Macrobrachium rosenbergii BFT culture system construction method based on SBOS-FOS composite carbon source

Abstract

The invention discloses a macrobrachium rosenbergii probiotic biofloc technology (BFT) culture system construction method based on an SBOS-FOS composite carbon source, and belongs to the technical field of aquaculture. The total ratio of SBOS (soybean oligosaccharide) and FOS (fructo-oligosaccharide) in the composite carbon source is 2%-10%, and the balance is glucose; and adding the composite carbon source into a macrobrachium rosenbergii culture system according to the C / N ratio of (10: 1)-(20: 1) according to the feed protein content in the culture water body. Through a specific ratio of SBOS to FOS, the content of functional probiotics in a culture water body is specifically increased, stable control of ammonia nitrogen and nitrite nitrogen and directional optimization of a carbon source-flora-host three-level regulation and control network in a BFT system are realized, the weight gain rate (WG), the specific growth rate (SGR) and digestive enzyme activity (trypsin and amylase) of macrobrachium rosenbergii are increased, and the survival rate of the macrobrachium rosenbergii is increased. The abundance of probiotics in the water body and the immune gene expression of the macrobrachium rosenbergii are obviously improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of aquaculture, and particularly relates to a Macrobrachium rosenbergii BFT (Biofloc Technology, BFT) probiotic breeding system construction method based on an SBOS-FOS composite carbon source, and more particularly to a breeding method taking a soybean oligosaccharide (SBOS) and fructo-oligosaccharide (FOS) composite carbon source as the core. BACKGROUND

[0002] As a globally important freshwater economic shrimp species, the breeding scale of Macrobrachium rosenbergii has been continuously expanding in recent years. According to industry statistics, the total output of Macrobrachium rosenbergii breeding in China reached about 180,000 tons in 2024, and the industry chain output value exceeded 20 billion yuan. The main production areas are concentrated in Guangdong, Jiangsu, and Zhejiang provinces, and the emerging production areas such as Hainan and Shaanxi have shown significant growth. With the popularization of intensive breeding modes such as "pond intensive culture" and "rice-fish rotation", the industry has expanded, but technical bottlenecks and ecological contradictions have become increasingly prominent, and it is urgent to achieve sustainable development through technological innovation.

[0003] In the aspects of germplasm resources and seedling breeding, the Macrobrachium rosenbergii industry in China faces severe challenges. Although improved varieties such as "South Taihu No. 2" and "South Taihu No. 3" have been bred (the latter has a 21.16% weight gain compared to the former generation, and its stress resistance has been significantly improved), the core germplasm still relies on imports from wild resources in Southeast Asia, and long-term inbreeding has led to germplasm degradation. There are problems such as extensive technology and large fluctuations in survival rate among seedling enterprises. Research shows that germplasm degradation directly weakens the disease resistance of shrimps and exacerbates disease risks. For example, cases of Streptococcus agalactiae infection have been found in India recently, and the virulence genes carried by the bacteria are highly resistant to conventional antibiotics. However, China has not established a complete SPF (specific pathogen-free) seed production system, leading to increased pressure on disease control.

[0004] Feed efficiency and water quality management are another key pain point. The crude protein content of current Macrobrachium rosenbergii feed varies significantly (38%-42%), and the mixing of fake feed by some enterprises has led to a decline in the immunity of shrimps and frequent deaths. Ammonia nitrogen emission is particularly problematic during the breeding process. The traditional water exchange mode not only wastes freshwater resources but also causes environmental pollution due to improper tail water treatment. Although the "three ponds and two dams" ecological treatment technology has been partially promoted, the cost and efficiency are still difficult to balance. In addition, although the bio-floc technology (BFT) can reduce ammonia nitrogen through microbial assimilation, its application in Macrobrachium rosenbergii breeding is still in the exploratory stage, and there is a lack of optimization standards for core parameters such as carbon source selection and microbial community regulation, which restricts the efficiency of the technology.

[0005] The core of BFT system is the regulation of microbial community driven by carbon source. Previous studies have shown that the type of carbon source directly affects the structure and function of the bacterial community in water: plant cellulose can enrich ammonia-oxidizing bacteria, and strengthen the biological transformation of ammonia nitrogen; cassava dregs can improve the water ecology by increasing the abundance of Rhodobacteraceae; starch carbon source can promote the proliferation of Cyanophyta, enhance photosynthesis and organic matter circulation. In addition, the choice of carbon source will also affect the health of farmed animals through the "water-intestinal microbe interaction". For example, sucrose can increase the abundance of Amaricoccus and Microbacterium in the intestines of shrimp, glucose promotes the proliferation of Rhodobacteraceae, and β-hydroxybutyric acid-β-hydroxyvaleric acid ester significantly increases the proportion of Fusobacteria. These microbial changes are closely related to the growth performance and immune response of the host: water probiotics regulate the structure of the intestinal flora by colonizing the intestinal tract, activate immune gene expression, and at the same time, the microbial enzymes in the biofloc and the host digestive enzymes synergistically enhance the efficiency of nutrient absorption. Therefore, the selection of suitable carbon source to optimize the interaction network between flora and host is the key to improving the efficiency of BFT system.

[0006] In recent years, oligosaccharides have shown unique potential as functional carbon sources. These short-chain carbohydrates composed of 2-10 monosaccharides are difficult for the host to digest, but can selectively promote the proliferation of beneficial bacteria, thereby improving intestinal health. Studies have shown that oligosaccharides as feed additives can significantly improve the growth performance and immunity of aquatic animals, for example: soybean oligosaccharides (SBOS, containing sucrose, raffinose, stachyose) can enhance the disease resistance of fish by regulating the intestinal flora; fructooligosaccharides (FOS) can inhibit pathogenic bacteria by enriching Lactobacillus, while also improving the antioxidant capacity of Litopenaeus vannamei. Notably, research on the application of oligosaccharides as carbon sources in BFT systems has also made progress: 2.5% SBOS replacing glucose can optimize the structure of the high-nitrogen water bacterial community, 1-5% SBOS addition can improve the growth performance of crucian carp and increase the number of beneficial bacteria in the intestines, and 2.5% FOS can enhance the stability of the system by promoting the proliferation of core functional bacteria. However, existing research has focused on the effects of a single oligosaccharide, and there is a lack of in-depth exploration of the combined application of multiple oligosaccharides and their synergistic effects, especially the mechanism of "water-intestinal" microbial regulation by complex carbon sources.

[0007] Based on this, the present invention proposes the use of SBOS and FOS as a complex carbon source in BFT systems for the culture of Macrobrachium rosenbergii. The raffinose and stachyose in SBOS can target the regulation of the host's intestinal flora and alleviate inflammation, while FOS can optimize nitrogen cycling by promoting the proliferation of functional bacteria in water, such as Rhodobacteraceae. The combined use of the two is expected to achieve multi-level gains in "water purification-flora regulation-host health". SUMMARY

[0008] In order to overcome the problem of ammonia nitrogen emission in the breeding process of the existing breeding technology, the immunity of shrimps is decreased, and the phenomenon of stealing death occurs frequently, and the purpose of the present application is to provide a construction method of BFT breeding system of Macrobrachium rosenbergii based on SBOS-FOS composite carbon source, which not only fills the research blank of oligosaccharide composite carbon source in BFT system, but also provides innovative technical support for green transformation of aquaculture.

[0009] The purpose of the present application is achieved by the following technical solutions:

[0010] Soybean oligosaccharides (SBOS), fructooligosaccharides (FOS) and glucose are mixed in proportion, wherein the total mass ratio of SBOS and FOS is 2%-10%, and the balance is glucose.

[0011] Preferably, the mass ratio of SBOS and FOS is 1:1, and the total ratio is 5%.

[0012] Further preferably, the SBOS is composed of sucrose, raffinose and stachyose, and the mass percentage of the three is 49.59%:9.52%:39.89%.

[0013] According to the crude protein content of the feeding feed, the carbon source demand is calculated, and the C / N ratio is 10:1 to 20:1.

[0014] Preferably, the C / N ratio is 15:1, the carbon source adding time is 2 hours after feeding every day, and after dissolving, it is uniformly sprayed into the breeding water body.

[0015] The breeding environment is controlled to be water temperature 30±1℃, pH 7.8-8.0, dissolved oxygen content 6-7.5 mg / L, and zero water change throughout the process;

[0016] Preferably, the initial aeration time of the breeding water body is 24-48 hours, and the dissolved oxygen saturation is greater than or equal to 90%;

[0017] Further preferably, the evaporation loss water is supplemented every 2 weeks, and the supplemented water is pretreated by aeration to be consistent with the original water parameters.

[0018] Compared with the prior art, the beneficial effects of the present application mainly lie in:

[0019] By the specific proportion of SBOS (soybean oligosaccharides containing sucrose, raffinose, stachyose) and FOS (fructooligosaccharides), the stable control of ammonia nitrogen and nitrite nitrogen is realized, and the directional optimization of the "carbon source-bacterial flora-host" three-level regulation network in the BFT system is achieved, while the weight gain rate (WG), specific growth rate (SGR) and digestive enzyme activity (trypsin, amylase) of Macrobrachium rosenbergii are improved, and the water body probiotic abundance and immune gene expression of Macrobrachium rosenbergii are significantly improved. The core value lies in breaking through the limitations of traditional technology focusing on water quality or growth alone, achieving multi-target synergistic gain through microbial ecological regulation, meeting the sustainable development needs of "reducing investment, reducing emissions and increasing efficiency" in aquaculture industry, and providing a replicable technical paradigm for green and efficient aquaculture of Macrobrachium rosenbergii. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 For the regulation effect of compound carbon source on nitrogen transformation and water quality stability in BFT system, figures (a-d) are the change trends of ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and total nitrogen, figure (e) is the change of floc volume, and figure (f) is the change of water turbidity;

[0021] Figure 2 For the promotion effect of different treatments on the digestion and metabolism of Macrobrachium rosenbergii, figure (a) is the trypsin activity, figure (b) is the lipase activity, and figure (c) is the amylase activity;

[0022] Figure 3 For the influence of compound carbon source on the antioxidant indexes of Macrobrachium rosenbergii liver, figure (a) is the catalase activity, figure (b) is the superoxide dismutase activity, and figure (c) is the malondialdehyde content;

[0023] Figure 4 For the influence of compound carbon source on the immune indexes of Macrobrachium rosenbergii serum, figure (a) is the phenol oxidase activity, figure (b) is the alkaline phosphatase activity, and figure (c) is the acid phosphatase activity;

[0024] Figure 5 For the relative expression level of immune genes and growth genes of Macrobrachium rosenbergii liver;

[0025] Figure 6 For the reconstruction of microbial community in BFT system by compound carbon source, figure (a) is the Venn diagram of water microbial community, and figure (b) is the Venn diagram of intestinal microbial community of Macrobrachium rosenbergii;

[0026] Figure 7 For the column chart of community structure at the door level, E represents the water environment microbial community, and I represents the intestinal microbial community of Macrobrachium rosenbergii;

[0027] Figure 8 For the heat map of community structure at the genus level, E represents the water environment microbial community, and I represents the intestinal microbial community of Macrobrachium rosenbergii;

[0028] Figure 9 PCoA analysis of microbial community, where figure (a) is the bacterial community in water, figure (b) is the intestinal microbial community of Macrobrachium rosenbergii;

[0029] Figure 10 Differences in the abundance of bacterial community in water;

[0030] Figure 11 Differences in the abundance of intestinal microbial community;

[0031] Figure 12 Bacterial community taxa with significant inter-group differences in water identified by LEfSe analysis;

[0032] Figure 13 Bacterial community taxa with significant inter-group differences in the intestine identified by LEfSe analysis. DETAILED DESCRIPTION

[0033] The technical solutions of the present application are further described below in conjunction with the detailed description. However, those skilled in the art should understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the present application. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if not specifically mentioned, are conventional products obtained through commercial channels.

[0034] Example 1 Construction of Macrobrachium rosenbergii BFT breeding system based on SBOS-FOS composite carbon source

[0035] This example was implemented in the Standardized Aquaculture Laboratory of Hu Zhou Normal College. In view of the problems of single carbon source and low nitrogen conversion efficiency of traditional biological flocculation technology (BFT), a composite carbon source of soybean oligosaccharides (SBOS) and fructooligosaccharides (FOS) was innovatively used to construct a probiotic high-efficiency breeding system. The specific implementation steps are as follows:

[0036] (1) Experimental materials and animals

[0037] Breeding container: food-grade high-density polyethylene (HDPE) material breeding tank (volume 300 L, length x width x height = 1.2 x 0.8 x 0.5 m) lined with water-proof cloth, 200 L of tap water treated by 48 hours aeration to remove chlorine was injected into each tank.

[0038] Experimental animals: 600 healthy Macrobrachium rosenbergii juvenile shrimps were purchased from Zhongyi Aquatic Seed Technology Co., Ltd. and acclimated in the laboratory recirculating water system for 14 days. During the acclimation period, the basic feed was fed to adapt to the experimental environment.

[0039] Basic feed: see Table 1

[0040] Table 1. Formula of basal diet

[0041]

[0042] Vitamin premix (mg / kg diet): vitamin A, 16000 IU; vitamin C, 150 mg; vitamin D3, 2000 IU; vitamin E, 180 mg; vitamin K3, 10 mg; vitamin B1, 16 mg; vitamin B2, 45 mg; vitamin B6, 20 mg; vitamin B12, 0.4 mg; pantothenic acid, 70 mg; nicotinic acid, 80 mg; folic acid, 5 mg; biotin, 1 mg; and inositol, 320 mg.

[0043] Mineral salt premix (mg / kg diet): potassium iodide, 1.77; sodium selenite, 0.57; cobalt carbonate, 0.71; magnesium sulfate heptahydrate, 3019.89; copper sulfate pentahydrate, 80.89; manganese sulfate monohydrate, 38.51; ferrous sulfate heptahydrate, 93.24; zinc sulfate heptahydrate, 280.30.

[0044] Carbon source: soybean oligosaccharides SBOS (purchased from Shanghai Yuan Ye Biotechnology Co., Ltd., main components: sucrose 49.59%, raffinose 9.52%, stachyose 39.89%); FOS (purchased from Shanghai Yuan Ye Biotechnology Co., Ltd., purity 95.2%); anhydrous glucose (purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd., analytical pure, purity 99.5%).

[0045] (2) Construction of breeding system

[0046] After domestication, 600 shrimps (initial weight 1.78 ± 0.17 g) were randomly allocated to 20 breeding boxes, with an average of 30 tails per box, and 5 treatment groups were set up, with 4 replicates in each group:

[0047] Table 2. Grouping of breeding system and carbon source feeding

[0048]

[0049] At 8:00 and 17:00 every day, 2 hours after feeding the basal diet (formula see Table 1), the carbon source requirement was calculated according to the crude protein content of the feed (43.99%) and the C / N = 15:1:

[0050]

[0051] The carbon content of SBOS ​​was 42.3% by weighted average, FOS was 45.1%, and glucose was 40.0%. The carbon source powder was dissolved in 1 L of 35℃ warm water (to avoid high temperature damaging the oligosaccharide structure), filtered through a 200-mesh filter, and then evenly sprinkled into the breeding tank. The bottom aeration device (HAILEA V60, air flow rate 5 L / min) was turned on and stirred for 10 minutes to ensure that the carbon source was fully dispersed.

[0052] The experiment lasted for 8 weeks with no water changes throughout. Every 14 days, water lost due to evaporation was replenished (approximately 5-8 L / tank). The replenished water underwent aeration pretreatment to match the parameters of the original water. During the experiment, all light sources were natural light. The water temperature was maintained at 30±1℃, the pH at 7.8-8.0 (monitored daily; if fluctuations exceeded 0.2, adjustment was made with 0.1 mol / L sodium bicarbonate solution), and the dissolved oxygen at 6-7.5 mg / L.

[0053] Experimental Example 1: The Regulatory Effect of Composite Carbon Sources on Nitrogen Transformation and Water Quality Stability in a BFT System

[0054] A 100 mL water sample was taken weekly and filtered through a 0.45 μm mixed cellulose ester membrane. Ammonia nitrogen (NH4) in the water was determined using a QC8500 flow injection water quality analyzer (Lachat, USA). + -N), nitrite nitrogen (NO2) - -N), nitrate nitrogen (NO3) - The content of nitrogen (N-N) and total nitrogen (TN) was measured. 1000 mL of water sample was taken from each tank and injected into the Inhofe conical tube. After standing for 30 minutes, the volume of settled flocs (mL / L) was recorded. Turbidity was measured using a WTW 430 turbidity meter (WTW, Germany), and the result was the average of three measurements.

[0055] The results are as follows Figure 1 As shown, during the experiment, the ammonia nitrogen and nitrite nitrogen levels in all experimental groups were within the healthy range. The ammonia nitrogen level generally showed a trend of first increasing and then decreasing, and stabilized at a low level after 3 weeks. The nitrite nitrogen level remained at a low level and relatively stable. Figure 1 -a, Figure 1 -b). The nitrate and total nitrogen contents in all experimental groups showed a trend of first decreasing and then stabilizing, with the overall concentration remaining at a low level. Figure 1 -c, Figure 1 -d). The overall trend of floc volume and turbidity in all experimental groups was increasing. Figure 1 -e, Figure 1 -f). The floc volume of the SBOS ​​group was significantly higher than that of the control group and the other experimental groups at 3 weeks (P<0.05). The turbidity of the GLU group was significantly higher than that of the other experimental groups at 6 and 7 weeks, and significantly higher than that of the other experimental groups except the SBOS ​​group at 8 weeks (P<0.05).

[0056] Effect of complex carbon sources on growth performance of Macrobrachium rosenbergii

[0057] At the end of the experiment, the fish were deprived of food for 24 hours, and the total weight of each tank was recorded along with the number of surviving individuals. Twenty individuals were randomly selected from each tank to calculate the growth performance indicators. The weight gain (WG), specific growth rate (SGR), survival rate (SR), and feed conversion ratio (FCR) were calculated using the following formulas:

[0058]

[0059]

[0060]

[0061]

[0062] where W0 is the initial weight of the shrimp (g), W is the final weight of the shrimp (g), S is the number of shrimp at the end of the culture, S0 is the number of shrimp at the beginning of the culture, F is the feed intake (g), and d is the number of days of feeding (d). t t

[0063] Table 3 Growth performance of Macrobrachium rosenbergii

[0064]

[0065] The growth performance indicators of Macrobrachium rosenbergii are shown in Table 3. There were no significant differences in survival rate and feed conversion ratio among all experimental groups (P>0.05). The final weight, weight gain rate, and specific growth rate of the FOS group were not significantly different from those of the GLU group (P>0.05), while the final weight, weight gain rate, and specific growth rate of the SBOS, FS1, and FS2 groups were significantly higher than those of the GLU group (P<0.05). In addition, there were no significant differences in final weight, weight gain rate, and specific growth rate between the FS1 and SBOS groups (P>0.05), but the final weight, weight gain rate, and specific growth rate of the FS2 group were significantly higher than those of the SBOS group (P<0.05).

[0066] Promoting effect of complex carbon sources on digestion and metabolism of Macrobrachium rosenbergii

[0067] ​​Each bucket to take 10 shrimp, with ice water anesthesia, peel the midgut to the hindgut section, rinse the contents with PBS buffer, then add pre-cooled physiological saline to the intestinal tissue at 1:9 (w / v), homogenize in ice bath and centrifuge (3500 rpm, 10 min), and measure the activities of trypsin (Trypsin, Nanjing Jiancheng, item number A080-2-2), lipase (Lipase, Nanjing Jiancheng, item number A054-1-1), and amylase (Amylase, Nanjing Jiancheng, item number C016-1-1) in the supernatant using a kit. The results are shown in Figure 2 Trypsin activity in the SBOS group was significantly higher than that in the GLU group (P<0.05) (a). Figure 2 There was no significant difference in lipase activity among the experimental groups (P>0.05) (b). Figure 2 Amylase activity in the FOS and FS1 groups was significantly higher than that in the control group (c). Figure 2

[0068] Test Example 4: Activation effect of complex carbon sources on the immune defense system of shrimp

[0069] Each bucket to take 10 shrimp, with ice water anesthesia, separate the liver, add pre-cooled physiological saline to the liver at 1:9 (w / v), homogenize in ice bath and centrifuge (3500 rpm, 10 min), and measure the contents of superoxide dismutase (SOD, Nanjing Jiancheng, item number A001-3-2), peroxidase (CAT, Nanjing Jiancheng, item number A084-3-1), and malondialdehyde (MDA, Nanjing Jiancheng, item number A003-1-2) in the supernatant using a kit. The results are shown in Figure 3 CAT activity in the SBOS, FOS, and FS2 groups was significantly higher than that in the GLU group, and CAT activity in the FS2 and SBOS groups was significantly higher than that in the FS1 group (a). Figure 3 There was no significant difference in SOD among all experimental groups (b). Figure 3 MDA content in the FS1 and FS2 groups was significantly higher than that in the control group and the other experimental groups (P<0.05) (c). Figure 3

[0070] Each bucket to take 10 shrimp, with ice water anesthesia, collect blood into an EDTA-K2 anticoagulant tube (Biyun Tian, ST002-50mL), centrifuge at 3500 rpm and 4°C for 10 minutes, and measure the activities of serum immune indicators phenol oxidase (PO, Jiangsu Enzyme, item number MEIMIAN-MM-0453H2), alkaline phosphatase (AKP, Nanjing Jiancheng, item number A059-1-1), and acid phosphatase (ACP, Nanjing Jiancheng, item number A060-1-1). The results are shown in Figure 4 PO content in the SBOS and FS2 groups was significantly higher than that in the GLU and FOS groups, and PO content in the FS1 group was significantly higher than that in the FOS group (P<0.05) (a).​​Figure 4 -a). AKP activity, FS2 group was significantly higher than the rest of the experimental groups, FS1 group was significantly higher than GLU, SBOS and FOS groups, SBOS group was significantly higher than GLU and FOS groups (P<0.05) Figure 4 -b). ACP activity was similar to AKP, SBOS group and FS1 group were significantly higher than the rest of the experimental groups, FS2 group was significantly higher than GLU and FOS groups (P<0.05) Figure 4 -c).

[0071] Ten shrimps were taken from each tank, anesthetized with ice water, and the livers were separated to extract RNA (Aidley, item number RN2801), reverse-transcribed into cDNA (Aidley, item number PC7002), and qPCR analysis was performed using the MonAmpTM SYBR® Green qPCR Mix kit (Gonghe Gene, item number MQ10101S). The CFX-96 real-time quantitative instrument (BioRad Laboratories, Inc., USA) was used, with a reaction system of 20 μL (SYBR Green Mix 10 μL, primers each 0.8 μL, cDNA 2 μL, ddH2O 6.4 μL), and the cycle conditions were 95℃ for 3 min; 95℃ for 15 s, 60℃ for 30 s, 40 cycles. A total of 6 immune-related genes (Propo, HSP70, NF-κB, TOLL-R, TOLL-1, IMD) and 2 growth-related genes (IGFI-R, ECR) were determined, and the primers were designed by Primer6 software. β-actin was used as an internal reference gene, and the relative expression level of target gene mRNA was calculated using 2 -ΔΔCt Method calculation, primer sequences are shown in Table 4.

[0072] Table 4 Primer sequences of genes in fluorescence quantitative PCR

[0073]

[0074] The results are shown in Figure 5 FS2 group and FS1 group immune-related genes were significantly up-regulated (P<0.05). The expression of Propo in FS1 group and FS2 group was significantly higher than that in GLU group and FOS group (P<0.05) -a). The expression of HSP70 in FS1 group was significantly higher than that in the rest of the experimental groups, and the expression in the experimental groups was significantly higher than that in GLU group -b). The expression of NF-κB in FOS group and FS1 group was significantly higher than that in SBOS group and FS2 group, and the expression in all experimental groups was significantly higher than that in GLU group (P<0.05) -c). TOLL-R, similar to Propo, the expression of Propo in FS1 group and FS2 group was significantly higher than that in GLU group and SBOS group (P < 0.05) -d). The expression of TOLL-1 in FOS group was significantly higher than that in GLU group (P < 0.05) -e). The FS1 group showed the highest expression of IMD, which was significantly higher than the rest of the experimental groups and the control group, and the SBOS group and the FS2 group were also significantly higher than the GLU group (P < 0.05) -f). In terms of growth genes, there was no significant difference between each experimental group (P > 0.05), but it was significantly higher than that in GLU group (P < 0.05) -g, -h).

[0075] Test Example 5 Reconstruction of microbial community in BFT system by composite carbon source

[0076] Take 10 shrimps from each barrel, anesthetize with ice water, and rinse with PBS to collect intestinal contents. Take 100 mL of water sample from each experimental tank. Filter the intestinal contents and water samples using a 0.22 μm filter membrane to the surface of the filter membrane with obvious attachments. Scrape the biofilm on the surface of the filter membrane into DNA preservation solution. Send the sample to Shanghai Meiji Biomedicine Technology Co., Ltd. for 16S rRNA and gene abundance sequencing analysis using Illumina to determine the bacterial community. Use primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') (SEQ ID NO. 19) and 806R (5'-GGACTACHVGGGTWTCTAAT-3') (SEQ ID NO. 20) to amplify the V3-V4 hypervariable region. The experimental results are presented at the phylum and genus levels, with the minimum taxonomic unit being OUT (a standard unit of classification in phylogenetic and population genetic studies, such as strain, genus, species, or grouping, etc.). OTU is clustered using 97% similarity. The experiment reflects community richness (Community richness) using Ace and Chao, community diversity (Community diversity) using Shannon and Simpson, and community coverage (Community coverage) using coverage.

[0077] The water body alpha diversity index results are shown in Table 5, and the intestinal alpha diversity index results are shown in Table 6. In the bacterial community of the water body, the OTU, Ace, and Chao indexes of each experimental group were significantly higher than those of the control group, the OTU of the FOS group was significantly higher than that of the GLU group (P < 0.05), and the OTU of the rest of the experimental groups had no significant difference compared with the GLU (P > 0.05). The Ace of the FOS group and the FS1 group was significantly higher than that of the GLU group (P < 0.05), and the Ace of the rest of the experimental groups had no significant difference compared with the GLU (P > 0.05). The Chao of the FS1 group was significantly higher than that of the GLU group (P < 0.05), and the Chao of the rest of the experimental groups had no significant difference compared with the GLU (P > 0.05). The shannon and simpson indexes had no significant difference (P > 0.05).

[0078] Table 5 Microbial diversity and abundance in water body

[0079]

[0080] Table 6 Microbial diversity and abundance in the intestine of Macrobrachium rosenbergii

[0081]

[0082] The microbial community Venn diagram results are shown in Figure 6 , and the unique bacterial species of each group had little difference. Figure 6 -a), and the Venn diagram results show that the unique bacterial species of each group had little difference Figure 6 -b).

[0083] The results of the bacterial community composition at the phylum level are shown in Figure 7, the core bacterial phyla in water were Proteobacteria, Actinobacteriota, Bacteroidota and Chloroflexi. Compared with E_GLU, the abundance of Actinobacteriota was significantly increased (P<0.05) and the abundance of Chloroflexi was decreased, but there was no significant difference (P>0.05) in the experimental groups. The core intestinal bacterial community was Proteobacteria, Firmicutes and Actinobacteriota. Compared with I_GLU, the abundance of Firmicutes was significantly reduced in I_FS1 and I_FS2 groups, and the abundance of Proteobacteria was significantly increased in I_FS1 group (P<0.05). At the same time, the abundance of Actinobacteriota was significantly increased in all experimental groups compared with I_GLU (P<0.05), which was consistent with the results of water bacterial community.

[0084] The results of bacterial community composition at genus level are shown in Figure 8 , the distribution of bacterial genera in water was relatively uniform, with rich species and little difference in the relative abundance of each genus. The core intestinal bacterial community was Lactococcus, Staphylococcus, unclassified_f_Enterobacteriaceae, unclassified_o_Micrococcales and Nakamurella. The results showed that I_FS2 group had the highest Nakamurella abundance in intestinal and water bacterial communities, and I_FOS group had the highest unclassified_o_Micrococcales abundance in intestinal and water bacterial communities. At the same time, I_FS2 group also had the highest bacillus abundance.

[0085] The results of PCoA are shown in Figure 9 , Figure 10 , there were extremely significant differences in water bacterial community and intestinal bacterial community. The results of water bacterial community analysis showed that compared with E_GLU, the abundance of Polynucleobacter and Nakamurella was significantly increased in E_SBOS group (P<0.05), and the abundance of Micrococcales, Rhodobacter and Micropruina was significantly increased in E_FOS group (P<0.05). Figure 10 -a) Figure 10- b), E_FS1 group significantly reduced the abundance of norank_f_Caldilineaceae and increased the abundance of Polynucleobacter (P < 0.05) Figure 10 - c), E_FS2 group significantly increased the abundance of Polynucleobacter, Nakamurella, unclassified_o_Micrococcales (P < 0.05) Figure 10 - d), compared with E_SBOS group, E_FOS group significantly reduced the abundance of Emticicia and increased the abundance of Rhodobacter Figure 10 - e), E_FS1 group significantly reduced the abundance of Emticicia and increased the abundance of Pedomicrobium Figure 10 - f), E_FS2 group significantly increased the abundance of Haliangium, Xanthomonadaceae (P < 0.05) Figure 10 - g), compared with E_FOS group, E_FS1 group significantly reduced the abundance of norank_f_Caldilineaceae and increased the abundance of Edaphobaculum Figure 10 - h), E_FS2 group significantly reduced the abundance of norank_o_Oligoflexales and increased the abundance of Hirschia (P < 0.05) (Figure 10-i). Compared with E_FS1 group, E_FS2 group significantly reduced the abundance of Legionella and increased the abundance of Hirschia (P < 0.05) Figure 10 - j).

[0086] The results of intestinal flora analysis are as follows Figure 11 - a), I_FOS group significantly increased the abundance of Nakamurella, Rhizobiales, Ensifer and Rhodobacteraceae Figure 11 - b), I_FS1 group significantly reduced the abundance of Enterococcus and increased the abundance of Shewanella Figure 11 - c), I_FS2 group significantly reduced the abundance of Staphylococcus, Enterococcus and increased the abundance of Nakamurella (P < 0.05) Figure 11 - d), compared with I_SBOS group, I_FOS group significantly increased the abundance of Lactococcus and significantly reduced the abundance of Rhizobiales Figure 11 - e), I_FS1 group significantly reduced the abundance of Enterococcus and increased the abundance of Shewanella Figure 11- e), I_FS1 group significantly reduced the abundance of Mycobacterium and unclassified_f_ Mycoplasmataceae (P<0.05) Figure 11 - f), I_FS2 group significantly reduced the abundance of Staphylococcus and significantly increased the abundance of Lactococcus (P<0.05) Figure 11 - g), I_FS1 group significantly reduced the abundance of Hyphomicrobium and Roseimicrobium compared with I_FOS group Figure 11 - h), I_FS2 group significantly increased the abundance of Aeromonas and reduced the abundance of Acinetobacter (P<0.05) Figure 11 - i), I_FS2 group significantly reduced the abundance of Chitinilyticum and increased the abundance of Roseimicrobium compared with I_FS1 group (P<0.05) Figure 11 - j).

[0087] The results of LEfSe analysis are shown in Figure 12 , Figure 13 The results of water microbiota showed that E_SBOS group was significantly enriched in Niveibacterium, E_FOS group was significantly enriched in Micrococcales, E_FS1 group was significantly enriched in Pedomicrobium, and E_FS2 group was significantly enriched in the most bacterial genera, including Nakamurella and Micropruina (P<0.05) Figure 12 The results of intestinal microbiota showed that I_SBOS group was significantly enriched in Carnobacterium, I_FOS group was significantly enriched in norank_p_ Firmicutes, I_FS1 group was significantly enriched in Chitinilyticum, and I_FS2 group was significantly enriched in Nakamurella (P<0.05) Figure 13 .

[0088] The experimental results show that this technology can effectively improve the water environment, promote the construction of beneficial microbial community, and significantly enhance the immune response and growth performance of shrimp. SBOS and FOS, whether used alone or in combination, can regulate the linkage mechanism of microbial metabolic network and host physiological function, achieving multi-dimensional optimization of water quality regulation, nutrient utilization and disease resistance. At the same time, SBOS and FOS have a superposition effect. The results show that 2.5% SBOS and 2.5% FOS together as the carbon source of the BFT system can enrich Nakamurella in the water environment and intestinal tract, effectively improving the growth and immune performance of Macrobrachium rosenbergii. Compared with traditional methods, this scheme has unique advantages in reducing environmental load and improving breeding efficiency, providing a theoretical basis and technical support for the green transformation of aquaculture industry, and has broad application potential.

[0089] Finally, it should be pointed out that the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit it. The basic principles and main features of the present application have been described above with specific embodiments, and some modifications or substitutions can be made on the basis of the present application, but these modifications or substitutions do not make the corresponding technical solutions deviate from the scope of the present application.

Claims

1. A method for constructing a Macrobrachium rosenbergii BFT culture system based on SBOS-FOS composite carbon source, characterized in that, The total proportion of SBOS and FOS in the composite carbon source is 2%-10%, and the balance is glucose; according to the feed protein content in the aquaculture water, the composite carbon source is added to the Macrobrachium rosenbergii aquaculture system at a C / N ratio of 10:1-20:

1.

2. The method of claim 1, wherein, The mass percentage of SBOS and FOS in the composite carbon source is 1:1, the total proportion is 5%, and the proportion of glucose is 95%.

3. The method of claim 1, wherein, The C / N ratio is 15:1, the carbon source is added 2 hours after daily feeding, and is uniformly sprayed into the aquaculture water after dissolution.

4. The method according to any one of claims 1 to 3, characterized in that, By regulating the composition ratio and adding strategy of the composite carbon source, the water quality is improved, the water microbial community structure is optimized, and the immune gene expression and growth performance of Macrobrachium rosenbergii are simultaneously improved.

5. The method of claim 4, wherein, The optimized water microbial community structure is specifically to promote the enrichment of Actinobacteria and functional bacteria Nakamurella in the water, and inhibit the proliferation of pathogenic bacteria.

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