Application of feed additive for changing fatty acid composition of poultry livers in feed

Abstract

The invention discloses application of a feed additive capable of changing fatty acid composition of poultry livers in feed. The fatty acid composition of poultry livers can be changed by adding glucurolactone into poultry feed, the fatty acid composition of poultry liver lipid is changed, the content of super-long carbon chain fatty acid of triglyceride in the poultry livers is regulated and controlled through feed containing glucurolactone, and the super-long carbon chain fatty acid is fatty acid with the carbon atom number larger than 18; by adding glucurolactone into the feed, the content of specific fatty acid deposited in triglyceride of poultry livers is regulated and controlled, the deposition amount of the triglyceride in the livers is changed, and the immunity and oxidation resistance of the poultry are improved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of a feed additive for changing the fatty acid composition of poultry liver in feed. Background Art

[0002] The fatty acid composition of the liver is closely related to the metabolism and health of the liver. Lipids in the liver include triglycerides, free fatty acids (FFAs), lysophosphatidylcholine, free cholesterol and bile acid. Among them, FFAs are the final product of triglyceride decomposition, which can be divided into short-chain fatty acids (C1-C5), medium-chain fatty acids (C6-C12), long-chain fatty acids (C14-C17) and very long-chain fatty acids (C18-C24) according to the length of the carbon chain. The most common in the liver are very long-chain fatty acids, which are divided into saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids according to the number of double bonds on their carbon chains. Saturated fatty acids such as oleic acid have strong cytotoxicity and can cause hepatocyte inflammation and oxidative stress through various mechanisms, and ultimately lead to cell apoptosis. Polyunsaturated fatty acids such as docosahexaenoic acid and eicosapentaenoic acid can reduce lipid deposition by inhibiting oxidative stress and inflammation. Therefore, the different types of fatty acids deposited in poultry livers are closely related to lipid deposition, oxidative stress and inflammation in poultry livers. Therefore, how to directionally regulate the fatty acid composition of liver lipids has also become a key technology for regulating poultry liver health.

[0003] Currently, laying hens are generally raised in cages with limited space for activities. In order to meet the long-term demand for egg production, laying hens have to eat a large amount of food. Therefore, the liver health of laying hens is extremely susceptible to being affected. Some even suffer from fatty liver disease, which leads to a decrease in the immunity and antioxidant capacity of laying hens, and a decrease in egg production rate, causing huge economic losses to poultry production. Therefore, it is necessary to find a new and effective feed additive to prevent and treat fatty liver disease in poultry.

[0004] Glucuronolactone is a natural chemical metabolite produced by the decomposition of glucose. After glucose is decomposed by the liver, it naturally exists in all connective tissues of the human body. Therefore, glucuronolactone has been studied for many years as a liver detoxification and functional beverage. However, the prior art does not disclose that glucuronolactone can solve the problem of long-term lipid metabolism disorder caused by poultry that already suffers from fatty liver disease during breeding by changing the fatty acid composition of poultry liver lipids. Summary of the invention

[0005] The purpose of the present invention is to address the problem that the fatty acid composition of poultry liver cannot be directional regulated in the prior art, and to provide a method for changing the fatty acid composition of poultry liver lipids, so as to be applied to the treatment and improvement of the long-term lipid metabolism disorder caused by poultry suffering from fatty liver disease.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] The invention discloses an application of a feed additive for changing the fatty acid composition of poultry liver in feed. The feed additive comprises glucuronolide. The changing of the fatty acid composition of poultry liver lipid comprises regulating the content of ultra-long carbon-chain fatty acids in triglycerides in poultry liver by feed containing glucuronolide, wherein the ultra-long carbon-chain fatty acids are fatty acids with carbon atoms greater than 18.

[0008] Furthermore, the fatty acid composition of the poultry liver lipids is changed to reduce the content of very long carbon chain fatty acids in triglycerides in the poultry liver.

[0009] Furthermore, the reducing the content of very long carbon chain fatty acids in triglycerides in poultry liver is reducing the content of very long carbon chain fatty acids at the sn-2 position of triglycerides.

[0010] Furthermore, the ultra-long carbon chain fatty acid is a fatty acid with 18 carbon atoms.

[0011] Furthermore, the method of changing the fatty acid composition of poultry liver lipids also includes regulating the content of long-chain fatty acids in poultry livers by feeding feed containing glucuronolactone, wherein the long-chain fatty acids are fatty acids with 14-17 carbon atoms.

[0012] Furthermore, the method of changing the fatty acid composition of poultry liver lipids also includes regulating the content of long carbon chain fatty acids in the sn-2 position of triglycerides in the poultry liver.

[0013] Furthermore, the method of regulating the content of long-chain fatty acids at the sn-2 position of triglycerides in poultry liver is to increase the content of long-chain fatty acids at the sn-2 position of triglycerides.

[0014] Furthermore, the content of glucuronolactone in the feed is 250-400 mg / kg.

[0015] Furthermore, the poultry is laying hens.

[0016] Furthermore, the formula of the feed includes: corn, soybean meal, soybean oil, limestone, dicalcium phosphate, methionine, sodium chloride, choline chloride, mineral premix, and vitamin additives.

[0017] The implementation of the present invention has the following beneficial effects:

[0018] Adding glucuronolide to feed changes the lipid composition and lipid structure of poultry liver. By regulating the specific fatty acid content of triglycerides in poultry liver, namely very long carbon chain fatty acids, it changes the deposition amount of triglycerides in the liver and improves the immunity and antioxidant capacity of poultry. Therefore, glucuronolide can be used as a feed additive to directionally regulate the fatty acid composition of poultry liver to regulate the health of poultry liver, and can also improve and alleviate the long-term lipid metabolism disorder problems caused by poultry with fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings used in the embodiments, wherein the same components are represented by the same reference numerals. Obviously, the drawings described below are only some embodiments of the present invention, and it is possible for ordinary technicians in this field to obtain other drawings based on these drawings without creative work.

[0020] Figure 1 is the production performance of laying hens with or without D-Glu added to their diets for 12 weeks, where A is the egg production rate of laying hens during the entire experimental period, B is the egg production of laying hens during the entire experimental period, C is the feed-egg ratio of laying hens during the entire experimental period, and D is the average egg weight of laying hens during the entire experimental period, where the * mark or p value indicates the statistical result of the 12-week mean.

[0021] Figure 2 The deposition amounts of triglycerides in egg yolk and liver of the control group and D-Glu group. The left figure shows the deposition amounts of triglycerides in liver, and the right figure shows the deposition amounts of triglycerides in egg yolk.

[0022] Figure 3 Representative liver pictures, liver weights, and liver index graphs of laying hens in each group.

[0023] Figure 4 Representative images of the liver of laying hens in each group after HE staining and Oil Red O staining (scale bar: 75 μm), as well as fatty liver score, Oil Red O stained lipid droplet area, abdominal fat percentage and abdominal fat band width.

[0024] Figure 5 The PCA score diagram of quality control results shows that the red dots represent QC samples and the other colored dots represent samples under different condition groups.

[0025] Figure 6 This is the quality control result-RSD distribution diagram, where the vertical axis on the left represents the proportion, the vertical axis on the right represents the specific quantity, and the horizontal axis is the RSD value range.

[0026] Figure 7Figure 1 is a graph of non-targeted liver lipidomics analysis by LC-MS / MS, where:

[0027] Figure A is the OPLS-DA score diagram of the liver lipids of laying hens in each group. The horizontal axis represents the first principal component explanation, and the vertical axis represents the second principal component explanation. The points represent samples, and the colors represent different groups.

[0028] Figure B is the OPLS-DA permutation test diagram. The R2 and Q2 points corresponding to the upper right corner represent the OPLS-DA model parameters of the actual sample grouping, and the left side represents the parameters of different sample grouping models after multiple permutation tests.

[0029] Figure C is a volcano plot of the differential lipids in the liver of laying hens in each group.

[0030] Figure D is a heat map of differential lipid clustering in laying hens, where the columns represent samples, the rows represent lipids, the clustering tree on the left is the differential lipid clustering tree, and the top is the sample clustering tree.

[0031] Figure E is a heat map of differential lipid Class classification of laying hens, where columns represent samples and rows represent lipid classes. The clustering tree on the left side of the figure is the lipid classification clustering tree, and the top is the sample clustering tree.

[0032] Panel F is a bar graph showing LION enrichment in laying hens.

[0033] Figure 8 The heat map of lipid structure characteristics of laying hens in each group, where the X-axis represents the carbon saturation level (number of double bonds) and the Y-axis represents the number of lipid carbon atoms.

[0034] Fig. 9 The heat map of lipid structure characteristics of TG and DG of laying hens in each group. A is the distribution diagram of TG in the liver of laying hens in each group and the distribution diagram of different types of fatty acids, and B is the distribution diagram of DG in the liver of laying hens in each group and the distribution diagram of different types of fatty acids.

[0035] Fig.10 The distribution diagram of fatty acids with different carbon chain lengths at the sn-2 site of TG in each group of laying hens. A is the total distribution of fatty acids with different carbon chain lengths at the sn-2 site of TG, and B, C and D are the distribution of 15, 16 and 18 carbon chain fatty acids at the sn-2 site of TG respectively. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments, and therefore cannot be understood as limiting the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0037] It should be noted that the terms "first", "second", etc. in the specification, claims and drawings of the present invention are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention can be implemented in an order other than the following diagrams or the following descriptions. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or server that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0038] This embodiment provides an application of a feed additive for changing the fatty acid composition of poultry liver in feed, wherein the feed additive includes glucuronolide, and changing the fatty acid composition of poultry liver lipids includes regulating the content of ultra-long carbon chain fatty acids in triglycerides in poultry livers through feed containing glucuronolide, wherein the ultra-long carbon chain fatty acids are fatty acids with carbon atoms greater than 18. Adding glucuronolide to feed can regulate the specific fatty acid content of triglycerides in poultry livers and change the deposition amount of triglycerides in the liver. Therefore, glucuronolide can be used as a feed additive to directionally regulate the fatty acid composition of poultry livers to regulate the health of poultry livers, and can also improve and alleviate the problem of long-term lipid metabolism disorders in poultry suffering from fatty liver disease.

[0039] Optionally, regulating the content of very long carbon chain fatty acids in triglycerides in poultry liver is to regulate the content of very long carbon chain fatty acids in triglycerides in poultry liver. Further, changing the fatty acid composition of poultry liver lipids is to reduce the content of very long carbon chain fatty acids in triglycerides in poultry liver. By reducing the content of very long carbon chain fatty acids in triglycerides in poultry liver, the deposition of very long carbon chain fatty acids in triglycerides in the liver is reduced, the liver health of poultry is improved, thereby improving the management of diseases in poultry.

[0040] Optionally, the ultra-long carbon chain fatty acid is a fatty acid with 18 carbon atoms. Studies have shown that the fatty acids with 18 carbon atoms in triglycerides in the liver of poultry suffering from fatty liver disease are significantly increased. By reducing the fatty acids with 18 carbon atoms in triglycerides, the fatty liver disease of poultry can be alleviated, thereby improving the disease management of poultry. Further, the ultra-long carbon chain fatty acid is a saturated fatty acid and / or unsaturated fatty acid with 18 carbon atoms. Preferably, the ultra-long carbon chain fatty acid is an unsaturated fatty acid with 18 carbon atoms. Further preferably, the ultra-long carbon chain fatty acid is linoleic acid and linolenic acid. Glucuronolactone reduces the degree of unsaturation of deposited fatty acids, making triglycerides more easily hydrolyzed by the body.

[0041] Optionally, reducing the content of very long carbon chain fatty acids in triglycerides in poultry liver is to reduce the content of very long carbon chain fatty acids at the site of triglycerides. Further, reducing the content of very long carbon chain fatty acids in triglycerides in poultry liver is to reduce the content of very long carbon chain fatty acids at the sn-2 position of triglycerides. By reducing the content of very long carbon chain fatty acids at the sn-2 position of triglycerides, the hydrolysis of triglycerides can be promoted and the liver health of poultry can be promoted. Optionally, regulating the content of very long carbon chain fatty acids in triglycerides in poultry liver is to increase the content of very long carbon chain fatty acids in triglycerides in poultry liver. Further, increasing the content of very long carbon chain fatty acids in triglycerides in poultry liver is to increase the content of very long carbon chain fatty acids at the site of triglycerides. Further, increasing the content of very long carbon chain fatty acids at the site of triglycerides is to increase the content of very long carbon chain fatty acids at the sn-2 position of triglycerides. Preferably, the very long carbon chain fatty acid is an unsaturated fatty acid with 18 carbon atoms. More preferably, the ultra-long carbon chain fatty acids are oleic acid and stearidonic acid.

[0042] Optionally, regulating the content of very long carbon chain fatty acids in poultry liver is to regulate the content of very long carbon chain fatty acids in the sn-1 and sn-3 positions of triglycerides in poultry liver. Further, regulating the content of very long carbon chain fatty acids in poultry liver is to increase the content of very long carbon chain fatty acids in the sn-1 and sn-3 positions of triglycerides in poultry liver. The increase in the content of very long carbon chain fatty acids in the sn-1 and sn-3 positions of triglycerides can promote the metabolism of triglycerides and reduce their deposition in the liver.

[0043] Optionally, changing the fatty acid composition of poultry liver lipids also includes regulating the content of long-chain fatty acids in poultry livers by feeding feed containing glucuronolactone, wherein the long-chain fatty acids are fatty acids with carbon atoms of 14-17. Further, the long-chain fatty acids are saturated fatty acids and / or unsaturated fatty acids with carbon atoms of 14-17. By regulating the content of long-chain fatty acids in poultry livers, the hydrolysis of triglycerides can be further promoted and the deposition of lipids in the liver can be reduced.

[0044] Optionally, regulating the content of long-chain fatty acids in poultry livers is to regulate the content of the sites of long-chain fatty acids in poultry livers in triglycerides. Further, regulating the content of long-chain fatty acids in poultry livers is to regulate the content of long-chain fatty acids in poultry livers at the sn-1, sn-2 and sn-3 positions of triglycerides. By regulating the content of sn-2 of long-chain fatty acids in poultry livers in triglycerides, triglycerides in the liver can be more easily hydrolyzed, the stability of triglycerides is reduced, and they are easily degraded in the liver.

[0045] Optionally, regulating the content of long-chain fatty acids in poultry liver is to regulate the content of long-chain fatty acids in poultry liver at the sn-2 position of triglycerides. Further, regulating the content of long-chain fatty acids in poultry liver at the sn-2 position of triglycerides is to increase the content of long-chain fatty acids at the sn-2 position of triglycerides. The increase in the content of long-chain fatty acids at the sn-2 position of triglycerides means that the content of ultra-long-chain fatty acids at the sn-1 and sn-3 positions of triglycerides increases, thereby promoting the metabolism of triglycerides and reducing their deposition in the liver. Preferably, the long-chain fatty acids are unsaturated fatty acids with a carbon number of 15-16. Further preferably, the long-chain fatty acids are pentadecanoic acid and palmitic acid.

[0046] Optionally, regulating the content of long-chain fatty acids at the sn-2 position of triglycerides in poultry liver is to reduce the content of long-chain fatty acids at the sn-2 position of triglycerides. Further, the long-chain fatty acids are saturated fatty acids with carbon atoms of 14-17. Preferably, the long-chain fatty acids are pentadecenoic acid and palmitoleic acid.

[0047] Optionally, regulating the content of long-chain fatty acids in poultry liver is to regulate the content of long-chain fatty acids in poultry liver at the sn-1 and sn-3 positions of triglycerides. Further, regulating the content of long-chain fatty acids in poultry liver is to reduce the content of long-chain fatty acids in poultry liver at the sn-1 and sn-3 positions of triglycerides. Reducing the content of long-chain fatty acids in poultry liver at the sn-1 and sn-3 positions of triglycerides can increase the content of very long-chain fatty acids in the sn-1 and sn-3 positions of triglycerides, and promote the metabolism of triglycerides containing very long-chain fatty acids.

[0048] Optionally, the poultry is laying hens. Laying hens are generally caged and have limited space for movement in the cages. In addition, laying hens have a large food intake, which makes them more susceptible to obesity and fatty liver disease. Therefore, using laying hens as test subjects is more reliable.

[0049] Specifically, the formula of the feed includes corn, soybean meal, soybean oil, limestone, dicalcium phosphate, methionine, sodium chloride, choline chloride, mineral premix, and vitamin additives. Feed is food used to feed animals. When used for livestock and poultry farming, feed is food for animals raised in agriculture or animal husbandry in a narrow sense. Glucuronolactone is stable in nature and can be directly mixed with feed for use. The preparation of reasonable feed is convenient, fast, time-saving and labor-saving. In addition, a reasonable feed ratio can avoid the problem of increased fat content in poultry due to high feed energy, and reduce the probability of poultry suffering from fatty liver disease. After the feed enters the body of poultry, it can improve the long-term lipid metabolism disorder of poultry suffering from fatty liver disease.

[0050] Specifically, the content of glucuron lactone in the feed is 250-400 mg / kg. The upper limit of the content of glucuron lactone can be but not limited to 400 mg / kg, 390 mg / kg, 380 mg / kg, etc., and the lower limit of the content of glucuron lactone can be but not limited to 250 mg / kg, 260 mg / kg, 270 mg / kg, etc.; it can be understood that the content of glucuron lactone can also be any point value within the above range, which is not enumerated here. Adding glucuron lactone in a specific range of content to the feed can effectively change the liver composition and lipid structure of poultry, so that the difference in lipid structure between poultry with fatty liver disease and poultry without fatty liver disease is more obvious, the reliability is improved, and the long-term lipid metabolism disorder of poultry with fatty liver disease is further improved, and the immunity and antioxidant capacity of poultry are improved. Further, the content of glucuron lactone in the feed is 250-350 mg / kg. Preferably, the content of glucuron lactone in the feed is 280 mg / kg.

[0051] Example 1: Grouping design of experimental animals

[0052] A total of 256 healthy 42-week-old Hy-Line brown chickens (egg-laying peak period) with similar egg-laying performance (egg-laying rate higher than 95%) were selected and randomly divided into 2 groups (8 replicates in each group, 16 chickens in each replicate), namely the control group (Control) and the glucuronolactone group (D-Glu group). After the control group and the glucuronolactone group were pre-fed with the basic feed for 1 week, the glucuronolactone group added 280 mg / kg of glucuronolactone to the basic feed, and the control group was still fed with the basic feed. The two groups continued to be fed for 12 weeks, and samples were collected on the last day of 12 weeks.

[0053] Example 2: Preparation of experimental basal diet and analysis of nutritional levels

[0054] The experimental basal diet and its nutritional level are shown in Table 1 and Table 2. The basal diet in each stage of the experiment was the same, and glucuronolactone was added to the diet of each group according to the experimental design.

[0055] Table 1: Composition of the basal diet.

[0056]

[0057] Table 2: Nutritional levels

[0058]

[0059]

[0060] Note: 1) Vitamin A, Vitamin D3, Vitamin E, Vitamin K3, Vitamin B1, Vitamin B2, Vitamin B6, Vitamin B 12 The contents are 13500IU, 3900IU, 30IU, 4.80mg, 3.00mg, 7.50mg, 6.00mg, and 0.024mg respectively; folic acid, biotin, niacin, and calcium pantothenate are 1.50mg, 0.18mg, 45mg, and 18mg respectively.

[0061] 2) In each kilogram of mineral premix: the contents of Fe, Zn, Mn, Se, I, Co and Cu are 144.00 mg, 144.00 mg, 120.00 mg, 0.70 mg, 0.96 mg, 1.00 mg and 20 mg respectively.

[0062] 3) Nutritional level: calculated according to NRC (1994).

[0063] Example 3: Effects of dietary glucuronolactone on growth performance and fat deposition in laying hens

[0064] Test method:

[0065] 1) On the last day of feeding each group of laying hens for 12 weeks, one laying hen was randomly selected from each group and euthanized by cutting the jugular vein for sample collection. Eggs from each laying hen were collected and the yolks were stored at -80℃. Blood samples from the wing vein were collected before slaughter for later testing.

[0066] 2) The liver tissue and abdominal fat of each group of laying hens were weighed and the organ index was calculated. Liver index = liver weight / live chicken weight * 100%; abdominal fat rate = abdominal fat weight / live chicken weight * 100%. Then, the width of the abdominal fat of the laying hens was measured at three locations with a vernier caliper, and the average value was taken at the same time. The liver image of each laying hen was collected. In addition, the determination kit of TG (triglyceride) content in liver and egg yolk was purchased from Nanjing Jiancheng Bioengineering Institute, China.

[0067] 3) Most of the liver tissues were immediately frozen in liquid nitrogen at -80°C, a portion of the liver tissue was mixed with 4% neutral paraformaldehyde buffer for hematoxylin-eosin (HE) staining, and the remaining liver tissue was cut into square sections and embedded in OCT for Oil Red O staining. The liver tissues used for staining were stored at -20°C.

[0068] Test results: Figure 1 As shown in the results, under the long-term egg-laying pressure, the egg-laying performance of laying hens in the peak egg-laying period showed a downward trend in the egg-laying rate, feed-egg ratio, and egg production during the 12-week trial period. The D-Glu group significantly increased the egg-laying rate and egg production of laying hens (P<0.05), and had a significant downward trend in the feed-egg ratio (P=0.061). Figure 2 As shown in the results, the addition of D-Glu significantly reduced the TG content in the liver (P<0.05) and had a trend of significantly reducing the TG content in the egg yolk (P=0.061), but had no significant effect on the cholesterol deposition in the liver and egg yolk (P>0.05).

[0069] In addition, representative images of laying hen livers in the control and D-Glu treated groups are shown in Figure 3 As shown in the figure, the liver of the control group was yellow, while the liver of the laying hens in the D-Glu group was normal red, and the addition of D-Glu had a tendency to reduce the fatty liver score and liver weight of laying hens ( Figure 4 ). The liver was further analyzed, and representative pictures of the liver HE staining results are shown in Figure 4 As shown in the figure, the liver tissue gaps in the control group were larger and there were more vacuolated cells, while the liver of laying hens in the D-Glu treatment group showed compact liver parenchyma and fewer vacuolated cells. Representative pictures of the results of liver oil red O staining are shown in the figure. Figure 4 As shown, there were more stained lipid droplets in the interstitial spaces of liver tissue in the control group, while there were fewer lipid droplets in the liver of laying hens in the D-Glu treatment group. Further quantitative analysis of lipid droplets showed that the addition of D-Glu significantly reduced the lipid droplet area in the liver (P < 0.05). At the same time, the addition of D-Glu significantly reduced the width of the abdominal fat band of laying hens (P < 0.05), but had no significant effect on the abdominal fat rate (P > 0.05).

[0070] In summary, in this experiment, we found that D-Glu can effectively reduce lipid deposition in the liver, yolk, and abdominal fat of laying hens. Lipid accumulation is not only a characteristic of MASLD, but also causes lipid peroxidation and liver damage. Our results in LMH cells and HE staining showed that D-Glu can also effectively reverse oxidative stress and hepatic steatosis caused by lipid accumulation. This shows that the health of laying hens' liver is positively correlated with their egg-laying performance, which is consistent with the previous research results on fatty liver in laying hens. In addition, we found that long-term addition of D-Glu to laying hen feed has a positive effect on laying hens.

[0071] Example 4: Effect of dietary glucuronolactone on lipid profile in the liver of laying hens

[0072] 1) LC-MS / MS non-targeted liver lipidomics analysis

[0073] Preparation of liver tissue lipid samples: First, the laying hen liver was accurately weighed into an EP tube (2 replicates), and then two small steel balls and methanol-water were added to the tube to obtain a mixed solution, and then the mixed solution was placed in a -40°C refrigerator for 2 minutes, and then ground to obtain an ultrasonic extract, and then the ultrasonic extract was placed in an ice water bath for 10 minutes, and then placed at -40°C for 2 hours, and then centrifuged at 4°C for 10 minutes, and the supernatant was filtered with a 0.22μm filter to obtain the sample to be tested, and the sample to be tested was stored at -80°C for LC-MS analysis.

[0074] Preparation of quality control samples (QC): A certain amount of liquid was drawn from each sample and mixed in the same centrifuge tube to obtain a quality control sample.

[0075] 2) Liver lipidomics analysis was performed using an ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) system (UPLC, Agilent 1290 system, USA; MS, AB6600 Sciex, USA). Liver lipidomics chromatographic conditions were set as follows: chromatographic column: Phenomenex Kinetex C18 column (2.1×100 mm, 1.7 μm), column temperature of 55°C, injector temperature of 8°C, flow rate of 0.30 mL / min, A: acetonitrile: water = 60:40 + 10 mM ammonium formate, B: isopropanol: acetonitrile = 90:10 + 10 mM ammonium formate, cation injection volume: 2 μL, anion injection volume: 2 μL. Mobile phase gradient: 0-1.5 min, 40% B; 1.5-10.5 min, 40%-85% B; 10.5-14 min, 85% B; 14-14.1 min, 85-100% B; 14.1-15 min, 100% B; 15-15.2 min, 100%-40% B; 15.2-18 min, 40% B.

[0076] The mass spectrometry conditions for liver lipidomics were as follows: nebulizer gas (GS1), 60 psi, auxiliary heating gas 2 (GS2), 60 psi, curtain gas (CUR), 30 psi, temperature, 600 °C; nebulizer voltage (ISVF), 5000 V and 4500 V in positive and negative modes, respectively; declustering potential (DP) 100 V. Data-dependent acquisition (DDA) method was used for MS / MS acquisition, and each acquisition cycle included a fast TOF MS survey scan (200 MS), followed by continuous acquisition of 11 product ion scans (50 MS each). For TOF MS measurement, the scan mass range was 200-2000 Da, and the collision energy (CE) was set to 10 V. For product ion scans, the mass range was 100-2000 Da, and the collision energy (CE) was set to 45 ± 25 V. Dynamic background exclusion was used and dynamic exclusion was used to remove unnecessary MS / MS information.

[0077] 3) Data processing and analysis;

[0078] The identified metabolites were annotated using the lipid structure database. The data were subjected to principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) using metaX software to obtain the variable importance projection value (VIP). The difference in metabolites between groups was screened with VIP>1 and P<0.05 as the standard. Student's t-test was used to further verify whether the difference in metabolites between groups was significant.

[0079] 4) Test results: First, the repeatability of the QC samples was used to evaluate the quality of the data obtained. The evaluation parameters included QC coincidence and relative standard deviation (RSD), such as Figure 5 As shown, principal component analysis (PCA) evaluated the system stability of LC-MS by 7 cross-validations (7 cycles of cross-validation), and the three closely overlapped on the PCA score graph, indicating that this test has good stability and repeatability, as shown in Figure 6 As shown, the proportion of characteristic peaks less than 30 in QC samples can reach 84.5%, indicating that the data are repeatable. The above analysis shows that the lipidomics analysis method has good stability and reliability throughout the process of sample detection and analysis.

[0080] A total of 1042 metabolites were screened in this test, and the lipid metabolites were analyzed using orthogonal partial least squares discriminant analysis (Orthogonal Projections to Latent Structures Discriminant Analysis, OPLS-DA). The permutation test showed the stability of the OPLS-DA model. Figure 7 As shown in A, the lipid profiles of the livers of laying hens in the control group and the D-Glu group were significantly different. Figure 7 As shown in C, based on the selection criteria of log2(FC) and P<0.05 in the volcano map, the relative peak intensity of lipid molecules between two different sample groups was compared, and a total of 87 differential lipid molecules were screened out in different control groups, of which 29 lipid molecules were upregulated compared with the control group, and 58 lipid molecules were downregulated compared with the control group. These results prove that the lipid molecules identified during the experiment have undergone significant changes. The quantitative results and cluster heat map of 87 differential metabolites are shown in Figure 7 As shown in D, different lipid molecules in the same lipid class may have opposite trends. In order to better verify the lipid differences altered by D-Glu, lipid cluster analysis was performed as shown in Figure 7 As shown in E, D-Glu significantly reduced the content of total TG and DG in lipidomics analysis data, and the results were consistent with the biochemical detection results ( Figure 2 ). Lipid functional enrichment analysis was performed on the metabolites with significant differences ( Figure 7 F), the results showed that the addition of D-Glu enriched the lipid metabolism of laying hens' liver most significantly in the glycerophospholipid ethanolamine metabolic pathway, mitochondrial metabolic pathway, 22-24 carbon fatty acid metabolism and bilayer membrane metabolism, and enriched the largest number of cell membrane components, glycophospholipid GP and endoplasmic reticulum ER metabolic pathways, and the related lipid expressions were significantly increased. The increased expression of GP and ER functions indicates that in the process of fatty acid metabolism, the addition of D-Glu promotes the formation of phospholipids by fatty acids and glycerol skeletons. A part of the phospholipids is secreted through bile and intestines and excreted through feces, and the other part of the phospholipids forms cell membrane components; through the above pathways, the deposition of triglycerides is reduced, that is, the synthesis and deposition of liver lipids are reduced.

[0081] It is generally believed that the liver of laying hens is more inclined to lipid deposition to meet the high demand of egg production, which is similar to the hallmark of MASLD in human clinics, namely the accumulation of TG in hepatocytes. Neutral glycerols are mainly composed of TG, diglycerides (DG) and monoglycerides (MG). In this experiment, D-Glu treatment increased the proportion of phospholipids (PLs) and reduced the proportion of neutral lipids (TG and DG). Studies have shown that PLs can significantly reduce the accumulation of lipids in the liver of NAFLD model rats induced by a high-fat diet. The most significant and enriched lipids are concentrated in metabolic pathways related to GP, ER and mitochondria, in which glycerophospholipids and ethanolamine derivatives are increased compared with the control group, which means that the metabolism of molecules and cellular bioenergetics in liver lipids is increased. Similar liver lipidomics studies have previously reported that the reduction of TG synthesis is accompanied by an increase in GP. We speculate that D-Glu may help to distribute energy and lipids in cellular bioenergetics instead of accumulating lipids in MASLD livers, which may also be important in hepatocyte reconstruction.

[0082] Example 5: Effect of the addition of glucuronolactone on the lipid structure of laying hens' liver

[0083] The structural analysis of the above differential lipids with chain length and saturation information was performed and displayed separately according to lipid classification, and the number of lipid carbon atoms and saturation level were calculated. The differential lipid structure analysis diagram shows that ( Figure 8 ), the differentially expressed DG types were significantly reduced, and the saturation of fatty acids in the reduced DG was low, while TG decreased and increased according to the type, among which the increased part was enriched in fatty acids with low saturation, while the reduced part was enriched in long-chain fatty acids with high saturation. The lipid structure enrichment of PC and PE also showed the same trend.

[0084] We further analyzed the detailed changes of D-Glu on the lipid species of TG and DG. T-test was performed on the total amount of TG and DG in lipids. The lipid group results showed that the addition of D-Glu reduced the TG content in the liver by nearly 5% ( Fig. 9 A), among which the most significant contributions to TG difference were TG (18:2-18:2-18:2), (18:1-18:2-16:0) (22:5-18:2-18:2) / (17:0-18:2-18:2) / (18:1-18:2-16:1) and (18:1-18:1-18:3), while the DG content decreased by nearly 1% ( Fig. 9B) (all of the above are percentages of the total amount of all lipids detected), among which DG (18:2-18:1), (18:2-18:2), and (18:3-16:1) contributed the most to the significant difference in DG. The above results show that the addition of D-Glu significantly reduced the deposition of 18-carbon fatty acids on the glycerol backbone, especially C18:1 and C18:2. After consulting relevant information, the fatty acids of C18:1 and C18:2 are oleic acid and linoleic acid.

[0085] In summary, we found that glucuronolide significantly reduced the deposition of 18-carbon chain fatty acids, especially 18:2 and 18:1, and had no effect on reducing the total deposition of long-chain fatty acids (14-17 carbon chains). We speculate that glucuronolide can specifically bind to 18-carbon chain fatty acids, promote their excretion with urine and feces, and reduce their deposition in the liver. The results of the study showed that the most abundant fatty acid in the liver of humans, rats or hens with MASLD is 18-carbon chain fatty acids. The above studies are similar to the results of this study, indicating that glucuronolide can indeed alleviate the long-term lipid metabolism disorders in laying hens with MASLD by affecting the deposition of 18-carbon chain fatty acids.

[0086] Example 6: Effect of dietary glucuronolactone on the deposition sites of fatty acids in liver triglycerides of laying hens

[0087] The distribution of fatty acids with different carbon chain lengths at the sn-2 site in triglycerides was statistically analyzed, such as Fig.10 As shown in A, the addition of D-Glu significantly reduced the deposition of 18-carbon chain fat at the sn-2 site, and significantly increased the deposition of 15-carbon chain and 16-carbon chain fatty acids at the sn-2 site. The saturation analysis of the fatty acids with significant changes in deposition was performed, and it was found that the addition of D-Glu increased the deposition of 15-carbon chain saturated fatty acids at the sn-2 site, and reduced the deposition of C15:1 (such as Fig.10 B); and 16-carbon chain fatty acids (such as Fig.10 C) and 18-carbon chain fatty acids (such as Fig.10 D) The same trend was also shown in the deposition of sn-2 sites, that is, the deposition of saturated fatty acids in the middle site of TG was increased and the deposition ratio of unsaturated fatty acids was reduced.

[0088] In summary, we analyzed the fatty acids deposited at the sn-2 position in liver triglycerides ( Fig.10), and found that glucuron lactone increased the deposition of very long-chain fatty acids at the sn-1 / 3 site and long-chain fatty acids at the sn-2 site. This means that after the addition of glucuron lactone, the triglycerides deposited in the liver will be more easily hydrolyzed, and the stability of triglycerides will be reduced. This may explain how glucuron lactone reduces the deposition mechanism of 18-carbon chain fatty acids. The addition of glucuron lactone has the effect of increasing the saturation of the fatty acid structure. The presence of double bonds means that it is more difficult to hydrolyze. Glucuron lactone reduces the unsaturation of deposited fatty acids, making them easier to be hydrolyzed by the body.

[0089] Example 7: Effect of dietary glucuronolactone on the antioxidant capacity of laying hens

[0090] 1) Liver samples were homogenized in PBS and centrifuged at 3000 rpm for 10 min at 4 °C to obtain 10% liver tissue homogenate. The protein concentration in the homogenate was determined by Coomassie Brilliant Blue (CBB) protein assay kit (A045-2-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China). After pretreatment, catalase (CAT; kit number: A007-1-1), total antioxidant capacity (T-AOC; kit code: A015-2-1) levels in liver homogenate were determined using a detection kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China).

[0091] 2) The test results are shown in Table 2. The content of catalase and total antioxidant capacity in the liver of the control group was significantly lower than that of the D-Glu group, indicating that the addition of D-Glu improved the antioxidant capacity of laying hens.

[0092] Table 2: Effect of D-Glu supplementation on the antioxidant capacity of laying hens

[0093]

[0094] Note: * indicates significant difference between the two groups. Same below.

[0095] Example 8: Effect of adding glucuronolactone to the diet on the immunity of laying hens

[0096] 1) Total RNA was isolated from liver using the TRIzol (TianGen, DP421, Beijing, China) method. First-strand cDNA was synthesized using a cDNA synthesis kit (TianGen, KR123, Beijing, China) and qRT-PCR was performed using SuperReal PreMix Plus (TianGen, FP205). 2 -ΔΔCtMethods Analysis Results Primers were designed by Primer 5 (as shown in Table 3 below), and β-actin, which is not affected by experimental factors, was used as an internal reference to normalize the differences in technical results.

[0097] Table 3: Primer sequence list.

[0098]

[0099] 2) The test results are shown in Table 4. The expression of pro-inflammatory factors in the liver of the control group was significantly lower than that of the D-Glu group, indicating that the addition of D-Glu improved the immunity of laying hens.

[0100] Table 4: Effect of D-Glu addition on the immunity of laying hens

[0101]

[0102]

[0103] From the above experiments, it can be seen that glucuronolide can alleviate and treat the long-term lipid metabolism disorders caused by MASLD by changing the deposition type, site and saturation degree of fatty acids on triglycerides, and improve the antioxidant capacity and immune capacity of laying hens. From the in vivo experiment, it can be found that the addition of glucuronolide reduces the TG content in the liver and yolk of laying hens; from the lipidomics analysis experiment, it can be found that the addition of D-Glu increases the proportion of phospholipids (PLs) and reduces the proportion of neutral lipids (TG and DG), which increases the expression of GP and ER functions, reduces the deposition of triglycerides, and reduces the synthesis and deposition of liver lipids; from the lipid structure experiment, it can be seen that the addition of D-Glu significantly reduces the deposition of 18-carbon fatty acids on the glycerol skeleton, which can prove that glucuronolide can affect 18-carbon chain fat The long-term lipid metabolism disorder in laying hens suffering from MASLD can be alleviated by reducing the deposition of acids; from the fatty acid deposition site test, it can be seen that glucuronolide increases the deposition of very long-chain fatty acids at the sn-1 / 3 site, increases the deposition of long-chain fatty acids at the sn-2 site, reduces the unsaturation degree of deposited fatty acids, and makes the triglycerides deposited in the liver easier to be hydrolyzed; from the antioxidant capacity test, it can be seen that the addition of glucuronolide significantly improves the antioxidant capacity of laying hens; from the immunity test, it can be seen that the addition of glucuronolide significantly improves the immunity of laying hens.

[0104] It can be seen from the above embodiments that the present invention has the following beneficial effects:

[0105] Adding glucuronolide to feed changes the lipid composition and lipid structure of poultry liver. By regulating the specific fatty acid content of triglycerides in poultry liver, namely very long carbon chain fatty acids, it changes the deposition amount of triglycerides in the liver and improves the immunity and antioxidant capacity of poultry. Therefore, glucuronolide can be used as a feed additive to directionally regulate the fatty acid composition of poultry liver to regulate the health of poultry liver, and can also improve and alleviate the long-term lipid metabolism disorder problems caused by poultry with fatty liver disease.

[0106] What is described above are only some embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art should understand that the present invention may be subject to various changes and improvements, and any modifications, equivalent substitutions and improvements made in accordance with the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A feed additive for changing the fatty acid composition of poultry liver, characterized in that: The feed additive includes glucuronolide, and the changing of the fatty acid composition of poultry liver lipids includes regulating the content of ultra-long carbon chain fatty acids in triglycerides in poultry livers through feed containing glucuronolide, wherein the ultra-long carbon chain fatty acids are fatty acids with more than 18 carbon atoms.

2. The use according to claim 1, characterized in that: The method of changing the fatty acid composition of poultry liver lipids is to reduce the content of very long carbon chain fatty acids in triglycerides in the poultry liver.

3. The use according to claim 2, characterized in that: The method of reducing the content of very long carbon chain fatty acids in triglycerides in poultry liver is to reduce the content of very long carbon chain fatty acids at the sn-2 position of triglycerides.

4. The use according to claim 1, characterized in that: The ultra-long carbon chain fatty acid is a fatty acid with 18 carbon atoms.

5. The use according to claim 1, characterized in that: The method of changing the fatty acid composition of poultry liver lipids further comprises regulating the content of long-chain fatty acids in the poultry liver by feeding feed containing glucuronolactone, wherein the long-chain fatty acids are fatty acids with 14-17 carbon atoms.

6. The use according to claim 1, characterized in that: The method of changing the fatty acid composition of poultry liver lipids also includes regulating the content of long carbon chain fatty acids at the sn-2 position of triglycerides in the poultry liver.

7. The use according to claim 6, characterized in that: The method of regulating the content of the long-chain fatty acids at the sn-2 position of triglycerides in the poultry liver is to increase the content of the long-chain fatty acids at the sn-2 position of triglycerides.

8. The use according to claim 1, characterized in that: The content of the glucuronolactone in the feed is 250-400 mg / kg.

9. The use according to claim 1, characterized in that: The poultry is laying hens.

10. The use according to claim 1, characterized in that: The formula of the feed includes: corn, soybean meal, soybean oil, limestone, dicalcium phosphate, methionine, sodium chloride, choline chloride, mineral premix and vitamin additives.

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