A chitosan oligosaccharide prepared composite microecological preparation and application thereof

Abstract

The application provides a kind of composite microecological preparation prepared by chitosan oligosaccharide and application thereof, the microecological preparation is prepared from chitosan oligosaccharide and food sinus weiss, and the preservation number of the food sinus weiss is CGMCC NO.32640.Colony composition analysis shows that after adding the composite microecological preparation in feed, the intestinal tract of soft-shelled turtles is enriched with bacteria that are beneficial to nutrient conversion, and the balance of bacterial flora is maintained.The immune enzyme activity analysis results prove that adding the composite microecological preparation in feed can improve the T-AOC activity, CAT activity, GSH-Px and SOD activity in the body of soft-shelled turtles, reduce the ACP and MDA enzyme activity, and improve the antioxidant and antibacterial capacity of soft-shelled turtles.Meanwhile, adding the composite microecological preparation can improve the intestinal digestive enzyme activity and the amino acid content in the muscle of soft-shelled turtles, thereby promoting the growth of soft-shelled turtles.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture technology, specifically relating to a compound microecological preparation made from chitosan oligosaccharide and its application. Background Technology

[0002] Soft-shelled turtles, also known as water turtles, are oviparous reptiles that live both in water and on land. Their meat is delicious and nutritious, possessing properties that clear heat and nourish yin, calm the liver and extinguish wind, and soften and disperse lumps. They are not only a delicacy but also a widely used tonic and traditional Chinese medicine ingredient. Currently, in the artificial breeding of soft-shelled turtles, the provided nutrients cannot meet their growth needs, and the turtles' nutrient intake cannot satisfy their actual requirements. This leads to a long-term imbalance in their intestinal flora, weak immunity, low survival rate, and poor muscle quality in adult turtles.

[0003] Microecological preparations, composed of various beneficial bacteria, are non-toxic, have no side effects, and leave no drug residues. They are a green water quality improver and additive that can significantly improve the aquaculture environment, enhance the disease resistance of turtles, and improve their quality, and have been increasingly accepted by aquaculture farmers. However, in practical applications, due to differences in product characteristics and manufacturing processes, these biological preparations have shown unsatisfactory effects, poor stability, and significant variations in performance.

[0004] Patent document CN118160814A discloses a compound microecological preparation and its application in the farming of juvenile soft-shelled turtles. The compound microecological preparation includes multiple probiotics such as lactic acid bacteria, Aspergillus niger, Saccharomyces cerevisiae, and Bacillus subtilis, as well as extracts of traditional Chinese medicine, adjuvants, and nutrients. Through the synergistic effect of probiotics and extracts of traditional Chinese medicine, it effectively improves the degradation capacity of organic matter, the water quality of the farming water, and the feed utilization rate of juvenile soft-shelled turtles. However, research on regulating the intestinal flora of soft-shelled turtles and improving their survival rate is still lacking.

[0005] Therefore, providing a method that can effectively regulate the balance of gut microbiota in soft-shelled turtles, improve their immunity and survival rate, and promote the digestion and absorption of nutrients is of great significance to the soft-shelled turtle farming industry. Summary of the Invention

[0006] To address the technical problems existing in current turtle farming, this invention provides a compound microecological preparation made from chitosan oligosaccharide and its application. The compound microecological preparation is composed of prebiotics and probiotics. Adding this compound microecological preparation to the feed of turtles can not only effectively improve the digestive and absorptive capacity and immunity of turtles, but also improve the water quality in which turtles live.

[0007] On the one hand, the present invention provides a compound microecological preparation, including *Westernella taurida*, the preservation number of which is CGMCC NO.32640.

[0008] Increased aquaculture density exacerbates the risk of disease outbreaks. Common diseases in aquaculture include bacterial septicemia caused by pathogens such as Aeromonas hydrophila. Aeromonas hydrophila easily infects both aquatic animals and humans, posing a significant danger to the aquaculture industry. The overuse of drugs has polluted the growth environment of aquatic organisms and induced drug resistance in animals. Therefore, finding and developing drug alternatives is urgently needed to improve the resistance of aquatic animals to intestinal pathogens and to enhance the quality of the aquatic environment in which they grow.

[0009] Antagonism is one of the ways in which bacteria or other microorganisms interact. Utilizing strains with strong antagonistic properties to inhibit the growth, reproduction, or pathogenicity of pathogenic bacteria is a method of biocontrol. This invention utilizes the inhibitory effect on Aeromonas hydrophila to screen three probiotics—Weissella cibaria, Enterococcus faecalis, and Lactococcus lactis—from the intestines of soft-shelled turtles using the dilution plating method. Furthermore, the three probiotics were co-cultured with Aeromonas hydrophila using the Oxford cup method, and the results showed that Weissella cibaria exhibited the strongest inhibitory effect on Aeromonas hydrophila. Moreover, this invention demonstrates through experiments that, compared to other sources of Weissella cibaria, the compound microecological preparations or soft-shelled turtle feed made from Weissella cibaria obtained from the intestines of soft-shelled turtles significantly improve the antioxidant capacity of soft-shelled turtles and the quality of the water used for turtle farming.

[0010] The *Westernella esculenta* strain provided by this invention was screened from the intestines of soft-shelled turtles and deposited on November 15, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO.32640.

[0011] Subsequent analysis of the gut microbiota structure of turtles revealed that adding *Weissella zedoaria* alone to the feed increased the diversity of gut microbiota, with the gut enriched by the genera *Turicibacter*, *Pediococcus*, and *Weissella*. Feeding with a compound microecological preparation significantly improved the diversity of the gut microbiota structure, enriching the gut with the genera *Paeniclostridium* and *Cellulosilyticum*. These two genera promote protein decomposition and cellulose degradation, thus facilitating nutrient conversion.

[0012] Furthermore, the compound microecological preparation also includes any one or more prebiotics selected from fructooligosaccharides, chitosan oligosaccharides, and β-glucan.

[0013] Prebiotics are indigestible components in food or feed that selectively stimulate the growth of beneficial bacteria in the digestive tract of host animals, thereby producing beneficial effects. These include oligosaccharides, microalgae, and natural plant extracts. In recent years, prebiotics have shown disease prevention and treatment efficacy in aquaculture. Various types of prebiotics, including xylooligosaccharides and galactooligosaccharides, have been used as antibiotic alternatives to promote gut health and prevent disease by selectively promoting the growth of beneficial bacteria. Chitosan oligosaccharides are natural alkaline polymers of glucosamine. They are oligosaccharide products with a degree of polymerization between 2 and 20, and a molecular weight ≤3200 Da, obtained by degrading chitosan using special bioenzymatic technology. They are low molecular weight products with good water solubility, significant functional effects, and high biological activity. They possess many unique functions, such as high solubility not found in chitosan, complete water solubility, and easy absorption and utilization by organisms. Due to their beneficial functions in regulating aquatic animal immunity, promoting growth, and anti-oxidation responses, they are used as feed additives, vaccine adjuvants, and preservatives for aquatic products. Another prebiotic used in aquaculture as an immunomodulator and antioxidant is β-glucan. Adding β-glucan to the diet can improve the immunity and antioxidant levels of soft-shelled turtles. Meanwhile, another prebiotic, fructooligosaccharides (FOS), can also maintain intestinal flora balance and enhance the body's immunity. FOS, also known as oligofructose or sucrose oligosaccharides, are derived from sucrose (beets, sugarcane, etc.) and inulin (chicory, Jerusalem artichoke). FOS is a natural active substance with a sweetness 0.3 to 0.6 times that of sucrose. It retains the pure sweetness of sucrose but has a more refreshing sweetness. It is a novel sweetener with functions such as regulating intestinal flora, promoting bifidobacteria growth, and immunomodulation, and is hailed as the most promising new generation of additives since the antibiotic era.

[0014] This invention further compared the effects of feeding different prebiotics separately. α-diversity analysis showed that the COS group (chitosan oligosaccharide) had slightly higher α-diversity than the FOS group (fructooligosaccharide), but the difference was not significant. β-diversity analysis showed that the gut microbiota structure of the turtles differed significantly between the two groups after prebiotic feeding compared to the control. The COS group (chitosan oligosaccharide) had slightly lower β-diversity dispersion than the FOS group (fructooligosaccharide), indicating a stronger ability to regulate the microbiota structure. In addition, the content of Cellulosilyticum, Helicobacter, and Solibacillus was higher in the chitosan oligosaccharide group, indicating that adding chitosan oligosaccharide or fructooligosaccharide to the feed can alter the composition of the turtle gut microbiome and differentially enrich specific bacteria.

[0015] Furthermore, the prebiotic is chitosan oligosaccharide.

[0016] Based on the comparative results of feeding turtles with individual components, it can be seen that different prebiotics or probiotics have different effects on improving the antioxidant and antibacterial capabilities of turtles. Therefore, the promoting effects of compound microecological preparations made from different prebiotics or probiotics may also differ. This invention demonstrates through comparative experiments that the compound microecological preparation made from chitosan oligosaccharide and *Wesleyania stolonifera* significantly improved total antioxidant capacity (T-AOC), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activity, while reducing acid phosphatase (ACP) and malondialdehyde (MDA) levels. Other groups had no significant effect on these indicators, making the compound microecological preparation made from chitosan oligosaccharide and *Wesleyania stolonifera* the preferred choice.

[0017] Furthermore, the content of *Westernella esculenta* is 1 × 10⁻⁶. 7 ~1×10 9 CFU / g, with a chitosan oligosaccharide content of 0.1–0.5 wt%.

[0018] In some methods, the content of *Westernella esculenta* is 1 × 10⁻⁶. 7 ~1×10 8 CFU / g, wherein the chitosan oligosaccharide content is 0.1-0.3 wt%.

[0019] In some methods, the content of *Westernella esculenta* is 1 × 10⁻⁶. 8 CFU / g, wherein the content of chitosan oligosaccharide is 0.3wt%.

[0020] On the other hand, the present invention provides a turtle feed, comprising the compound microecological preparation described above.

[0021] The compound microecological preparation provided by this invention can be supplemented with appropriate ingredients, such as fish meal, rice bran, wheat bran, soybean cake, binder, additives, vitamins, inorganic salts, etc., as needed, without compromising the effectiveness of this invention. Various feeds or products with the same effect prepared based on this invention are all within the protection scope of this invention.

[0022] In another aspect, the present invention provides a method for raising soft-shelled turtles, using the feed described above.

[0023] In another aspect, the present invention provides the use of a composition for preparing a reagent for regulating the balance of intestinal flora in turtles, the composition comprising *Westernella esculenta* and chitosan oligosaccharides.

[0024] Studies have shown that the composition of intestinal flora varies after adding different prebiotics, probiotics, or compound microecological preparations. The colony diversity of the probiotic group is significantly higher than that of the prebiotic group. The compound microecological preparation group, which is made from Weissella tamariscina (probiotic) and chitosan oligosaccharide (prebiotic), is enriched with bacteria that are beneficial to the conversion of nutrients and maintains the balance of the flora.

[0025] In another aspect, the present invention provides the use of a composition for preparing a reagent to enhance the immunity of soft-shelled turtles, the composition comprising *Westernella esculenta* and chitosan oligosaccharides.

[0026] Furthermore, through analysis of antioxidant and non-specific immune indicators in turtle liver tissue, this invention found that the catalase (CAT) activity in the chitosan oligosaccharide group was significantly higher than that in the fructooligosaccharide group, indicating that chitosan oligosaccharide is superior to fructooligosaccharide in improving antioxidant capacity. At the same time, the superoxide dismutase (SOD) activity in the compound microecological preparation group was significantly higher than that in the chitosan oligosaccharide group, indicating that the compound microecological preparation is superior to chitosan oligosaccharide in improving antioxidant capacity. In addition, the compound microecological preparation significantly reduced the levels of acid phosphatase (ACP) and malondialdehyde (MDA), improving the antioxidant capacity of turtles. This demonstrates that adding compound microecological preparations to feed can significantly improve the immunity of turtles.

[0027] In another aspect, the present invention provides the use of a composition for preparing a reagent that promotes the digestion and absorption of nutrients, the composition comprising *Westernella esculenta* and chitosan oligosaccharides.

[0028] Furthermore, intestinal digestive enzyme activity analysis demonstrated that *Westernella stolonifer*, *Enterococcus faecalis*, and the compound microecological preparation significantly increased trypsin activity, with *Westernella stolonifer* showing the strongest effect. Chitosan oligosaccharide increased the activity of lipase and amylase, with better effects than fructooligosaccharide. Simultaneously, the addition of chitosan oligosaccharide or the compound microecological preparation to the feed resulted in a dramatic increase in the number and length of colonic villi in turtles, indicating that the use of the compound microecological preparation containing *Westernella stolonifer* and chitosan oligosaccharide can promote the digestion and absorption of nutrients in the turtle's intestines.

[0029] In another aspect, the present invention provides the use of a composition for preparing a reagent that increases the amino acid content of turtle muscle, the composition comprising *Westernella esculenta* and chitosan oligosaccharides.

[0030] Studies have shown that adding the prebiotics, probiotics, or compound microecological preparations provided by this invention to the feed can increase the content of umami amino acids in turtle muscle. Furthermore, adding the compound microecological preparations to the feed significantly increases the content of glutamic acid, aspartic acid, and glycine.

[0031] In another aspect, the present invention provides a feed for improving the water quality of turtle farming water, comprising Weissella tamariscina, the preservation number of which is CGMCC NO: 32640.

[0032] Furthermore, the feed also includes any one or more prebiotics selected from fructooligosaccharides, chitosan oligosaccharides, and β-glucan.

[0033] Furthermore, the prebiotic is chitosan oligosaccharide.

[0034] Furthermore, the content of *Westernella esculenta* is 1 × 10⁻⁶. 7 ~1×10 9 CFU / g, with a chitosan oligosaccharide content of 0.1–0.5 wt%.

[0035] In another aspect, the present invention provides a method for improving water quality when raising soft-shelled turtles, by feeding the turtles with the feed described above.

[0036] Furthermore, the feed is given to the turtles by sprinkling, and the feed enters the water body at the same time. The aquatic flora improves the water quality by decomposing the feed and the probiotics or prebiotics in the feed regulating the aquatic flora.

[0037] In another aspect, the present invention provides the use of feed for preparing a reagent to reduce the content of ammonia nitrogen, total phosphorus and chemical oxygen demand in turtle farming water, wherein the feed includes Weissella tamariscina, the preservation number of which is CGMCC NO.32640.

[0038] In another aspect, the present invention provides the use of feed for preparing a reagent to increase the dissolved oxygen content in turtle aquaculture water, the feed comprising Weissella tamariscina, the preservation number of Weissella tamariscina being CGMCC NO.32640.

[0039] In another aspect, the present invention provides the use of feed for preparing a reagent to maintain the balance of the microbial community in the water body for turtle farming, wherein the feed includes Weissella tamariscina, and the accession number of Weissella tamariscina is CGMCC NO.32640.

[0040] Because the compound microecological preparation is mixed into the feed before being given to the turtles, some of the feed enters the water along with the turtles' intestines. Furthermore, since the feed ingested by the turtles, like their intestinal flora, is also excreted into the water, both the turtle's intestinal flora and the aquatic flora are affected by the administered microbial agent. Some bacteria in the aquatic environment produce ammonia nitrogen after decomposing the turtles' food residue or feces, which can stress the turtles' growth. Therefore, the administered microbial agent needs to suppress the abundance of ammonia-producing bacteria. Verification results show that adding the compound microecological preparation to the turtles' water resulted in higher dissolved oxygen levels, lower ammonia nitrogen and sulfide content, slightly acidic water, and better water quality. Simultaneously, the abundance of beneficial bacteria increased, while the abundance of harmful bacteria (Proteobacteria) decreased, maintaining the bacterial balance of the aquaculture water.

[0041] Furthermore, the feed also includes any one or more prebiotics selected from fructooligosaccharides, chitosan oligosaccharides, and β-glucan.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] 1. Feeding soft-shelled turtles with the compound microecological preparation provided by this invention regulates the balance of the intestinal flora, increases the abundance of bacterial genera related to nutrient conversion efficiency, and is beneficial to the synthesis of nutrients and their absorption by the body.

[0044] 2. Soft-shelled turtles fed with compound microecological preparations showed high antioxidant enzyme activity and low MDA content, which enhanced their non-specific immunity and reduced their mortality rate.

[0045] 3. The intestinal villi of turtles fed with compound microecological preparations are longer and more numerous, which is conducive to the digestion and absorption of nutrients in the intestines, and at the same time increases the total content of umami amino acids in the turtle muscle.

[0046] 4. The compound microecological preparation simultaneously regulates the abundance of bacteria in the water, effectively improving water quality. Attached Figure Description

[0047] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0048] Figure 1 This is a diagram of probiotic culture in the laboratory of this invention;

[0049] Figure 2 This is a diagram illustrating the structural composition of the gut microbiota for β-diversity analysis in this invention.

[0050] Figure 3 The super-index (A) and Shannon index (B) for the gut microbiota α-diversity analysis of this invention are shown.

[0051] Figure 4 This invention provides a phylum-level analysis diagram of the gut microbiota structure.

[0052] Figure 5 This invention relates to a horizontal analysis of the gut microbiota diagram;

[0053] Figure 6 This is a comparative analysis diagram of multiple groups of intestinal flora in this invention;

[0054] Figure 7 This is a LefSe score diagram of the gut microbiota in this invention;

[0055] Figure 8 This is an anatomical diagram of the turtle intestinal tissue in this invention;

[0056] Figure 9 This invention relates to the effects of the present invention on the antioxidant capacity and immune-related enzyme activity of turtle liver;

[0057] Figure 10 This is a diagram showing the activity of digestive enzymes in the intestine of turtles in this invention;

[0058] Figure 11 The following are the analytical results of the crude protein content (A), essential amino acid content (B), umami amino acid content (C), glutamic acid content (D), glycine content (E), aspartic acid content (F) and alanine content (G) in the muscle of turtle in this invention;

[0059] Figure 12 The following are the analytical results of chemical oxygen demand (A), nitrite nitrogen content (B), sulfide content (C), ammonia nitrogen content (D), dissolved oxygen content (E), and pH (F) of the water body in this invention;

[0060] Figure 13 This is a diagram illustrating the structural composition of aquatic microbial communities used in the β-diversity analysis of this invention.

[0061] Figure 14 This is a super-index diagram of aquatic microbial communities for α-diversity analysis according to the present invention;

[0062] Figure 15 This invention provides a phylum-level analysis diagram of aquatic microbial communities.

[0063] Figure 16 This invention relates to a horizontal analysis diagram of aquatic microbial communities;

[0064] Figure 17 This is a comparative analysis diagram of multiple groups of water microbial communities according to the present invention;

[0065] Figure 18 This is a LefSe diagram for the differential analysis of water microbial communities in this invention. Detailed Implementation

[0066] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0067] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0068] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0069] Example 1: Preparation of feed using the compound microecological preparation provided by the present invention.

[0070] 1. Preparation of *Westernella esculenta* bacterial suspension

[0071] The *Westernella esculenta* bacterial suspension from the cryopreservation tubes was inoculated into MRS liquid medium at a 2% inoculum size and cultured at 37°C for 24 hours. After two subcultures, fresh bacterial suspension was obtained. One portion of the fresh bacterial suspension was centrifuged at 5000 rpm for 5 minutes, and the supernatant was gently transferred to another sterile centrifuge tube to obtain the bacterial supernatant. The centrifuged bacterial cells were washed twice with sterile PBS, and then the OD of the strain was further reduced using PBS buffer. 600 The nm value was adjusted to 1.0 to obtain a bacterial suspension. Then, 10 mg / g of the basal diet (purchased from Zhejiang Jindadi Agricultural Technology Co., Ltd.) was added. 8 The colony count of CFU was calculated by weighing the bacterial suspension. The basal diet formulation is shown in Table 1.

[0072] Table 1 Nutritional composition of the basal diet

[0073]

[0074] 2. Add 0.003g of chitosan oligosaccharide powder (purchased from Zhejiang Jinke Pharmaceutical Co., Ltd., model SLKG) per gram of basal diet, i.e., 0.3wt%;

[0075] 3. Add the bacterial culture and chitosan oligosaccharide powder to the basic diet, mix well and it is ready to use.

[0076] Example 2: Screening and Isolation of Probiotics

[0077] Multiple strains of bacteria were screened from the intestines of healthy soft-shelled turtles using the dilution plating method. Three strains that showed significant inhibitory effects on Aeromonas hydrophila, a common pathogenic bacterium in soft-shelled turtles, were selected using the Oxford cup method. These strains were Weissella cibaria, Enterococcus faecalis, and Lactococcus lactis.

[0078] The specific steps for screening three probiotics using the Oxford cup method are as follows:

[0079] (1) Prepare nutrient agar medium (NA)

[0080] Prepare NA and MRS plates in advance and allow them to air dry. The NA and MRS culture media were purchased from Qingdao Haibo Biotechnology.

[0081] (2) Preparation of Aeromonas hydrophila bacterial culture

[0082] Streaking was performed on NA agar plates to activate Aeromonas hydrophila (strain source: previously isolated from the intestines of diseased turtles in our laboratory). Single colonies were then picked and transferred to NB medium and cultured aerobically at 37°C for 24 hours. The colonies were then transferred to new NB medium at a ratio of 2% and cultured aerobically at 37°C for 24 hours to obtain fresh bacterial culture.

[0083] (3) Preparation of fermentation broth from 3 probiotic strains

[0084] The probiotic culture from the cryopreserved tubes was inoculated into MRS liquid medium at a 2% inoculation rate and cultured at 37°C for 24 hours. After two subcultures, fresh culture was obtained. One portion of the fresh culture was centrifuged at 5000 rpm for 5 minutes, and the supernatant was gently transferred to another sterile centrifuge tube to obtain the bacterial supernatant. The centrifuged bacteria were washed twice with sterile PBS, and then the bacterial OD was further reduced using PBS buffer. 600 After adjusting nm to 1.0, a bacterial resuspension was obtained.

[0085] (4) Oxford Cup Antibacterial Test

[0086] Prepare NA medium and sterilize it; after the temperature drops below 47℃, add Aeromonas hydrophila culture and shake well; pour it onto a plate, and after solidification, place an Oxford cup on it. Add 100μL of probiotic fermentation broth, supernatant after centrifugation of fermentation broth, bacterial resuspension, and PBS (blank control) to each well. Incubate at 37℃ for 24h and observe for the presence or absence of inhibition zones.

[0087] turn out( Figure 1The inhibitory effect of *Westernella stolonifer* on *Aeromonas hydrophila* was the strongest. The *Westernella stolonifer* strain screened from the intestines of turtles was deposited on November 15, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO.32640.

[0088] Example 3: Analysis of Gut Microbial Composition and Diversity

[0089] Based on the characteristics that prebiotics and probiotics can maintain intestinal flora balance and improve the body's immunity, this embodiment further explores the effects of prebiotics, probiotics, and the compound microecological preparation provided by the present invention on intestinal flora diversity.

[0090] Eight paddy fields (approximately 40m × 10m in area, each field divided into three smaller plots) were selected for the experiment. 36-month-old Chinese soft-shelled turtles were randomly divided into eight groups (six experimental groups + two control groups). The initial average weight was 1 ± 0.2 kg, and the average carapace length was 20.2 cm. They were fed a designated diet for four weeks. The components and dosages added to the diet of the six experimental groups are shown in Table 2. The control group included a basal diet and a commercially available microbial agent (purchased from Zhejiang Hemudu Biotechnology Co., Ltd.). The basal diet formula and its approximate composition are shown in Table 1. The turtles were fed twice daily (8:30 and 17:30). Sampling was conducted two weeks after feeding, with six turtles randomly selected from each group (two from each small paddy field). Their weight was measured. Blood samples were collected from the jugular vein, centrifuged at 4℃ (3500 × g) for 15 min, and serum samples were collected. Hind leg muscle tissue, liver tissue, intestinal tissue, and colon contents were collected and frozen in liquid nitrogen.

[0091] 16S rRNA gene sequencing was used to align the gut microbiota in intestinal contents. α-diversity indices, including the Chao1 and Shannon indices, were calculated using QIIME2 to assess microbial community complexity (21). β-diversity analysis was performed to investigate intra- and inter-group differences based on principal coordinate analysis (PCoA) and the R-vegetarian package. Furthermore, microbial function prediction was achieved using PICRUSt2. Functional pathway annotation was performed based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. All data processing and analysis were performed using OmicShare tools (https: / / www.omicshare.com / tools).

[0092] Table 2. Components and dosages added to different groups

[0093]

[0094] β-diversity analysis showed that ( Figure 2 Compared with the control group, the intestinal flora structure of the WC group (Westernella esculenta) and the CWC group (compound microecological preparation) was significantly different, indicating that the intestinal flora structure changed after feeding with the compound microecological preparation. The COS group (chitosan oligosaccharide) had slightly higher α-diversity than the FOS group (fructooligosaccharide), but the difference was not significant, and the β-diversity dispersion was slightly lower, indicating that the abundance of intestinal flora in the COS group was higher than that in the FOS group, and the intra-group flora differences were smaller. α-diversity analysis ( Figure 3 The results showed that the α-diversity of the WC and CWC groups was slightly higher than that of the control group, but the difference was not significant. These results indicate that feeding turtles with chitosan oligosaccharides, *Westernella septemlobus*, or compound microecological preparations resulted in a more stable gut microbiota structure.

[0095] Analysis at the gut microbiota level ( Figure 4 The study found that both the COS and FOS groups showed increased Firmicutes / Bacteriota values ​​compared to the control group, indicating that both chitosan oligosaccharides and fructooligosaccharides improved the ability of soft-shelled turtles to absorb nutrients and store energy, with chitosan oligosaccharides showing a better effect; analysis at the gut microbiota genus level... Figure 5 The study found that the COS and FOS groups were enriched with more *Cetobacterium* compared to the control group. Increased *Cetobacterium* levels can enhance vitamin B12 synthesis, which is crucial for normal nerve function, erythrocyte formation, and other physiological processes. The WC and CWC groups were enriched with more *Romboutsia* compared to the control group. *Romboutsia* helps break down food, promotes nutrient absorption, assists the body in producing short-chain fatty acids beneficial to the gut, provides energy for intestinal cells, and maintains the health of the intestinal mucosa.

[0096] Results of difference analysis ( Figure 6 and Figure 7 The results showed that the gut microbiota composition differed among the different experimental groups. The COS group showed an abundance of *Dielma* bacteria in its gut, while the WC group showed an abundance of *Turicibacter*, *Pediococcus*, and *Weissella*. The CWC group showed an abundance of *Paeniclostridium* and *Cellulosilyticum*. *Paeniclostridium* and *Cellulosilyticum* promote protein breakdown and cellulose degradation, thus facilitating nutrient conversion. This indicates that adding *Weissella oblongata* or a compound microecological preparation to the feed can significantly improve gut microbiota diversity and maintain gut microbiota balance.

[0097] Example 4: Intestinal Morphology Analysis

[0098] To further investigate the changes in the intestinal morphology of turtles after feeding in the eight groups shown in Table 2 of Example 3, this example observes the histological morphology of intestinal tissues from different groups collected in Example 3 after feeding. Colonic tissue was washed with phosphate-buffered saline (PBS) and rapidly fixed in 4% paraformaldehyde. After 24 hours, it was embedded in paraffin. Sections were longitudinally sectioned to a thickness of 4 μm, dehydrated using an automatic dehydrator, and stained with hematoxylin and eosin (H&E). Histological morphology was observed using a digital pathology scanner (Kfbio, China) and an optical microscope (Nikon, Japan).

[0099] like Figure 8 As shown, the addition of chitosan oligosaccharide and compound microecological preparations to the feed led to a sharp increase in both the number and length of colonic villi in soft-shelled turtles, with chitosan oligosaccharide showing a stronger effect than the compound microecological preparations. Conversely, the addition of fructooligosaccharide resulted in a sharp decrease in intestinal villi length. There was no significant difference in intestinal villi between the *Enterococcus faecalis* and *Westernella esculenta* groups, while the intestinal villi length of *Lactococcus lactis* increased. These results indicate that adding chitosan oligosaccharide or compound microecological preparations to the feed can significantly improve the digestion and absorption of nutrients in soft-shelled turtles, thereby promoting their growth.

[0100] Example 5: Immunoenzyme Activity Analysis

[0101] This embodiment further analyzes the immunomodulatory indicators of the liver tissues of turtles collected in Example 3 after feeding in different groups. Five indicators of antioxidant activity in the liver were detected according to the manufacturer's instructions using commercially available kits (Nanjing Jiancheng Bioengineering Institute, China): total antioxidant capacity (T-AOC), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) levels. T-AOC and GSH-Px activities were determined colorimetrically, CAT activity was determined by visible light method, SOD activity was determined by WST-1 method, and MDA content was determined by thiobarbituric acid (TBA) method. In addition, three indicators of non-specific immunity were measured, including liver lysozyme (LZM), alkaline phosphatase (ALP), and acid phosphatase (ACP) levels. LZM activity was determined by turbidimetric method, and ALP and ACP activities were determined by microenzyme labeling method. All reactions were performed in 96-well plates, and absorbance was measured using a microplate reader (Thermo Scientific). TM 5580, China) read.

[0102] The formation of MDA can cause cross-linking and polymerization of biomolecules such as proteins and nucleic acids, and it is cytotoxic; excessively high levels can be detrimental to the body. CAT, on the other hand, is a reactive oxygen species scavenger that can decompose reactive oxygen species such as hydrogen peroxide produced during metabolism. Meanwhile, SOD is an antioxidant metalloenzyme present in the body that catalyzes the disproportionation of superoxide anion free radicals to produce oxygen and hydrogen peroxide, playing a crucial role in the body's oxidation-antioxidant balance. Therefore, increased CAT and SOD activity indicates an improved antioxidant capacity. Analysis of liver sample enzyme activity results showed ( Figure 9 Compared with the control, the SOD levels in the COS group (chitosan oligosaccharide) and FOS group (fructooligosaccharide) were increased, while other indicators showed no significant changes. The T-AOC activity, CAT activity, GSH-Px and SOD activity in the WC group (Westernella esculenta) and CWC group (compound microecological preparation) were significantly higher than those in other groups, and CWC was more effective. This indicates that the antioxidant capacity of Weisternella esculenta is superior to that of the other two probiotics, and the addition of compound microecological preparation can significantly improve the body's antioxidant capacity.

[0103] In addition, ACP, an acidic hydrolase abundant in lysosomes and considered a marker for identifying lysosomal subcellular components, is a non-specific immune indicator, and its elevation is associated with various diseases. ALP can serve as a natural anti-infective substance with bactericidal activity. Compared to the control group, the ACP activity and MDA enzyme activity in the CWC group were significantly reduced. Several prebiotics and probiotics also reduced ACP levels but had no significant effect on MDA activity, indicating that the use of a microecological compound preparation can enhance the antibacterial effect of turtles. ALP enzyme activity showed no significant difference among the groups. In summary, the use of a compound microecological preparation can further improve the antioxidant and antibacterial capabilities of turtles.

[0104] Example 6: Digestive Enzyme Activity Analysis

[0105] The intestines were dissected and immediately frozen in liquid nitrogen, then temporarily stored at -80°C for later use. Following the manufacturer's instructions, the digestive enzyme activities, including trypsin, lipase, and amylase, of the turtle intestinal tissues collected in Example 3 after feeding were determined using a commercial kit (Nanjing Jiancheng Bioengineering Institute).

[0106] like Figure 10 As shown, compared with the control group, there was no significant difference in lipase and amylase activities among the six experimental groups. However, *Westernella esculenta*, *Enterococcus faecalis*, and the compound microecological preparation significantly increased trypsin activity, with *Westernella esculenta* showing a significantly greater effect than the other two groups.

[0107] Example 7: Amino acid composition analysis for improved muscle

[0108] The changes in crude protein, essential amino acids, umami amino acids, and four key amino acids in the hind leg muscles of turtles from eight groups after feeding, collected in Example 3, were detected using a Hitachi L8900 fully automated amino acid analyzer. One-way ANOVA revealed the following: Figure 11 Adding chitosan oligosaccharides and compound microecological preparations increased the crude protein content in turtle muscle, and adding Lactococcus lactis increased the content of essential amino acids. Adding chitosan oligosaccharides, fructooligosaccharides, Enterococcus faecalis, Weissella esculenta, or compound microecological preparations all increased the content of umami amino acids, but the effect of adding compound microecological preparations was more significant. Furthermore, adding compound microecological preparations also significantly increased the content of glutamic acid, aspartic acid, and glycine. Therefore, compound microecological preparations are preferred.

[0109] Example 8: Water Quality Adjustment Analysis

[0110] Since prebiotics, probiotics, or compound microecological preparations are added to the feed and then sprinkled on top, both the intestinal flora and aquatic flora of the turtles are affected by the administered microbial agents. The aquatic flora decomposes food residue or feces, altering water quality; some colonies decompose substances to produce ammonia nitrogen, which is detrimental to the growth of the turtles. To investigate the water quality changes after feeding the eight groups shown in Table 1 of Example 3, this example used a water quality analyzer (Greencare) to measure the chemical oxygen demand, total phosphorus, nitrite nitrogen, ammonia nitrogen, and sulfides.

[0111] Chemical oxygen demand (COD) measures the total amount of reducing substances (mainly organic matter) in water that can be oxidized by strong oxidants. An elevated COD value indicates water pollution. Nitrite nitrogen and sulfur compounds are harmful substances in water, deteriorating water quality and affecting the growth and health of aquatic animals. Ammonia nitrogen can lead to eutrophication and is a major oxygen-consuming pollutant in water. Excessive phosphorus content can cause excessive algal growth, leading to eutrophication, algal blooms, or red tides, disrupting the water's balance. Higher dissolved oxygen levels are beneficial for aquatic animals to absorb oxygen. Figure 12 As shown, compared with the control, feeding with chitosan oligosaccharide alone resulted in significantly higher COD, nitrite nitrogen, and sulfur compound content in the water, and lower dissolved oxygen levels, indicating poor water quality. However, feeding with probiotics or compound microecological preparations did not result in significant changes in COD, nitrite nitrogen, and sulfur compound content in the water, but the ammonia nitrogen content in the compound microecological preparation group was significantly lower, the dissolved oxygen content was higher, and the water was more acidic, indicating better water quality.

[0112] like Figure 13 and 14As shown, the abundance of Gemmatimonadota bacteria was high in the CK group. Studies have found that some Gemmatimonadota species may produce harmful substances, such as toxins or pathogenic factors, leading to infection or disease. At the phylum level, the abundance of Proteobacteria was reduced in the COS, FOS, and WC groups compared to the control group. This phylum contains many pathogens that may disrupt the intestinal microecological balance and induce intestinal infection, indicating that adding chitosan oligosaccharides, fructooligosaccharides, or Weissella esculenta can reduce the risk of disease in turtles. The abundance of Bacteroidota bacteria was increased in the COS, LL, and CWC groups compared to the control group. Figure 15 Studies have found that Bacteroidota can help break down food, especially complex carbohydrates, and can also form a protective biofilm in the gut, helping to prevent the invasion of harmful microorganisms and strengthening the intestinal barrier function. Analysis of LefSe results ( Figure 16 The CK group was enriched with *Terrisporobacter* and *Limnobacter*, the COS group was enriched with *Epulopiscium* and *OLB12*, the FOS group was enriched with *Mycobacterium*, *Flavobacterium*, and *Fluviicola*, the WC group was enriched with *Paeniclostridium*, and the CWC group was enriched with *Romboustsia*, *Turicibacter*, and *Terrimonas*. Figure 17-18 As shown, the CWC group and the commercial protease group reduced community diversity, indicating that adding microecological preparations to the feed for turtles simultaneously altered the microbial community structure in the water.

[0113] Example 9: Comparison of Compound Microecological Preparations with Different Components

[0114] The results of Examples 3-8 show that different prebiotics or probiotics have differences in intestinal flora composition, intestinal morphology, immune capacity, digestive capacity, and water quality regulation. Therefore, the comprehensive performance of compound microecological preparations composed of different prebiotics or probiotics should also differ. To further compare the differences in compound microecological preparations prepared from different components and to screen for the optimal combination, this example uses two prebiotics, chitosan oligosaccharide and fructooligosaccharide, and three probiotics, *Westernella esculenta*, *Lactococcus lactis*, and *Enterococcus faecalis*, to prepare four compound microecological preparations as shown in Table 3. The preparation method is the same as in Example 1. After adding the four compound microecological preparations to the feed, the animals were fed according to the method in Example 3. The control group was the basal diet. Then, the immune indicators in the liver tissue of turtles were analyzed using the same method as in Example 5. The results are shown in Tables 4 and 5.

[0115] Table 3 Formulations of different compound microecological preparations

[0116]

[0117] Table 4 Results of Antioxidant Index Measurement

[0118]

[0119] Table 5 Results of Non-Specific Immunological Indicators Measurement

[0120]

[0121] As shown in Tables 4 and 5, the four compound microecological preparations had no significant effect on the activities of T-AOC and GSH-Px. Compound microecological preparations 2, 3, and 4 did not significantly promote the activities of the five enzymes related to antioxidant activity and the three enzymes related to non-specific immunity. However, compound microecological preparation 1 increased CAT activity, significantly increased SOD activity, and simultaneously decreased MDA levels. Furthermore, compound microecological preparation 1 also reduced ACP levels. This indicates that adding compound microecological preparation 1 to the feed can significantly improve the antioxidant and antibacterial capabilities of turtles and enhance their immunity. Therefore, the compound microecological preparation prepared from chitosan oligosaccharide and *Westernella esculenta* is preferred.

[0122] Example 10: Comparison of Weissella esculenta from different sources

[0123] Existing studies have isolated *Westernella stolonifera* from various sources, such as mandarin fish. The effects of compound microecological preparations made from different *Westernella stolonifera* and chitosan oligosaccharides may vary. To further verify the differences in compound microecological preparations made from *Westernella stolonifera* from different sources, this example uses *Westernella stolonifera* from mandarin fish and chicken, respectively, to prepare compound microecological preparations with chitosan oligosaccharides, using the same preparation method as in Example 1. These two compound microecological preparations, along with compound microecological preparation 1 from Example 9, were added to the feed and fed according to the method in Example 3. The control group served as the basal diet. Immunological indicators in the liver tissue of the turtles were then analyzed using the same analytical method as in Example 5. The results showed that feeding with the compound microecological preparation made from *Westernophora sinensis* derived from mandarin fish increased the activities of T-AOC, CAT, and GSH-Px enzymes, but not significantly, while decreasing ALP levels, and the water quality of the turtle farming water did not change significantly. Feeding with the compound microecological preparation made from *Westernophora sinensis* derived from chicken showed little change in the five antioxidant indicators, a slight decrease in ACP levels, and a decrease in total phosphorus and ammonia nitrogen content in the turtle farming water. However, feeding with compound microecological preparation 1 significantly increased the activities of T-AOC, CAT, and SOD, decreased MDA activity, and reduced ACP and ALP levels, while significantly reducing ammonia nitrogen and chemical oxygen demand in the turtle farming water and increasing dissolved oxygen content. Therefore, the compound microecological preparation provided by this invention is preferred.

[0124] In summary, the above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make improvements and modifications without departing from the core technology of the present invention, and such improvements and modifications should also fall within the patent protection scope of the present invention. Any changes within the meaning and scope equivalent to the claims of the present invention should be considered as included within the scope of the claims.

Claims

1. A composite microecological preparation, characterized in that, Weissella cibaria with the preservation number of CGMCC NO.32640, the content of which is 1x10 7 ~1x10 8 CFU / g, and chitooligosaccharide, the content of which is 0.1~0.3 wt%.

2. A feed for feeding soft-shelled turtles, characterized by, The composite microecological preparation as claimed in claim 1.

3. A method for culturing soft-shelled turtles, characterized by, The feed of claim 2 is used for feeding.

4. Use of a composition for the manufacture of a reagent for modulating the intestinal flora balance of a fish, characterized in that, The composition comprises Weissella cibaria with a preservation number of CGMCC NO.32640, and chitooligosaccharide, wherein the content of the Weissella cibaria is 1x10 7 ~1x10 9 CFU / g, and the content of the chitooligosaccharide is 0.1~0.5 wt%.

5. The use of a composition for preparing an agent for improving the immunity of soft-shelled turtles, characterized in that, The composition comprises Weissella cibaria with a preservation number of CGMCC NO.32640, and chitooligosaccharide, wherein the content of the Weissella cibaria is 1x10 7 ~1x10 9 CFU / g, and the content of the chitooligosaccharide is 0.1~0.5 wt%.

6. Use of a composition for the manufacture of an agent for facilitating the digestive absorption of nutrients, characterized in that, The composition comprises Weissella cibaria with a preservation number of CGMCC NO.32640, and chitooligosaccharide, wherein the content of the Weissella cibaria is 1x10 7 ~1x10 9 CFU / g, and the content of the chitooligosaccharide is 0.1~0.5 wt%.

7. Use of a composition for the manufacture of an agent for increasing the amino acid content of the muscle of a turtle, characterized in that, The composition comprises Weissella cibaria with a preservation number of CGMCC NO.32640, and chitooligosaccharide, wherein the content of the Weissella cibaria is 1x10 7 1x10 9 CFU / g, and the content of the chitooligosaccharide is 0.1-0.5 wt%.

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