Application of Klebsormidum sp. GXU-A8 as aquatic animal feed additive

Abstract

The invention provides an application of Klebsormidum sp.GXU-A8 as an aquatic animal feed additive, and belongs to the technical field of aquatic animal feeds. According to the invention, the krill GXU-A8 is continuously cultured for 10 days under the condition of 0.75 g / L nitrogen, so that the krill cells are obtained. By adding 5% of GXU-A8 and 10% of GXU-A8 into the feed, the FBW, WGR and SGR of the tilapia can be remarkably improved, and the levels of VSI and serum ALT, AST, TG, CHO and LDL are reduced; the GSH-PX activity of the nile tilapia liver is improved, the MDA content is reduced, the liver is protected, the digestion function is optimized, and the enteric microorganism richness is increased. The GXU-A8 synergistically improves the health level of tilapia mossambica by regulating and controlling lipid metabolism and immune regulation and control pathways of nile tilapia mossambica, relieving oxidative stress, enhancing iron metabolism and antibacterial immunity, optimizing lipid metabolism and enhancing intestinal barrier, nutrient absorption and immunocompetence.

Description

Technical Field

[0001] This invention belongs to the field of aquatic animal feed technology, and particularly relates to a... Klebsormidium sp . Application of GXU-A8 as a feed additive for aquatic animals. Background Technology

[0002] genus Kerria ( Klebsormidium Kerneliae, a filamentous microalga belonging to the phylum Streptophyta, is mostly aerial. Its algal cells are cylindrical, forming single-chain, unbranched filamentous structures. Studies have found that Kerneliae is rich in anti-inflammatory small molecule compounds and can synthesize high-value-added metabolites such as palmitic acid, linoleic acid, and linolenic acid, which can significantly promote the growth, antioxidant, and anti-inflammatory capabilities of Litopenaeus vannamei. Furthermore, Kerneliae has a high growth rate and unique nutritional composition and metabolites, making it necessary to evaluate its potential as an aquatic feed additive. However, current research on Kerneliae is limited; for example, studies on its effects on fish are not yet reported, and the effects of its various active ingredients need further exploration. Summary of the Invention

[0003] In view of this, the object of the present invention is to provide a Klebsormidium sp . Application of GXU-A8 as a feed additive for aquatic animals.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides Kerria. Klebsormidium sp . Application of GXU-A8 as a feed additive for aquatic animals, specifically the *Kreecia* algae. Klebsormidium sp . GXU-A8 was deposited at the China Center for Type Culture Collection on June 16, 2025, with accession number CCTCC NO: M 20251401.

[0005] This invention also provides Kerria. Klebsormidium sp . Application of GXU-A8 in improving the growth performance, antioxidant capacity, or digestive performance of aquatic animals, the aforementioned Kerria. Klebsormidium sp . GXU-A8 was deposited at the China Center for Type Culture Collection on June 16, 2025, with accession number CCTCC NO: M 20251401.

[0006] Preferably, the Kerneliae Klebsormidium sp . The addition amount of GXU-A8 in aquatic animal feed is 5-10%.

[0007] Preferably, the Kerneliae Klebsormidium sp . The initial nitrogen concentration for GXU-A8 culture was 0.25~3 g / L.

[0008] Preferably, the aquatic animal includes Nile tilapia.

[0009] Compared with the prior art, the present invention has the following beneficial effects: The biomass of *Ceratophyllum demersum* GXU-A8, as described in this invention, reached 4.85 g / L after 8 days of cultivation under 0.75 g / L nitrogen conditions, significantly higher than other nitrogen concentration groups. Furthermore, continuous cultivation of GXU-A8 in batches of 10 days yielded approximately 150 g of microalgae per batch. This process was repeated to obtain algal cells for feed production.

[0010] Adding 5% and 10% GXU-A8 to the feed of this invention significantly improved FBW, WGR, and SGR in tilapia, while reducing VSI and serum ALT, AST, TG, CHO, and LDL levels. The 5% GXU-A8 group showed significantly increased GSH-PX activity and significantly decreased MDA content in the liver of Nile tilapia, and microscopic morphological observation revealed that 5% GXU-A8 exhibited better liver protection. The 5% addition group also showed significantly increased LIP, AMY, and PEP activities, which, along with increased intestinal villus length, confirm that GXU-A8 optimizes digestive function. Furthermore, with increasing GXU-A8 addition, intestinal microbial richness increased, regulating intestinal flora structure and increasing Firmicutes abundance while decreasing Fusobacterium abundance.

[0011] This invention utilizes GXU-A8 to primarily regulate lipid metabolism, immune regulation, and signal transduction pathways in Nile tilapia. It reduces oxidative stress by downregulating hepatic IGF1R, IGF2R, C3, FAS, and CAT genes, and upregulates the hamp gene to enhance iron metabolism and antibacterial immunity. Simultaneously, GXU-A8 upregulates intestinal lipid metabolism genes (LCLAT1 and APOB), digestive enzyme genes (CTRL, CELA2A, Prss2), and immune-related genes (IGHV4-28, TLR5, sptssa), optimizing lipid metabolism, strengthening the intestinal barrier, nutrient absorption, and immune capacity, synergistically improving the health of tilapia. The 5% addition group showed more significant regulatory effects on gene expression and gut microbiota structure, while the 10% addition group demonstrated greater advantages in enhancing microbial diversity. Attached Figure Description

[0012] Figure 1These are morphological images of GXU-A8 in different states (where A is the cell morphology of microalgae, B is GXU-A8 cultured on a large scale in a flat plate photobioreactor, C is GXU-A8 algae after filtration, and D is GXU-A8 algal powder after drying). Figure 2 It is a phylogenetic tree based on ITS sequences; Figure 3 It shows the biomass accumulation of GXU-A8 under different initial nitrogen concentrations; Figure 4 The effect of adding GXU-A8 to the feed on the production performance of Nile tilapia (where A is the final body weight, B is the weight gain rate, C is the specific growth rate, and D is the visceral body index). Figure 5 The effect of adding GXU-A8 to the feed on the physicochemical parameters of Nile tilapia plasma (where A is ALT, B is TG, C is HDL, D is AST, E is CHO, and F is LDL). Figure 6 The effect of adding GXU-A8 to the feed on the activity of antioxidant enzymes in the plasma of Nile tilapia (where A is SOD, B is MDA, and C is GSH-PX). Figure 7 The effect of adding GXU-A8 to the feed on the antioxidant index of Nile tilapia liver (where A is SOD, B is MDA, C is GSH-PX, and D is T-AOC). Figure 8 This study investigated the effects of dietary supplementation with GXU-A8 on the intestinal and liver tissue structure of Nile tilapia (where A and D: control group (CK); B and E: supplemented with 5% GXU-A8 (K5); C and F: supplemented with 10% GXU-A8 (K10). Figures AC show intestinal morphology, and Figure DF shows liver morphology; scale bar: liver 100µm, intestine 200µm). Figure 9 The gut microbiota of Nile tilapia fed with diets containing 0%, 5%, and 10% GXU-A8 for 8 weeks are shown in the following figures: (A is a Venn diagram based on the Nile tilapia gut microbiota profile; B is based on Bray diversity (PCoA) analysis; C is the community composition and relative abundance of the top 10 bacteria at the phylum level on the 16S rDNA sequence; and D is the community composition and relative abundance of the top 10 bacteria at the 16S rDNA sequence sequencing level). Figure 10 This is a gut microbiota function prediction analysis and interaction network (where A is the relative abundance stacking plot of the top 10 pathways for predicting gut microbiota function in Nile tilapia, B is the gut microbiota function prediction heatmap analysis based on the top 20 KEGG pathways, C is the gut microbiota correlation network analysis at the order level, and D is the positive and negative correlation statistics in the correlation network). Figure 11 It is a multi-group differential scatter plot (where A is the differential scatter plot of the intestinal transcriptome and B is the differential scatter plot of the liver transcriptome). Figure 12 This is a bubble map of GO enrichment in the gut of tilapia (where A is the K5 vs CK group, B is the K10 vs CK group, and C is the K5 vs K10 group). Figure 13 This is a bubble map of GO enrichment in tilapia livers (where A is the K5 vs CK group, B is the K10 vs CK group, and C is the K5 vs K10 group). Figure 14 This is a secondary bar chart of GO enrichment classification in the gut of tilapia; Figure 15 This is a secondary bar chart of differential GO enrichment classification in tilapia livers; Figure 16 This is a statistical chart of differential KEGG enrichment numbers in the intestines of tilapia; Figure 17 This is a statistical chart of differential KEGG enrichment numbers in tilapia livers; Figure 18 This is a trend analysis of differentially expressed genes in the liver of tilapia; Figure 19 This is a heatmap of gene expression in tilapia livers; Figure 20 This is a trend analysis of differential gene expression in the gut of tilapia; Figure 21 This is a heatmap of gene expression in the gut of tilapia; Figure 22 It is the correlation between tilapia gut microbiota and growth parameters; Figure 23 It is a correlation between tilapia gut microbiota and digestive enzyme activity; Figure 24 It is a correlation between tilapia gut microbiota and liver genes; Figure 25 It is a correlation between tilapia gut microbiota and liver immune genes; Figure 26 It is the correlation between tilapia gut microbiota and gut genes; Figure 27 It is a correlation between tilapia gut microbiota and gut fat metabolism genes.

[0013] Biological Preservation Instructions

[0014] The Kernel Algae provided by this invention Klebsormidium sp .GXU-A8 is deposited at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M 20251401, deposited on June 16, 2025, at Wuhan University, Wuhan, China. Detailed Implementation

[0015] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0016] Example 1

[0017] Isolation, purification, culture and identification of microalgae

[0018] I. Purification and Cultivation of Microalgae

[0019] The GXU-A8 used in this experiment was obtained from wild dragon blood trees in Longzhou area of ​​Guangxi ( Dracaena It was isolated from filamentous algae growing on the surface of tree bark (sp.); specifically, the following steps were included: scraping algae from the surface of tree bark under aseptic conditions, inoculating them into BG-11 liquid medium, and incubating at 25°C and a light intensity of 100 μmol·L⁻¹. ²· ¹, Cultured for 3-5 days under 12 h light / 12 h dark conditions; after the culture medium turns uniformly green, single filamentous algae are picked using capillary microscopy and transferred to fresh BG-11 medium for further culture; subsequently, the culture is repeatedly transferred to BG-11 medium containing 100 mg / L ampicillin to remove associated bacteria and protozoa; after continuous subculturing and microscopic examination to confirm the absence of contamination by bacteria and other algae, a pure culture is obtained and named GXU-A8.

[0020] The culture medium used was a modified version of BG-11 freshwater algae medium. The medium formulation was as follows: per liter of BG-11 medium, add 75 mg MgSO4·7H2O, 36 mg CaCl2·2H2O, 20 mg Na2CO3, 40 mg K2HPO4, 3.15 mg FeCl3·2H2O, 4.36 mg Na2EDTA·2H2O, 6.0 mg citric acid, and 1 mL A5mix (A5mix formulation: 2.86 g H3BO3, 1.81 g MnCl2·4H2O, 222 mg ZnSO4·7H2O, 79 mg CuSO4·5H2O, 0.39 g NaMoO4·2H2O, 49.4 mg Co(NO3)2·6H2O, and 1 mL 98% H2SO4. Finally, add water to a final volume of 1 L).

[0021] II. Observation of microalgal morphology using an optical microscope

[0022] After preparing the microalgae culture slide, place a drop of cedarwood oil on the coverslip and observe the microalgae cell morphology using an oil immersion microscope.

[0023] Experimental results: such as Figure 1 As shown. GXU-A8 cells are filamentous, unbranched, and cylindrical in shape with blunt ends. The cells are arranged in a single row of short chains and contain a large number of green chloroplasts arranged along the long axis. The dark green chloroplasts are concentrated at the edge of the cell wall. The cell wall is thin and uniform. The cells are plump and the surface is slightly rough, showing texture and wrinkles. They are covered with a thin gelatinous sheath or outer membrane. The cell diameter is about 3-5 μm.

[0024] III. Second-generation sequencing and raw data filtering

[0025] The obtained microalgal cells were flash-frozen in liquid nitrogen and stored at -80°C for later use. Total DNA was extracted from the microalgae using the CTAB method, and after passing quality testing, it was sent to BGI Genomics Co., Ltd. in Shenzhen for whole-genome sequencing on the Illumina HiSeq 4000 platform.

[0026] To ensure the reliability of the data used in subsequent analyses, the raw sequencing data was filtered using FastQC software. Reads with adapter contamination exceeding 5 bp, reads containing more than 5% N, and reads with low-quality bases (Q≤19) exceeding 20% ​​of the total length were removed sequentially. This yielded 4.48 Gb of high-quality clean reads.

[0027] IV. Assembly of the complete microalgal ribosome sequence and phylogenetic analysis of the ITS sequence

[0028] Using the GetOrganelle software, the whole ribosomal RNA sequence was assembled de novo from the filtered data, with references to the embplant_nr and fungus_nr databases (using the get_organelle_from_reads.py workflow, setting parameters -R 10; -k 21,65,105). After manual inspection, the whole ribosomal genome sequence was obtained, and then the ITS sequence of the ribosomes was extracted using the ITSX software (https: / / github.com / ncbi / ITSx).

[0029] ITS sequences were aligned online using NCBI BLAST, and phylogenetic trees were constructed from ITS sequences of closely related species downloaded based on the alignment results. MAGE 11 software was used for ITS multiple sequence alignment, and the optimal model was predicted using maximum likelihood (ML) with a site coverage limit set to 85% and other parameters default. Based on BIC scores, the ML phylogenetic tree was constructed using the predicted optimal model and two base substitution ratios (R, transition / transversion ratio). The bootstrap test for each branch was set to 1,000, the No. of Discrete Gamma Categories was set to 5, and the Partial Deletion method was selected for Gaps / Missing processing with a site coverage limit set to 85%. The Branch Swap Filter was set to a moderate filtering intensity, and other parameters were default.

[0030] Experimental results: The complete ribosome genome size of GXU-A8 was 13,419 bp, with a GC content of 52.52%, and all structures included SSU, ITS1, 5.8S, ITS2, and LSU sequences. The ITS sequence extracted from microalgal ribosomes was 717 bp in length, with a GC content of 60.11%.

[0031] A phylogenetic tree was constructed based on the complete ITS sequence (as shown in SEQ ID NO:1, specifically). Figure 2 As shown, GXU-A8 and Kreliidae ( Klebsormidiophyceae ) species clustered together on one branch, and with Klebsormidium sp.K43 and Klebs ormidium sp. strain CCALA 1187 is the closest relative, with 100% similarity. It has been preliminarily identified as an undetermined species of the genus *Ceratophyllum* in the class *Ceratophyllum* of the phylum Chlorophyta, and named *Ceratophyllum* GXU-A8. Klebsormidium sp.strain GXU-A8).

[0032] Example 2

[0033] Kree Klebsormidium Application of sp.strain GXU-A8

[0034] I. Algal strains and cultivation

[0035] 1. Growth optimization of Kerria GXU-A8 under different initial nitrogen concentrations

[0036] Kerria GXU-A8 was inoculated at four different initial nitrogen concentrations (0.25, 0.75, 1.5 and 3.0 g L). –1 The culture medium (containing NaNO3) was prepared in BG-11 medium, with the same formulation as in Example 1. The culture was carried out in a columnar photobioreactor measuring Ø4 cm × 60 cm, with an initial inoculum density (OD750) of 0.5 ± 0.01, a culture temperature of 25 ± 0.2 °C, and a light intensity of 300 µmol / m². 2 Every two days, algal samples were taken to determine microalgal biomass. The biomass was determined using the dry weight method: 10 mL of algal solution was filtered through a 0.45 μm fiber membrane, washed with deionized water, and dried at 80°C to constant weight. The weight was then weighed and converted to g·m⁻¹. ¹.

[0037] Experimental results: such as Figure 3 As shown. At a normal nitrogen concentration (1.5 g·L⁻¹) -1 Under culture conditions, the biomass of GXU-A8 continued to increase, reaching its peak at 4.68 g·L⁻¹ on day 10. -1 Subsequently, growth slowed and plateaued. Under low nitrogen (0.25 g·L⁻¹) conditions... -1 Under these conditions, the plant grew rapidly in the first 8 days, reaching a biomass of 3.29 g·L⁻¹. -1 Subsequently, growth plateaus. However, under high nitrogen (3 g·L⁻¹) conditions... -1 Under these conditions, its growth was actually inhibited, and its growth trend in the first 12 days was significantly lower than that of other nitrogen concentration groups. And at 0.75 g·L⁻¹ -1 Nitrogen concentration reached its peak on day 8, at 4.85 g·L⁻¹. -1 This indicates that at this initial nitrogen concentration, biomass can accumulate rapidly in a short period of time.

[0038] 2. Large-scale cultivation of Kerria griseus GXU-A8

[0039] The results showed that at an initial nitrogen concentration of 0.75 g·L⁻¹, -1After 8 days of cultivation under specific conditions, the algal cell biomass reached its peak. Based on this, large-scale cultivation was carried out using a rectangular flat-plate photobioreactor with dimensions of 110 cm (length) × 120 cm (height) × 6 cm (width), an operating volume of 60 L, an initial inoculum density (OD750) of 0.5 ± 0.01, and a cultivation temperature controlled at 25 ± 0.2℃. After 10 days of cultivation, approximately 50 L of algal culture was harvested and filtered to obtain algal cells. The remaining 10 L of algal culture was then added to a final volume of 60 L of fresh BG11 medium, and cultivation continued for another 10 days, followed by filtration to obtain algal cells. This continuous cultivation process was repeated to obtain algal cells, which were then dried and pulverized to obtain Kerria biomass, which was used as a feed additive for later use.

[0040] Experimental results: such as Figure 1 As shown. This algal strain has a single-chain, unbranched filamentous structure, and the algal cells are cylindrical with blunt, rounded ends. GXU-A8 was cultured in a rectangular flat-plate photobioreactor with an operating volume of 60 L. After 10 days of culture, approximately 50 L of algal solution was harvested, filtered to obtain 156 g of algal biomass, and the remaining 10 L of algal solution was added to fresh BG11 medium to a final volume of 60 L and cultured for another 10 days. The biomass was then obtained by filtration. Two batches were cultured using the above method, and the algal cells were filtered and dried for later use.

[0041] II. Farming and Physiological Indicator Measurement of Nile Tilapia

[0042] This aquaculture experiment included three different treatment groups: CK group (control), K5 group (supplemented with 5% GXU-A8 Kernel), and K10 group (supplemented with 10% GXU-A8 Kernel). The source of the experimental fish, feeding experiments, growth performance, plasma immune parameters, antioxidant capacity, histological observation, gut microbiota, feed formulation, and data statistics and analysis methods were based on patent CN116218679A - A strain of *Hygrophytes spp.* and its cultivation method and the prepared functional feed.

[0043] Experimental results: such as Figure 4 As shown. After eight weeks of rearing, the tilapia fed with GXU-A8, K5, and K10 showed significantly higher FBW, WGR, and SGR than the CK group. P <0.01), VSI was significantly lower than that of the CK group ( P The difference was <0.01), but there was no significant difference between the K5 and K10 treatment groups. The results indicate that adding GXU-A8 to the feed is beneficial to the growth of tilapia and increases the growth rate, and there is no significant difference in effect between the two addition amounts.

[0044] like Figure 5 As shown, after eight weeks of feeding, ALT, AST, CHO, LDL, and TG levels in groups K5 and K10 were significantly reduced. P<0.05), but there was no difference between the K5 and K10 groups. The HDL level in the K5 group was significantly lower than that in the CK group ( P The difference was <0.01%, and there was no difference compared to the K10 group. This indicates that adding 5% and 10% GXU-A8 to the feed can improve the plasma biochemical parameters of tilapia.

[0045] like Figure 6 As shown, in terms of plasma antioxidant enzyme activity, there was no significant difference in plasma SOD activity between the K5 and K10 groups compared to the CK group; however, MDA levels in both the K5 and K10 groups were significantly reduced. P <0.01); plasma GSH-PX in the K5 group was significantly higher than that in the CK group ( P <0.05), but there was no significant difference between the CK and K10 groups. The results showed that adding GXU-A8 to the diet did not affect the SOD activity in the plasma of the experimental fish, but adding 5% GXU-A8 could significantly reduce the MDA content in the plasma of the experimental fish, while increasing GSH-PX activity, which was consistent with the results of liver antioxidant capacity.

[0046] Further analysis was conducted on the activity of antioxidant enzymes and the tissue structure of the liver. For example... Figure 7 As shown, regarding liver antioxidant enzyme activity, there was no difference in SOD activity between the K5 and K10 groups compared to the CK group, but the SOD activity in the K5 group was significantly higher than that in the K10 group. P <0.05. The MDA content in both K5 and K10 groups was significantly reduced ( P <0.01); GSH-PX activity in group K5 was significantly higher than that in groups CK and K10 ( P <0.01); there was no significant difference in T-AOC activity among the groups. The results indicate that adding 5% GXU-A8 to the feed can increase liver SOD activity, reduce MDA content, and increase GSH-PX activity, thereby improving liver health.

[0047] After eight weeks of rearing, the experimental fish in the CK group showed obvious lesions in their liver tissue structure, characterized by a large amount of lipid deposition. Figure 8 In contrast, no obvious inflammatory cells were observed in the livers of the K5 and K10 groups; the cell structures were clear, with fewer lipid vacuoles and smaller lipid droplets, indicating a healthier state. Figure 8 (B and C in the text). This indicates that adding 5% and 10% GXU-A8 to the feed can significantly promote the health of fish livers.

[0048] III. Determination of Intestinal Digestive Capacity in Nile Tilapia

[0049] Intestinal tissue samples were rinsed thoroughly with pre-cooled physiological saline. The tissue was then minced and homogenized on ice to obtain a 10% intestinal tissue homogenate. The homogenate was centrifuged at 4°C (3000 rpm, 10 min), and the supernatant was used as the enzyme solution. The activities of intestinal lipase (LIP), amylase (AMY), pepsin (PEP), and trypsin (TRY) in the enzyme solution were measured using kits from the Nanjing Jiancheng Biotechnology Institute, China.

[0050] Regarding intestinal morphology, as shown in Table 1, the length of villi on the intestinal wall was significantly increased in both the K5 and K10 groups compared to the CK group. P <0.01). The mean villus heights of the CK, K5, and K10 groups were 232.08±9.85μm, 360.11±19.02μm, and 396.68±6.15μm, respectively, but there was no significant difference between the K5 and K10 groups.

[0051] Table 1. Effects of GXU-A8 supplementation in feed on intestinal villus height and muscle layer thickness in Nile tilapia.

[0052] Note: Completely different superscript letters in the same column indicate significant differences. P <0.05).

[0053] The study measured the activity of digestive enzymes in the fish intestines, as shown in Table 2. It was found that the LIP activity in the K5 group was higher than that in the CK group (…). P <0.05%. Compared with the CK group, the AMY and PEP activities of K5 and K10 were significantly increased ( P <0.05%, TRY activity was significantly reduced ( P <0.05). The results showed that adding 5% GXU-A8 to the feed could increase the activities of LIP, AMY and PEP in the experimental fish, reduce TRY activity, and promote intestinal digestion and absorption.

[0054] Table 2. Effects of GXU-A8 supplementation on digestive enzyme activity in Nile tilapia.

[0055] Note: Completely different superscript letters in the same column indicate significant differences. P <0.05).

[0056] IV. Transcriptome Sequencing of Nile Tilapia Liver and Intestine

[0057] Liver and intestinal tissues were rapidly frozen in liquid nitrogen and then stored at -80°C to prevent RNA degradation. 50 mg of tissue was placed in a pre-chilled RNase-free centrifuge tube, 1 mL of TRIzol reagent was added, and the tissue was homogenized on ice until completely broken down. After homogenization, the homogenate was allowed to stand for 5 min, chloroform was added, and the RNA was separated by centrifugation (12000 rpm, 15 min, 4°C). 500 µL of the supernatant was transferred to an RNase-free centrifuge tube, 500 µL of isopropanol was added, and the mixture was allowed to stand for 10 min, then centrifuged to remove the supernatant (12000 rpm, 10 min, 4°C). 1 mL of 75% ethanol was added to the precipitate to wash the RNA precipitate, the supernatant was removed, and 50 µL of RNase-free water was added to dissolve the RNA precipitate (10000 rpm, 3 min, 4°C). RNA purity was determined using a spectrophotometer (such as NanoDrop) (OD260 / OD280>2.0, OD260 / OD230>1.8).

[0058] The reagents required for reverse transcription of cDNA are as follows; Table 3. cDNA reaction system (10 μL)

[0059] The reaction system was then incubated in a metal bath (50℃) for 30 min, followed by incubation at 85℃ for 5 min. cDNA was diluted to 1 μg / μL and PCR amplification was performed, with each sample repeated three times. The qPCR kit used was Novizan's ChamQ UniversalSYBR qPCR Master Mix. Fragment size and concentration of the library were detected. High-throughput sequencing was performed using the Illumina platform (NovaSeq 6000). Sequencing data quality was checked using FastQC; low-quality sequences were filtered and aligned with a reference genome. Gene expression levels were quantified using Gffcompare (version: gffcompare-0.10.1), StringTie (version: stringtie-1.3.3b), and Ballgown (version: ballgown-2.4.3).

[0060] 1. Effects of Kerria GXU-A8 on the richness and diversity of gut microbiota in Nile tilapia

[0061] (1) The impact of the diversity index

[0062] Nine samples were filtered by OTUs < 97%, yielding a total of 1,069,776 sequences. The number of OTUs detected in each sample ranged from 766 to 944, totaling 7,632. CK, K5, and K10 contained 388, 427, and 448 OTUs respectively, with a common number of 236 OTUs. The remaining OTUs were unique to CK, K5, and K10, with 94, 96, and 125 OTUs respectively. Figure 9 (A) Group K5 had the highest number of OTUs, at 871. The results indicate that adding 5% GXU-A8 to the diet can improve the gut microbiota richness of Nile tilapia. Table 4 shows that the OTUs and Shannon index of the gut microbiota in groups K5 and K10 were higher than those in the CK group, while group K10 had the highest Shannon and Chao indices, at 5.39 and 490.60, respectively. Therefore, Nile tilapia fed with GXU-A8 exhibited the highest gut microbiota diversity and richness.

[0063] Table 4. Changes in the diversity index of Nile tilapia

[0064] Note: Completely different superscript letters in the same column indicate significant differences. P <0.05).

[0065] (2) Influence of gut microbiota composition

[0066] pass Figure 9 As shown in Figure B, groups K5 and K10 are relatively close. On the first principal coordinate (PCo1), the treatment group fed CK is farther away from the treatment groups fed K5 and K10, while the K5 and K10 groups are closer together. This indicates that there are differences in gut microbiota diversity between the fish fed GXU-A8 and CK groups. However, different amounts of GXU-A8 feed additive have little effect on the gut microbiota.

[0067] The top ten colony compositions and abundances at each level of species abundance are as follows: Figure 9 As shown in C), the dominant bacterial colony types were largely the same, but the abundance of the dominant bacterial species changed. At the phylum level, the dominant phylum in each group was Fusobacterium (…). Fusobacteria ), followed by Proteobacteria ( Proteobacteria ) and Firmicutes ( FirmicutesFusobacteria accounted for 65.41%, 56.02%, and 57.04% of the flora in CK, K5, and K10, respectively; Proteobacteria accounted for 11.55%, 9.94%, and 14.12% of the flora in CK, K5, and K10, respectively; and Firmicutes accounted for 6.60%, 15.42%, and 13.25% of the flora in CK, K5, and K10, respectively. Compared with the CK group, the K5 and K10 groups had a higher proportion of Firmicutes and a lower proportion of Fusobacteria and Proteobacteria. This indicates that the addition of GXU-A8 reduces the abundance of Fusobacteria and Proteobacteria in the tilapia gut, while increasing the colonization of Firmicutes.

[0068] Community composition and relative abundance of the top 10 bacteria at the microscopic level, as follows: Figure 9 As shown in D in the diagram. Among all groups, the dominant family is Fusobacteriaceae (…). Fusobacteriales ), followed by Clostridium ( Clostridiales ) and Verruciformes ( Verrucomicrobiales The proportion of Clostridium orders was higher in the CK group than in the K5 and K10 groups, while the proportions of Clostridium and Verruciformis orders were higher in the K5 and K10 groups than in the CK group.

[0069] In summary, the addition of GXU-A8 significantly altered the structure and abundance of the gut microbiota. At the phylum level, the abundance of Firmicutes significantly increased, while the abundance of Fusobacterium and Proteobacterium significantly decreased; at the order level, the abundance of Fusobacterium also significantly decreased, while the abundance of Clostridium and Verruciformes significantly increased. These results suggest that the addition of GXU-A8 can modulate the composition of the gut microbiota and may have a positive impact on the host's digestive and metabolic health.

[0070] (3) Functional prediction analysis and interaction network of gut microbiota

[0071] KEGG functional prediction of 16S rRNA sequences was performed using the Tax4Fun algorithm, and the abundance information of each pathway was calculated. The results showed significant differences among the groups for the 37 gene pathways predicted in the KEGG database. A stacked plot of the relative abundance of the top 10 pathways is shown below. Figure 10 In group A, the expression levels of metabolism-related pathways were dominant across all treatment groups. The three pathways with the highest expression levels were carbohydrate metabolism, membrane transport, and amino acid metabolism. The expression levels of the top 20 pathways in each treatment group are shown below. Figure 10As shown in B, 55% (11 pathways) are related to metabolism. Although the expression of metabolic pathways was basically the same across different experimental groups, their expression levels differed significantly. The expression levels of lipid metabolism, terpenoid and polyketide metabolism, and endocrine system pathways in the K5 and K10 groups were significantly lower than those in the CK group. In contrast, the expression levels of cell growth and death and cell motility pathways in the K5 and K10 groups were significantly higher than those in the CK group.

[0072] At the order level, species correlation analysis was performed. Circular nodes represent bacterial communities, size indicates correlation strength, green lines indicate positive relationships, and red lines indicate negative relationships. Figure 10 C in the middle). Proteobacteria ( Proteobacteria ) and Actinobacteria ( Actinobacteria The order containing the most orders is 25 and 12 respectively. Among them, the order Micrococcidales is positively correlated with Proteobacteria. Micrococcales ), Xanthomonadales ( Xanthomonadales ), Lellaellales ( Reyranellales ), Desulfovibrioles ( Desulfovibrionales Rickettsia ( Rickettsiales ) and Elsterales ( Elsterales The bacteria with a negative correlation include the SAR11 clade and the β-proteobacteria order ( ). Betaproteobacteriales ) and Alternating Monocystes ( Alteromonadales Bacteria positively correlated with the phylum Actinobacteria include the orders Micrococci and Pseudomonas (…). Pseudomonadales ) and Actinomycetes ( Actinomycetales Bacteria with a negative correlation include those in the order Propionibacterium ( Propionibacteriales ) and Rhodotorula ( Coriobacteriales The bacterial groups showed mainly positive correlations, while negative correlations were less common and mainly associated with Proteobacteria.

[0073] The gut microbiota correlation network at the target level identified 169 pairs of positive correlations and 34 pairs of negative correlations. Figure 10 (D in the text). Among them, Micrococcidales, Pseudomonasales, and Xanthomonasales contain 15, 14, and 13 pairs of significant positive correlations, respectively, and no significant negative correlations.

[0074] 2. Effects of Kerria GXU-A8 on the liver and intestinal transcriptome of Nile tilapia

[0075] (1) Transcriptome analysis

[0076] As shown in Table 5, a total of 134,495,518 raw reads were obtained from the intestinal samples. After preprocessing and removing low-quality sequences, 133,450,318 clean reads were obtained, accounting for 99.16%–99.28% of each sample. The clean reads were aligned to the tilapia genome Ensembl_release110 (https: / / ftp.ensembl.org / pub / release-110 / gtf / oreochromis_niloticus / ), detecting 26,852 known genes, with an alignment rate of 85.78%–86.79%.

[0077] A total of 144,064,928 raw reads were obtained from liver samples. After processing, 141,612,044 clean reads were obtained, accounting for 96.55%–99.45% of each sample. After comparing the clean reads, 26,852 known genes were detected, with an alignment rate of 88.04%–89.92%.

[0078] Table 5. Transcriptome sequencing data of Nile tilapia gut and liver

[0079] Note: CK, control group; K5, supplemented with 5% GXU-A8; K10, supplemented with 10% GXU-A8. G: intestinal transcriptome; L: liver transcriptome.

[0080] (2) Differentially expressed genes (DEGs) analysis

[0081] Using |log2(Fold change)|>1 and FDR<0.05 as thresholds, differentially expressed genes in the liver and intestines of K5 vs. CK, K10 vs. CK, and K5 vs. K10 were compared, and represented by multiple differential scatter plots (e.g.) Figure 11 (As shown in the figure), where orange represents the number of upregulated genes and blue represents the number of downregulated genes. The results showed that, in intestinal transcriptome expression, compared with the CK group, a total of 3064 differentially expressed genes were screened in the K5 group, of which 2650 genes were significantly upregulated and 414 genes were significantly downregulated; compared with the CK group, a total of 1792 differentially expressed genes were screened in the K10 group, of which 1046 genes were significantly upregulated and 746 genes were significantly downregulated; compared with the K10 group, a total of 2948 differentially expressed genes were screened in the K5 group, of which 2201 genes were significantly upregulated and 747 genes were significantly downregulated.

[0082] In liver transcriptome expression, compared with the CK group, 7084 differentially expressed genes were screened in the K5 group, of which 6770 genes were significantly upregulated and 314 genes were significantly downregulated; compared with the CK group, 6561 differentially expressed genes were screened in the K10 group, of which 6014 genes were significantly upregulated and 547 genes were significantly downregulated; compared with the K10 group, 2079 differentially expressed genes were screened in the K5 group, of which 1466 genes were significantly upregulated and 613 genes were significantly downregulated. In summary, the positive feedback regulation of intestinal and liver gene expression was more pronounced in the K5 group, and its effect on tilapia gene expression was more significant, indicating that 5% GXU-A8 supplementation has a stronger effect on the regulation of tilapia gene expression. Furthermore, there were significant differences in the effects of 5% and 10% GXU-A8 on tilapia intestinal and liver gene expression, indicating that the amount of GXU-A8 supplementation has a dose-dependent effect on the regulation of tilapia gene expression.

[0083] (3) Study on differential gene function

[0084] GO and KEGG enrichment analyses were performed on differentially expressed genes (DEGs) in the gut and liver of tilapia. The top 20 significantly enriched GO terms and KEGG pathways were selected based on FDR values, and bubble charts were generated (e.g., ...). Figure 12 and Figure 13 (As shown). The size of the bubble indicates the number of differentially enriched genes in the pathway, and the color intensity represents the significance level of the enrichment.

[0085] In intestinal samples, differentially expressed genes between the K5 and CK groups were primarily enriched in the Group II intron splicing pathway; differentially expressed genes between the K10 and CK groups showed the most significant enrichment in the Group II intron splicing pathway, and also had the highest number of enriched genes in the catalytic activity pathway; differentially expressed genes between the K5 and K10 groups also showed the most significant enrichment in the Group II intron splicing pathway. Furthermore, GO classification secondary bar charts (such as...) Figure 14 and 15 Analysis (as shown) revealed that the differentially regulated genes between the K10 and CK groups, the K5 and CK groups, and the K5 and K10 groups were most abundant in pathways such as cellular process, metabolic process, binding, cellular anatomical entity, and catalytic activity.

[0086] In liver samples, differentially expressed genes between the K5 and CK groups primarily participated in the protein-binding pathway; differentially expressed genes between the K10 and CK groups were most significantly enriched in the cell activation pathway, and had the highest number of enriched genes in the intracellular anatomical structure pathway; differentially expressed genes between the K5 and K10 groups were most significantly enriched in the RNA processing pathway, and also had the highest number of enriched genes in the metabolic pathway. Furthermore, the upregulated and downregulated differentially expressed genes between the K10 and CK groups, the K5 and CK groups, and the K5 and K10 groups were significantly enriched in pathways involving cellular processes, binding, cellular anatomy, metabolism, biological regulation, and catalytic activity.

[0087] GO enrichment analysis of liver and intestinal samples revealed highly similar enrichment patterns across the three comparative groups. At the biological process level, the most significantly enriched GO terms were "cellular processes" and "metabolic processes"; at the molecular function level, the most significantly enriched categories were "binding" and "catalytic activity"; and at the cellular component level, the most significantly enriched category was "cellular anatomy." Further analysis showed that several GO terms in intestinal samples were associated with transcriptional regulation, while several GO terms in liver samples were associated with immune responses. For example, in the liver K5 and K10 groups, the expression levels of the *hamp* and *IGHV3-49* genes were significantly downregulated, while the expression level of the *VMO1* gene was significantly upregulated. Furthermore, in the intestines of tilapia supplemented with GXU-A8, the expression levels of the *Mrc2*, *gal*, and *IGHV3-33* genes were downregulated, while the expression levels of the *sptssa* and *IGHV4-28* genes were upregulated.

[0088] Furthermore, based on the KEGG database, differentially expressed genes in the gut and liver are annotated into five categories: Metabolism, Organismal Systems, Genetic Information Processing, Cellular Processes, and Environmental Information Processing.

[0089] like Figure 16 and Figure 17As shown in the figure. By analyzing the gene number distribution through bar charts at different classification levels, it was found that in intestinal samples, the differentially expressed genes in the K5-VS-CK and K10-VS-CK groups were mainly enriched in the signal transduction and immune system regulatory network pathways; while the differentially expressed genes in the K5-VS-K10 group were mainly enriched in the signal transduction and global and overview maps pathways.

[0090] In liver samples, differentially expressed genes in the K5-VS-CK and K10-VS-CK groups were mainly enriched in the signal transduction and immune system regulatory network pathways; differentially expressed genes in the K5-VS-K10 group were mainly enriched in the signal transduction and global and overview maps pathways.

[0091] 3. Expression patterns of Nile tilapia GXU-A8 in liver and gut-related genes

[0092] (1) Liver

[0093] like Figure 18 As shown in the figure. In-depth analysis of the differentially expressed gene trends in the liver revealed that the amount of GXU-A8 added significantly affected gene expression patterns, exhibiting a clear dose-response relationship. Specifically, in Profile 2 and Profile 5, the number of genes expressed in the K5 group was the lowest and highest, respectively; while in Profile 0 and Profile 7, gene expression levels decreased or increased with increasing GXU-A8 content. Analysis of differentially expressed genes in Profile 1, Profile 2, and Profile 0 revealed that significantly altered genes were mainly enriched in pathways related to immune response, metabolic regulation, and intercellular communication.

[0094] like Figure 19 As shown. Further analysis of liver gene expression among the groups revealed that, compared with the CK group, the GXU-A8-added group showed significantly downregulated expression levels of growth-related genes (IGF1R, IGF2R, igf2, IGFBP3), immune and inflammation-related genes (TLR1 / 2 / 5, C3, FAS), cell transport and metabolism-related genes (VAMP8, VAMP727, IGHV3-49), and antioxidant genes (CAT), while the expression levels of genes involved in immune responses (hamp, NIrc3) were significantly upregulated. P<0.05).

[0095] (2) Intestines

[0096] like Figure 20 As shown in the figure, analysis of differentially expressed gene trends in the gut revealed that the amount of GXU-A8 added significantly affected gene expression patterns, exhibiting a clear dose-response relationship. The differentially expressed genes in Profile 2 and Profile 5 were the lowest and highest in the K5 group, respectively; while in Profile 0 and Profile 7, gene expression levels decreased or increased with increasing GXU-A8 content. Analysis of differentially expressed genes in Profile 2, Profile 1, and Profile 4 revealed that significantly altered genes were mainly enriched in pathways related to immune response, metabolic regulation, maintaining cell stability, and regulatory nodes.

[0097] like Figure 21 As shown. Among lipid metabolism-related genes, LPL expression in both the K5 and K10 groups was significantly lower than that in the CK group (as shown). P <0.05), while LCLAT1 and APOB expression were significantly increased in the K5 group ( P <0.05. Digestive enzyme genes (CTRL, CELA2A, Prss2) were significantly upregulated in the K10 group. P <0.05. In the GXU-A8 group, the expression of Sptssa, which regulates membrane structure, was significantly enhanced. Immune-related genes (IGHV4-28, C3, TLR5) and growth-regulating genes (IGF1R, GRB10) were significantly upregulated in the K10 group, while the expression of antioxidant genes (cat, VAMP727) was significantly increased in the K5 group. P <0.05).

[0098] 4. Correlation analysis of gut microbiota

[0099] (1) Correlation between gut microbiota and growth phenotype

[0100] like Figure 22 As shown in the figure. Correlation analysis was performed based on gut microbiota and tilapia growth phenotypes. The red line represents a highly significant correlation between gut microbiota and related tilapia indicators. P <0.01, the green line represents a significant correlation ( P <0.05), the thicker the line, the stronger the correlation; blue indicates a negative correlation between gut microbiota, and red indicates a positive correlation. It was found that the body length, body width, WGR, SGR, VSI, and HSI of Nile tilapia were all correlated with those of *Cetobacter* spp. (…). Cetobacterium ), genus Rombutz ( Romboutsia ), genus *Pseudomonas* Plesiomonas Clostridium genus 1 ( Clostridium_sensu_stricto_1 ) and Burkholderia ( Bosea ) are positively correlated ( P <0.01). Furthermore, *Bacteroides* genus ( Macellibacteroides ) is significantly positively correlated with VSI ( P <0.01).

[0101] (2) Correlation analysis between gut microbiota and digestive enzyme activity

[0102] like Figure 23 As shown. Correlation analysis revealed that LPL enzyme activity was correlated with that of *Rhomboidella* spp. (…). Romboutsia ), Akkermania ( Akkermansia ) and Mycobacterium showed a significant positive correlation ( P <0.01). AMY enzyme activity and Cetacea spp. ( Cetobacterium ), Bacteroides genus ( Macellibacteroides Clostridium genus 1 ( Clostridium sensu_stricto_1 ), genus *Pseudomonas* Plesiomonas ) and Burkholderia ( Bosea ) showed a significant positive correlation ( P <0.01]. PEP enzyme activity and Cetacea ( Cetobacterium ), genus Rombutz ( Romboutsia Clostridium genus 1 ( Clostridium_sensu_stricto_1 ), genus *Pseudomonas* Plesiomonas Burkholderia ( ) Bosea ) showed a significant positive correlation ( P <0.01).

[0103] (3) Association analysis between gut microbiota and liver-related genes

[0104] like Figure 24 and Figure 25 As shown. Based on liver trend analysis, differentially expressed genes were extracted from Profile 1, Profile 2, and Profile 0 for association analysis. The results showed that gut microbiota was highly correlated with genes such as ENSONIG00000039512 (hamp), MSTRG.20984 (Nlrc3), ENSONIG00000012865 (cat), ENSONIG00000015757 (IGF2R), ENSONIG00000027234 (VAMP8), ENSONIG00000015389 (VAMP727), and ENSONIG00000039552 (igf2). Further analysis selected highly correlated immune genes (|Pearson r|>0.8 or...). PSubsequent functional analysis was performed on samples with a value <0.05, and the results showed that the liver immune genes IGF1R, IGF2R, VAMP8, and VAMP727 were all associated with *Citrus aurantium* (Citrus spp.). Aurantimicrobium Highly significant correlation ( P <0.01); both IGF2 and IGFBP3 immune genes are associated with Clostridium spp. 1 ( Clostridium_sensu_stricto_1 ), genus *Pseudomonas* Plesiomonas Highly significant correlation ( P <0.01).

[0105] (4) Association analysis between gut microbial diversity and gut-related genes

[0106] like Figure 26 and 27 As shown. Differentially correlated genes extracted from Profile 1, Profile 2, and Profile 0 based on liver trend analysis were subjected to association analysis. The results showed that gut microbiota were associated with ENSONIG00000006129 (APOB), ENSONIG00000015389 (VAMP727), ENSONIG00000002741 (CTRL), ENSONIG00000011424 (Prss2), and ENSONIG00000002140 (CELA2A). Furthermore, highly correlated lipid metabolism genes (|Pearson r|>0.8 or...) were selected. P Subsequent functional analysis of samples <0.05 revealed that the intestinal lipid metabolism genes LPL and LCLAT1 are associated with Akkermansia spp. ( Akkermansia Highly significant correlation ( P <0.01).

[0107] This invention introduces GXU-A8, rich in polyunsaturated fatty acids, into the feed of aquatic animals to promote their growth and digestion. GXU-A8 is rich in metabolites such as L-phenylalanine, L-tyrosine, L-methionine, maltotriose, citric acid, and D-malic acid, which can promote tilapia growth by directly participating in protein synthesis or indirectly enhancing intestinal nutrient absorption. Kernel algae are rich in linolenic acid and linoleic acid; therefore, feeding GXU-A8 can reduce ALT, AST, and TG levels, improve the health of tilapia, and reduce the possibility of liver disease. GXU-A8 can enhance tilapia plasma GSH-PX activity because the ortholeucine and phenylalanine in GXU-A8 can improve oxidative stress and enhance antioxidant activity. GXU-A8 can significantly enhance the antioxidant capacity of tilapia liver; this effect stems from the synergistic effect of multiple active ingredients in Kernel algae: lutein exerts its antioxidant function by directly scavenging free radicals; the fatty acid system composed of palmitic acid, linolenic acid, and linoleic acid jointly regulates oxidative stress responses; and phenylalanine has antioxidant activity. β-D-ethylglucuronide in GXU-A8 is a glucuronic acid conjugate that helps aquatic animals excrete toxins from the liver, improving immunity and providing better liver protection. Since *Kreecia* contains up to 59% lipids, and linoleic acid is its main fatty acid component, the fish intestine's digestibility of polyunsaturated fatty acids is superior to that of saturated fatty acids. Therefore, after feeding GXU-A8, the LIP, AMY, and PEP activities of Nile tilapia were significantly higher than the control group, improving the fish's digestive and absorptive capacity. GXU-A8 can significantly increase the diversity of the tilapia gut microbiota and reduce the abundance of potentially pathogenic intestinal phyla such as Fusobacterium and Proteobacterium. Furthermore, the abundance of Firmicutes increases significantly with increasing GXU-A8 addition, which may be related to the lipid-rich nature of GXU-A8; increased Firmicutes abundance under high-fat diet conditions promotes nutrient absorption. GXU-A8 affects host growth and immunity by regulating cell growth and death and cell motility, improving intestinal lipid accumulation and reducing oxidative damage.

[0108] GXU-A8 is rich in ethyl glucuronide. Adding GXU-A8 to feed can regulate the activity of tilapia liver cells, enhance immune defense and endocrine regulation, promote tissue repair, metabolic waste removal and environmental adaptation, improve the metabolic efficiency of tilapia, enhance stress adaptability and maintain normal liver physiological function, thereby improving the health status and farming performance of tilapia.

[0109] GXU-A8 enhances the intestinal barrier function and immune defense capabilities of tilapia by synergistically regulating the expression of immune-related genes through multiple pathways. GXU-A8's regulation of immune-related genes exhibits multi-target and multi-pathway characteristics: it regulates innate immune responses through the C3 gene, influences adaptive immune responses through IGHV family genes, and participates in cell signal transduction via the sptssa-mediated sphingolipid metabolism pathway.

[0110] Specific gut microbiota are significantly associated with key physiological functions in tilapia and may influence host health and growth performance through synergistic or competitive interactions among microbes. GXU-A8 affects fat deposition and energy utilization efficiency in tilapia by regulating the expression of lipid metabolism-related genes.

[0111] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Kernel Klebsormidium sp . The application of GXU-A8 as a feed additive for aquatic animals is characterized by... The Kernel Klebsormidium sp . GXU-A8 was deposited at the China Center for Type Culture Collection on June 16, 2025, with accession number CCTCC NO: M 20251401.

2. Kernel Klebsormidium sp . The application of GXU-A8 in improving the growth performance, antioxidant capacity, or digestive performance of aquatic animals is characterized by... The Kernel Klebsormidium sp . GXU-A8 was deposited at the China Center for Type Culture Collection on June 16, 2025, with accession number CCTCC NO: M 20251401.

3. The application according to claim 1 or 2, characterized in that, The Kernel Klebsormidium sp . The addition amount of GXU-A8 in aquatic animal feed is 5-10%.

4. The application according to claim 1 or 2, characterized in that, The Kernel Klebsormidium sp . The initial nitrogen concentration for GXU-A8 culture was 0.25~3 g / L.

5. The application according to claim 1 or 2, characterized in that, The aquatic animals mentioned include Nile tilapia.

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