Use of beta-glucan in promoting growth of ricefield eel and / or enhancing disease resistance of ricefield eel

Abstract

The invention belongs to the technical field of monopterus albus breeding, and particularly relates to application of beta-glucan in promoting monopterus albus growth and / or enhancing disease resistance of monopterus albus. It is found and verified that beta-glucan can significantly improve the growth of ricefield eel and the activity of digestive enzyme in the intestinal tract and improve the tissue structure of the intestinal tract; besides, the intestinal metagenome shows that by adding the beta-glucan, the proportion of beneficial bacteria (such as proteobacteria flora and / or spirillum flora) in the intestinal tracts of the ricefield eels can be increased, the proportion of harmful bacteria (such as desulfurization vibrio) can be reduced, then the intestinal environment is optimized, the growth of the ricefield eels is promoted, and the disease resistance of the ricefield eels is enhanced.

Description

Technical Field

[0001] The invention belongs to the technical field of rice field eel breeding, and particularly relates to the use of beta-glucan in promoting the growth of rice field eels and / or enhancing the disease resistance of rice field eels. Background Art

[0002] The eel (Monopterus albus) has a scaleless epidermal structure and a short digestive tract (the length of the intestine is only 0.6-0.8 times that of the body length), which results in a significantly lower absorption efficiency of exogenous active ingredients than conventional fish. The thickness of the epidermal mucus layer (50-80 μm) forms a natural permeability barrier, while the retention time of chyme in the intestine is short (≤4 hours) and the activity of digestive enzymes is low (amylase activity is less than 20% of that of cyprinid fish), which further limits the effect of functional additives. Although the currently commonly used technical solutions (such as microencapsulation embedding process) attempt to prolong the intestinal action time, they are not adapted to the unique digestive kinetics of the eel, and the actual bioavailability is less than 30%. In addition, heavy metal ions (such as Cu2+) in the aquaculture water are easily combined with additive ingredients, resulting in a loss rate of effective ingredients as high as 40%-50%.

[0003] In intensive eel farming, there is an inherent correlation between growth performance improvement and disease prevention and control needs: on the one hand, high-density farming (>200 eels / m 3 ) increases the risk of pathogen transmission (such as Edwardsiella infection rate increased by 60%); on the other hand, the dynamic balance between nutrient metabolism intensity and hepatopancreas detoxification capacity is easily broken. Studies have shown that there is a cross-inhibition effect between the TOR signaling pathway (regulating protein synthesis) and the NF-κB pathway (mediating pathogen response) of the eel, and a single technical solution is difficult to achieve synergistic activation of the two, resulting in a comprehensive increase in growth rate and disease resistance of less than 15%. Summary of the invention

[0004] The present invention discovers and verifies that beta-glucan can promote the growth of rice field eels and enhance the resistance of rice field eels to diseases.

[0005] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0006] In one aspect, the present invention provides a use of β-glucan, which includes:

[0007] (i) Use of β-glucan in promoting the growth of rice field eel;

[0008] (ii) Use of β-glucan in enhancing the disease resistance of rice field eel.

[0009] Preferably, the above uses include:

[0010] (a) Use of β-glucan in improving intestinal digestive enzyme activity;

[0011] (b) Use of β-glucan in improving intestinal tissue structure;

[0012] (c) Use of β-glucan in increasing beneficial intestinal bacteria and reducing harmful bacteria.

[0013] More preferably, among the above uses, use of β-glucan in improving intestinal tissue structure includes: use of β-glucan in increasing the length of intestinal villi.

[0014] More preferably, in the above use, the beneficial bacteria include Proteobacteria and / or Lachnospira; and / or the harmful bacteria include Desulfovibrio.

[0015] More preferably, in the above use, the Proteobacteria group includes Methylobacterium.

[0016] More preferably, in the above use, the β-glucan is derived from Euglena.

[0017] The beneficial effects of the present invention include: the present invention discovered and verified that β-glucan can significantly improve the growth and intestinal digestive enzyme activity of the eel, and improve the intestinal tissue structure; in addition, the intestinal metagenome shows that: adding β-glucan can increase the proportion of beneficial bacteria (such as Proteobacteria and / or Lachnospira) in the intestine of the eel, reduce the proportion of harmful bacteria (such as Desulfovibrio), and then optimize the intestinal environment, thereby promoting the growth of the eel and enhancing its resistance to disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The effect of β-glucan on the digestive enzyme activity in the intestine of the eel. In the bar graph, the same letters indicate no significant difference (P>0.05), and different letters indicate significant difference (P<0.05).

[0019] Figure 2 The effect of β-glucan on the intestinal tissue morphology of the eel, where (a) to (e) are the intestinal tissue morphology of the eel treated with different concentrations of β-glucan, (f) is the length of the intestinal villi of the eel treated with different concentrations of β-glucan, scale: 200μm; the bar graphs marked with different letters indicate significant differences among different treatments (P<0.05);

[0020] Figure 3 The distribution and composition of species in the intestine of each group of yellow eels;

[0021] Figure 4 It is a heat map of species abundance clustering;

[0022] Figure 5 This is the sample community distribution map of the species evolution tree. DETAILED DESCRIPTION

[0023] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0024] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0025] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0026] In the following examples, β-glucan is derived from euglena powder, that is, extracted from euglena, and is algae-derived β-glucan. The actual measured β-glucan content is 70%. The rice field eel and basic pellet feed (see Table 1) are all from eel breeding bases.

[0027] Table 1 Experimental feed formula and main nutrients (% dry matter basis)

[0028] Ingredients Composition Fishmeal 45 Soybeanmeal 12 Corn protein powder 5 Wheat flourWheatstarch 25 <![CDATA[Compound protein 1 > 2 Shrimpmeal 1.5 Soybean oil 2 Soybeanlecithin 2 <![CDATA[Premix Vitamin and mineral premix 2 > 1 Earthwormmeal 2 Dicalciumphosphate 2 Choline 0.3 Phytase 0.2 Nutritional level Proximate composition MoistureMoisture 12.37 Crude protein 44.78 Crudelipid 7.92 Ash 11.64

[0029] In the following example, the experimental diet is obtained by grinding the basic pellet feed into powder and then adding a corresponding amount of powdered β-glucan to mix; after fully mixing, pure water is added in a ratio of 5:1 and kneaded into a pancake-shaped dough.

[0030] 1. Experimental Design

[0031] (I) Group test

[0032] (1) The experimental eels were first raised for one week in aeration pond water with natural light, and the water temperature was maintained at 22-30°C. The eels were fed a basic diet that met their growth and comprehensive nutritional needs.

[0033] (2) After one week of feeding, 300 healthy eels (12.38±0.50g) were selected, 20 in each box, and randomly placed in 15 indoor breeding boxes (length 105cm, width 75cm, height 50cm); a total of 5 treatment groups were set up, with 3 replicates in each group; the treatment groups included: A1 group, A2 group, A3 group, A4 group and A5 group, among which, A1 group (control group) was fed with an experimental diet without β-glucan added to the basic diet, A2 group was fed with an experimental diet with 250mg / kg β-glucan added to the basic diet, A3 group was fed with an experimental diet with 500mg / kg β-glucan added to the basic diet, A4 group was fed with an experimental diet with 1000mg / kg β-glucan added to the basic diet, and A5 group was fed with an experimental diet with 2000mg / kg β-glucan added to the basic diet. The feeding amount each time was 5% of their body weight. The breeding water was fully aerated, naturally illuminated pond water, and the water temperature was maintained at 22-30℃; in addition, an eel nest was placed in each breeding box as a shelter, and the five groups of eels were fed the corresponding experimental diet once at 6 pm every day for 8 weeks.

[0034] (II) Sample collection

[0035] After being fed for 8 weeks, the eels were starved for 24 hours, and the terminal body weight (FBW) and body length were measured, and the weight gain rate (WGR), specific growth rate (SGR), survival rate (SR) and feed conversion rate (FCR) were calculated. After dissection, the weights of organs and liver were measured, and the condition factor (CF), liver-somatic index (HSI) and organ-somatic index (VSI) were calculated. Three intestinal samples of eels were randomly selected from each group to make sections for histomorphological observation. In addition, the remaining intestine was taken out and placed on ice, the intestine was cut open longitudinally, and the contents were scraped and placed in a 1.5 ml centrifuge tube and stored at -80°C for the determination of digestive enzyme activity and intestinal microorganisms.

[0036] In addition, the intestinal digestive enzyme activity was determined according to the instructions of the relevant kits, and the kits used in the experimental determination were purchased from Nanjing Jiancheng Bioengineeing Institute (Nanjing Jiancheng Bioengineeing Institute, China).

[0037] The intestinal tissue morphology observation method includes: preparing paraffin tissue sections, staining with hematoxylin and eosin (H&E), observing and photographing under a microscope, analyzing intestinal morphological parameters; and measuring the length of foregut villi using ImageJ software.

[0038] The intestinal microorganisms were determined using 16S rRNA gene sequencing technology. The gene sequence information of different bacteria was obtained through PCR amplification and sequencing, and then the types of bacteria were determined. The experiment was commissioned to Wuhan Servicebio Technology Co., Ltd.

[0039] 2. Experimental Results

[0040] 1. Effects of β-glucan on the growth of rice field eel

[0041] After the eels were continuously fed with different concentrations of β-glucan for 8 weeks, there was no disease or death. The growth results showed (see Table 2) that compared with the control group (A1), under the same initial body weight, the FBW, WGR, SGR and HSI of the eels in the β-glucan group were significantly increased (P<0.05), and the FCR was significantly reduced (P<0.05). That is, it can be seen from the above that the addition of β-glucan to the feed has no significant effect on SR, HIS and CF (P>0.05). In addition, the WGR and SGR of the eels in the A4 group reached the highest.

[0042] Table 2 Effects of β-glucan on the growth performance of rice field eel

[0043]

[0044]

[0045] (II) Effects of β-glucan on digestive enzyme activities in the intestine of rice field eel

[0046] The digestive enzyme activities of the eels in each group are shown in Figure 1 As shown in the figure, the results showed that among the related indicators of intestinal digestive enzyme activity, compared with the control group, trypsin activity and amylase activity in the intestines of eels in groups A3, A4 and A5 were significantly increased (P<0.05); lipase activity was only significantly increased in the intestines of eels in group A4 (P<0.05). In addition, the activities of the three digestive enzymes shown in the figure all reached the highest level in group A4.

[0047] (III) Effects of β-glucan on intestinal tissue morphology of rice field eel

[0048] The intestinal tissue morphology of each group of eels is as follows Figure 2 As shown, the results showed that compared with the control group (A1) ( Figure 2 a) Compared with A3( Figure 2 c) A4( Figure 2 d) and A5( Figure 2 The intestinal cross-sectional area of ​​the A4 group was larger and the number of intestinal villi was greater. In addition, the length of intestinal villi in the A4 group was significantly increased compared with the A1 group (P<0.05), while that in the A2( Figure 2 b), the length of intestinal villi of groups A3 and A5 had no significant change (P<0.05) (2f). In addition, the thickness of the muscular layer and goblet cells of the intestine of the eel had no significant effect among the groups (P>0.05).

[0049] (IV) Intestinal microbial status

[0050] (1) Effects of β-glucan on species distribution in the intestine of rice field eel

[0051] The distribution of species in the intestine of each group of eels is as follows Figure 3 As shown, the results showed that Firmicutes, Proteobacteria, Bacteroidota, Desulfobacterota, Actinobacteriota and Cyanobacteria were identified as dominant bacteria; the abundances of the five dominant phyla were 94.4%, 99.6%, 98.2%, 85.2% and 84.7% in groups A1, A2, A3, A4 and A5, respectively; among them, compared with group A1, the abundance ratio of Proteobacteria in groups A2, A3, A4 and A5 increased, and the abundance ratio of Desulfobacterota decreased. In addition, at the genus level, Clostridium was the dominant species in groups A1, A2 and A3. Compared with group A1, the abundance ratio of Methylobacterium in group A4 increased, while that of Clostridium decreased; the abundance ratio of Buchnera in group A5 increased, while that of Clostridium decreased.

[0052] (2) Species abundance cluster analysis Effects of adding β-glucan to feed on the intestinal microbiota of rice field eels

[0053] The community heat map was used to analyze the abundance of the top 30 genera in each group. Figure 3 As shown ( Figure 3In the figure, the sample and group information are displayed horizontally, and the species taxonomy is displayed vertically; the cluster tree on the left represents the clustering of species, while the cluster tree above represents the similarity of community composition between samples; the values ​​in the heat map are standardized to obtain the Z value of the relative abundance of species in each row; the heat map color gradient: from blue to red, it means that the relative proportion of a species in different samples is from low to high, which can only be used for comparison between samples, not for comparison between species). The results showed that compared with group A1, the relative abundance of Escherichia in group A2 increased, and the relative abundance of Plesiomonas in group A3 increased; in group A4, 7 bacteria: Cetobacterium, Akkermansia, Lachnospiraceae, Methylobacterium, Mucispirillumhe, Alistipes, unclassified The abundance of Oscillospiraceaewas increased. In addition, the abundance of 7 bacteria including Sphingomonas, Bacillus, Aeromonas, Buchnera, Candidatus, and unclassified Rhizobiaceae increased in group A5.

[0054] (3) Effects of β-glucan on the intestinal microbial community of rice field eels

[0055] The sample community distribution diagram of the species evolution tree (see Figure 4 In the figure, the legend in the upper right corner is the species name at the phylum level, the inner circle is the species evolutionary tree, and the same phylum in the inner circle shows the same color; the outer circle indicates the relative abundance ratio of the species in different samples / groups) provides information on the evolutionary relationship and relative abundance ratio between species. Among them, Firmicutes and Bacteroidoeta have the farthest evolutionary relationship. Firmicutes, Actinobacteriota, Desulfobacterota, and Bacteroidoeta have a large relative abundance ratio in group A4, and Proteobacteria has a large relative abundance ratio in group A5.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. The uses of β-glucan include: (i) Use of β-glucan in promoting the growth of rice field eel; (ii) Use of β-glucan in enhancing the disease resistance of rice field eel.

2. The use according to claim 1, characterized in that include: (a) Use of β-glucan in improving intestinal digestive enzyme activity; (b) Use of β-glucan in improving intestinal tissue structure; (c) Use of β-glucan in increasing beneficial intestinal bacteria and reducing harmful bacteria.

3. The use according to claim 2, characterized in that: The use of β-glucan in improving intestinal tissue structure includes: the use of β-glucan in increasing the length of intestinal villi.

4. The use according to claim 2, characterized in that: The beneficial bacteria include Proteobacteria and / or Lachnospira; and / or the harmful bacteria include Desulfovibrio.

5. The use according to claim 4, characterized in that The Proteobacteria group includes Methylobacterium.

6. The use according to any one of claims 1 to 5, characterized in that β-Glucan is derived from Euglena.

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