium* sp. strain WY(Y)3 was added at a rate of 1×10⁻⁶ to the basal feed. 6 cfu / g.
8. The heterotrophic nitrifying-aerobic denitrifying Bacillus as described in claim 1 or the method described in any one of claims 3-7, in promoting fish growth or in fish farming where there are allometric growth rates.