Application of inulin in preparation of product for relieving chicken liver injury caused by OTA

Abstract

The invention discloses a new application of inulin, and particularly relates to an application of inulin in preparation of a product for relieving liver injury caused by chick ingestion of ochratoxin A. The product can be a medicine, a feed or a feed additive. The medicine, the feed or the feed additive takes inulin as an active ingredient, and the inulin (in a solution form) is added into a basic ration of the chicks according to 1mL / kg of the weight of the chicks. After the chicks eat the feed polluted by OTA by mistake, the inulin can remarkably relieve pathological damage of liver tissues of the chicks caused by OTA poisoning, regulate intestinal flora structures of the chicks, improve the immunity of the chicks and reduce the death rate of the chicks through multiple ways of regulating intestinal flora, inhibiting oxidative stress and inflammatory response and the like. The invention provides a safe, effective and residue-free solution of natural medicine, feed or feed additive for preventing and controlling liver injury caused by OTA poisoning of poultry, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to the application of inulin in the preparation of products for relieving liver damage caused by OTA in chicks. BACKGROUND

[0002] Ochratoxin A (OTA) is mainly produced by Aspergillus and Penicillium fungi during metabolism, and is most likely to be generated in humid and warm environmental conditions. OTA is widely present in moldy grains, foods and feeds, and fungi are prone to breed and produce OTA in environments that are humid, high in temperature and improperly stored. After chicks, especially those with an immune system and organs that have not fully developed, ingest feed contaminated with OTA, the toxin enters the body through the digestive tract, and the liposolubility of OTA allows it to be absorbed by the gastrointestinal mucosa and enter the blood, so the intestinal tract is the first organ to be damaged by OTA, and the enterotoxicity of OTA manifests as damage to the integrity of the intestinal barrier and the intestinal microecology. OTA is transported through the blood and is widely distributed in tissues and organs such as the liver and kidneys, and has strong hepatotoxicity, causing changes in the shape, size and color of the liver. In addition, OTA also has immunosuppressive and teratogenic effects, causing growth retardation, diarrhea and even death. Given its serious harmfulness and its impact on health, OTA has become one of the mycotoxins that has attracted attention after aflatoxin.

[0003] Common physical detoxification methods for OTA include high-temperature degradation, radiation treatment and adsorption by adsorbents. High-temperature treatment can reduce or destroy the activity of some mycotoxins, but it is only suitable for product types that are resistant to high temperatures. The method of using adsorbents to remove OTA is relatively environmentally friendly and easy to operate, but some adsorbents may adsorb nutrients in the feed while adsorbing mycotoxins.

[0004] Chemical detoxification methods use chemicals such as acids, bases and ozone to destroy the chemical structure of mycotoxins and reduce their toxicity, but due to the destruction of feed nutrients and the possibility of chemical residues, their application in actual production is relatively limited.

[0005] Biological detoxification methods mainly use microbial fermentation and enzyme preparations to degrade mycotoxins into non-toxic or low-toxic metabolic products, and are currently considered the most environmentally friendly and efficient. However, biological detoxification methods also have certain limitations, such as the activity of bacteria and enzymes being affected by environmental conditions such as temperature, humidity and pH, and the actual production and application process may face problems such as low activity, slow effect and poor stability.

[0006] In recent years, inulin as a prebiotic has corresponding application value in the fields of food, medicine and animal breeding. In the field of food, the water solubility of inulin enables it to be used as a thickening agent, emulsifier and stabilizer in dairy products, beverages and other products to improve the taste. In the field of medicine, inulin is found to be used as a stabilizer for protein drugs and vaccines due to its low hygroscopicity, low crystallization rate and high glass transition temperature. In the field of animal breeding, the use of inulin in specific alleviation of OTA-induced liver damage in chicks has not been reported. SUMMARY

[0007] The purpose of the present application is to provide an application of inulin in the preparation of a product for alleviating liver damage caused by OTA in chicks, so as to provide a safe, efficient and harmless product for alleviating liver damage caused by OTA in chicks.

[0008] According to a first aspect of the present application, there is provided an application of inulin in the preparation of a product for alleviating liver damage caused by OTA in chicks. Thus, through the application, liver damage caused by the intake of OTA in chicks can be efficiently and safely alleviated, and the mortality rate of chicks can be reduced. Moreover, inulin is used as an effective component of the product for alleviating liver damage caused by OTA in chicks, which is natural, safe and residue-free, and has good application prospects.

[0009] In some embodiments, the product comprises a pharmaceutical product, a feed or a feed additive.

[0010] According to a second aspect of the present application, there is provided a non-therapeutic method for preventing OTA poisoning in chicks, which comprises adding inulin to the feed of chicks or adding inulin to the drinking water of chicks or directly gavage with an inulin solution. Thus, through the method, liver damage caused by the intake of OTA in chicks can be efficiently and safely alleviated, and the mortality rate of chicks can be reduced. Moreover, the method is natural, safe and residue-free, and has good application prospects.

[0011] In some embodiments, the inulin is used in an amount of 1 mL / kg, and the concentration of inulin is 0.8 g / mL.

[0012] According to a third aspect of the present application, there is provided a pharmaceutical product containing inulin, wherein the effective component of the product is inulin, and the product is used to prevent and treat liver damage caused by OTA poisoning in chicks. Thus, by using inulin as a pharmaceutical product for alleviating liver damage caused by OTA in chicks, liver damage caused by the intake of OTA in chicks can be efficiently and safely alleviated, and the mortality rate of chicks can be reduced, which has good application prospects.

[0013] According to a fourth aspect of the present application, a feed containing inulin is provided, which can be used for preventing and treating liver damage caused by OTA poisoning in chicks. Thus, by using inulin as a feed for alleviating liver damage caused by OTA in chicks, liver damage caused by OTA in chicks can be effectively and safely alleviated, and the mortality of chicks can be reduced, which has a good application prospect.

[0014] According to a fifth aspect of the present application, a feed additive containing inulin is provided, which can be used for preventing and treating liver damage caused by OTA poisoning in chicks. Thus, by using inulin as a feed additive for alleviating liver damage caused by OTA in chicks, liver damage caused by OTA in chicks can be effectively and safely alleviated, and the mortality of chicks can be reduced, which has a good application prospect.

[0015] According to a sixth aspect of the present application, the use of inulin in improving intestinal flora imbalance caused by OTA poisoning in chicks is provided. Thus, by feeding chicks with inulin, the intestinal flora imbalance caused by OTA poisoning in chicks can be effectively improved, and the immunity of chicks can be improved.

[0016] According to a seventh aspect of the present application, the use of inulin in preparing a product for improving intestinal flora imbalance caused by OTA poisoning in chicks is provided. Thus, by feeding chicks with the product prepared from inulin, the intestinal flora imbalance caused by OTA poisoning in chicks can be effectively improved, and the immunity of chicks can be improved.

[0017] In some embodiments, the product includes a pharmaceutical product, a feed or a feed additive.

[0018] The present application discloses a new use of inulin, and particularly relates to the use of inulin in preparing a product for alleviating liver damage caused by OTA in chicks, which can be a pharmaceutical product, a feed or a feed additive. The pharmaceutical product, the feed or the feed additive contains inulin as an effective component, which is added to the basic daily ration of chicks in a solution form at a dosage of 1 mL / kg of the body weight of the chicks. When chicks mistakenly eat feed contaminated by OTA, inulin can significantly reduce the pathological damage to the liver tissue of chicks caused by OTA poisoning, regulate the intestinal flora structure of chicks, improve the immunity of chicks, and reduce the mortality of chicks, by regulating intestinal flora, inhibiting oxidative stress and inflammatory response, and other multiple pathways. The present application provides a safe, effective and residue-free natural pharmaceutical product, feed or feed additive solution for preventing and controlling liver damage caused by OTA poisoning in poultry, which has a good application prospect.

[0019] The inulin is used for preparing a product for relieving liver damage of chicks caused by OTA, so as to solve the problems of poor prevention and control effect of OTA pollution in feed, safety hidden trouble and the like in the prior art, and the inulin has the following beneficial effects:

[0020] 1. High efficiency of relieving: through synergistic effect of multiple mechanisms (flora regulation, antioxidant, anti-inflammatory), the liver toxicity of OTA is relieved in a targeted manner.

[0021] 2. Safety and no residue: the inulin is a natural plant component, is safe to animals, has no drug residue problem, and meets the requirements of green breeding.

[0022] 3. Low cost and convenient to use: the inulin can be directly used for mixing feed, and complex equipment is not needed, so the inulin is easy to popularize in farms.

[0023] 4. Nutritional and health care functions: while detoxifying, the inulin as a prebiotic can promote intestinal health of chicks and improve overall growth performance. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The figure is a weight change curve of chicks in different treatment groups, wherein Control represents a control group fed with a basic diet, OTA represents a group fed with the basic diet and fed with an OTA solution, Inulin represents a group fed with the basic diet and fed with an inulin solution, and O+Inulin represents a group fed with the basic diet and fed with both the OTA and inulin solutions.

[0025] Figure 2 The figure is a pathological change result of livers of chicks in different treatment groups, wherein Control represents a control group fed with a basic diet, OTA represents a group fed with the basic diet and fed with an OTA solution, Inulin represents a group fed with the basic diet and fed with an inulin solution, and O-Inulin represents a group fed with the basic diet and fed with both the OTA and inulin solutions.

[0026] Figure 3 The figure is a result of serum biochemical indexes of chicks in different treatment groups, wherein A in the figure represents an ALT result, B in the figure represents an AST result, C in the figure represents an ALP result, and D in the figure represents a TG result, Control represents a control group fed with a basic diet, OTA represents a group fed with the basic diet and fed with an OTA solution, Inulin represents a group fed with the basic diet and fed with an inulin solution, O-Inulin represents a group fed with the basic diet and fed with both the OTA and inulin solutions, ns represents no significant difference P>0.05, ** represents significant difference P<0.01, and **** represents extremely significant difference P<0.0001. Figure 3 Figure 3 Figure 3 Figure 3

[0027] ​​​​Figure 4 The liver tissue oxidative stress enzyme index level results chart of different treatment groups of chicks: among them, Figure 4 A in the above table represents the MDA results, Figure 4 B in the above table represents the T-SOD results, Figure 4 C in the above table represents the CAT results, Figure 4 D in the above table represents the GSH results, Control represents the control group fed only with the basic diet, OTA represents the group fed with the basic diet and gavaged with OTA solution, Inulin represents the group fed with the basic diet and gavaged with inulin solution, O-Inulin represents the group fed with the basic diet and gavaged with OTA and inulin solution at the same time, ns represents no significant difference P>0.05, * represents significant difference P<0.05, ** represents extremely significant difference P<0.01;

[0028] Figure 5 The relative transcription level results chart of pro-inflammatory factors in the liver tissue of different treatment groups of chicks: among them, Figure 5 A in the above table represents the IL-1β mRNA expression amount, Figure 5 B in the above table represents the IL-6 mRNA expression amount, Figure 5 C in the above table represents the TNF-α mRNA expression amount, Control represents the control group fed only with the basic diet, OTA represents the group fed with the basic diet and gavaged with OTA solution, Inulin represents the group fed with the basic diet and gavaged with inulin solution, O-Inulin represents the group fed with the basic diet and gavaged with OTA and inulin solution at the same time, ns represents no significant difference P>0.05, * represents significant difference P<0.05, ** represents significant difference P<0.01, *** represents extremely significant difference P<0.001, **** represents extremely significant difference P<0.0001;

[0029] Figure 6 The results chart of the influence of inulin on the intestinal microbial changes of chicks caused by OTA poisoning: among them Figure 6 A-D in the above table are the β and α diversity analysis results of chicks in different treatment groups, Figure 6 E in the above table is the top 15 column chart of the relative abundance of species at the genus level of intestinal bacteria of chicks in different treatment groups, Figure 6 F-G in the above table are the species composition column charts at the genus level of intestinal bacteria of chicks in different treatment groups, Control represents the control group fed only with the basic diet, OTA represents the group fed with the basic diet and gavaged with OTA solution, O-Inulin represents the group fed with the basic diet and gavaged with OTA and inulin solution at the same time. DETAILED DESCRIPTION

[0030] The application will be described in detail below with specific examples. The following examples will help those skilled in the art to further understand the application, but do not limit the application in any form. It should be noted that those skilled in the art can make several changes and improvements without departing from the concept of the application, which are within the scope of the application. Chemicals, proteins, enzymes or kits used in the application, unless otherwise specified, can be obtained commercially.

[0031] 1. Preparation of main reagents:

[0032] (1) NaHCO3 solution: Dissolve NaHCO3 of analytical purity in double distilled water to obtain a NaHCO3 solution with a concentration of 0.1 mol / L, and store at 4°C.

[0033] (2) OTA solution: Dissolve pure crystalline OTA in 0.1 mol / L NaHCO3 solution to obtain an OTA stock solution with a concentration of 1 mg / mL, and store at -20°C. Before use, dilute the OTA stock solution with NaHCO3 solution according to the body weight of each chicken to prepare the OTA working solution.

[0034] (3) Inulin solution: Dissolve inulin in double distilled water to prepare an inulin solution with a concentration of 0.8 g / mL, and store at 4°C. When used, according to the body weight of each chicken, 1 mL / kg of inulin solution is administered intragastrically. Inulin is purchased from Yuanye Biotechnology Co., Ltd. with item number 9005-80-5.

[0035] 2. Experimental animals and experimental design.

[0036] Healthy ephedrine chickens (1 day old, female) were purchased from Foshan Nanhai Poultry Co., Ltd. and were raised in a stable environment with suitable temperature (33±1°C for the first three days, then reduced by 2.5±0.5°C per week and the final temperature maintained at 27°C), suitable humidity (65±5%), and constant light and dark alternation (light for 18 hours and dark for 6 hours) per day. The chickens had free access to food and water. The basic diet was purchased from Beijing Kaocuo Fei Feed Co., Ltd. to meet the nutritional needs of the chickens during growth. The feeding environment and feeding materials were sterilized and disinfected in advance. The feeding apparatus, drinking apparatus and fecal trays were cleaned every day to meet the experimental environment requirements. After 7 days of adaptive feeding, the subsequent experiments were conducted on the 8th day.

[0037] Twenty-four 1-day-old healthy ephedrine chickens were randomly divided into Control group, OTA group, Inulin group, and O-Inulin group, with 6 chickens in each group.

[0038] Control group (control group): fed with basic diet;

[0039] OTA group (OTA treatment group): fed with basal diet and gavaged with 0.3 mg / kg OTA solution;

[0040] Inulin group (inulin alone treatment group): fed with basal diet and gavaged with 1 mL / kg inulin solution;

[0041] O-Inulin group (OTA + inulin co-treatment group): fed with basal diet and gavaged with 0.3 mg / kg OTA and 1 mL / kg inulin solution at the same time.

[0042] The gavage amount of OTA solution and inulin solution was calculated according to the body weight of each chick.

[0043] The administration time lasted for 14 days, and the chicks were gavaged at fixed time every day, and the body weight and state of the chicks before and after the experiment were recorded.

[0044] The body weight results of each test group are shown in Table 1: Figure 1 The results showed that there was no significant difference in the body weight of chicks in each group (P>0.05). The above results showed that neither OTA nor inulin had obvious effect on the body weight of chicks.

[0045] 3. Sample collection.

[0046] After the administration period, each chicken was weighed and blood was collected. The blood sample was left at room temperature for 1 h, then centrifuged at 4°C, 1200g for 10 min to obtain the upper serum, which was divided into centrifuge tubes and stored in a -20°C refrigerator for subsequent detection. After each chicken was sacrificed, the cecum was quickly separated, the end was cut open, the cecal contents were squeezed out and divided into sterilized centrifuge tubes, and placed in a -80°C refrigerator for standby. Then 0.5 cm 3 of liver tissue was cut from each chicken, washed with PBS and fixed in 4% formaldehyde solution, and the rest was washed with PBS and collected in a -80°C refrigerator for subsequent experiments.

[0047] 4. Hematoxylin-eosin (H&E) staining and detection.

[0048] H&E staining was used for morphological analysis of the liver of chicks in each group, and the main steps were as follows:

[0049] (1) Fixing and trimming: the liver tissue was fixed with 4% paraformaldehyde for 24 h to ensure that the tissue was fully fixed. The fixed liver tissue was cut into tissue blocks of appropriate size and placed in embedding frames.

[0050] (2) Washing and dehydrating: the tissue blocks were washed with PBS for 3 times, 10 min each time, to remove excess paraformaldehyde. The washed tissue blocks were immersed in 70% ethanol at room temperature overnight. The next day, ethanol gradient dehydration was performed.

[0051] (3) Transparency: The dehydrated tissue block is immersed in xylene, xylene I (30 min)→xylene II (30 min), and whether the tissue block is completely transparent is observed (transparency is the premise of paraffin immersion).

[0052] (4) Wax immersion and embedding: The transparent tissue block is moved into melted paraffin (usually 56-58°C), paraffin I (1 h)→paraffin II (1 h), and it is ensured that the paraffin completely penetrates the tissue.

[0053] (5) Embedding: The wax-immersed tissue block is placed into an embedding mold, melted paraffin is added, and the position of the tissue block is adjusted. Cooling to paraffin solidification forms a wax block.

[0054] (6) Sectioning and spreading: The wax block is cut into a 4-5 μm thick section using a microtome, and the section is floated in a 37°C water bath to make the section fully spread. The spread section is picked up with a glass slide and placed in a 37°C oven for drying, so that the section is firmly attached to the glass slide.

[0055] (7) De-waxing and hydration: The paraffin section is placed in xylene for de-waxing, 5-10 min, and repeated twice. It is sequentially passed through 100%, 95%, 90%, 80%, 70% ethanol, 2-3 min for each grade, and finally washed with distilled water.

[0056] (8) Hematoxylin staining: The section is immersed in hematoxylin staining solution for 5-10 min. It is washed with distilled water to remove excess staining solution.

[0057] (9) Differentiation: The section is immersed in acid alcohol (1% hydrochloric acid alcohol) for several seconds until the nucleus is clear, and then immediately washed with distilled water.

[0058] (10) Bluing: The section is washed with distilled water to make the nucleus blue, about 5-10 min, and then washed with distilled water again.

[0059] (11) Eosin staining: The section is immersed in eosin staining solution for 1-3 min. It is washed with distilled water to remove excess staining solution.

[0060] (12) Dehydration: It is sequentially passed through 70%, 80%, 90%, 95%, 100% ethanol, 2-3 min for each grade.

[0061] (13) Transparency: The section is immersed in xylene for transparency, 5-10 min, and repeated twice.

[0062] (14) Mounting: It is mounted with neutral gum and covered with a cover glass. It is photographed under a magnification of 100 times using an upright optical microscope and the picture is saved.

[0063] It is found through observation of the H&E staining section of the liver tissue that there are significant differences in the pathological morphology of each treatment group, and the test results are as followsFigure 2 The liver structure of the control group (Control group) and the inulin single treatment group (Inulin group) was complete, and the liver cord was arranged in a radial and orderly manner with the central vein as the center, and the cell morphology was normal. However, the OTA treatment group caused severe damage to the liver tissue structure, which was characterized by deformation of the liver cord, disordered arrangement, and a large number of inflammatory cell infiltration. However, the OTA+inulin co-treatment group (O-Inulin group) effectively alleviated the liver damage induced by OTA, and it was observed that the inflammatory cell infiltration was significantly reduced, the disordered liver cord structure was reconstructed, and tended to restore normal arrangement. It shows that inulin can effectively alleviate the liver damage caused by OTA.

[0064] 5. Detection of serum biochemical indicators.

[0065] The serum ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase), and TG (triglyceride) were determined using a full-automatic biochemical analyzer according to the instructions of the kit (Shenzhen Mindray Biomedical Co., Ltd.).

[0066] The results of serum biochemical indicators of each group are shown in Table 2. Figure 3 As shown in Table 2: there was no significant difference in each indicator between the control group (Control group) and the inulin single treatment group (Inulin group) (P>0.05); compared with the control group (Control group), the OTA group significantly increased the activities of ALT, AST, and ALP in the serum of chicks, and the content of TG (P<0.0001). Compared with the OTA group, in the O-Inulin group, due to the supplementation of inulin, the activity of ALP was significantly reduced (P<0.01), and the activities of ALT and the content of TG were extremely significantly decreased (P<0.0001, P<0.0001).

[0067] Since ALT, AST, ALP, and TG are important biochemical indicators reflecting liver function, they are commonly used for clinical liver function tests, and correspond to different physiological functions of the liver. Generally, their increase reflects different types of liver tissue damage. Therefore, the above results show that the supplementation of inulin can alleviate the liver function damage of chicks caused by OTA to a certain extent.

[0068] 6. Detection of oxidative stress products and antioxidant enzyme content in liver tissue.

[0069] (1) The liver tissue was taken out from -80℃, and 0.1 g of tissue sample was weighed on ice, then 900 saline and 3 grinding beads were added, mixed, and homogenized in a high-throughput tissue grinder.

[0070] (2) After homogenization, centrifuge at 4℃, 3000g for 15min in a high-speed refrigerated centrifuge, use a pipette to transfer the supernatant to a new set of centrifuge tubes, and store in a -20℃ refrigerator for standby.

[0071] (3) The protein concentration of the supernatant was detected using Pierce™ BCA Protein Assay Kits. First, the standard samples with known concentrations were gradient diluted according to the operation instructions. Then, an appropriate amount of the supernatant of the sample to be tested was diluted 10 times to prevent the protein concentration from being too high to exceed the detection range of the kit. In each well of a 96-well plate, 20 μL of the supernatant of the sample to be tested with unknown concentration and the standard sample gradient dilution liquid were added, and then 200 μL of the pre-prepared BCA working solution was added. After shaking and mixing, the plate was placed in a 37°C incubator for 30 min. After incubation, the plate was placed at room temperature, and the OD value of each well was detected at a wavelength of 562 nm using a microplate reader. The protein concentration standard curve was prepared according to the OD value of the gradient diluted standard sample with known concentration, and the sample OD value was substituted into the standard curve to finally calculate the protein concentration of the sample to be tested.

[0072] (4) An appropriate amount of the supernatant of the sample to be tested was taken, and the operation was performed according to the instructions of the MDA (Malondialdehyde), GSH (Glutathione), T-SOD (Total Superoxide Dismutase) and CAT (Catalase) assay kits (Nanjing Jiancheng Biological Engineering Institute). Then, the OD value of each well was measured at 532, 405, 550 and 405 nm using a spectrophotometer, and finally the measured protein concentration and OD value were substituted into the formula in the instructions to calculate the enzyme content of each tissue.

[0073] The results of the oxidative stress enzyme activity indicators of different treatment groups are shown in Table 2: Figure 4 Compared with the control group (Control group), the MDA in the OTA group was significantly increased (P<0.05), and the GSH enzyme activity was significantly decreased (P<0.01), and the T-SOD showed a slight downward trend. Compared with the OTA group, the GSH in the O-Inulin group showed an upward trend. The above results show that the supplementation of inulin regulates the content of oxidation and antioxidant indicators, and can alleviate the oxidative stress of liver tissue induced by OTA.

[0074] 7. Detection of the relative transcription level of liver pro-inflammatory factors.

[0075] (1) Total RNA extraction: Take out the liver tissue from -80℃, weigh 0.1 g on ice, and add 1 mL Transzolup, 0.2 ml RNA Extraction Agent and 3 grinding beads to the centrifuge tube containing the tissue according to the instructions of TransZol Up enhanced RNA extraction kit. Mix well and homogenize in a high-throughput tissue grinder. After sufficient homogenization, vortex for 5 min to ensure RNA extraction effect. Centrifuge at 4℃, 10000g for 15 min using a high-speed refrigerated centrifuge. The sample after centrifugation is divided into three layers: upper layer (colorless aqueous phase), middle layer, and lower layer (pink organic phase). The tissue RNA is mainly in the upper layer. Carefully pipette 500 μL of the upper aqueous phase and transfer it to a new labeled 1.5 mL RNase-free centrifuge tube. Add 500 μL of pre-cooled isopropanol, mix well, and incubate at room temperature for 10 min. Then centrifuge at 4℃, 10000g for 10 min using a high-speed refrigerated centrifuge. After centrifugation, slowly pour off the supernatant to obtain the white gelatinous precipitate at the bottom of the tube. Add 1 mL of 75% ethanol prepared with RNase-free water, vortex vigorously on a vortex shaker, and then centrifuge at 4℃, 7500g for 5 min using a high-speed refrigerated centrifuge. After centrifugation, slowly pour off the supernatant to obtain the white gelatinous precipitate at the bottom of the tube. Room temperature and air dry until the precipitate becomes translucent. Add 80 μL of RNA lysis solution, mix well, and dissolve after shaking. Then water bath at 60℃ for 10 min. Finally, obtain the RNA sample and store it in a -80℃ refrigerator for future use.

[0076] (2) RNA concentration and purity detection and reverse transcription: Take 1 μL of the RNA sample and use a ultramicro UV spectrophotometer to detect and record the concentration, OD260 and OD280 ratio of the RNA sample, to ensure the availability of the RNA. Reverse transcription: according to the determined RNA concentration and One-Step gDNA Removal and cDNA Synthesis SuperMix (Beijing Quanshijin Biotechnology Co., Ltd.) reverse transcription application system instructions to calculate the amount of RNA and reverse transcription reagents for each sample to be tested. Perform reverse transcription reaction according to the kit usage instructions, and store the completed cDNA sample at -80℃ for future use.

[0077] (3) Primer design and synthesis: The internal reference gene of this experiment is glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In the NCBI database, the sequence of the target gene (IL-1β, IL-6, TNF-α) was searched with chicken (Gallus galus) as the species, and the CDS region was found. The upstream and downstream primers were designed using Primer 5.0 and synthesized by Shanghai Shengong Biological Engineering Co., Ltd. The primer sequences are shown in Table 1. The synthesized primer dry powder was prepared into a working solution with a concentration of 10 μM using RNase-free water and stored in a -20°C refrigerator for use.

[0078] Table 1 Primer sequences

[0079]

[0080] (4) qRT-PCR reaction mixture preparation: According to the instructions of ChamQ Universal SYBP qPCR MasterMix kit (Beijing Zhenbenjin Biotechnology Co., Ltd.), the amount of each reagent was calculated, and the total volume of the qRT-PCR reaction mixture in each reaction well was 20 μL in a RNase-free 1.5 mL centrifuge tube.

[0081] (5) qRT-PCR amplification program: The reaction mixture of the sample to be tested was added to the 96-well plate in order, and then the amplification reaction program was performed on the qTower3G real-time fluorescent quantitative gene amplifier. The relative expression of the gene was calculated by 2 -ΔΔCt method.

[0082] The expression levels of pro-inflammatory factors in the liver tissues of chicks were analyzed, and the results are shown in Figure 5 Compared with the control group (Control group), the OTA group promoted the accumulation of pro-inflammatory factors, showing that the relative gene mRNA levels of IL-6, IL-1β and TNF-α were extremely significantly increased (P<0.0001). However, compared with the OTA group, the O-Inulin group supplemented with inulin significantly reduced the relative gene mRNA levels of IL-6, IL-1β and TNF-α (P<0.0001). The above results show that inulin can alleviate the release of pro-inflammatory factors and inflammatory response in the liver tissues of chicks induced by OTA.

[0083] 8. Cecal content 16s rDNA sequencing analysis

[0084] The collected cecal contents were sent to Beijing Nuowoziyuan Technology Co., Ltd. for DNA extraction, and then the processes of gene library construction and sequencing were completed in sequence to obtain raw data. The raw data was processed by Nuowoziyuan Company to obtain the corresponding OTUs, Shannon, Simpson, Chao1 index and other intestinal flora related data.

[0085] To investigate the effect of inulin on the intestinal flora of chicks caused by OTA poisoning, 16S rRNA sequencing analysis was performed on the cecal contents, and the results are shown in Figure 6 PCoA results showed that the intestinal flora structure of the control group (Control group), OTA group and O-Inulin group was obviously separated at the ASV level, indicating that there were differences in microbial composition between groups (A in Figure 6 Alpha diversity analysis further revealed that compared with the OTA group, the richness and evenness of the flora in the O-Inulin group were significantly increased (B-D in Figure 6 At the level of phylum and genus, there were significant differences in the flora structure of each group (E in Figure 6 Among them, Lactobacillus, Escherichia-Shigella and Clostridia_UCG-014 were the core genera in the cecum of chicks. Further comparison found that compared with the control group (Control group), OTA exposure led to significant enrichment of the relative abundance of 3 genera (Escherichia-Shigella, Clostridia_UCG-014 and Erysipelotrichaceae), and at the same time, it led to the decrease of the abundance of 3 genera (Lactobacillus, Ruminococcus_torques_group and Clostridia_vadinBB60_group). However, inulin intervention effectively reversed this imbalance: in the O-Inulin group, the abundance of Lactobacillus, Ruminococcus_torques_group, Erysipelotrichaceae and Clostridia_vadinBB60_group was significantly higher than that in the OTA group, while the abundance of Escherichia-Shigella and Clostridia_UCG-014 was significantly lower (F-K in Figure 6

[0086] ​In summary, the application discloses a new use of inulin, and particularly relates to application of inulin in preparation of a product for relieving liver damage of chicks caused by intake of OTA, wherein the product can be a medicine, a feed or a feed additive. The medicine, the feed or the feed additive takes inulin as a main component, and the inulin (in a solution form) is added into a basic daily ration of the chicks at 1 mL / kg of a body weight of the chicks. When the chicks mistakenly eat the feed contaminated by the OTA, the inulin can significantly reduce pathological damage of liver tissue of the chicks caused by OTA poisoning, regulate intestinal flora structure of the chicks, improve immunity of the chicks, and reduce mortality of the chicks through multiple ways such as regulating intestinal flora, inhibiting oxidative stress and inflammatory response. The application provides a safe, effective and residue-free natural medicine, feed or feed additive solution for preventing and controlling liver damage caused by poultry OTA poisoning, and has a good application prospect.

Claims

1. Use of inulin in the preparation of a product for alleviating liver damage caused by OTA in chicks.

2. The use according to claim 1, wherein, The product comprises a medicine, a feed or a feed additive.

3. A non-therapeutic method of preventing OTA intoxication in young chickens, wherein, The method is achieved by adding inulin to the feed of chicks or by adding inulin to the drinking water of chicks or by directly gavage of inulin solution.

4. The method as recited in claim 3, wherein, The use amount of inulin is 1 mL / kg, and the concentration of inulin is 0.8 g / mL.

5. A pharmaceutical product comprising inulin, wherein, The effective component of the medicine is inulin, and the medicine is used for preventing and treating liver damage caused by OTA poisoning in chicks.

6. A feed comprising inulin, wherein, The feed contains inulin, and the feed can be used for preventing and treating liver damage caused by OTA poisoning in chicks.

7. A feed additive comprising inulin, wherein, The feed additive contains inulin, and the feed additive can be used for preventing and treating liver damage caused by OTA poisoning in chicks.

8. Use of inulin in improving intestinal flora imbalance caused by OTA poisoning in chicks.

9. Use of inulin in the preparation of a product for improving intestinal flora imbalance caused by OTA poisoning in chicks.

10. Use according to claim 9, wherein, The product comprises a medicine, a feed or a feed additive.

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