CN112970977A - Feed additive for promoting intestinal microecological recovery of fish, preparation method and application - Google Patents
Feed additive for promoting intestinal microecological recovery of fish, preparation method and application Download PDFInfo
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Images
Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
- A01K61/13—Prevention or treatment of fish diseases
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/20—Animal feeding-stuffs from material of animal origin
- A23K10/26—Animal feeding-stuffs from material of animal origin from waste material, e.g. feathers, bones or skin
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/105—Aliphatic or alicyclic compounds
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention discloses a feed additive for promoting intestinal microecological restoration of fish, a preparation method and application, and belongs to the technical field of microorganism application. The feed additive comprises intestinal contents separated from fish intestinal tissue, and the thallus content of the intestinal contents is 1 × 1010‑1×1012one/mL. The application of the feed additive comprises any one of the following components: 1) repairing damage of fish intestinal structures caused by antibiotics; 2) fish sausage for improving qualityThe structure of the tract flora. The feed additive prepared by extracting intestinal contents from the intestinal tissues of the fishes is added into the fish feed, so that the damage of the intestinal structures of the fishes and the disturbance of intestinal flora caused by using antibiotics can be improved, the immunity of the fishes is improved, the use of the antibiotics can be reduced, and the sustainable development of aquaculture or ornamental fishery is realized.
Description
Technical Field
The invention relates to the technical field of microorganism application, and relates to a feed additive for promoting intestinal microecological recovery of fish, a preparation method and application thereof.
Background
In recent years, the aquaculture industry in China is rapidly developed, and great economic and social benefits are generated. However, as the scale of aquaculture continues to expand, diseases caused by various pathogens frequently occur, and pose a great threat to the sustainable development of the aquaculture industry. At present, the use of antibiotics for preventing and treating bacterial diseases of fishes is still taken as a main prevention and treatment way, but the long-term use of the antibiotics can cause the problems of drug residues, drug resistance of pathogenic bacteria and the like. Moreover, the use of antibiotics can also obviously influence the composition structure and diversity of intestinal microorganisms, thus causing intestinal microecological disorder and creating favorable conditions for the invasion and proliferation of pathogenic bacteria. In addition, one of the characteristics of the aquatic animal medicine is the group medication, when the antibiotics are used for treating diseases, most healthy individuals are exposed to the medicine environment with therapeutic dose, so that the side effects of the antibiotics can occur not only in the diseased individuals but also in the healthy individuals. Therefore, the problem of finding antibiotic alternatives for the treatment of diseases in aquatic animals is imminent.
The intestinal tract of an animal is an important organ in the body, in which a large number of microorganisms are attached. These microorganisms constitute a complex ecosystem that maintains the homeostasis of the gastrointestinal tract and functions by producing bioactive metabolites. Research shows that the functions of the intestinal microorganisms include decomposing nutrients and providing the host with essential substances for vital activities such as enzymes, amino acids and vitamins. In addition to this, numerous studies have demonstrated that: the intestinal microorganisms play a great role in protecting the health of the host, can occupy attachment sites and consume nutrient resources, and provide a physical barrier against pathogen invasion through competitive exclusion. However, currently, there is no relevant application of animal intestinal contents in feed additives in aquaculture industry.
Disclosure of Invention
The invention aims to provide a feed additive for promoting intestinal microecological restoration of fishes, a preparation method and application thereof, and aims to solve the problems in the prior art.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a feed additive for promoting the intestinal microecological recovery of fish, which comprises intestinal contents separated from intestinal tissues of fish, wherein the viable bacteria content of the intestinal contents is 1 multiplied by 1010-1×1012one/mL. The main genera and proportion thereof contained therein are 16.0-53.0% of cetacea (Cetobacterium), 21.5-31.0% of Streptococcus (Streptococcus), 4.7-24.0% of Vibrio (Vibrio), 2.2-10.0% of Aeromonas (Aeromonas), 0.053-4.6% of Rosemobacter (Rothia), 0-3% of Porphyromonas (Porphyromonas), 0.13-2.9% of Clostridium (Fusobacterium), 0.061-2.4% of Actinomyces (Actinomyces), 0.0079-2.0% of Acinetobacter (Acinetobacter), 0.071-2.0% of Pseudomonas (Pseudomonas), 0.024-1.8% of Haemophilus (Haemophilus), 0.021-1.8% of Streptococcus (Shteus), 0.098% of Geophilus (Gevorella), 0.7-1.8% of Pseudomonas (Gevorax).
Preferably, the intestinal content is derived from healthy koi that has lost ornamental value.
The invention also provides a preparation method of the feed additive for promoting intestinal microecological recovery of fish, which comprises the following steps:
s1: collecting fish intestinal tissues, placing the fish intestinal tissues in a precooled PBS solution, and removing intestinal surface fat, blood and redundant tissues;
s2: extruding out the content of the intestinal tract under the aseptic condition of the intestinal tract tissue treated by the S1, and homogenizing to obtain homogenate;
s3: mixing the homogenate with a sterile PBS solution, filtering, and collecting intestinal content filtrate;
s4: adding glycerol into the obtained intestinal content filtrate, and packaging to obtain feed additive.
Preferably, the homogenization conditions in S2 are: homogenizing at 50-100rpm at 4 deg.C for 30-90 s.
Preferably, the homogenate of S3 and the sterile PBS solution are mixed in a volume ratio of 1-2: 1 and mixing.
Preferably, the final concentration of glycerol in S4 is 10-15%.
The invention also provides application of the feed additive for promoting intestinal microecological restoration of fish, which comprises any one of the following components:
1) repairing damage of fish intestinal structures caused by antibiotics;
2) improving the structure composition of fish intestinal flora.
Preferably, the method for applying is as follows: diluting the feed additive with normal saline, uniformly spraying the diluted feed additive into feed, and uniformly mixing the feed additive and the feed for feeding fishes.
The invention discloses the following technical effects:
according to the invention, intestinal contents of healthy fishes are extracted to prepare the feed additive, and then the feed additive is added into feed according to a certain proportion and used for feeding fishes, so that intestinal mucosa repair can be accelerated, intestinal mucosa permeability is reduced, and repair of intestinal injury of fishes is realized; can also improve the structure composition of fish intestinal flora and promote the recovery of disturbed intestinal microecology of fish to normal level. Experiments prove that the invention can improve the damage of the intestinal structure and the intestinal flora disturbance of fishes caused by using antibiotics, improve the immunity of the fishes, reduce the use of the antibiotics and realize the sustainable development of aquaculture or ornamental fishery.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 shows the effect of florfenicol on the morphology and barrier function of koi intestinal mucosa; a: morphological structure of foregut, midgut and hindgut of control group (CG-7); b: morphology of the front, middle and rear intestines of the Florfenicol (FL) -treated group; c: a statistical map of intestinal histology scores; d: intestinal tissue muscularis thickness; p <0.05, P < 0.01;
FIG. 2 is a graph of the number of OTU of koi intestinal flora Veen in florfenicol group and control group;
FIG. 3 is a graph of the effect of florfenicol on koi gut microbial alpha-diversity index; a: chao1 index group difference boxplot; b: shannon index interclass difference boxplot; c: ACE index inter-group difference boxplot; d: simpson index interclass difference boxplot;
FIG. 4 is a graph of the effect of florfenicol on koi gut microbial beta-diversity; a: a master coordinate analysis chart; b: a non-metric multi-dimensional scale; c: a UPGMA distance map;
FIG. 5 is a graph of the effect of florfenicol on the gut microbiome composition structure of koi carps; a: a microbial building block at the phylum level; b: at the genus level, the constituent structure of the microorganism;
FIG. 6 shows the results of the difference analysis of LEfSe; a: a differential flora clade graph; b: LDA value distribution histogram;
FIG. 7 shows the recovery effect of feeding feed additive on the appearance and barrier function of intestinal mucosa of koi; a: morphogram of foregut, midgut and hindgut of control group (CG-14); b: morphology of the front, middle and rear intestine of the natural recovery (NC) group; c: morphological structure of front, middle and rear intestine of feed additive (FM) group; d: a bowel histology score map; e: intestinal muscle layer thickness. P <0.05, P < 0.01;
FIG. 8 is a Veen graph showing the distribution of koi intestinal microorganisms OTU in the feed additive group, the natural recovery group and the control group;
FIG. 9 is the effect on the alpha-diversity index of koi intestinal microorganisms after feeding with a feed additive; a: chao1 index group difference boxplot; b: shannon index interclass difference boxplot; c: ACE index inter-group difference boxplot; d: simpson index interclass difference boxplot;
FIG. 10 is a graph showing the effect on the beta-diversity of koi intestinal microorganisms after feeding a feed additive; a: a master coordinate analysis chart; b: a non-metric multi-dimensional scale; c: a UPGMA distance map;
FIG. 11 shows the effect of feeding a feed additive on the composition of koi intestinal microorganisms; a: at the phylogenetic level, the microbial building blocks; b: at the genus level the microbial constitutive structure;
FIG. 12 shows groups of significant differences in abundance among the LEfSe assay feed additive groups, the natural restoration groups, and the control groups; a: a group with significantly different abundances of the feed additive group and the natural recovery group; b: a group with remarkably different abundances between the natural recovery group and the control group; c: and (3) a group with remarkably different abundances between the feed additive group and the control group.
Detailed Description
The present invention will now be described in detail by way of examples, which should not be construed as limiting the invention but as providing more detailed descriptions of certain aspects, features and embodiments of the invention.
The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
The fancy carps used in the following examples are fancy carps without ornamental value, which are offered by the cultivation base of the ornamental fish in the refined Wuzhen district of Xiqing, Tianjin, and the selection criteria of the fancy carps without ornamental value are as follows: selecting the fancy carps which do not have ornamental value, such as the fancy carps which are active in swimming, quick in action and positive in eating, and have dark and impure body surface color and luster, and blurred color block edges.
Example 1 analysis of the flora composition structure of intestinal contents of healthy koi, comprising the following steps:
firstly, 9 healthy fancy carps without ornamental value are taken. Narcotizing Cyprinus carpiod with MS-222, wiping body surface with 75% ethanol, cutting abdominal cavity from anus along abdomen with ophthalmic scissors, ligating intestine ends with sterile cotton thread, separating intestinal tract tissue, placing in culture dish containing PBS (precooling at 4 deg.C), and cleaning intestinal tract surface fat, blood and excessive tissue.
And respectively extruding and collecting the contents in each fish intestinal tract, then cutting open the intestinal tract, and scraping the intestinal mucosa. The intestinal contents of every 3 fishes are combined into a sample, put into a 50mL centrifuge tube and mixed evenly, and stored at-80 ℃ for later use.
Extracting the genome DNA of the intestinal microorganisms by a conventional method, detecting by using 1% agarose gel electrophoresis, and then carrying out 16S rDNA high-throughput sequencing. The PCR amplification product was purified using a SanPrep column DNA gel extraction kit (Biotech, USA). Finally, the PCR products were sequenced using Illumina MiSeq platform. And performing pedigree classification on the effective sequences, and counting the dominant bacterial groups of the intestinal flora and the relative abundance of the dominant bacterial groups at different classification levels. The identified main genera are cetacea (Cetobacterium), 21.5-31.0% Streptococcus (Streptococcus), 4.7-24.0% Vibrio (Vibrio), 2.2-10.0% Aeromonas (Aeromonas), 0.053-4.6% Rosemobacter (Rothia), 0-3% Porphyromonas (Porphyromonas), 0.13-2.9% Clostridium (Fusobacterium), 0.061-2.4% Actinomyces (Actinomyces), 0.0079-2.0% Acinetobacter (Acinetobacter), 0.071-2.0% Pseudomonas (Pseudomonas), 0.024-1.8% Haemophilus (Haemophilus), 0.021-1.8% Streptococcus (Nutoella), 0.41-1.8% Pseudomonas (Germinatum), 0.095-1.8% Pseudomonas (Pseudomonas), 0.095-1.8% Pseudomonas sp), and 0.095-1.8% Pseudomonas sp.
Example 2 preparation method and application of feed additive for promoting intestinal microecological recovery of fish
The preparation method comprises the following steps:
firstly, healthy koi with no ornamental value is used as an intestinal content donor. Narcotizing Cyprinus carpiod with MS-222, wiping body surface with 75% ethanol, cutting abdominal cavity from anus along abdomen with ophthalmic scissors, ligating intestine ends with sterile cotton thread, separating intestinal tract tissue, placing in culture dish containing PBS (precooling at 4 deg.C), and cleaning intestinal tract surface fat, blood and excessive tissue.
Subsequently, the intestinal tissue was transferred to an ultraclean bench and the contents were squeezed out into a 50mL centrifuge tube. Homogenizing with homogenizer at 4 deg.C and 50-100rpm for 30s-1.5min (preferably 100rpm for 1 min). The homogenized intestinal contents were transferred as soon as possible to an anaerobic incubator, after which the contents were homogenized: PBS volume ratio 2: 1 dilute the homogenate and mix well.
The homogenate diluted and mixed with PBS was filtered twice with a single layer of sterile gauze and then filtered through a 80-150 mesh (preferably 100 mesh) stainless steel mesh to remove undigested particulate matter and minimize loss of functional components associated with intestinal contents.
The above procedures are carried out at 20-25 deg.C (preferably 25 deg.C), and the preparation process minimizes the exposure time of the contents to air. In order to improve the survival rate of microorganisms in the intestinal contents of the koi in the process of cryopreservation, glycerol is added into the obtained intestinal content filtrate, the final concentration of the glycerol is 10% -15% (preferably 12%), and the content range of viable bacteria in the obtained intestinal contents is ensured to be 1 x 10 by a microscopic direct counting method10-1012And (4) packaging the filtrate per mL according to the use requirement. The obtained mixture is the feed additive for promoting the intestinal microecological restoration of the fish. Then storing at-80 ℃ for later use.
The application method comprises the following steps: repeated freezing and thawing should be avoided during application, the feed additive thawed at 37 deg.C is inverted and mixed well before feeding, after normal saline is diluted properly, the mixture is sprayed into feed uniformly by using spray can (viable bacteria content is 1 × 10)7-108CFU/kg), appropriate stirring may be used for feeding.
Example 3 Effect of antibiotics (florfenicol) on the mucosal Structure of the intestinal tract of Koi
(1) Preparation of antibiotic (containing florfenicol) feed: the florfenicol dry powder is accurately weighed, then the dosage is selected according to the recommended dosage in the specification and added into common feed, after being uniformly mixed and stirred, the mixture is prepared into granulated feed with the particle size of 3mm in a preparation machine, and after being air-dried, the granulated feed is subpackaged by sealing bags and is stored in a shade place.
(2) Grouping experiments and feeding: dividing the fancy carps into two groups at random, feeding common feed (CG-7) to the control group, feeding Feed (FL) containing florfenicol to the treatment group, sampling on the 7 th day, taking 11 fancy carps which are fed with the common feed and added with the florfenicol feed, separating intestinal contents, and preserving intestinal tissues. The feeding is carried out twice a day in a feeding amount of 2% of the weight of the fish during the duration of the culture experiment.
(3) Intestinal mucosa microstructure: according to the method described by Sun et al (2019), the intestinal tissue is divided into anterior, medial and posterior 3 sections, each of which takes 3 segments. Placed in Bouin's fixative for histological HE staining. Followed by gradient dehydration, paraffin embedding, sectioning and staining. During microscopic examination, 3 tissue slices are selected from each intestinal section, 3 visual fields are selected from each slice to measure the intestinal wall thickness, and the damage degree of intestinal mucosa of different treatment groups is scored according to the damage scoring standard of the intestinal tissue.
The results of the florfenicol effect on the structure of the intestinal mucosa are shown in figure 1. The results show that: koi intestinal mucosa developed significant lesions after 7 days of Florfenicol (FL) feeding compared to control (CG-7) (see fig. 1A-B). Among them, the most significant are: a large number of neutrophils and other inflammatory cells infiltrate the mucosa and submucosa of the anterior, middle and posterior intestines; the mucosal layers of the foregut and hindgut were slightly separated from the lamina propria and the lamina propria collapsed. In addition, the villi in the middle and rear intestine are loosely arranged, accompanied by dulling, dissolution and shedding of intestinal villi. Proves that after the florfenicol is fed, the intestinal mucosa of the koi has certain damage.
Similarly, as can be seen from fig. 1C, the florfenicol group had a tissue damage score of 5, and the above results all demonstrated that the koi intestinal mucosa was damaged to some extent after feeding florfenicol. Furthermore, as can be seen from fig. 1D, the koi intestinal tissue muscle layer thickness was significantly decreased in the florfenicol group compared to the control group (P <0.01), indicating that intestinal mucosal permeability was increased after the florfenicol was fed.
Example 4 Effect of antibiotics (florfenicol) on the gut micro-ecology of koi
(1) Collecting samples: wiping the body surface with 75% ethanol, dissecting the abdominal cavity from the anus along the abdomen, ligating the two ends of the intestine with sterile cotton threads, separating the intestine, placing in a petri dish, extracting the contents of the intestine into a 50mL centrifuge tube, diluting with sterile PBS, mixing, and storing at-80 deg.C.
(2) Analyzing the diversity of the intestinal flora: extracting the genome DNA of the koi intestinal tract microorganism according to the instruction of the kit, performing 16S rDNA sequence amplification, performing high-throughput sequencing, and analyzing the change of the koi intestinal tract flora after feeding florfenicol.
The results of the intestinal flora analysis are shown in FIGS. 2-6, specifically:
as shown in FIG. 2, 3261 OTUs (97% similarity) were obtained after two groups of Koi intestinal tract samples were sequenced and clustered, and 2590 and 2127 OTUs were obtained in the control group (CG-7) and the florfenicol group (FL), wherein the number of the OTUs in the two groups was 1134 and 671, respectively, and the number of the OTUs in the two groups was 1456. Compared with a control group, the number of koi intestinal tract OTUs is remarkably reduced after the florfenicol is fed (P < 0.05).
Species abundance and diversity are expressed as alpha-diversity index (Chao1, ACE, Shannon and Simpson index). As can be seen from FIG. 3, the diversity and abundance of koi intestinal flora were significantly reduced (P <0.01) compared to the control group when florfenicol was fed.
The beta-diversity analysis includes principal coordinate analysis (PCoA) of nonlinear structure which can reflect ecological data and NMDS analysis which reflects community structure difference. As can be seen from FIG. 4, the FL group and CG-7 group samples were clustered and away from each other. And after the UPGMA method is used for carrying out cluster analysis on the samples, the obtained result is consistent with PCoA and NMDS, and the CG-7 group and the FL group are respectively gathered into one group. This indicates that, compared with the control group, the koi intestinal flora structure is significantly changed after feeding florfenicol.
The analysis results of the microbial composition structure are shown in FIG. 5, and the dominant phyla of intestinal microbes of Koi in CG-7 group and FL group are the same. Although florfenicol has no effect on the major dominant phyla species, the relative abundance of the dominant phyla has changed. The relative abundance of the group FL proteobacteria, Bacteroides and firmicutes was reduced and the relative abundance of the fusobacteria was increased compared to CG-7. In the main genus composition structure, two groups of results are changed, and vibrio, aeromonas and unidentified Cyanobacteria are CG-7 main dominant genera; while cetacea, Pseudomonadaceae and Schoenbergii are the major dominant genera of the FL group.
The key differential cohorts at phylum and genus levels after feeding florfenicol-containing feed were further analyzed using a linear differential significance algorithm (LEfSe, LDA >4), see fig. 6A, B. The results show that the relative abundance of bacteroidetes, firmicutes and cyanobacteria is significantly reduced after florfenicol administration; and the abundance of the genera faecalis, unidentified Cyanobacteria, aeromonas, bacteroides and vibrio in the FL group is also obviously reduced.
The results show that the abundance and diversity of koi intestinal flora are remarkably reduced and the flora structure is also remarkably changed after feeding the feed containing florfenicol for 7 days, which indicates that the florfenicol causes disorder of the koi intestinal microecological structure.
Example 5 feed additive repair of intestinal mucosal Structure of Koi
(1) Grouping experiments and feeding: the rest fancy carps fed to the florfenicol-containing group are randomly divided into two groups, namely a feed additive group (FM) and a natural recovery group (NC). Feed additive containing feed additive for fancy carp of feed additive group (viable bacteria content is 5 × 10)7CFU/kg), feeding common feed and normal saline by a natural recovery group, and continuously culturing until the 14 th day for sampling. Taking the rest fancy carps of the control group fed with common feed as blank control (CG-14), continuously feeding according to the unchanged condition, similarly sampling at 14 days, taking 18 fancy carps in each group, separating intestinal contents, and preserving intestinal tissues. The fish were fed twice daily for the duration of the breeding experiment at a feed rate of 2% of the weight of the fish.
(2) Intestinal mucosal microstructure: according to the method described by Sun et al (2019), the intestinal tissue is divided into anterior, medial and posterior 3 sections, each of which takes 3 segments. Placed in Bouin's fixative for histological HE staining. Followed by gradient dehydration, paraffin embedding, sectioning and staining. During microscopic examination, 3 slices are selected for each intestinal section, 3 visual fields are selected for each slice to measure the intestinal wall thickness, and the damage degree of intestinal mucosa of different treatment groups is scored according to the damage scoring standard of intestinal tissues.
As shown in FIG. 7, the damage degree was recovered in the FM group and NC group, but the NC group was slightly damaged, and the villus was irregularly arranged, as compared with the CG-14 group (control) (see FIGS. 7A, B, and C). The CG-14 tissue injury score was 0, and the average tissue injury score (foregut: 4; midgut 4; hindgut: 3 points) in the NC group was significantly higher than that in the FM group (foregut: 3; midgut 3; hindgut: 2 points) (P < 0.01; see FIG. 7D), indicating that the feed additive was more effective in alleviating the damage caused to the intestinal mucosa by florfenicol. In addition, according to histopathological result analysis, the thickness of muscle layers of the foregut, midgut and hindgut of koi in FM group was significantly increased (P <0.01) compared to NC group (see fig. 7E), indicating that the permeability of koi intestinal tract was decreased after feeding with the feed additive.
Example 6 Effect of feed additives on restoration of intestinal micro-ecological homeostasis of Koi
(1) Collecting samples: the same as in (1) of example 3;
(2) analyzing the diversity of the intestinal flora: the same as in (2) of example 3.
The results of the intestinal flora analysis are shown in fig. 8-12, specifically:
as can be seen from FIG. 8, 2754 OTUs (97% similarity) were obtained after sequencing and clustering koi intestinal microorganism samples in CG-14 group, FM group and NC group. Wherein 1618 OTUs are obtained in the CG-14 group, 2292 OTUs are obtained in the NC group, and 1451 OTUs are obtained in the FM group. The CG-14, NC and FM groups had a total number of OTUs of 929 and unique numbers of OTUs of 225, 696 and 155, respectively. Therefore, the number of the intestinal microorganisms OTUs in the NC group is higher than that in the CG-14 group and the FM group, and the CG-14 group and the FM group have no obvious difference.
Comparison of the α -diversity indices found (see FIG. 9): the indexes of Chao1, ACE, Shannon and Simpson in the NC group are significantly higher than those in the CG-14 group and the FM group (P <0.01), and no significant difference exists between the CG-14 group and the FM group (P > 0.05). This indicates that the feed additive is more beneficial to restoring the stable state of the intestinal flora structure.
In the β -diversity analysis, it is known from the PCoA analysis and the NMDS analysis (see fig. 10): the samples of the CG-14 and FM groups are clustered and are far from the majority of the samples of the NC group. While the samples from the NC group are more scattered in the graph, which is consistent with the clustering results in the UPGMA graph (fig. 10C), which shows all samples from the CG-14 and FM groups, and 6 samples from the NC group grouped into one; while the remaining 12 samples of the NC group are scattered over each other. Therefore, it is further proved that only a small part of individual samples are gathered together with the control group sample after the florfenicol is fed for 7 days through natural recovery (without adding feed additives), and the intestinal flora of the small part of samples is presumed to be recovered; and the samples fed with the feed additive are recovered, which shows that the feed additive can accelerate the recovery of the koi intestinal flora steady state.
Analysis of the microbial composition structure, as shown in fig. 11A: the dominant phyla of the 14-day control group (CG-14), the NC group and the FM group are all fusobacteriales, proteobacteria, bacteroidetes, firmicutes and actinomycetes, and the sum of the relative abundances accounts for more than 90% of the abundance of each phylum. However, the proportion of the dominant bacteria in each group is not the same. Compared with the control group and the FM group, the relative abundance of each phylum has no obvious difference; however, in the control group and the FM group, the relative abundance of other dominant phyla is significantly lower than that of the NC group, except that the relative abundance of the Clostridia is significantly higher than that of the NC group. As shown in fig. 11B, at the genus level, the main constitutional structures of the three groups of genera are significantly different.
The LEfSe analysis results are shown in fig. 12, showing: compared with the NC group, the FM group has higher relative abundance of cetacean, pseudoxanthomonas, scheimpfera and the like, and has lower relative abundance of streptococcus, bifidobacterium, sphingomonas and the like; compared with CG-14 group, the relative abundance of the FM group Shen's bacillus, the H.phagemid, the pseudo-red bacillus and the red bacillus is obviously increased, and the abundance of the Bacteroides, the Shewanella, the Aeromonas and the Vibrio is obviously reduced; compared with CG-14, the relative abundance of the NC group Shewanella, whale bacillus and the like is low, and the relative abundance of the stenotrophomonas, helicobacter, Bacteroides and the like is high.
The results show that after the feed additive is fed for 7 days, florfenicol-induced intestinal flora disturbance of the koi is reversed, so that the composition structure of intestinal flora is restored to a level similar to that of normal koi, and the diversity among treated samples is homogenized. Most of the intestinal micro-ecological states of the koi which is not fed with the feed additive are not recovered to the normal level. The analysis of microbial flora structure shows that the colonization of pathogenic microorganisms in the intestinal tract can be reduced by feeding the feed additive. Therefore, the feed additive can change the function of intestinal flora by changing the structure of the intestinal flora and further promote the steady-state recovery of intestinal micro-ecology of fishes.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (8)
1. A feed additive for promoting intestinal microecological recovery of fish, which comprises intestinal contents separated from intestinal tissues of fish, wherein the thallus content of the intestinal contents is 1 × 1010-1×1012one/mL.
2. The feed additive for promoting intestinal microecological restoration of fish according to claim 1, wherein said intestinal contents are derived from healthy koi that has lost ornamental value.
3. A method for preparing a feed additive for promoting intestinal microecological restoration of fish according to claim 1 or 2, which comprises the steps of:
s1: collecting fish intestinal tissues, placing the fish intestinal tissues in a precooled PBS solution, and removing intestinal surface fat, blood and redundant tissues;
s2: extruding out the content of the intestinal tract under the aseptic condition of the intestinal tract tissue treated by the S1, and homogenizing to obtain homogenate;
s3: mixing the homogenate with a sterile PBS solution, filtering, and collecting intestinal content filtrate;
s4: adding glycerol into the obtained intestinal content filtrate, and packaging to obtain feed additive.
4. The method for preparing a feed additive for promoting intestinal microecological restoration of fish according to claim 3, wherein the homogenizing conditions in S2 are: homogenizing at 50-100rpm at 4 deg.C for 30-90 s.
5. The method for preparing a feed additive for promoting intestinal microecological restoration of fish according to claim 3, wherein said homogenate of S3 and said sterile PBS solution are mixed in a volume ratio of 1-2: 1 and mixing.
6. The method for preparing a feed additive for promoting intestinal microecological restoration of fish according to claim 3, wherein the final concentration of glycerol in S4 is 10-15%.
7. The application of the feed additive for promoting intestinal microecological restoration of the fishes is characterized by comprising any one of the following components:
1) repairing damage of fish intestinal structures caused by antibiotics;
2) improving the structure composition of fish intestinal flora.
8. The application of claim 7, wherein the method of applying is: diluting the feed additive with normal saline, uniformly spraying the diluted feed additive into feed, and uniformly mixing the feed additive and the feed for feeding fishes.
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Application publication date: 20210618 Assignee: TIANJIN AOLV SHENNONG TECHNOLOGY Co.,Ltd. Assignor: TIANJIN AGRICULTURAL University Contract record no.: X2024980002951 Denomination of invention: Feed additives, preparation methods, and applications for promoting the restoration of fish gut microbiota Granted publication date: 20220322 License type: Common License Record date: 20240315 |