Application of tannin in preparation of drugs and / or functional products for eliminating displacement and / or recessive infection of salmonella in livers and lungs of poultry

Abstract

The invention belongs to the technical field of animal feed, and particularly relates to application of tannin in preparation of drugs and / or functional products for eliminating displacement and / or recessive infection of salmonella in livers and lungs of poultry. The montanin disclosed by the invention can effectively inhibit displacement, colonization and recessive infection of salmonella in liver and lung of poultry, alleviate continuous injury of the liver and lung caused by the salmonella, reduce the recurrence rate after salmonella infection of the poultry, and improve the health level and growth performance of the poultry.

Description

Technical Field

[0001] The invention belongs to the technical field of animal feed, and in particular relates to the application of tannin in the preparation of medicines and / or functional products for eliminating the translocation and / or latent infection of salmonella in the liver and lungs of poultry. Background Art

[0002] Salmonella is a common pathogen in poultry farming and an important pathogen of zoonotic diseases, which seriously threatens poultry and human health. Clinically, Salmonella infection can cause poultry diseases such as white diarrhea, typhoid and paratyphoid. The morbidity and mortality of young poultry are both high. Adult poultry, especially laying poultry, are often latent and continue to carry and excrete the bacteria after being infected with Salmonella, which contaminates eggs and seriously endangers human food safety. The course of livestock and poultry infection with Salmonella is generally 4 to 7 days. Animals that are not treated in time after infection but survive will continue to carry the bacteria in their bodies. However, studies have found that Salmonella will shift and lurk in the liver and lungs, which not only seriously impairs growth performance, but more importantly, it continues to excrete toxins, contaminating livestock and poultry products, seriously affecting food safety and public health, and has become a major problem in breeding and food hygiene.

[0003] Tannins, also known as tannic acid, are polyphenolic secondary metabolites widely found in a variety of plants (such as legumes, sorghum seeds, grapes, persimmons, oak trees, etc.). Tannins easily interact with proteins in the digestive tract of animals, producing a bitter taste, affecting the palatability of food and reducing the absorption efficiency of nutrients. Therefore, tannins have always been considered as "anti-nutritional factors" in feed. In recent years, researchers have found that tannins have astringent and anti-diarrhea effects on animal intestines, can inhibit intestinal bacteria and viruses, and enhance the antioxidant and anti-cancer capabilities of animals. Adding an appropriate amount of tannins to the feed can effectively improve the health level and production performance of livestock and poultry.

[0004] At present, under natural breeding conditions, the problems of latent infection and high recurrence rate of Salmonella in poultry and strong antibiotic resistance have not been solved. There are no reports on the elimination of organ displacement of the gut-liver axis and gut-lung axis and persistent latent infection after Salmonella infection. The elimination of organ displacement of the gut-liver axis and gut-lung axis and persistent latent infection after Salmonella infection is of great significance for reducing the harm of Salmonella clinically. Summary of the invention

[0005] The purpose of the present invention is to provide the use of tannin in the preparation of a drug and / or functional product for eliminating the translocation and / or latent infection of Salmonella in the liver and lungs of poultry. The tannin of the present invention can effectively inhibit the translocation and latent infection of Salmonella in the liver and lungs of poultry.

[0006] The invention provides application of tannin in preparing medicine and / or functional product for eliminating translocation and / or latent infection of salmonella in poultry liver and lung.

[0007] As a preferred embodiment, the source of the tannin includes plant extracts.

[0008] As a preferred embodiment, the plant-derived extract includes chestnut extract.

[0009] As a preferred embodiment, the extraction method of the plant-derived extract includes organic solvent extraction, ultrasonic extraction, steam distillation, organic solvent extraction, supercritical fluid extraction or enzyme conversion.

[0010] The present invention also provides a poultry feeding method, comprising the step of adding tannin to poultry feed and then feeding the poultry, wherein the amount of tannin added to the feed is 17.5-450 mg / kg.

[0011] As a preferred solution, the tannin is added in the form of a tannin premix, and the tannin content in the tannin premix is ​​35wt% to 90wt%.

[0012] As a preferred embodiment, the tannin premix comprises chestnut extract, and the tannin content in the chestnut extract is 50wt% to 90wt%.

[0013] As a preferred embodiment, the content of tannin in the chestnut extract is 60wt% to 80wt%.

[0014] As a preferred embodiment, the feeding cycle is 21 to 60 days.

[0015] As a preferred embodiment, the poultry includes chickens, ducks and geese.

[0016] Beneficial effects: The present invention provides the use of tannin in the preparation of a drug and / or functional product for eliminating the translocation and / or latent infection of Salmonella in the liver and lungs of poultry. The tannin of the present invention can effectively inhibit the translocation, colonization and latent infection of Salmonella in the liver and lungs of poultry, alleviate the continuous damage of the liver and lungs caused by Salmonella, reduce the recurrence rate of Salmonella infection in poultry after tolerance, and improve the health level and growth performance of poultry.

[0017] The tannin application method provided by the present invention can inhibit the colonization of bacteria in the liver and lungs of young poultry after salmonella resistance, reduce the continuous immune damage caused by bacteria to the liver and lungs of young poultry, reduce the use of antibiotics, reduce the risk of recurrence of salmonella infection after resistance, improve their health status, improve growth performance, and reduce salmonella contamination in meat and egg products. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0019] Figure 1 The figure shows the effect of chestnut tannin on the morphological structure of lung and ileum of broiler chickens infected with Salmonella in Experimental Example 1, wherein A is the macroscopic appearance of ileum and H&E staining of pathological sections (original magnification ×100); B is the macroscopic appearance of lung and H&E staining of pathological sections (original magnification ×300); C is villus length (VH, μm); D is crypt depth (CD, μm); E is villus crypt ratio (VH:CD); F is MPO content in lung; G is LPS content in serum;

[0020] Figure 2 The figure shows the effect of chestnut tannin on the intestinal barrier function induced by Salmonella in Experimental Example 1, where A is the immunofluorescence double staining of Claudin-1 and MUC-2 (original magnification ×100); BC are the fluorescence intensity statistics of Claudin-1 and MUC-2, respectively; DH are the relative mRNA expressions of Claudin-1, Occludin, ZO-1, MUC-2 and SIgA in ileal tissues, respectively;

[0021] Figure 3 This is a diagram showing the effect of chestnut tannin on Salmonella-induced ileal inflammation in broiler chickens in Experimental Example 1, where A is the immunofluorescence of IL-1β and TNF-α in ileal tissue of broiler chickens (original magnification ×100); BC are the statistics of fluorescence intensity of IL-1β and TNF-α, respectively; DH are the relative mRNA expressions of IL-1β, TNF-α, IL-6, IFN-γ and IL-4, respectively;

[0022] Figure 4 The effect of chestnut tannin on the ileal microbiota of broiler chickens infected with Salmonella in Experimental Example 1, where A is the Venn diagram of intestinal microorganisms; BC are Shannon and Simpson indexes, respectively; D is PCoA for β diversity analysis; E is the composition of the microbiota at the phylum level; F is the comparison of the dominant phylum levels of CON, SE and SE+TA(L) groups at the phylum level;

[0023] Figure 5 The effect of chestnut tannin on the microbial composition at the order level and genus level in the ileum of broiler chickens infected with Salmonella in Experimental Example 1, wherein A is the microbial community composition at the order level; B is the comparison of the dominant bacteria in the CON, SE and SE+TA(L) groups at the order level; C is the microbial community composition at the genus level; D is the comparison of the dominant bacteria in the CON, SE and SE+TA(L) groups at the genus level;

[0024] Figure 6This is the effect of chestnut tannin on the lung microbial composition of broiler chickens infected with Salmonella in Experimental Example 1, where A is the Venn diagram of intestinal microorganisms; BC are Shannon and Simpson indexes, respectively; D is the PCoA of β diversity analysis; E is the microbial community composition at the phylum level; F is the comparison of dominant bacteria at the phylum level;

[0025] Figure 7 This is a diagram showing the effect of chestnut tannin on the microbial composition at the order level and genus level in the lungs of broiler chickens infected with Salmonella in Experimental Example 1, wherein A is the microbial community composition at the order level; B is the comparison of dominant bacteria at the order level; C is the microbial community composition at the genus level; and D is the comparison of dominant bacteria at the genus level;

[0026] Figure 8 The effect of chestnut tannin on lung inflammation induced by Salmonella infection in Experimental Example 1, wherein A is the immunofluorescence detection of NF-κB in the lung (original magnification × 100), B is the fluorescence intensity statistics of NF-κB; CE are the relative mRNA expressions of TLR4, NF-κB and MYD88, respectively; F is the immunofluorescence detection of IL-1β and TNF-α in the lung (original magnification × 100); GH are the fluorescence intensity statistics of IL-1β and TNF-α, respectively; IQ are the relative mRNA expressions of IL-1β, TNF-α, IL-4, IL-6, IL-17, IL-22, IL-10, TGF-β and IFN-γ, respectively;

[0027] Fig. 9 The effect of chestnut tannin on Salmonella-induced oxidative lung injury and macrophage polarization in Experimental Example 1, wherein A is immunofluorescence double staining of CD11C and CD206 (original magnification ×100); BC is fluorescence intensity statistics of CD11C and CD206; DG is mRNA expression of INOS, ARG1, CCL2 and CCL4; H is immunofluorescence detection of ROS in the lung (original magnification ×400); I is fluorescence intensity statistics of ROS; J is MDA content in the lung; K is CAT content in the lung; LN is mRNA expression of NRF2, HO-1 and NQO1;

[0028] Fig.10 The effect of chestnut tannin on serum biochemistry and jejunal morphology of broilers infected with Salmonella in Experimental Example 2, wherein A is serum ALT; B is serum AST; C is serum IgG; D is serum IgM; E is histopathology of jejunal tissue; F is villus length; G is crypt depth; H is villus-crypt ratio; I is goblet cell count;

[0029] Fig.11The figure is the effect of chestnut tannin on the jejunal barrier function of broiler chickens infected with Salmonella in Experimental Example 2, wherein A is the immunofluorescence staining analysis of Cluadin-1 and MUC2 protein expression in jejunal tissue; B is the image analysis result of Cluadin-1 immunofluorescence staining; C is the image analysis result of MUC2 immunofluorescence staining; DH are the mRNA expression levels of jejunal ZO-1, Occludin, Claudin-1, MUC2, and IL-1β, respectively;

[0030] Fig.12 The effect of chestnut tannin on the morphological structure of the liver of broiler chickens infected with Salmonella in Experimental Example 2, where A is macroscopic photography of the liver, histopathological variation of liver tissue, inflammatory cell infiltration (red arrow); red blood cells (black arrow); B is liver index; C is liver LPS; D is liver lysozyme;

[0031] Fig.13 The effect of chestnut tannin on the jejunal microbiota of broiler chickens infected with Salmonella in Experimental Example 2, where A is a VENN diagram showing the unique and shared OTUs in each group of jejunal microbiota; B is the Chao index; C is the Shannon index; D is the ACE index; E is the principal coordinate analysis (PCoA) of OUT; F is the composition analysis of the intestinal microbiota at the phylum level;

[0032] Fig.14 The effect of chestnut tannin on the phylum and genus level bacterial flora composition of jejunum of broiler chickens infected with Salmonella in Experimental Example 2, where A is the abundance level of Firmicutes at the phylum level; B is the abundance level of Bacteroidetes; C is the ratio of Firmicutes to Bacteroidetes; D is the composition of microorganisms at the genus level; E is the abundance of Bacteroidetes, Shigella and Lactobacillus at the genus level;

[0033] Fig.15 The co-culture system of poultry Bacteroides and chestnut tannin in Experimental Example 2, wherein A is a flow chart of the co-culture system; B is the isolation of Bacteroides in the BEE culture medium plate; C is the developmental tree of the isolates; D is the growth of Bacteroides after treatment with different concentrations of chestnut tannin; E is the concentration of tannic acid in the co-cultured bacterial solution; F is the concentration of gallic acid in the co-cultured bacterial solution;

[0034] Fig.16 The effect of chestnut tannin on the liver microbial composition of broiler chickens infected with Salmonella in Experimental Example 2, wherein A is the bacterial culture of liver tissue in LB and MAC medium; B is the colony count in MAC medium; C is a VENN diagram showing the unique and shared OUTs in each group of liver microbiota; D is the principal coordinate analysis (PCoA) of OUTs; E is the Pielou index; F is the Shannon index; G is the Simpson index;

[0035] Fig.17The effect of chestnut tannin on the microbial composition at the family level and genus level in the liver of broiler chickens infected with Salmonella in Experimental Example 2, wherein A is the percentage of the abundance of the microbial community at the family level; B is the percentage of the abundance of the microbial community at the genus level; C is the abundance of the genus Salmonella in each group; D is the abundance of Escherichia-Shigella in each group;

[0036] Fig.18 The effects of chestnut tannin on Salmonella-induced liver immune damage in broiler chickens in Experimental Example 2, wherein A is the expression of CD80 and CD206 proteins in the liver analyzed by immunofluorescence staining; B is the immunofluorescence intensity of CD80 in the liver; C is the immunofluorescence intensity of CD206 in the liver; D is the immunofluorescence staining analysis of IL-1β protein expression; E is the immunofluorescence staining analysis of TNF-α protein expression in the liver; F is the immunofluorescence intensity of IL-1β in the liver; G is the immunofluorescence intensity of TNF-α in the liver; HQ are the mRNA expression levels of IFN-γ, TNF-α, IL-18, IL-6, IL-1, INOS, IL-1β, CCL4, CCL1, and IL-10, respectively. DETAILED DESCRIPTION

[0037] The invention provides application of tannin in preparing medicine and / or functional product for eliminating translocation and / or latent infection of salmonella in poultry liver and lung.

[0038] The tannin source of the present invention includes plant-derived extracts; the extraction methods of the extracts include organic solvent extraction, ultrasonic extraction, steam distillation, organic solvent extraction, supercritical fluid extraction or enzyme conversion; the plant-derived extract of the present invention includes chestnut extract; the chestnut tannin (Castaneatannins) of the present invention is a natural plant tannin substance in chestnut (Castanea spp.) wood, which is widely present in the bark, wood and leaves of chestnut trees, and has active functions such as anti-oxidation, antibacterial and antiviral.

[0039] As a specific embodiment, adding chestnut tannins to feed can reduce intestinal inflammation induced by Salmonella infection and alleviate the inflammatory response and oxidative damage induced by Salmonella in the lungs; chestnut tannin treatment can significantly alleviate liver damage caused by Salmonella and increase the richness and diversity of species in the liver; Salmonella colonization in the liver can destroy immune function and stimulate inflammation, and chestnut tannins can alleviate this situation.

[0040] The present invention also provides a poultry feeding method, comprising feeding the poultry after adding tannin to the feed, wherein the amount of tannin added to the feed is 17.5-450 mg / kg. In a specific embodiment, the amount of tannin added can be 17.5 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 110 mg / kg, 120 mg / kg, 130 mg / kg, 140 mg / kg, 150 mg / kg, 160 mg / kg, 170 mg / kg, 180 mg / kg, 190 mg / kg, 200 mg / kg, 210 mg / kg, 220 mg / kg , 230mg / kg, 240mg / kg, 250mg / kg, 260mg / kg, 270mg / kg, 280mg / kg, 290mg / kg, 300mg / kg, 310mg / kg, 320mg / kg, 330mg / kg, 340mg / kg, 350mg / kg, 360mg / kg, 370mg / kg, 380mg / kg, 390mg / kg, 400mg / kg, 410mg / kg, 420mg / kg, 430mg / kg, 440mg / kg or 450mg / kg. As a specific embodiment, adding tannin to feed can prevent Salmonella infection and its intestinal-liver axis and intestinal-lung axis displacement, prevent and eliminate latent infection of Salmonella, and reduce Salmonella contamination of eggs.

[0041] The tannin described in the embodiment of the present invention is added in the form of a tannin premix, and the content of tannin in the tannin premix is ​​35wt% to 90wt%; in a specific embodiment, the content of tannin can be 35wt%, 40wt%, 45wt%, 50wt%, 55wt%, 60wt%, 65wt%, 70wt%, 75wt%, 76.4wt%, 80wt%, 85wt% or 90wt%; as a specific embodiment, the tannin premix includes chestnut extract; as a specific embodiment, adding chestnut tannin to feed can significantly alleviate Salmonella-induced intestinal and lung lesions in broilers, and adding chestnut tannin to broiler diet can significantly improve the morphological structure of the lungs.

[0042] The feeding cycle of the present invention is 21 days to 60 days. In a specific embodiment, the feeding cycle can be 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 61 days, 62 days, 63 days, 64 days, 65 days, 66 days, 67 days, 68 days, 69 days, 70 days, 71 days, 72 days, 73 days, 74 days, 75 days, 76 days, 77 days, 78 days, 79 days, 80 days 5 days, 56 days, 57 days, 58 days, 59 days or 60 days; the poultry described in the present invention includes chickens, ducks and geese; as a specific embodiment, the addition of chestnut tannins in the feed significantly improves the growth performance of broilers 10 days after infection with Salmonella, and significantly reduces the colonization of Salmonella in the liver and lungs. Chestnut tannins can intervene and regulate the composition of the intestinal microbiota, enhance the integrity of the intestinal barrier, inhibit the colonization of pathogens in the liver and lungs and the polarization of M1 macrophages, and reduce inflammatory damage to the liver and lungs.

[0043] In order to further illustrate the present invention, the application of the tannin provided by the present invention in the preparation of a drug and / or functional product for eliminating the translocation and / or latent infection of Salmonella in the liver and lungs of poultry is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Unless otherwise specified, the present invention has no special requirements for the preparation raw materials, and commercially available products known to those skilled in the art can be used.

[0045] Example 1

[0046] The specific steps of extracting tannins by organic solvent extraction are as follows: Raw material preparation: Select fresh chestnut wood, dry it in a constant temperature drying oven at 60°C, crush it, pass it through a 60-mesh sieve after crushing, obtain chestnut wood powder, extract it with methanol, weigh 100g chestnut wood powder, add methanol according to the solid-liquid ratio of 1:4 (g:mL), extract at 60°C for 40min, and concentrate under reduced pressure to obtain the extract. Use a rotary evaporator to evaporate the solvent from 100mL of the extract and dry it at 40°C to obtain a dried crude extract. The dried crude extract was dissolved with 10 mL of 50% ethanol, and then 40 mL of anhydrous ethanol was slowly added to make the final V(C2H5OH):V(H2O)=9:1. After sufficient stirring, it was allowed to stand at room temperature for 24 hours, centrifuged (4200 r / min, 10 min), and ethanol was recovered at 40°C to obtain a concentrate from which impurities such as polysaccharides and proteins were removed. The concentrate was extracted 3 times with an equal volume of petroleum ether, the aqueous phase was collected, the aqueous phase was extracted 3 times with an equal volume of chloroform, the aqueous phase was collected, the aqueous phase was extracted 6 times with an equal volume of ethyl acetate, the ethyl acetate was recovered at 40°C, and chestnut tannin was obtained by constant temperature drying at 40°C.

[0047] According to the standard LY / T 1082-2008 "Test Method for Tannin Analysis", the tannin content in chestnut tannin was detected using the skin powder method. The analysis steps include: weighing 6.25g of air-dried chrome skin powder equivalent to absolute dry chrome skin powder, accurate to 0.01g, and the mass m of air-dried cheese skin powder was calculated according to formula A;

[0048]

[0049] Where X0 is the moisture content of chrome powder (mass fraction), %.

[0050] Put the weighed air-dried chrome skin powder into a 250mL wide-mouth bottle, add (26.25-m)mL distilled water and seal it for 30 minutes, then draw the fully stirred chestnut tannin extract into the wide-mouth bottle containing the skin powder and shake it to make the extract fully contact with the skin powder; filter the skin powder and chestnut tannin extract, mix the filtrate with 1% gelatin-sodium chloride solution and heat it to 60°C to observe whether turbidity or precipitation occurs. If it is obviously turbid or precipitated, it indicates that the concentration of the analysis solution exceeds the specified value, and the sample volume should be readjusted. At the same time, draw 50mL of filtrate for evaporation, drying and weighing, and its mass is m3; blank test: use distilled water instead of the extract to carry out a blank test to correct the results, and measure the blank residue of chrome skin powder, and its mass is m4; calculate the non-tannin and tannin content according to the weight of the evaporation residue of non-tannin and tannin and the blank test results.

[0051] The soluble matter content X1 is calculated according to formula (2):

[0052]

[0053] Where m is the mass of the sample, g; m1 is the mass of the dry residue of 50 mL of soluble matter, g; X2 is the moisture content of the sample.

[0054] The non-tannin content X3 is expressed as mass fraction (%) and is calculated according to formula (3):

[0055]

[0056] Wherein 1.2 is the volume ratio of the diluent to the original solution; m3 is the mass of non-tannin, g; m4 is the mass of the blank residue of chrome powder, g; m is the mass of the sample, g; X2 is the moisture content of the sample, %; 0.075 is the allowable correction value of the blank test of chrome powder, g.

[0057] The tannin content X4 is expressed as mass fraction (%) and is calculated according to formula (4):

[0058] x4=x1-x3………(4)

[0059] Test results: Tannin content is 76.4wt%

[0060] Example 2

[0061] The complete feed for broilers was formulated in accordance with the Chinese broiler feeding standard (NY / T 33-2004). Table 1 details the formula of the complete feed for broilers and the nutritional level of broilers. Chestnut tannin was added to the complete feed for 1-day-old broilers at a ratio of 400 mg / kg and fed until 21 days of age.

[0062] Table 1 Composition and nutritional level of complete feed for experimental broiler chickens (%)

[0063] raw material Content of each raw material (%) Nutritional Levels Content of each nutrient (%) corn 54.00 Metabolizable Energy 12.65 Soybean meal 38.00 Crude Protein 21.09 Soybean Oil 4.00 calcium 1.00 Calcium Hydrogen Phosphate 0.60 Available Phosphorus 0.45 Stone powder 1.99 Lysine 1.10 salt 0.24 Methionine 0.50 DL-Methionine 0.11 Methionine + Cysteine 0.90 L-Lysine 0.08 Choline chloride 0.1 <![CDATA[Vitamin premix 1 > 0.02 <![CDATA[Mineral premix 2 > 0.86 total 100.00

[0064] Note: 1 Each kilogram of feed is provided by premix: vitamin A 8870IU, vitamin D 2840IU, vitamin E23.6IU, vitamin K 2.36mg, vitamin K 2.36mg, vitamin B12.36 mg, vitamin B27.10 mg, vitamin B30.33mg, vitamin B120.03 mg, D-pantothenic acid 2.36mg, biotin 0.12mg, niacinamide 37.82mg, and folic acid 1.18mg. 2 Each kilogram of feed is provided by premix: 75.00 mg zinc, 5.30 mg copper, 114.60 mg iron, 121.40 mg manganese, and 0.09 mg selenium.

[0065] Example 3

[0066] Add chestnut tannin at a ratio of 200 mg / kg to the complete feed of one-day-old broiler chickens and feed until 21 days of age.

[0067] Example 4

[0068] Chestnut tannin was added to the premix of laying hens at a ratio of 8 g / kg, and the premix was added to the complete feed of laying hens at a ratio of 5%, and the chickens were fed until 2 months of age.

[0069] Example 5

[0070] Chestnut tannin was added to the premix at a ratio of 8 g / kg, and the premix was added to the complete feed of growing laying hens at a ratio of 5%, and fed from 2 to 4 months of age.

[0071] Example 6

[0072] Chestnut tannin was added to the premix at a ratio of 4 g / kg, and the premix was added to the complete feed of growing laying hens at a ratio of 5%, and fed from 2 to 4 months of age.

[0073] Example 7

[0074] Chestnut tannin was added to the premix at a ratio of 3 g / kg, and the premix was added to the complete feed for laying hens at a ratio of 5%.

[0075] Example 8

[0076] Chestnut tannin is added to the concentrated feed of laying hens at a ratio of 600 mg / kg, and the concentrated feed is prepared into complete feed for laying hens at a ratio of 30-50%.

[0077] Example 9

[0078] Chestnut tannin was added to the premix at a ratio of 4 g / kg, and the premix was added to the complete feed for laying ducks at a ratio of 5%.

[0079] Example 10

[0080] Chestnut tannin is added to concentrated feed at a ratio of 600 mg / kg, and the concentrated feed is prepared into complete feed for meat ducks at a ratio of 30-50%.

[0081] Embodiment 11

[0082] The chestnut tannin is added into the concentrated feed of goose at a ratio of 800 mg / kg, and the concentrated feed is prepared into the complete feed of goose at a ratio of 30-50%.

[0083] Example 12

[0084] Chestnut tannin was added to the premix at a ratio of 6 g / kg, and the premix was added to the complete feed of goose at a ratio of 5%.

[0085] Test Example 1

[0086] 250 broiler chickens of similar body weight were randomly divided into 5 groups, with 5 replicates in each group and 10 chickens in each replicate. There was no significant difference in the initial body weight of the chickens in each group (P>0.05). (1) CON: fed with complete feed; (2) SE: fed with 3×10 8CFU dose of Salmonella (strain number CICC 21510 purchased from China Industrial Microbiological Culture Collection Center); (3) SE+TA (L): 200 mg / kg chestnut tannin was supplemented in the complete feed (chestnut tannin was supplemented in the manner of Example 3), and Salmonella was orally administered as in the SE group; (4) SE+TA (H): 400 mg / kg chestnut tannin was added to the complete feed (chestnut tannin was supplemented in the manner of Example 2), and Salmonella was orally administered as in the SE group; (5) TA (H): 400 mg / kg chestnut tannin was added to the complete feed (chestnut tannin was supplemented in the manner of Example 3). During the feeding process, broilers in each group had free access to food and water, and the experimental period was 21 days. After 21 days of feeding, blood was collected from the wing vein of broilers and then the cervix was broken. The ileum and lung tissues were collected and fixed with paraformaldehyde. After dehydration, transparency, wax immersion and other steps, the fixed tissues were embedded and observed with hematoxylin and eosin (H&E) staining. Subsequent index detection was carried out through biochemical detection, histopathological detection, molecular biology experiments and microbial diversity analysis.

[0087] As shown in Table 2, on day 21, the body weight of the SE group was significantly lower than that of the CON group (P<0.01). The body weight of the SE+TA(L) and SE+TA(H) groups increased significantly compared with the SE group (P<0.01). As shown in Table 2, the average daily gain of broilers infected with Salmonella was significantly reduced compared with the CON group (P<0.01), while the average daily feed intake did not show a significant difference. The addition of chestnut tannins to the diet significantly increased the average daily gain. In addition, the SE+TA(L) group reduced the feed-to-gain ratio (P<0.05). These results indicate that the addition of chestnut tannins to the diet can improve the growth performance decline induced by Salmonella in broilers.

[0088] Table 2 Effects of chestnut tannin on growth performance of broilers infected with Salmonella

[0089] Growth performance CON SE SE+TA(L) SE+TA(H) TA(H) Average weight at 21 days of age (g) 480.4±10.70** 401.9±5.90 500.1±12.94** 482.0±4.83** 514.0±5.55** Average daily weight gain from 1 to 21 days of age (g / D) 21.52±0.53** 17.64±0.30 22.50±0.65** 21.59±0.24** 23.19±0.28** Average daily feed intake (g / D) from 1 to 21 days old 29.05±1.78 28.40±0.73 29.82±0.81 31.06±0.23 31.40±0.71 Material weight ratio (g / g) 0.35±0.09* 1.61±0.05 1.32±0.03* 1.44±0.02* 1.35±0.04*

[0090] Note: Compared with SE group, *P<0.05, **P<0.01, the same below.

[0091] The effects of chestnut tannins on Salmonella-induced intestinal and lung lesions in broiler chickens were evaluated using H&E staining. Figure 1 As shown in Figure A, the ileum of the SE group showed obvious bleeding spots, and the intestinal villi were destroyed and arranged irregularly. Compared with the SE group, the addition of chestnut tannins in the feed significantly alleviated these lesions (P<0.05). On day 21, the ileal crypt depth of broiler chickens in the SE group was significantly greater than that in the CON group, while SE+TA(L) and SE+TA(H) both significantly reduced the crypt depth (P<0.05). Figure 1In addition, compared with the CON group, the SE group showed alveolar collapse, interstitial thickening, and extensive infiltration of inflammatory cells. However, the addition of chestnut tannins to the diet significantly improved the morphological structure of the lung ( Figure 1 (middle B).

[0092] like Figure 2 As shown, the expression levels of Claudin-1 and MUC-2 in the ileum of broiler chickens were analyzed by immunofluorescence. Figure 2 As shown in Figures AC. The fluorescence intensity of Claudin-1 and MUC-2 in the ileum was downregulated by Salmonella infection, while the addition of 200 mg / kg chestnut tannin to the diet significantly upregulated the fluorescence intensity of Claudin-1 and MUC-2 (P<0.05). qPCR results ( Figure 2 This result was further confirmed by the results of the DH. The mRNA expression levels of Claudin-1, Occludin, ZO-1, MUC-2, and SIgA in the ileum tissue of the SE group were significantly lower than those of the CON group (P<0.05). Chestnut tannin treatment significantly upregulated the mRNA expression of Claudin-1, Occludin, MUC-2, and SIgA (P<0.05); however, it had no significant effect on the mRNA expression level of ZO-1.

[0093] Chestnut tannins can reduce intestinal inflammation induced by Salmonella infection. Figure 3 As shown in Figure A, Salmonella infection led to an increase in the fluorescence intensity of proinflammatory cytokines IL-1β and TNF-α, while chestnut tannin treatment significantly downregulated the fluorescence intensity (P<0.05). qPCR analysis further confirmed this result ( Figure 3 BC). The mRNA expression levels of IL-6, IFN-γ, and IL-4 in the ileum. The mRNA expression levels of IL-6, IFN-γ, and IL-4 in the SE group were significantly higher than those in the CON group (P<0.01). In contrast, the mRNA expression levels of IL-4, IL-6, and IFN-γ in the SE+TA(L) group were significantly downregulated (P<0.01) ( Figure 3 (in DH).

[0094] To accurately evaluate the effect of chestnut tannin on the composition of intestinal flora after Salmonella infection, ileal contents of CON, SE, and SE+TA(L) groups were selected for 16S rRNA sequencing. Figure 4 As shown in Figure A, the common and unique OUTs between samples are plotted into a Venn diagram, which can intuitively show the similarities and differences between groups of samples. It can be found that the number of OUTs of samples in each group is different. The diversity and richness of the ileal microbiota were evaluated by alpha diversity using the Shannon and Simpson index ( Figure 4Compared with the SE group, both indexes in the SE+TA(L) group were significantly increased (P<0.05). Figure 4 In the PCoA diagram of β diversity analysis shown in D, the clustering angles between the groups are dense, the overlap of the confidence ellipses is high, and the sample differences between the groups are small. Figure 4 As shown in Figure EF, at the phylum level, the main bacteria in the three groups were Firmicutes, Bacteroidota, and Proteobacteria, among which the SE group showed a decrease in the abundance of Firmicutes and an increase in the abundance of Proteobacteria. The relative abundance of Desulfobacterota in the SE group was significantly higher than that in the other two groups (P<0.05). Figure 5 As shown in the results, at the order level, Salmonella infection led to an increase in the relative abundance of Burkholderiales in the ileum of broiler chickens, while dietary tannic acid supplementation reduced the colonization of Burkholderiales in the ileum. The relative abundance of Desulfovibrionales was significantly increased in the SE group (P<0.05). Ruminococcus_torques_group was relatively reduced in the SE group, while Pelomonas, Escherichia-Shigella, and Ralstonia were increased. Notably, chestnut tannin treatment led to a decrease in the abundance of Pelomonas, Escherichia-Shigella, and Ralstonia.

[0095] The lung microbial composition of the CON, SE, and SE+TA(L) groups was analyzed using 16S rRNA gene sequencing. Figure 6 As shown in Figure A, the number of OUT samples in the three groups was different. The Shannon and Simpson indices associated with α diversity tended to decrease in the SE group, while chestnut tannin treatment significantly increased the Shannon index of lung microorganisms in broiler chickens infected with Salmonella (P<0.05) ( Figure 6 The beta diversity of the lung microbiome was analyzed. The PCoA graph showed that there was a large difference between the samples of the SE group and the CON group, and the overlap of the ellipses was low, indicating that there was a large difference in the composition structure of the lung microbiome between the two groups ( Figure 6 D). However, the difference between the samples of SE+TA(L) and CON group was smaller. Compared with SE group, the lung microbial composition of SE+TA(L) group was more similar to that of CON group. Figure 6 As shown in Figure EF, at the phylum level, the dominant bacteria in the three groups were Firmicutes, Bacteroidota, and Proteobacteria. The SE group showed an increase in the abundance of Proteobacteria and a decrease in the relative abundance of Bacteroidota. In addition, the microbial composition of the SE+TA(L) group was found to be more similar to that of the CON group. Figure 7 As shown in the figure, at the order level, significant differences (P < 0.05) were observed in Chitinophagales, Cytophagales, Tepidisphaerales, Myxococcales, and Polyyangiales among the CON, SE, and SE+TA(L) groups as determined by the Kruskal-Wallis rank sum test. Figure 7 As shown in Figures CD, at the genus level, the relative abundance of Enterococcus, Acinetobacter, Escherichia-Shigella, and Salmonella increased in the SE group compared with the other two groups.

[0096] The effects of Salmonella infection on lung inflammation signaling pathways were examined. Figure 8 As shown in Figures AE, the fluorescence intensity of NF-κB was significantly increased in the SE group (P<0.01), and the mRNA expression levels of TLR4, MYD88, and NF-κB were significantly upregulated (P<0.01). Chestnut tannin significantly reduced the mean fluorescence intensity of NF-κB (P<0.01), and the mRNA expression of MYD88 and NF-κB was significantly downregulated (P<0.01). Next, the protective effect of tannic acid on Salmonella-induced lung injury was evaluated. Figure 8 The results of FH showed that the fluorescence intensity of IL-1β and TNF-α in the lungs of the SE group was significantly increased compared with CON (P<0.01). In contrast, the fluorescence intensity of IL-1β and TNF-α was significantly reduced in the SE+TA(L) group. Salmonella infection induced a significant upregulation of proinflammatory cytokines (including IL-1β, TNF-α, IL-6, IL-17, IL-22, and IFN-γ) (P<0.01), while leading to a significant downregulation of the mRNA levels of anti-inflammatory cytokines (such as IL-4 and TGF-β) (P<0.01). Tannic acid treatment significantly reversed these changes (P<0.01) ( Figure 8 Medium IQ).

[0097] like Fig. 9 As shown in Figures AC, the mean fluorescence intensity of CD11C, which indicates M1 macrophages in the lung, was significantly higher in the SE group than in the CON group (P<0.01). Compared with the SE group, SE+TA(L) significantly decreased the mean fluorescence intensity of CD11C and increased the mean fluorescence intensity of CD206, a marker of the M2 phenotype (P<0.01). Compared with the other groups, the mRNA expression level of INOS was significantly upregulated in the SE group (P<0.01; Fig. 9 In the SE+TA(L) group, the mRNA expression of AGR1, a marker of M2 macrophages, was further increased (P<0.01)( Fig. 9 In addition, the mRNA expressions of CCL2 and CCL4 were significantly increased in the SE group, and tannic acid significantly reversed these changes (P<0.01) ( Fig. 9 FG). ROS detection showed that Salmonella infection led to increased ROS levels in the lungs, and the MDA content in the lungs also increased significantly (P<0.05). TA treatment reduced ROS accumulation and MDA content. Compared with CON, CAT content in the SE group was significantly reduced, while that in the SE+TA(L) group was significantly increased (P<0.05) ( Fig. 9 In addition, the mRNA expression levels of the Nrf2 / HO-1 signaling pathway in the lung were detected. The mRNA expression of Nrf2, HO-1, and NQO1 was significantly downregulated in the SE group (P<0.05). In contrast, the mRNA expression of these genes was upregulated in SE+TA(L) (P<0.05) ( Fig. 9 Medium LN).

[0098] Test Example 2

[0099] The broilers treated in the same manner as in Example 1 were subjected to cervical dislocation and jejunal and liver tissues were collected. After being fixed with paraformaldehyde, the fixed tissues were embedded and observed by hematoxylin and eosin (H&E) staining through steps such as dehydration, transparency, and wax immersion. The protective effect of chestnut tannin on the liver of broilers infected with Salmonella was studied by means of biochemical detection, histopathological detection, molecular biological experiments, and microbial diversity analysis.

[0100] like Fig.10 As shown in Figure 2, serum AST and ALT levels were significantly increased in the SE group ( Fig.10 In addition, compared with the CON, SE+TA(L), and SE+TA(H) groups, the levels of serum IgG and IgM in the SE group were significantly decreased ( Fig.10C, D) (P<0.05). Histopathological changes were observed by H&E staining. The jejunal tissue morphology of the control group was normal, with clear boundaries and neatly arranged epithelial cells. In contrast, SE-induced jejunal injury was characterized by the destruction and curling of the intestinal villi and a significant decrease in the length of the intestinal villi. Compared with the control group, the crypt depth in the SE group was aggravated and the villus / crypt ratio (VCR) was significantly reduced; however, this injury was alleviated after chestnut tannin treatment, especially in the SE+TA(L) group ( Fig.10 F, H) (P < 0.05). Similar results were observed by PAS staining. The intestinal mucosa of the control group was covered with a thick and continuous mucin layer containing a large number of purple-blue goblet cells. After exposure to Salmonella, the mucin layer became thinner and the number of goblet cells was significantly reduced, while the addition of chestnut tannin showed signs of recovery (P < 0.05).

[0101] like Fig.11 As shown in Figure 2, the immunofluorescence levels of claudin-1 and MUC2 were significantly downregulated in the SE group compared with the control group ( Fig.11 The mRNA levels of occludin, ZO-1, claudin-1, MUC2, and inflammatory factor IL-1β in the SE group were significantly lower than those in the blank group; however, these levels were reversed in the SE+TA(L) group, which was consistent with the immunofluorescence results (P<0.05).

[0102] The surface of the liver in the SE group showed a more obvious yellow color. Correspondingly, the histopathological results showed that the liver in the SE group showed inflammatory cell infiltration (red arrows) and portal red blood cell aggregation (black arrows) compared with the control group ( Fig.12 In addition, the liver index of the SE+TA(H) group was significantly lower than that of the other groups. The liver LPS level of the SE group was significantly increased, and the lysozyme level was significantly decreased, which confirmed that the broilers in the SE group had severe liver damage, and tannic acid treatment significantly alleviated this damage (P<0.05, Fig.12 (C, D).

[0103] like Fig.13 As shown in Figure 2, the SE group showed significantly reduced species richness and diversity compared to the other groups. PCoA highlighted the changes in the gut microbial community. The results showed that the gut microbial composition of broiler chickens in the SE group was significantly different from that of the CON and SE+TA(L) groups. At the phylum level, chestnut tannins altered the abundance of Firmicutes and Bacteroidetes ( Fig.13 ).like Fig.14As shown in the Figure 2, the abundance of Firmicutes was significantly increased (P<0.05) and the abundance of Bacteroidota was also significantly increased (P<0.05) compared with the SE group, while the ratio of Firmicutes / Bacteroidetes (F / B) was not significantly different. At the genus level, the addition of chestnut tannin affected the abundance of Bacteroides, Escherichia-Shigella, and Lactobacillus. Further visual analysis showed that TA significantly increased the relative abundance of the beneficial bacteria Bacteroides and Lactobacillus (P<0.05). In this study, Lactobacillus was shown to enhance the host immune response and reduce the relative abundance of Shigella (P>0.05).

[0104] The abundance of Bacteroides was significantly higher in the SE and SE+TA(L) groups. Therefore, it is hypothesized that Bacteroides in the intestine can degrade tannic acid into more effective substances. Bacteroides isolated from the intestine were co-cultured with chestnut tannins ( Fig.15 A). The target strain was isolated using BEE selective medium ( Fig.15 (B). This sample was closest to strain KP944128.13-1294, with a shorter branch length and a confidence level of 66%. Among the three concentrations of LTB (14μg / mL), MTB (28μg / mL) and HTB (36μg / mL), a high concentration of tannic acid that was more conducive to promoting the growth of Bacteroides was selected for co-culture. After 16 to 18 hours of co-culture, the results showed that the tannic acid concentration in the HTB group was reduced compared with the blank group (HT) without bacteria (P<0.05). In contrast, the gallic acid concentration was significantly increased (P<0.05).

[0105] Tissue bacterial culture and microbiome analysis were performed on liver samples, and the results were as follows Fig.16 As shown. The liver tissues of the three groups were ground and cultured in LB and MAC medium, respectively. The total aerobic colony count in the SE group was significantly higher than that in the other groups. The colony count in MAC medium showed that the Salmonella colonies in the SE group were more abundant than those in the SL and SE+TA(L) groups (P<0.05)( Fig.16 The CON, SE, and SE+TA(L) groups had 434, 119, and 284 OTUs, respectively. PCoA showed that the microbial composition of the liver of mice in the SE group was significantly different from that in the SE+TA(L) group ( Fig.16 (D) Salmonella reduced species richness, while chestnut tannins altered species richness and diversity in the liver. Fig.17As shown in Figure 2, at both the phylum and genus levels, Salmonella was observed to be present in the genus-level abundance in both the SE and SE+TA(L) groups. Addition of tannic acid significantly reduced the abundance of Salmonella and pathogenic Escherichia coli Shigella (P<0.05). Notably, the abundance of Salmonella, Enterobacteraceae, and Proteobacteria increased significantly in the SE group.

[0106] To further investigate whether Salmonella colonization in the liver induces immune damage and whether the addition of chestnut tannins can alleviate such damage, immunofluorescence and qRT-PCR were used to show that Fig.18 As shown. The immunofluorescence intensity of the M1 macrophage marker CD80 in the SE group was significantly higher than that in the CON and SE+TA(L) groups. In contrast, the immunofluorescence intensity of the M2 macrophage marker CD206261 in the SE+TA(L) group was significantly increased compared with the SE group (P<0.05). In addition, the immunofluorescence results of the inflammation-related cytokines IL-1β and TNF-α in the liver showed that the fluorescence intensity in the SE group was significantly higher than that in the CON group. This increase was reversed after chestnut tannin treatment ( Fig.18 DG). For verification, qRT-PCR was performed for M1 and M2-related inflammatory cytokines in the liver. The mRNA expression levels of IFN-γ, TNF-α, IL-18, iNOS, IL-1, IL-6, IL-1β, CCL4, and CCL1 were significantly increased in the SE group (P<0.05). In contrast, these inflammatory factors were significantly reduced after treatment with chestnut tannins. Meanwhile, the mRNA level of IL-10 was significantly decreased in the SE group (P<0.05). These results suggest that Salmonella colonization in the liver can impair immune function and stimulate inflammation, which can be alleviated by chestnut tannins.

[0107] It can be seen that the addition of chestnut tannins to feed significantly reduced the colonization of Salmonella in the liver and lungs of broiler chickens 10 days after infection with Salmonella. Chestnut tannins can intervene and regulate the composition of intestinal microbiota, protect the intestinal barrier, inhibit the colonization of pathogens in the liver and lungs and the polarization of M1 macrophages, and reduce inflammatory damage to the liver and lungs.

[0108] Experimental Example 3

[0109] Two hundred white-feathered broilers of similar weight were randomly divided into four groups, with 50 chickens in each group. (1) Control group: fed with complete feed; (2) Salmonella group: fed with 3×10 8CFU dose of Salmonella was gavaged; (3) Salmonella + chestnut tannin group: Salmonella was gavaged on the 10th and 11th days of age, and 200 mg / kg chestnut tannin was supplemented in the complete feed; (4) Salmonella + enrofloxacin group (enrofloxacin was purchased from Henan Nanhua Qianmu Biotechnology Co., Ltd., item number: 6988202173347): Salmonella was gavaged on the 10th and 11th days of age, and 10% enrofloxacin soluble powder was used starting from the 10th day of age, 0.6 g per liter of drinking water, and continued drinking water for 5 days. During the feeding process, broilers in each group had free access to food and water. The experimental period was 35 days. During the experiment, the diarrhea rate and mortality rate from 10 to 35 days of age were counted. After 35 days, the growth performance was counted, and the Salmonella in feces, liver and lungs were detected.

[0110] The results are shown in Table 3. The addition of 200 mg / kg chestnut tannins to the feed can effectively reduce the mortality and diarrhea rate of broiler chickens after Salmonella infection, reduce the recurrence rate after tolerance (after 21 days of age), reduce the detection rate of Salmonella in the liver, lungs and feces at 35 days of age, and effectively improve production performance. Compared with antibiotics (enrofloxacin), the addition of chestnut tannins to the diet has a better effect on eliminating the liver and lung colonization of Salmonella and reducing its detoxification and recurrence rate. These results show that the addition of chestnut tannins to the diet can improve the growth performance decline of broiler chickens induced by Salmonella.

[0111] Table 3 Comparison of the application effects of chestnut tannin and enrofloxacin in broilers infected with Salmonella

[0112] project Control group Salmonella group Salmonella + chestnut tannin group Salmonella + enrofloxacin group Material weight ratio 1.69 1.89 1.78 1.76 Weight (g) 1705 1536 1650 1647 mortality rate(%) 2 10 4 4 Diarrhea rate at 10-21 days of age (%) 4 42 8 6 Diarrhea rate after 21 days of age (%) 0 16 0 4 Detection rate of Salmonella in liver (%) 2 50 2 10 Detection rate of Salmonella in lungs (%) 0 80 0 6 Fecal Salmonella detection rate (%) 0 10 0 0

[0113] It can be seen that the tannins of the present invention can effectively inhibit the translocation, colonization and latent infection of Salmonella in the liver and lungs of poultry, alleviate the persistent damage of the liver and lungs caused by Salmonella, reduce the recurrence rate of Salmonella infection in poultry after tolerance, and improve the health level and growth performance of poultry.

[0114] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of tannin in the preparation of medicines and / or functional products for eliminating the translocation and / or latent infection of Salmonella in the liver and lungs of poultry.

2. The use according to claim 1, characterized in that: The sources of the tannins include plant-derived extracts.

3. The use according to claim 2, characterized in that: The plant-derived extract includes chestnut extract.

4. The use according to claim 2, characterized in that: The extraction method of the plant-derived extract includes organic solvent extraction, ultrasonic extraction, steam distillation, organic solvent extraction, supercritical fluid extraction or enzyme conversion.

5. A poultry feeding method, characterized in that: The method comprises adding tannin to poultry feed and then feeding the poultry, wherein the added amount of tannin in the feed is 17.5-450 mg / kg.

6. The poultry feeding method according to claim 5, characterized in that: The tannin is added in the form of a tannin premix, and the content of tannin in the tannin premix is ​​35wt% to 90wt%.

7. The poultry feeding method according to claim 6, characterized in that: The tannin premix comprises a chestnut extract, and the content of tannin in the chestnut extract is 50wt% to 90wt%.

8. The poultry feeding method according to claim 7, characterized in that: The content of tannin in the chestnut wood extract is 60wt% to 80wt%.

9. The poultry feeding method according to claim 5, characterized in that: The feeding cycle is 21 to 60 days.

10. The poultry feeding method according to claim 5, characterized in that: The poultry include chickens, ducks and geese.

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