Application of chitosan oligosaccharide in aquatic feed additive and feed

Abstract

The invention belongs to the technical field of aquaculture, and discloses application of chitosan oligosaccharide in preparation of fish feed for promoting fish growth and improving disease resistance of grass carp bodies. Chitosan oligosaccharide is found to be capable of promoting fish growth and improving the disease resistance of grass carp bodies. In the embodiment, chitosan oligosaccharide is added as an immunopotentiator, so that non-specific antioxidant enzyme expression of the liver of the grass carp can be effectively induced, and the autoimmunity of the grass carp is improved. The chitosan oligosaccharide can improve the intestinal flora structure of the grass carp. In the embodiment, the relative abundance of the clostridium of the 3% chitosan oligosaccharide addition group is remarkably higher than that of the control group, and the 3% chitosan oligosaccharide addition group is the most important bacterial genus of the clostridium, so that the chitosan oligosaccharide has a remarkable effect on improving the intestinal flora structure of the grass carp.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture, in particular to application of chitosan oligosaccharide in aquatic feed additives and feed. Background Art

[0002] The liver is the main digestive and immune organ of fish, and it undertakes the important physiological functions of secreting digestive juices, detoxification and immunity. The intestine is not only an important organ for fish digestion and absorption, but also has important immune functions. Therefore, maintaining a healthy and stable liver and intestine state is crucial for the normal life activities of fish.

[0003] In recent years, with the intensive and large-scale development of aquaculture, the breeding density has increased significantly, resulting in the deterioration of the water environment and waste of feed. At the same time, breeding diseases have occurred frequently, seriously restricting the sustainable development of aquaculture. As the country's management of aquaculture becomes increasingly strict, it is becoming more and more urgent to seek alternatives to antibiotics to treat aquatic animal diseases. At present, both at home and abroad, the use of probiotics, natural products of biological origin, etc. as immune enhancers for aquatic animals is explored. By compounding with compound feed, it can regulate the intestinal microecological balance of aquatic animals, protect the liver and intestines, enhance the biological immunity of the subjects, and thus prevent and control the occurrence of diseases.

[0004] Grass carp is one of the aquatic animals with the highest farmed output in my country, with a national output of 5.941 million tons in 2023. However, with the development of intensive farming, diseases such as grass carp hemorrhagic fever and Aeromonas hydrophila infection have occurred frequently, which has brought serious adverse effects on grass carp farming. Oligochitosan (OCS) is an oligosaccharide obtained by enzymatic or chemical methods, in which 2-10 glucosamines are connected by β-1,4-glycosidic bonds. It has the characteristics of small molecular weight, easy absorption and good water solubility. It can be effectively absorbed by the intestines, can inhibit the growth of fungi and bacteria, and is an ideal substitute for antibiotics.

[0005] At present, existing patents use chitosan oligosaccharide as one of the composite raw materials to prepare compound feeds for aquatic animal immune enhancement. Cao Tingfeng et al. have developed a chitosan oligosaccharide aquatic feed and used for shrimp farming. Although it has a certain promotion to shrimp growth, it has no report on intestinal protection and immunity enhancement (CN106901065A). In addition, current work is basically to prepare aquatic feed after adopting multiple substances to compound, and chitosan oligosaccharide is only as one of the components, and its function cannot be independently displayed, and the chitosan oligosaccharide physicochemical properties added are also unclear and unified, lacking the reference standard for subsequent use, and current work lacks the influence research of chitosan oligosaccharide on the intestinal flora of farmed animals.

[0006] In view of the above problems, the present invention prepares a grass carp compound feed containing only a single component of chitosan oligosaccharide as an immune enhancer, and the chitosan oligosaccharide used has clear physical and chemical characteristics. The research results show that chitosan oligosaccharide (OCS) is non-toxic and harmless to farmed animals, and can not only promote the growth of grass carp, but also improve the nonspecific immune activity of grass carp liver, improve the intestinal microecological environment, directionally change the intestinal flora structure, and increase the proportion of beneficial bacteria. It is a suitable immune enhancer for aquatic animals. Summary of the invention

[0007] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an application of chitosan oligosaccharide in aquatic feed additives and feed.

[0008] The technical solution adopted by the present invention to solve its technical problem is:

[0009] Application of chitosan oligosaccharide in the preparation of fish feed that promotes fish growth and simultaneously improves the disease resistance of grass carp.

[0010] Application of chitosan oligosaccharide in the preparation of fish feed that promotes fish growth and improves the intestinal flora structure of grass carp.

[0011] The application of chitosan oligosaccharide in the preparation of fish feed that can simultaneously improve fish immunity, maintain fish liver and intestine health and stabilize intestinal microecology.

[0012] Furthermore, the fish is grass carp.

[0013] A fish feed prepared by using chitosan oligosaccharide, which can improve fish immunity, maintain fish liver and intestine health and intestinal microecological stability, and the preparation method thereof comprises the following steps:

[0014] The chitosan oligosaccharide is fully dissolved in an ethanol solution with a volume concentration of 75%, and a sterile PBS buffer solution with a molar concentration of 0.01M and a pH of 7.2-7.4 is added to a dissolving solution with a volume of 50 mL to prepare a chitosan oligosaccharide mother solution; the chitosan oligosaccharide mother solution is added to a common feed dry powder, and after being fully mixed, 1%-7% of chitosan oligosaccharide by mass percentage is added to a basic feed to prepare a chitosan oligosaccharide isonitrogen and isolipid feed; before the feed is prepared, all raw materials are crushed through a 60-mesh sieve, and the raw materials are fully mixed for 10 minutes according to the feed formula, and after adding distilled water and mixing for 10 minutes, a sinking expanded feed with a particle size of 5 mm and a length of 10 mm is prepared by an expander to obtain a fish feed.

[0015] Furthermore, the formula of the common feed dry powder is as follows:

[0016] Element content(%) Element content(%) Fishmeal 9 Rice bran 8 Soybean meal 22.5 Cottonseed Meal 15 Rapeseed Meal 9 Soybean Oil 2 Bran 7 Calcium Hydrogen Phosphate 1.5 flour 12 Premix 1 corn 13 Crude fat Crude Protein Coarse ash Feed nutritional level 6.02％ 32.83％ 7.54％

[0017] Among them, the above percentages are all mass percentages.

[0018] Furthermore, the basic indicators of the chitosan oligosaccharide are as follows:

[0019] index Numeric index Numeric Moisture 5.85％ Convertible glucosamine content 81.83％ Ash 0.23％ Average molecular weight of chitosan oligosaccharide 1231 Water insoluble matter 0.07％ Monosaccharide content 1.3％ pH 5.42

[0020] Among them, the above percentages are all mass percentages.

[0021] Application of the fish feed as described above in grass carp breeding.

[0022] The advantages and positive effects achieved by the present invention are:

[0023] 1. The present invention found that chitosan oligosaccharide can promote fish growth and improve the disease resistance of grass carp. For example, chitosan oligosaccharide added as an immunopotentiator in the examples can effectively induce the expression of nonspecific antioxidant enzymes in the liver of grass carp and improve the autoimmunity of grass carp.

[0024] 2. The present invention found that chitosan oligosaccharide can improve the intestinal flora structure of grass carp. As shown in the example, the relative abundance of intestinal Fusobacteria in the group with 3% chitosan oligosaccharide addition was significantly higher than that in the control group. As the most important genus of Fusobacteria, it can be seen that chitosan oligosaccharide has a significant effect on improving the intestinal flora structure of grass carp.

[0025] 3. The present invention found that chitosan oligosaccharide can improve the homeostasis and health level of grass carp intestinal flora. As shown in the embodiment, the ratio of Firmicutes / Bacteroidetes in the 3% chitosan oligosaccharide addition group was significantly increased compared with the blank control group, and this ratio is one of the important indicators of intestinal homeostasis.

[0026] Compared with traditional antibiotics, it is safer, greener and more efficient, with no obvious toxicity or side effects.

[0027] 4. The purpose of the present invention is to discover and utilize new uses of chitosan oligosaccharides in aquaculture to solve the problems existing in the prior art, such as improving fish immunity, maintaining fish liver and intestine health and intestinal microecological stability.

[0028] 5. The present invention clarifies the applicability of chitosan oligosaccharides as fish feed additives; provides the appropriate amount of chitosan oligosaccharides added as fish feed additives; and proves through experiments that parthenolide has the effect of significantly enhancing the maintenance of liver and intestine health and intestinal microecological stability of grass carp. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a graph showing changes in grass carp weight in the present invention;

[0030] Figure 2 This is a graph showing changes in SOD activity in the liver of grass carp in the present invention;

[0031] Figure 3 This is a graph showing changes in CAT activity in the liver of grass carp in the present invention;

[0032] Figure 4 This is a graph showing changes in LZM activity in the liver of grass carp in the present invention;

[0033] Figure 5 This is a graph showing changes in AKP activity in the liver of grass carp in the present invention;

[0034] Figure 6 This is a graph showing changes in the Shannon-Wiener index of intestinal microorganisms of grass carp in the present invention;

[0035] Figure 7 This is a diagram of the main dominant categories of intestinal microorganisms of grass carp in the present invention;

[0036] Figure 8 This is the enrichment heat map of the main dominant categories of grass carp intestinal microorganisms in the present invention. DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0038] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0039] Application of chitosan oligosaccharide in the preparation of fish feed that promotes fish growth and simultaneously improves the disease resistance of grass carp.

[0040] Application of chitosan oligosaccharide in the preparation of fish feed that promotes fish growth and improves the intestinal flora structure of grass carp.

[0041] The application of chitosan oligosaccharide in the preparation of fish feed that can simultaneously improve fish immunity, maintain fish liver and intestine health and stabilize intestinal microecology.

[0042] Preferably, the fish is grass carp.

[0043] A fish feed prepared by using chitosan oligosaccharide, which can improve fish immunity, maintain fish liver and intestine health and intestinal microecological stability, and the preparation method thereof comprises the following steps:

[0044] The chitosan oligosaccharide is fully dissolved in an ethanol solution with a volume concentration of 75%, and a sterile PBS buffer solution with a molar concentration of 0.01M and a pH of 7.2-7.4 is added to a dissolving solution with a volume of 50 mL to prepare a chitosan oligosaccharide mother solution; the chitosan oligosaccharide mother solution is added to a common feed dry powder, and after being fully mixed, 1%-7% of chitosan oligosaccharide by mass percentage is added to a basic feed to prepare a chitosan oligosaccharide isonitrogen and isolipid feed; before the feed is prepared, all raw materials are crushed through a 60-mesh sieve, and the raw materials are fully mixed for 10 minutes according to the feed formula, and after adding distilled water and mixing for 10 minutes, a sinking expanded feed with a particle size of 5 mm and a length of 10 mm is prepared by an expander to obtain a fish feed.

[0045] Preferably, the formula of the common feed dry powder is as follows:

[0046]

[0047]

[0048] Among them, the above percentages are all mass percentages.

[0049] Preferably, the basic indicators of the chitosan oligosaccharide are as follows:

[0050] index Numeric index Numeric Moisture 5.85％ Convertible glucosamine content 81.83％ Ash 0.23％ Average molecular weight of chitosan oligosaccharide 1231 Water insoluble matter 0.07％ Monosaccharide content 1.3％ pH 5.42

[0051] Among them, the above percentages are all mass percentages.

[0052] Application of the fish feed as described above in grass carp breeding.

[0053] Specifically, the relevant preparation and detection are as follows:

[0054] 1. Feed preparation and experimental grouping

[0055] (1) Feed preparation: In this experiment, different doses of chitosan oligosaccharide (OCS) were fully dissolved in a 75% ethanol solution, and a sterile PBS buffer solution (pH = 7.2-7.4) with a molar concentration of 0.01M was added to a volume of 50 mL of the dissolved solution to prepare an OCS mother solution. The OCS mother solution was added to a common feed dry powder (its specific formula is shown in Table 1), and after being fully mixed, 0, 1%, 3%, 5% and 7% of OCS were added to the basic feed to prepare isonitrogenous and lipid feeds with different OCS addition gradients. Before feed preparation, all raw materials were crushed through a 60-mesh sieve, and the raw materials were fully mixed for 10 minutes according to the feed formula. After adding an appropriate amount of distilled water and mixing for 10 minutes, a sinking expanded feed with a particle size of 5 mm and a length of about 10 mm was prepared using an extruder. After the experimental feed was air-dried at room temperature, it was packaged in a black sealed bag and stored at -20°C for later use.

[0056] Table 1 Basic feed formula for grass carp (percentages are by mass percentage.)

[0057]

[0058]

[0059] Table 2 Basic indicators of chitosan oligosaccharides used (percentages are all mass percentages.)

[0060] index Numeric index Numeric Moisture 5.85％ Convertible glucosamine content 81.83％ Ash 0.23％ Average molecular weight of chitosan oligosaccharide 1231 Water insoluble matter 0.07％ Monosaccharide content 1.3％ pH 5.42

[0061] (2) Grouping and management of experimental fish: Healthy and lively grass carp with no lesions on the body surface were selected for the experiment. The grass carp had a body length of 11.81±0.91cm and a body weight of 24.8±1.33g. Before the formal experiment began, the grass carp was placed in a recirculating aquaculture system with a water temperature of 25±1℃, dissolved oxygen ≥6mg / L, and pH 7.3-7.5 for 7 days. After the temporary culture, the 120 grass carps were randomly divided into 5 groups, with 24 parallels in each group. The 5 groups of grass carp were fed with feeds containing 0 (CK), 1%, 3%, 5% and 7% OCS respectively. The entire breeding experiment period was 28 days. During the breeding experiment, the daily feeding amount was 2% of the fish body weight, and it was fed once at 8:00 and 17:00 every day.

[0062] 2. Sample Collection

[0063] At the beginning of the experiment and every 7 days thereafter, grass carp were weighed (n=3), and liver samples of grass carp in each experimental group (n=3) were taken to measure nonspecific immune activity indicators. Based on the experimental results and cost analysis, 3% was determined to be the most economical amount of OCS. At the beginning and end of the experiment, the intestinal microbial composition of grass carp in the CK group and the 3% group was analyzed (n=3).

[0064] 3. Determination of liver nonspecific antioxidant enzyme activity

[0065] The collected grass carp liver tissue was added with 9 volumes of sterile saline (the mass concentration of saline was 0.9%) at a ratio of weight (g): volume (ml) = 1:9 to prepare a 10% tissue homogenate. Centrifuge (3000r, 10min, 4°C) and take the supernatant. The activity of superoxide dismutase (SOD), catalase (CAT), lysozyme (LZM) and alkaline phosphatase (AKP) was detected using the Nanjing Jiancheng kit.

[0066] 4. Analysis of intestinal microbial composition and structure

[0067] Intestinal samples of grass carp from the CK group and the 3% group were collected at the beginning and end of the experiment. The specific operation was to squeeze the hindgut contents of each grass carp into a sterile centrifuge tube, collect and store them in a -80℃ ultra-low temperature refrigerator for intestinal microbiota analysis. The samples were sent to Novogene Technology Co., Ltd. (Tianjin) for 16s amplicon sequencing.

[0068] Related test results:

[0069] 1. Weight changes of grass carp

[0070] In each experimental group, the weight of grass carp increased continuously as the experiment progressed. Among them, the weight growth trend of grass carp in the group with 3% chitosan oligosaccharide addition was more obvious, and the weight gain was significantly higher than that of other experimental groups, indicating that 3% is the most suitable addition amount to promote the growth of grass carp ( Figure 1 ).

[0071] 2. Nonspecific antioxidant enzyme activity in grass carp liver

[0072] The above method was used to determine the activities of nonspecific antioxidant enzymes SOD, CAT, LZM and AKP in grass carp liver. The experimental results are as follows: Figure 2-5 As shown. The results showed that when chitosan oligosaccharides were added as an immune enhancer in compound feed, the SOD activity of grass carp liver did not change significantly, but the activities of CAT, LZM and AKP were significantly improved. The CAT activity of the 5% addition group reached a maximum of 45.43U / mgprot, which was 4.42 times that of the control group, the LZM activity of the 3% addition group reached a maximum of 0.18U / mgprot, which was 3.8 times that of the control group, and the AKP activity of the 5% addition group reached a maximum of 5.44U / gprot, which was 1.8 times that of the control group. This shows that chitosan oligosaccharides can effectively induce the expression of nonspecific antioxidant enzymes in the liver of grass carp and improve the autoimmunity of grass carp. Combined with the above results, since 3% chitosan oligosaccharides have the best effect on promoting the growth of grass carp, and considering the economic cost factor, 3% is the optimal addition amount of chitosan oligosaccharides in compound feed.

[0073] 3. Composition and structure of grass carp intestinal microorganisms

[0074] As the experiment progressed, the effect of chitosan oligosaccharide on the diversity of grass carp intestinal microbial communities gradually emerged. Compared with the control group, the Shannon-Wiener index of grass carp intestinal microbial communities in the experimental group with 3% chitosan oligosaccharide was significantly reduced on the 28th day of the experiment ( Figure 6 ), indicating that chitosan oligosaccharides caused a decrease in the diversity of intestinal flora in grass carp.

[0075] During the experiment, the main dominant phyla of intestinal microorganisms of grass carp in each experimental group were Proteobacteria, Fusobacteriota, Firmicutes, Antinobacteriota and Bacteroidetes. At the beginning of the experiment, Proteobacteria dominated the intestinal flora of grass carp, followed by Fusobacteria and Firmicutes. However, as the experiment progressed, the relative abundance of the dominant phyla of intestinal microorganisms of grass carp in each experimental group changed significantly. On the 28th day, Fusobacteria dominated, especially in the 3% chitosan oligosaccharide treatment group, where the dominance of Fusobacteria was more obvious. Fusobacteria is an important microorganism to maintain the intestinal health of grass carp. In addition, the relative abundance of Proteobacteria decreased sharply, especially in the 3% chitosan oligosaccharide treatment group, while the relative abundance of Firmicutes in each experimental group increased. ( Figure 7 ).

[0076] As the experiment progressed, the intestinal flora of grass carp treated with 3% chitosan oligosaccharide was significantly enriched with Firmicutes microorganisms. At the end of the experiment (28 days), the ratio of Firmicutes to Bacteroidetes reached 63.6 times that of the control group. This ratio is an important indicator of intestinal homeostasis and health. The higher the value, the higher the intestinal homeostasis and health level. The results show that chitosan oligosaccharide can significantly improve the intestinal homeostasis and health level of grass carp ( Figure 8 ).

[0077] Although there are many studies on the use of chitosan oligosaccharides as immune enhancers in existing patent technologies, there are few reports on the effects on the intestinal microbial community structure of farmed animals, especially on the directional induction of beneficial bacteria. Therefore, the present invention has a groundbreaking innovative significance.

[0078] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. The application of chitosan oligosaccharide in the preparation of fish feed that promotes fish growth and improves the disease resistance of grass carp.

2. The application of chitosan oligosaccharide in the preparation of fish feed that promotes fish growth and improves the intestinal flora structure of grass carp.

3. The application of chitosan oligosaccharides in the preparation of fish feed that can simultaneously improve fish immunity, maintain fish liver and intestine health and intestinal microecological stability.

4. The use according to any one of claims 1 to 3, characterized in that: The fish is grass carp.

5. A fish feed prepared by using chitosan oligosaccharide, which improves fish immunity, maintains fish liver and intestine health and intestinal microecological stability, characterized by: The preparation method thereof comprises the following steps: The chitosan oligosaccharide is fully dissolved in an ethanol solution with a volume concentration of 75%, and a sterile PBS buffer solution with a molar concentration of 0.01M and a pH of 7.2-7.4 is added to a dissolving solution with a volume of 50 mL to prepare a chitosan oligosaccharide mother solution; the chitosan oligosaccharide mother solution is added to a common feed dry powder, and after being fully mixed, 1%-7% of chitosan oligosaccharide by mass percentage is added to a basic feed to prepare a chitosan oligosaccharide isonitrogen and isolipid feed; before the feed is prepared, all raw materials are crushed through a 60-mesh sieve, and the raw materials are fully mixed for 10 minutes according to the feed formula, and after adding distilled water and mixing for 10 minutes, a sinking expanded feed with a particle size of 5 mm and a length of 10 mm is prepared by an expander to obtain a fish feed.

6. The fish feed according to claim 5, characterized in that: The formula of the common feed dry powder is as follows: Among them, the above percentages are all mass percentages.

7. The fish feed according to claim 5 or 6, characterized in that: The basic indicators of the chitosan oligosaccharide are as follows: Among them, the above percentages are all mass percentages.

8. Use of the fish feed according to any one of claims 5 to 7 in grass carp breeding.

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