Babylonia areolata functional feed containing plant polysaccharide and application of Babylonia areolata functional feed

Abstract

The embodiment of the invention discloses a babylonia areolata functional feed containing plant polysaccharide and application thereof, and belongs to the technical field of aquaculture and aquatic animal nutrition. The babylonia areolata functional feed containing the plant polysaccharide is composed of a basal feed and the plant polysaccharide, wherein the plant polysaccharide comprises astragalus polysaccharide, lycium barbarum polysaccharide and lentinan. Growth performance determination, vibrio harveyi challenge test and intestinal tract and substrate microorganism high-throughput sequencing analysis verify that the functional feed can significantly improve the growth performance and disease resistance of Babylonia areolata, and synchronously optimize the micro-ecological structure of the intestinal tract and culture substrate of Babylonia areolata; and the feed can partially replace antibiotics, accords with the development trend of green breeding, is suitable for various breeding modes, and has a good industrial popularization prospect.

Description

Technical Field

[0001] This invention relates to the fields of aquaculture and aquatic animal nutrition technology, specifically to a functional feed for the spotted snail containing plant polysaccharides and its application. Background Technology

[0002] The spotted whelk is one of the important farmed shellfish species in the southern coastal areas of my country, especially in Hainan, characterized by its rapid growth, good meat quality, and high economic value. However, under factory-style, high-density farming conditions, the spotted whelk commonly suffers from the following problems:

[0003] 1. Uneven growth and insufficient weight gain rate

[0004] Due to the frequent occurrence of extreme weather events such as typhoons in coastal areas, the actual farming of spotted worm snails is subject to frequent stress. Furthermore, there is still considerable room for improvement in the growth rate and weight gain of spotted worm snails, especially under conditions of high-density farming and limited water exchange, which can easily lead to problems such as slow growth and inconsistent size.

[0005] 2. Outbreaks are prone to occur due to pathogens such as Vibrio harveyi.

[0006] Vibrio harveyi is a significant pathogen in the bald worm and various marine aquaculture animals, causing large-scale mortality and severe economic losses. Especially after typhoons in coastal areas, the disturbance of seabed silt leads to a surge in Vibrio levels in seawater. Traditional control measures mainly rely on antibiotics and chemical drugs, which have problems such as drug resistance, drug residues, and environmental pollution, and are inconsistent with the current development direction of green, antibiotic-free aquaculture.

[0007] 3. Imbalance in the gut microbiota and aquaculture substrate

[0008] Under high-density aquaculture conditions, uneaten feed, feces, and mucus from the spotted worm snail can easily lead to sediment deterioration and the accumulation of pathogenic bacteria. Simultaneously, the gut microbiota structure of the spotted worm snail is easily imbalanced by both feed and environmental factors, thus affecting digestion, absorption, and immune function. Existing technologies are mostly focused on species such as fish, shrimp, and crabs, with very limited research on the integrated gut-sediment microecological regulation of the spotted worm snail.

[0009] There is an urgent need to develop a plant polysaccharide functional feed suitable for the spotted worm snail, so as to achieve healthy, efficient and green farming of the spotted worm snail while reducing the use of drugs. Summary of the Invention

[0010] Therefore, this invention provides a functional feed for the spotted worm snail containing plant polysaccharides and its application, in order to solve the problems of insufficient growth rate, easy outbreak of Vibrio harveyi, and imbalance of intestinal and bottom microecology in the existing technology of spotted worm snail farming.

[0011] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0012] According to a first aspect of the present invention, the present invention provides a functional feed for the spotted worm snail containing plant polysaccharides, the functional feed for the spotted worm snail being composed of a basic feed and plant polysaccharides.

[0013] In some preferred embodiments, the plant polysaccharides include astragalus polysaccharide, wolfberry polysaccharide, and shiitake mushroom polysaccharide.

[0014] In some preferred embodiments, the amount of plant polysaccharides added is 2-4 g / kg based on the weight of the basal feed.

[0015] In some preferred embodiments, the amount of plant polysaccharide added is 2 g / kg.

[0016] In some preferred embodiments, the basic feed includes fishmeal, plant protein sources, carbohydrates, oils, vitamins and minerals premix.

[0017] In some preferred embodiments, the basic feed comprises, by weight percentage, the following components: 60% mackerel meat paste, 12% soy protein isolate, 9% α-starch, 4% meat and bone meal, 4% fish oil, 4.5% squid viscera powder, 3% calcium dihydrogen phosphate, 1% mixed vitamins, 0.5% choline chloride, and 2% binder.

[0018] In some preferred embodiments, the adhesive is edible gelatin.

[0019] According to a second aspect of the present invention, the present invention provides the application of the functional feed for the spotted worm snail as described above in improving the growth performance of the spotted worm snail, its resistance to Vibrio harveyi infection, and optimizing the microbial community structure of the gut and the culture substrate.

[0020] The embodiments of the present invention have the following advantages:

[0021] 1. Multi-indicator integrated functional feed evaluation system

[0022] This invention is the first to construct an evaluation system for the *Bambusa squarrosa* that integrates "growth performance, pathogenic Vibrio challenge test, intestinal microbial community and substrate microbial community analysis". This system can comprehensively and objectively reflect the effects of plant polysaccharides in actual aquaculture systems, and greatly improve the scientificity and reliability of the evaluation of feed functional additives.

[0023] 2. Specialized plant polysaccharide feed technology for the spotted snail (Bellamya alatus).

[0024] This invention selects plant polysaccharides such as Astragalus polysaccharide, Lycium barbarum polysaccharide, and Lentinus edodes polysaccharide, which have immune-enhancing and microecological regulation functions, and systematically verifies their application effects on the spotted roe snail. This results in a special functional feed program for the spotted roe snail, filling the gap in existing research on functional feeds for this shellfish species.

[0025] 3. Simultaneously enhances growth and disease resistance

[0026] The results of the examples show that polysaccharide functional feed can not only significantly improve the growth indicators (final weight, specific growth rate, etc.) of the spotted snail, but also significantly reduce the mortality rate and increase the survival rate after Vibrio harveyi challenge, achieving the dual effects of promoting growth and resisting disease, which is conducive to improving breeding yield and survival rate.

[0027] 4. An innovative model for the synergistic regulation of the gut-solid microecology

[0028] This invention, through the addition of feed polysaccharides, on the one hand, reshapes the intestinal flora structure of the spotted snail, promotes the enrichment of beneficial bacteria, inhibits potential pathogens, and improves the body's digestion, absorption, and immune function; on the other hand, by changing the microbial composition of excrement and uneaten feed, it indirectly optimizes the microecology of the aquaculture substrate, reduces substrate deterioration and pathogen accumulation, and constructs an integrated individual-environment healthy aquaculture model.

[0029] 5. Reduces drug dependence and aligns with the development direction of green aquaculture.

[0030] This invention uses natural polysaccharides derived from plants as functional additives, which can partially replace the use of antibiotics and chemical drugs, reduce the risk of drug resistance and drug residues, and are in line with the current development trend of antibiotic-free, green and sustainable aquaculture, and have good prospects for promotion and application.

[0031] 6. It has good industrialization and promotion value.

[0032] The polysaccharide functional feed of this invention is compatible with existing compound feed production processes and is suitable for various aquaculture models such as factory-scale recirculating aquaculture, ponds, and high-density net cages. It is particularly suitable for large-scale aquaculture of the spotted snail in southern coastal areas such as Hainan, providing a feasible technical path to improve aquaculture efficiency and reduce aquaculture risks. Attached Figure Description

[0033] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0034] Figure 1A comparative bar chart showing the weight gain effect of the square-spotted Dongfeng provided by the present invention;

[0035] Figure 2 A bar chart comparing the initial and final weight of the square-spotted roe snail provided by this invention;

[0036] Figure 3 A comparative bar chart of specific growth rates of the body weight of the spotted snail provided by the present invention;

[0037] Figure 4 A bar chart comparing the average mortality time in the challenge experiment with Vibrio harveyi from the spotted snail provided by this invention;

[0038] Figure 5 Survival curve of Vibrio harveyi in challenge experiment provided by the present invention;

[0039] Figure 6 A bar chart of the phylum-level community structure of the intestinal microorganisms of the spotted snail provided by this invention;

[0040] Figure 7 A bar chart illustrating the phylum-level community structure of substrate microorganisms in the culture of the spotted snail provided by this invention. Detailed Implementation

[0041] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] Please refer to the applicant's previous research results (CN114208977A) for the basic feed and its preparation method used in the following content. Specifically, the formula is as follows: 60% mackerel meat paste, 12% soy protein isolate, 9% α-starch, 4% meat and bone meal, 4% fish oil, 4.5% squid viscera powder, 3% calcium dihydrogen phosphate, 1% mixed vitamins, 0.5% choline chloride, 2% edible gelatin, and 15ml water.

[0043] The preparation method is as follows:

[0044] 1) Fish paste: Thaw the mackerel naturally from -20℃ to 4℃. Remove the head, skin and bones from the thawed mackerel and grind it in a meat grinder to make fish paste. Take 120 grams.

[0045] 2) Feed Mixed Dry Powder: Put soy protein isolate (24g), α-starch (18g), meat and bone meal (8g), squid viscera powder (9g), mixed vitamins (2g) and choline chloride (1g) into a grinder and grind them into powder.

[0046] 3) Carrageenan solution: Add 4 grams of edible gelatin to 10 ml of ice water to make a carrageenan solution.

[0047] 4) Calcium dihydrogen phosphate solution: Add an equal weight of 6 grams of calcium dihydrogen phosphate to 5 ml of ice water to make a calcium dihydrogen phosphate solution.

[0048] 5) Place 1) and 2) into a mixer and mix for 5 minutes. Then add the carrageenan solution and mix for 1 minute. Continue to add fish oil (8 grams) and calcium dihydrogen phosphate solution and mix for 2 minutes to make it evenly mixed. Mix the above evenly, weigh and divide into portions, place in a -20℃ refrigerator, take it out and let it cool to room temperature before feeding.

[0049] Example 1: Effects of plant polysaccharide functional feed on the growth and disease resistance of the spotted roe snail.

[0050] 1. Experimental Design

[0051] Experimental subjects: Healthy spotted snails with an initial average weight of approximately 2.47 ± 0.06 g were randomly divided into 4 groups, with 50 snails in each group and three replicates in each group, with the same number of snails in each replicate.

[0052] Experimental Groups:

[0053] (1) Control group: fed with basal diet without added polysaccharides;

[0054] (2) Astragalus polysaccharide group (APS): Astragalus polysaccharide was added to the basic feed at a rate of 2g / kg feed.

[0055] (3) Lycium barbarum polysaccharide group (LBP): Lycium barbarum polysaccharide was added to the basic feed at a rate of 2g / kg of feed;

[0056] (4) Lentinan group (LP): Lentinan was added to the basic feed at a rate of 2g / kg feed.

[0057] 2. Breeding conditions and feeding management

[0058] Aquaculture system: a continuous flow water aquaculture system, with water volume, salinity, temperature, dissolved oxygen, pH, etc., controlled within the suitable range for the spotted snail;

[0059] Breeding cycle: Continuous feeding for 2 months;

[0060] Feeding schedule: Feed once a day, and adjust the amount of feed according to the feeding situation to ensure that the food is basically eaten and a small amount is left over;

[0061] Water quality management: Regularly monitor various water quality indicators, and maintain the following standards: salinity 30.00±1.00‰, temperature 23.00±0.65℃, dissolved oxygen 6.05±0.05mg / L, pH 8.44±0.02; at the same time, change the water regularly to ensure water quality stability.

[0062] 3. Growth index measurement

[0063] At the end of the breeding season, 50 snails from each group were tested to measure their weight, shell height, and other indicators.

[0064] The formulas for calculating specific growth rate (SGR) and weight gain rate are as follows:

[0065] Weight Gain Rate (WGR) = (W t -W0) / W0×100%,

[0066] Where: W0: initial average weight (g); W t Final average weight (g).

[0067] Specific Growth Rate (SGR) = (ln W) t - ln W0) / t×100%,

[0068] Where: W0: initial average weight (g); W t Final average weight (g); t: number of days of rearing (d).

[0069] Results are expressed as mean ± standard deviation. Statistical analysis was used to compare differences between groups. Results are shown in [Figure 1]. Figures 1-3 .

[0070] The results showed that compared with the basic diet without added plant polysaccharides (control group), the treatment groups fed with functional diets containing Astragalus polysaccharides, Lycium barbarum polysaccharides and Lentinus edodes polysaccharides significantly increased the body weight of the spotted snail (P<0.05). The results indicate that functional diets containing plant polysaccharides can effectively improve the growth performance of the spotted snail.

[0071] 4. Vibrio harveyi challenge test

[0072] At the end of the rearing period, the survival rate of each group was 100%. After the rearing period, 20 individuals in good condition were randomly selected from each group and placed in independent containers for a Vibrio harveyi challenge experiment, at a ratio of 10:1. 8 Intramuscular injection of CFU / mL was administered to challenge the virus.

[0073] Record the time and number of deaths in each group, calculate the cumulative mortality rate, until all *Bellamya spp.* snails in the challenge experiment have died. Results are presented as curves or bar charts. Figures 4-5 .

[0074] The results showed that the average mortality time of the control group after challenge was significantly shorter than that of the three plant polysaccharide treatment groups (P < 0.05), while the average mortality time of the three treatment groups was significantly longer. The control group showed the fastest decline in survival rate, reaching 0% approximately 48 hours after challenge, while the plant polysaccharide treatment groups showed a significantly flatter decline and a significantly longer survival time (especially the APS group, which took nearly 88 hours to reach 0%). These results indicate that functional feed containing plant polysaccharides can effectively enhance the resistance of *Vibrio harveyi* to *Bellamya spp.* and strengthen its disease resistance.

[0075] 5. Intestinal microbiome testing

[0076] At the end of the rearing period and before the challenge test, 10 healthy individuals were randomly selected from each group for dissection and aseptic collection of intestinal tissue. The 16S rRNA gene sequences of intestinal microorganisms were determined using high-throughput sequencing to analyze community diversity. The relative abundance changes of beneficial and potentially pathogenic bacteria genera were compared between the control group and each polysaccharide group. The results are shown in [Table missing]. Figure 6 .

[0077] The results showed that, compared with the control group, treatment with the three polysaccharide immune enhancers significantly altered the dominant genera composition and community structure of the gut microbiota of *Bambusa angularis* (P < 0.05). In the control group, *Mycoplasma* was the dominant genus, accompanied by a certain proportion of *Nautella* and *Acinetobacter*. However, after polysaccharide treatment, significant replacement and rearrangement of dominant groups occurred: *Shimia* was significantly enriched in the APS group; the LBP group showed *Nautella* dominance with a high level of *Mycoplasma*; and the LP group showed extremely high *Nautella* dominance accompanied by an increase in *Acinetobacter*, while *Shimia* significantly decreased. These results indicate that plant polysaccharides, as functional immune enhancers, can significantly reshape the gut microecological structure of *Bambusa angularis* and alter the dominant bacterial genera's occupancy patterns.

[0078] 6. Substrate microbial testing

[0079] At the end of the rearing period, bottom sediments from each group of rearing ponds were collected, and DNA was extracted and 16S rRNA sequencing analysis was performed using the same procedure. Microbial diversity indices and dominant bacterial genera were statistically analyzed, with a focus on changes in functional microbiota related to organic matter decomposition, nitrogen and phosphorus cycling, and pathogenic bacteria. Results are shown below. Figure 7 .

[0080] The results showed that, compared with the control group without added polysaccharide immune enhancers, all three treatments significantly rearranged the dominant microbial composition of the substrate (sand) in the aquaculture tank. The substrate microbiota in the control group consisted of multiple heterotrophic degradation-related groups, with a relatively dispersed distribution of dominant genera (families) and a high proportion of "others." In contrast, the polysaccharide-treated groups showed a more significant enrichment of functional groups related to particle / biofilm attachment lifecycles and the degradation of complex organic matter (especially polysaccharides): the relative abundance of *Paracoccaceae* and *Ruegeria* was higher in the APS and LP groups, while *Bacteroidia* had a more prominent proportion in the LBP group. This suggests that polysaccharide input may have enhanced the key link of "attached heterotrophic bacteria - hydrolysis of high-molecular-weight organic matter" in the substrate. The results indicate that polysaccharides from different sources not only act on the host immune level but may also significantly shape the niche allocation and metabolic processes of substrate microorganisms by altering the carbon source structure and microenvironmental conditions of the substrate after uneaten feed / feces deposition, thus providing an important foundation for the microecological stability and health management of the aquaculture system.

[0081] 7. Results

[0082] Compared with the control group, the three polysaccharide treatment groups showed varying degrees of improvement in growth indicators such as final body weight and specific growth rate, all of which were statistically significant (P < 0.05).

[0083] In the challenge experiment, the cumulative mortality rate of the polysaccharide group was significantly lower than that of the control group, the survival rate was significantly improved (P<0.05), and the peak of disease incidence was delayed;

[0084] Gut microbiota analysis showed that plant polysaccharides significantly reshaped the gut microecological structure of *Bellamya spp.* (P < 0.05) and altered the dominant bacterial species' occupancy patterns.

[0085] Substrate microbial analysis showed that the polysaccharide group significantly shaped the ecological niche allocation and metabolic processes of the substrate microorganisms.

[0086] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A functional feed for the spotted roe snail containing plant polysaccharides, characterized in that, The functional feed for the spotted snail is composed of basic feed and plant polysaccharides.

2. The functional feed for the spotted snail according to claim 1, characterized in that, The plant polysaccharides include astragalus polysaccharide, wolfberry polysaccharide, and shiitake mushroom polysaccharide.

3. The functional feed for the spotted snail according to claim 1, characterized in that, Based on the weight of the aforementioned basic feed, the amount of plant polysaccharides added is 2-4 g / kg.

4. The functional feed for the spotted snail according to claim 1, characterized in that, The amount of plant polysaccharide added is 2g / kg.

5. The functional feed for the spotted snail according to claim 1, characterized in that, The basic feed includes fishmeal, plant protein sources, carbohydrates, oils, vitamins, and mineral premixes.

6. The functional feed for the spotted snail according to claim 5, characterized in that, The basic feed, by weight percentage, includes the following ingredients: 60% mackerel meat paste, 12% soy protein isolate, 9% α-starch, 4% meat and bone meal, 4% fish oil, 4.5% squid viscera powder, 3% calcium dihydrogen phosphate, 1% mixed vitamins, 0.5% choline chloride, and 2% binder.

7. The functional feed for the spotted snail according to claim 6, characterized in that, The adhesive is edible gelatin.

8. The application of the functional feed for the spotted worm snail as described in claim 1 in improving the growth performance of the spotted worm snail, its resistance to Vibrio harveyi infection, and optimizing the microbial community structure of the intestine and the culture substrate.

Images