Premix for improving egg quality and preparation method and application thereof

Abstract

The invention relates to the technical field of chicken feed, in particular to a premix for improving egg quality and a preparation method and application thereof. The invention provides a premix for improving egg quality. The premix comprises the following components in parts by mass: 10-55 parts of mulberry twig fiber, 5-40 parts of flaxseeds, 10-20 parts of a vitamin and mineral mixture and 10-60 parts of rice chaff, and the premix is mixed with corn, soybean meal, soybean oil, stone powder and calcium hydrophosphate to prepare a complete feed. The complete feed can reduce the accumulation of liver fat of laying hens, effectively control the risk of laying hens suffering from fatty liver, increase the content of unsaturated fatty acid in egg yolk, reduce the content of saturated fatty acid and improve intestinal microorganisms of laying hens, so that the quality of eggs is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of chicken feed, and in particular relates to a premix for improving egg quality, a preparation method and an application thereof. Background Art

[0002] Eggs are one of the best sources of nutrition for humans. In addition to high-quality protein, minerals and vitamins, they are also rich in beneficial fatty acids. At present, with the improvement of people's living standards, the quality of eggs has attracted more and more attention.

[0003] Fatty liver disease, also known as fatty liver syndrome (FLS) or fatty liver hemorrhagic syndrome, is a common nutritional and metabolic disease in laying hens. It primarily involves excessive fat accumulation within liver cells, which impairs liver function. In severe cases, it can even cause liver cell rupture, ultimately leading to intrahepatic hemorrhage and death. Chickens with fatty liver disease experience difficulty achieving peak egg production, with egg production typically reaching around 85% before gradually declining.

[0004] Therefore, while ensuring egg production rate, improving egg quality and reducing the risk of eggs suffering from fatty liver are issues that need to be addressed urgently. Summary of the Invention

[0005] The purpose of the present invention is to provide a premix for improving egg quality and a preparation method thereof. The premix can be used to prepare a diet for improving the metabolic performance of laying hens' livers, improving egg quality and improving the intestinal microorganisms of laying hens.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a premix for improving egg quality. The premix comprises the following components in parts by mass: 10 to 55 parts of mulberry branch fibers, 5 to 40 parts of flax seeds, 10 to 20 parts of a vitamin and mineral mixture, and 10 to 60 parts of rice husks.

[0008] Preferably, the ingredients include the following parts by weight: 20-45 parts of mulberry branch fiber, 15-30 parts of flax seeds, 13-17 parts of vitamin and mineral mixture, and 20-50 parts of rice husk.

[0009] Preferably, the preparation method of the mulberry branch fiber is: drying and crushing the mulberry branch to obtain the mulberry branch fiber;

[0010] The drying temperature is 50-60° C., the drying time is 20-28 hours, and the particle size of the mulberry branch fiber is 100-500 μm.

[0011] Preferably, the vitamin and mineral mixture comprises the following components at final concentrations: vitamin A 800,000-1,000,000 IU / kg, vitamin D3 220,000-500,000 IU / kg, vitamin E 1300-2300 IU / kg, vitamin K3 140-480 mg / kg, vitamin B1 180-280 mg / kg, vitamin B2 300-500 mg / kg, vitamin B6 300-500 mg / kg, vitamin B12 1-3 mg / kg, nicotinamide 2000-4000 mg / kg, D-pantothenate calcium 1000-3000 mg / kg, folic acid 55-75 mg / kg, D-biotin 15-35 mg / kg, choline chloride 280-480 mg / kg, Fe 5000-7000 mg / kg, Cu 700-900 mg / kg, Mn 5000-7000 mg / kg, Zn 5000~7000mg / kg, I 30~40mg / kg, Se 12~48mg / kg, Ca 6000~18000mg / kg.

[0012] The present invention also provides a preparation method of the premix, comprising the following steps: mixing mulberry branch fiber, flax seeds, vitamins, mineral mixture, and rice husks to obtain the premix.

[0013] The present invention also provides the use of the premix in preparing a diet for improving the metabolic performance of laying hens' livers, improving the quality of eggs, and improving the intestinal microorganisms of laying hens.

[0014] The present invention also provides a complete feed for improving egg quality, comprising the following components by weight: 5-10 parts of premix, 55-65 parts of corn, 20-30 parts of soybean meal, 0.5-4 parts of soybean oil, 9-10 parts of stone powder, and 0.5-1 part of calcium hydrogen phosphate;

[0015] The premix is ​​the premix.

[0016] The present invention also provides a method for preparing the complete feed, comprising the following steps: mixing premix, corn, soybean meal, soybean oil, rock powder and calcium hydrogen phosphate to prepare the complete feed.

[0017] The present invention also provides the use of the complete feed in improving the metabolic performance of laying hens' livers, improving egg quality and improving the intestinal microorganisms of laying hens.

[0018] Beneficial effects of the present invention:

[0019] The present invention provides a premix for improving egg quality, comprising the following components: mulberry branch fiber, flaxseed, a vitamin and mineral mixture, and rice husk. The premix is ​​mixed with corn, soybean meal, soybean oil, rock powder, and calcium hydrogen phosphate to prepare a complete feed. The complete feed reduces the fat content of laying hen livers, increases the activity of enzymes related to lipid decomposition (lipase, lipoprotein lipase, hepatic lipase, and total lipase) in the laying hen livers, reduces the activity of enzymes related to fatty acids (fatty acid synthase and acetyl-CoA carboxylase) in the laying hen livers, and has a positive impact on the intestinal bacterial composition of laying hen, thereby reducing the accumulation of liver fat and effectively controlling the risk of laying hen suffering from fatty liver. The complete feed affects the diversity and composition of laying hen intestinal viral communities, thereby further regulating the biosynthesis of short-chain fatty acids. The complete feed increases the content of unsaturated fatty acids and reduces the content of saturated fatty acids in egg yolks, thereby improving the quality of eggs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The contents of egg yolk fatty acids, fatty acid difference analysis, and comparison of egg yolk polyunsaturated fatty acid content between the CK group and the MF5 group were analyzed.

[0021] Figure 2 is the saturated fatty acid content of egg yolk in the CK and MF5 groups;

[0022] Figure 3 is the monounsaturated fatty acid content of egg yolk in the CK and MF5 groups;

[0023] Figure 4 The lipid indexes and enzyme activities related to lipid metabolism in the liver of laying hens in each group;

[0024] Figure 5 These are the light microscopic results of Oil Red O staining of laying hen livers in each group;

[0025] Figure 6Figure 5 is the test results of the changes in the intestinal microbiome of laying hens in each group, where a is the α-diversity of the intestinal bacterial and viral communities of laying hens (Chao1 index: Chao index evaluates the number of OTUs in a sample. The larger the Chao index, the more OTUs there are, indicating that the sample has more species; Shannon index: the larger the Shannon value, the higher the community diversity); b is the Bray-Curtis distance of the intestinal bacterial and viral communities of laying hens tested by PCoA and Adonis (Adonis, multivariate analysis of variance, R2 indicates the degree of explanation of the grouping method for the differences between samples. The larger the R2, the higher the degree of explanation of the grouping method for the differences; P < 0.05 indicates that the difference is significant); c is the Bray-Curtis distance of the intestinal bacterial and viral communities of laying hens; d is the relative abundance of Proteobacteria in the cecal contents of laying hens; e is the relative abundance of Ligamenovirales and Ortervicales in the cecal contents of laying hens;

[0026] Figure 7 are the test results of changes in the functions of the intestinal microbiota of laying hens in each group, wherein a is the Bray-Curtis distance of the intestinal microbial functions of laying hens supplemented with the complete feeds described in Examples 1 to 4 and Comparative Example 1 based on PCoA and adonis tests, b is the Bray-Curtis distance of the intestinal microbial functions of laying hens supplemented with the complete feeds described in Examples 1 to 4 and Comparative Example 1, c is the intestinal microbial functional pathways with different abundances in laying hens supplemented with the complete feeds described in Examples 1 to 4 and Comparative Example 1, and d is the correlation between the functional pathways and bacterial and viral genera in the cecal contents of laying hens supplemented with the complete feeds described in Examples 1 to 4 and Comparative Example 1;

[0027] Figure 8 The following are the test results of the metabolism of the intestinal microbiota of laying hens in each group, where a is the Bray–Curtis distance of SCFA composition in the cecal contents of laying hens supplemented with complete feed in the CK group and the MF5 group based on PCoA and adonis tests (Adonis, multivariate analysis of variance, R2 represents the degree of explanation of the grouping method for the differences between samples; the larger the R2, the higher the degree of explanation of the grouping method for the differences; when P < 0.05, the difference is significant); b is the SCFAs content in the cecal contents of laying hens supplemented with complete feed in the CK group and the MF5 group; c is the Bray–Curtis distance of amino acid composition in the cecal contents of laying hens supplemented with complete feed in the CK group and the MF5 group based on PCoA and adonis tests; d is the AAs content in the cecal contents of laying hens supplemented with complete feed in the CK group and the MF5 group; e is the abundance of genes related to SCAs biosynthesis and urea cycle in the cecal contents of laying hens supplemented with complete feed in the CK group and the MF2 to MF5 groups;

[0028] Figure 9This is the regulatory mechanism of the complete feed of the present invention on laying hens. DETAILED DESCRIPTION

[0029] The present invention provides a premix for improving egg quality, comprising the following components in parts by weight: 10 to 55 parts, preferably 20 to 45 parts, and more preferably 32.5 parts, of mulberry branch fiber;

[0030] 5-40 parts of flax seeds, preferably 15-30 parts, more preferably 22.5 parts;

[0031] 10-20 parts of vitamin and mineral mixture, preferably 13-17 parts, more preferably 15 parts;

[0032] Rice husks are 10 to 60 parts, preferably 20 to 50 parts, and more preferably 35 parts.

[0033] In the present invention, the preparation method of the mulberry branch fiber is as follows: drying and crushing the mulberry branch to obtain the mulberry branch fiber;

[0034] The drying temperature is 50-60° C., preferably 55° C.; the drying time is 20-28 hours, preferably 24 hours; and the particle size of the mulberry branch fiber is 100-500 μm, preferably 300 μm.

[0035] In the present invention, before the pulverization, the dried mulberry branches are passed through a primary sieve and a magnetic separator to remove impurities.

[0036] In the present invention, the vitamin and mineral mixture comprises the following components at final concentrations: vitamin A 800,000 to 1,000,000 IU / kg, preferably 900,000 IU / kg;

[0037] Vitamin D3 220,000 to 500,000 IU / kg, preferably 360,000 IU / kg;

[0038] Vitamin E 1300-2300 IU / kg, preferably 1800 IU / kg;

[0039] Vitamin K3140-480 mg / kg, preferably 310 mg / kg;

[0040] Vitamin B1 180-280 mg / kg, preferably 230 mg / kg;

[0041] Vitamin B2 300-500 mg / kg, preferably 400 mg / kg;

[0042] Vitamin B6 300-500 mg / kg, preferably 400 mg / kg;

[0043] Vitamin B12 1-3 mg / kg, preferably 2 mg / kg;

[0044] Nicotinamide 2000-4000 mg / kg, preferably 3000 mg / kg;

[0045] D-calcium pantothenate 1000-3000 mg / kg, preferably 2000 mg / kg;

[0046] Folic acid 55-75 mg / kg, preferably 65 mg / kg;

[0047] D-biotin 15-35 mg / kg, preferably 25 mg / kg;

[0048] Choline chloride 280-480 mg / kg, preferably 380 mg / kg;

[0049] Fe 5000-7000 mg / kg, preferably 6000 mg / kg;

[0050] Cu 700-900 mg / kg, preferably 800 mg / kg;

[0051] Mn 5000-7000 mg / kg, preferably 6000 mg / kg;

[0052] Zn 5000-7000 mg / kg, preferably 6000 mg / kg;

[0053] I 30-40 mg / kg, preferably 35 mg / kg;

[0054] Se 12-48 mg / kg, preferably 30 mg / kg;

[0055] Ca 6000-18000 mg / kg, preferably 12000 mg / kg.

[0056] The present invention also provides a preparation method of the premix, comprising the following steps: mixing mulberry branch fiber, flax seeds, vitamins, mineral mixture, and rice husks to obtain the premix.

[0057] The present invention also provides the use of the premix in preparing a diet for improving the metabolic performance of laying hens' livers, improving egg quality, and improving the intestinal microorganisms of laying hens.

[0058] In the present invention, the premix reduces the fat content of the laying hen's liver, increases the activity of enzymes related to lipid decomposition (lipase, lipoprotein lipase, hepatic lipase, total lipase) in the laying hen's liver, reduces the activity of enzymes related to fatty acids (fatty acid synthase, acetyl-CoA carboxylase) in the laying hen's liver, and has a positive effect on the intestinal bacterial composition of the laying hen, thereby reducing the accumulation of liver fat and effectively controlling the risk of laying hens suffering from fatty liver.

[0059] The premix reduces the diversity of the intestinal viral community of laying hens and increases the intestinal viral community of laying hens, thereby further regulating the biosynthesis of short-chain fatty acids;

[0060] The premix increases the content of unsaturated fatty acids in egg yolks and reduces the content of saturated fatty acids, thereby improving the quality of eggs.

[0061] The present invention also provides a complete feed for improving egg quality, comprising the following components in parts by weight: 5 to 10 parts, preferably 12.5 parts, of a premix;

[0062] 55-65 parts corn, preferably 60 parts;

[0063] 20-30 parts of soybean meal, preferably 25 parts;

[0064] 0.5-4 parts soybean oil, preferably 2.25 parts;

[0065] 9-10 parts of stone powder, preferably 9.5 parts;

[0066] 0.5-1 part of calcium hydrogen phosphate; preferably 0.75 part

[0067] The premix is ​​the premix.

[0068] The present invention also provides a method for preparing the complete feed, comprising the following steps: mixing premix, corn, soybean meal, soybean oil, rock powder and calcium hydrogen phosphate to prepare the complete feed.

[0069] The present invention also provides the use of the complete feed in improving the metabolic performance of laying hens' livers, improving egg quality and improving the intestinal microorganisms of laying hens.

[0070] In the present invention, the complete feed reduces the fat content of the laying hen's liver, increases the activity of enzymes related to lipid decomposition (lipase, lipoprotein lipase, hepatic lipase, total lipase) in the laying hen's liver, reduces the activity of enzymes related to fatty acids (fatty acid synthase, acetyl-CoA carboxylase) in the laying hen's liver, and has a positive effect on the intestinal bacterial composition of the laying hen, thereby reducing the accumulation of liver fat and effectively controlling the risk of laying hens suffering from fatty liver.

[0071] In the present invention, the complete feed reduces the fat content of the laying hen's liver, increases the activity of enzymes related to lipid decomposition (lipase, lipoprotein lipase, hepatic lipase, total lipase) in the laying hen's liver, reduces the activity of enzymes related to fatty acids (fatty acid synthase, acetyl-CoA carboxylase) in the laying hen's liver, and has a positive effect on the intestinal bacterial composition of the laying hen, thereby reducing the accumulation of liver fat and effectively controlling the risk of laying hens suffering from fatty liver.

[0072] The complete feed affects the diversity and composition of the intestinal viral community of laying hens, thereby further regulating the biosynthesis of short-chain fatty acids;

[0073] The complete feed increases the content of unsaturated fatty acids in egg yolks and reduces the content of saturated fatty acids, thereby improving the quality of eggs.

[0074] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0075] Example 1

[0076] Preparation of mulberry branch fiber: harvested mulberry branches are dried at 55°C for 24 hours to obtain dried mulberry branches; the dried mulberry branches are passed through a primary sieve and a magnetic separator to remove impurities, enter a crushing bin, and then enter a crusher through a feeder and crushed to a particle size of 500 μm to obtain mulberry branch fiber;

[0077] Preparation of premix: 10 kg of mulberry fiber, 38 kg of flax seeds, 12 kg of vitamin and mineral mixture, and 55 kg of rice husks are mixed to prepare a premix;

[0078] A complete feed for improving egg quality:

[0079] A complete feed was prepared by mixing 9 kg of premix, 55 kg of corn, 25 kg of soybean meal, 2 kg of soybean oil, 10 kg of rock powder, and 0.6 kg of calcium hydrogen phosphate.

[0080] The vitamin and mineral mixture includes the following components at final concentrations: vitamin A 800,000 IU / kg, vitamin D3 220,000 IU / kg, vitamin E 2300 IU / kg, vitamin K3 140 mg / kg, vitamin B1 180 mg / kg, vitamin B2 300 mg / kg, vitamin B6 300 mg / kg, vitamin B1 21 mg / kg, niacinamide 2000 mg / kg, D-pantothenate calcium 1000 mg / kg, folic acid 55 mg / kg, D-biotin 35 mg / kg, choline chloride 280 mg / kg, Fe 5000 mg / kg, Cu 700 mg / kg, Mn 5000 mg / kg, Zn 5000 mg / kg, I 30 mg / kg, Se 12 mg / kg, and Ca 6000 mg / kg.

[0081] Example 2

[0082] Preparation of mulberry fiber Reference Example 1;

[0083] Preparation of premix: 28 kg of mulberry fiber, 40 kg of flax seeds, 10 kg of vitamin and mineral mixture, and 40 kg of rice husk are mixed to prepare a premix;

[0084] A complete feed for improving egg quality:

[0085] A complete feed was prepared by mixing 6 kg of premix, 60 kg of corn, 20 kg of soybean meal, 1 kg of soybean oil, 9.5 kg of rock powder, and 0.8 kg of calcium hydrogen phosphate.

[0086] The vitamin and mineral mixture includes the following components at final concentrations: vitamin A 900,000 IU / kg, vitamin D3.5 million IU / kg, vitamin E 2000 IU / kg, vitamin K3200 mg / kg, vitamin B1280 mg / kg, vitamin B2350 mg / kg, vitamin B6400 mg / kg, vitamin B122 mg / kg, niacinamide 2500 mg / kg, D-calcium pantothenate 2500 mg / kg, folic acid 50 mg / kg, D-biotin 30 mg / kg, choline chloride 300 mg / kg, Fe 7000 mg / kg, Cu 750 mg / kg, Mn 7000 mg / kg, Zn 6000 mg / kg, I 35 mg / kg, Se 20 mg / kg, and Ca 9000 mg / kg.

[0087] Example 3

[0088] Preparation of mulberry fiber Reference Example 1;

[0089] Preparation of premix: 42 kg of mulberry fiber, 20 kg of flax seeds, 15 kg of vitamin and mineral mixture, and 45 kg of rice husks are mixed to prepare a premix;

[0090] A complete feed for improving egg quality:

[0091] A complete feed was prepared by mixing 8 kg of premix, 52 kg of corn, 23 kg of soybean meal, 4 kg of soybean oil, 9.4 kg of rock powder, and 0.5 kg of calcium hydrogen phosphate.

[0092] The vitamin and mineral mixture includes the following components at final concentrations: the vitamin and mineral mixture includes the following components at final concentrations: vitamin A 800,000 IU / kg, vitamin D3.4 million IU / kg, vitamin E 2000 IU / kg, vitamin K3 150 mg / kg, vitamin B1 250 mg / kg, vitamin B2 350 mg / kg, vitamin B6 350 mg / kg, vitamin B12 1.5 mg / kg, niacinamide 3500 mg / kg, D-calcium pantothenate 1500 mg / kg, folic acid 60 mg / kg, D-biotin 20 mg / kg, choline chloride 300 mg / kg, Fe 5500 mg / kg, Cu 750 mg / kg, Mn 5500 mg / kg, Zn 6500 mg / kg, I 35 mg / kg, Se 15 mg / kg, and Ca 8000 mg / kg.

[0093] Example 4

[0094] Preparation of mulberry fiber Reference Example 1;

[0095] Preparation of premix: 50 kg of mulberry fiber, 25 kg of flax seeds, 20 kg of vitamin and mineral mixture, and 20 kg of rice husk are mixed to prepare a premix;

[0096] A complete feed for improving egg quality:

[0097] A complete feed was prepared by mixing 10 kg of premix, 65 kg of corn, 30 kg of soybean meal, 3 kg of soybean oil, 9.6 kg of rock powder, and 0.5 kg of calcium hydrogen phosphate.

[0098] The vitamin and mineral mixture includes the following components at final concentrations: the vitamin and mineral mixture includes the following components at final concentrations: vitamin A 1 million IU / kg, vitamin D3.5 million IU / kg, vitamin E 2000 IU / kg, vitamin K3480 mg / kg, vitamin B1190 mg / kg, vitamin B2450 mg / kg, vitamin B6500 mg / kg, vitamin B123 mg / kg, niacinamide 4000 mg / kg, D-calcium pantothenate 2000 mg / kg, folic acid 75 mg / kg, D-biotin 35 mg / kg, choline chloride 400 mg / kg, Fe 7000 mg / kg, Cu 900 mg / kg, Mn 5600 mg / kg, Zn6500 mg / kg, I 40 mg / kg, Se48 mg / kg, and Ca 7000 mg / kg.

[0099] Comparative Example 1

[0100] A complete feed:

[0101] A complete feed was prepared by mixing 65 kg of corn, 30 kg of soybean meal, 3 kg of soybean oil, 9.6 kg of rock powder, and 0.5 kg of calcium hydrogen phosphate.

[0102] Experimental Example 1 Egg production performance

[0103] The laying hens (Yunnan Yunling Guangda Yukou Poultry Company) were randomly divided into 5 groups, each with 10 chickens, and fed with the complete feed of Example 1 (MF2), Example 2 (MF3), Example 3 (MF4), Example 4 (MF5), and Comparative Example 1 (CK), respectively, at a feeding amount of 110 g / hen / d.

[0104] Egg production efficiency

[0105] During the feeding period, the number of eggs, egg weight, daily feed intake and final body weight of the laying hens were counted every day. The final body weight, egg production rate, egg weight, egg weight and feed efficiency of the laying hens after feeding the complete feed described in Examples 1 to 4 and Comparative Example 1 were calculated. The calculation results are shown in Table 1.

[0106] Egg production rate (%) = number of eggs laid / number of test chickens * 100%;

[0107] Egg weight = total egg quantity / total number of eggs;

[0108] Egg quality = average daily egg production per laying hen * egg weight;

[0109] Feed efficiency = daily feed intake / egg weight.

[0110] Table 1 Effect of complete feed on egg production efficiency

[0111]

[0112] As can be seen from Table 1, the complete feeds of Examples 1 to 4 of the present invention had no significant effect on the final body weight, egg production rate, egg quality, and egg weight of laying hens (p>0.05), but had a significant quadratic effect on feed efficiency (p<0.05).

[0113] Egg quality

[0114] After the feeding period, two eggs were collected from each group, and the major diameter (length), minor diameter (width), and eggshell thickness of the eggs were measured using a vernier caliper (the eggshell thickness at the air chamber, equator, and tip were measured separately, and the average value was calculated). The eggshell strength was measured using an egg force analyzer (Robotmation Model-III, Tokyo, Japan). The egg weight, Haugh unit, albumen height, and yolk color number were determined using an egg quality analyzer. The yolks were separated and weighed using an electronic balance. The egg shape index and yolk ratio were calculated according to the formula. The statistical and calculated results of the above egg quality indices are shown in Table 2.

[0115] Egg shape index (%) = major diameter / minor diameter;

[0116] Yolk ratio (%) = yolk weight / egg weight.

[0117] Table 2 Statistics and calculation results of egg quality

[0118]

[0119] As can be seen from Table 2, the complete feed described in Examples 1 to 4 of the present invention had no significant effect on average egg weight, egg shape index, eggshell thickness, eggshell strength, albumen height, Haugh unit, yolk weight, yolk ratio and yolk color score (p>0.05), but had a significant linear effect on eggshell thickness (p<0.05).

[0120] The results showed that the complete feed of the present invention had no significant effect on the egg-laying performance of laying hens.

[0121] Experimental Example 2 Egg Yolk Fatty Acid Composition

[0122] After the feeding was completed, one egg was randomly collected from each group to determine the yolk fatty acid composition. The yolk fatty acid composition of the MF5 and CK groups was analyzed with reference to GB5009.168-2016 "National Standard for the Determination of Fatty Acids in Foods", and the total saturated fatty acids (SFA), dodecanoic acid (C12:0), tetradecanoic acid (C14:0), pentadecanoic acid (C15:0), palmitic acid (C16:0), heptadecanoic acid (C17:0), octadecanoic acid (C18:0), eicosanoic acid (C20:0), heneicosanoic acid (C21:0), docosanoic acid (C22:0), tricosanoic acid (C23:0), tetracosanoic acid (C24:0), total monounsaturated fatty acids (USFA), monounsaturated fatty acids (MUFA) and saturated fatty acids (SFA) in the yolk were calculated. The content of fatty acids (MUFA), myristoleic acid (C14:1), palmitoleic acid (C16:1), oleic acid (C18:1n9c), arachidonic acid (C20:1); docosenoic acid (C22:1n9), nervonic acid (C24:1), polyunsaturated fatty acids (PUFA), linoleic acid (C18:2n6c), α-linolenic acid (C18:3n3), γ-linolenic acid (C18:3n6), eicosadienoic acid (C20:2), linolenic acid (C20:3n6), arachidonic acid (C20:4n6), docosadienoic acid (C22:2), and docosahexaenoic acid (C22:6n3). The test results are as follows: Figures 1 to 3 shown.

[0123] from Figure 1As can be seen from the results, the complete feed in Example 4 significantly reduced the SFA content and increased the USFA content in the egg yolk compared with the CK group (Student's t test, p < 0.05, Figure 1 a); The increase in USFA was mainly caused by the increase in PUFA in egg yolk (Student's t test, p < 0.05, Figure 1 In addition, the fatty acid composition of egg yolks was found to be significantly different between the MF5 and CK groups (adonis test, p < 0.05, Figure 1 b); Further, the complete diet described in Example 4 significantly increased the concentrations of some SFAs (pentadecanoic acid, C15:0; heptadecanoic acid, C17:0; and tricosanoic acid, C23:0) in egg yolk, while palmitic acid (C16:0) and eicosanoic acid (C21:0) were significantly decreased (Student's t test, p < 0.05, Figure 2 ); for MUFA, palmitoleic acid (C16:1), oleic acid (C18:1n9c), and neuraminic acid (C24:1) in the MF5 group were significantly lower than those in the CK group (Student's t test, p<0.05, Figure 3 ); on the contrary, the contents of various PUFAs (linoleic acid, C18:2n6c; α-linolenic acid, C18:3n3; γ-linolenic acid, C18:3n6; eicosadienoic acid, C20:2; docosadienoic acid, C22:2; docosahexaenoic acid, C22:6n3) in the MF5 group were significantly higher than those in the CK group (Student's t test, p < 0.05, Figure 1 c) It can be seen that the complete feed of the present invention can improve the quality of eggs, including increasing the content of PUFA in egg yolk.

[0124] Experimental Example 3 Lipid Metabolism in the Liver

[0125] One laying hen was randomly selected from each group and sacrificed by exsanguination, and the liver and cecal contents were immediately transferred into liquid nitrogen and then stored at −80 °C for further analysis.

[0126] Lipid index in the liver:

[0127] One gram of liver from laying hens in the MF2-MF5 and CK groups was mixed with pre-cooled saline solution at a ratio of 1:9 (wt / vol) in a glass tissue homogenizer (Yizhe 10 mL, Yangzhou, Jiangsu Province, China) to obtain a homogenate. The homogenate was centrifuged at 12,000 rpm at 4°C for 10 min to obtain a supernatant.

[0128] The levels of total protein (BCA), triglyceride (TAG), and total cholesterol (TCH) in the supernatant, as well as the activities of hepatic lipase (HTGL), lipoprotein lipase (LPL), and lipase (LIP) were measured using commercial colorimetric kits (purchased from Nanjing Jiancheng Bioengineering Institute); the activities of fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) in the supernatant were determined using commercial enzyme-linked immunosorbent assay kits (purchased from Nanjing Jiancheng Bioengineering Institute);

[0129] 1 g of liver from laying hens in the MF2-MF5 and CK groups was mixed with anhydrous ethanol and ground, then centrifuged at 12,000 rpm for 10 min at 4°C to obtain the supernatant; LDL-C levels were detected using a commercial kit (purchased from Nanjing Jiancheng Bioengineering Institute).

[0130] Calculation of total esterase (TES): total esterase (TES) = hepatic lipase (HTGL) + lipoprotein lipase (LPL).

[0131] The test results of TAG, TCH, LDL-C, HTGL, LPL, LIP, FAS, ACC, and TES are as follows: Figure 4 shown.

[0132] from Figure 4 It can be seen that the complete feeds described in Examples 1 to 4 significantly reduced the fat content of laying hens' livers, including TAG, TCH and LDL-C (Tukey's HSD test, p < 0.05, Figure 4 a); In addition, the complete feeds described in Examples 1 to 4 increased the activities of enzymes related to lipid decomposition (including LIP, HTGL, LPL, and TES) in the liver of laying hens and inhibited the activities of enzymes related to fatty acid synthesis (FAS and ACC) (Tukey's HSD test, p < 0.05, Figure 4 b).

[0133] Oil red staining:

[0134] The liver tissues of laying hens in the MF2-MF5 and CK groups were collected and fixed in 4% paraformaldehyde. The fixed livers were processed routinely, sliced ​​at 5 μm, and stained with Oil Red O. The stained slices were observed under an optical microscope and photographed. Figure 5 The ratio of positive area to tissue area was calculated and analyzed using Aipathwell software, as shown in Figure 4 "OROPR" in a.

[0135] from Figure 5 It can be seen that compared with the CK group, the positive rate of Oil Red O staining of laying hens' livers in MF2-MF5 groups was significantly reduced (Tukey's HSD test, p<0.05).

[0136] The above results show that the complete feed of the present invention can reduce the accumulation of liver fat, thereby effectively controlling the risk of laying hens suffering from fatty liver.

[0137] Experimental Example 4: Changes in Intestinal Biota

[0138] Total microbial DNA was extracted from the cecal contents of laying hens in the MF2-MF5 and CK groups using the Fecal FastDNA Spin Kit (MP Biomedicals, USA). The quality and concentration of total microbial DNA were assessed using 1.5% agarose gel electrophoresis and a NanoDrop 1000 micro-spectrophotometer (Thermo Fisher Scientific, Inc., USA). 1 μg of total microbial DNA was used as a sample for sequencing preparation using a Ultra sequencing library generation TM Illumina DNA library preparation kits (NEB, USA) were used, with index codes added to the sequences of each sample according to the kit instructions, and PCR amplification was performed. PCR products were purified using the AMPure XP system to generate sequencing libraries. The library size distribution was analyzed using an Agilent 2100 Bioanalyzer, and sequencing was performed using the Illumina NovaSeq6000 platform. A sample containing sterile water was used as a control for library preparation and sequencing. After sequencing, low-quality raw data (phred scores below 30, ambiguous bases, and sequences shorter than 150 bp) were removed using the NGS QC Toolkit to generate clean data.

[0139] The microbial community composition was determined using Kraken2, with all parameters using default settings. This method maps metagenomic reads to the genome catalog currently spanning bacterial and viral phylogenies, thereby obtaining taxonomic classifications and their corresponding reads at the phylum to species level. The results were divided into bacteria and viruses according to the taxonomy, and the relative abundance of each group was calculated to obtain two abundance tables for bacteria and viruses for further statistical analysis. The results are shown in Figure 2. Figure 6 shown.

[0140] from Figure 6 It can be seen that the Chao1 index of intestinal bacteria and virus communities in laying hens in groups MF2 to MF5 was significantly lower than that in laying hens in group CK (Tukey's HSD test, p < 0.05, Figure 6 a). At the same time, the Shannon index of the intestinal virus community of laying hens in the MF2-MF5 groups was found to be significantly lower than that of laying hens in the CK group, but no significant difference was found in the bacterial community (Tukey's HSD test, p < 0.05, Figure 6a). Changes in intestinal bacterial and viral communities in laying hens in the MF2-MF5 and CK groups (adonis test, p<0.05, Figure 6 b), it can be seen that the intestinal bacterial and viral communities in laying hens in the CK group were clustered and separated from those in the MF2 to MF5 groups ( Figure 6 b). In addition, the inter-group variation of intestinal bacterial and viral communities in laying hens in the MF2-MF5 groups increased compared with those in the CK groups (Tukey's HSD test, p<0.05, Figure 6 c) The above conclusions indicate that the complete feed of the present invention has a significant effect on the diversity and composition of the intestinal microbiota of laying hens.

[0141] Bacteroidetes was the dominant phylum in the cecal digestive tract of laying hens, followed by Firmicutes, Proteobacteria, and Actinobacteria. Among these major intestinal bacterial phyla, only Proteobacteria was significantly affected by the complete feed described in Examples 1 to 4. Compared with CK, the relative abundance of Proteobacteria in the cecal contents of laying hens in the MF2 to MF5 groups was significantly reduced (Tukey's HSD test, p < 0.05, Figure 6 d). At the genus level, Bacteroides was the dominant genus, followed by Barnesiella, Paenibacillus, Alistipes, and Faecalibacterium. Several bacterial genera were found to be significantly affected by the complete diet described in Examples 1 to 4, with a decrease in Desulfovibrio, Flavonifractor, Intestinimonas, and Pseudomonas, but an enrichment in Muribaculum and Prevotella. Regarding enteroviruses, Caudovirales was the predominant genus, followed by Herpesvirales. Significant changes in the relative abundance of viruses were only found in two relatively rare orders, namely Ligamenvirales (Filovirales) and Ortervirales (Panretrovirales), where the number of viruses was reduced after adding the complete feed described in Examples 1 to 4 (Tukey's HSD test, p < 0.05, Figure 6e). Regarding viral genera, Alphabaculovirus was the most abundant, followed by D3virus, Pandoravirus, and T4virus. Six viral genera were significantly affected by the complete diet described in Examples 1-4, with Alphabaculovirus, D3virus, and T4virus enriched, while Cytomegalovirus, Mimivirus, and Varicellovirus were reduced.

[0142] Experimental Example 5: Changes in intestinal biota function

[0143] Based on the microbial and viral sequencing results of Experimental Example 4, functional changes were further analyzed. Before annotating the functions, the metagenomic reads were assembled into contigs using MEGAHIT with default parameters. After assembly, only contigs with a length of more than 500 bp were used for further analysis. Potential functional genes were predicted from the contigs using MetaGeneMark and default parameters. CD hit was then used to remove redundant sequences in the samples. In each sample, clean reads were used to map back to the predicted genes using BBMap to obtain the accurate abundance of each gene. To annotate functional genes, the predicted genes were mapped to the KEGG database using BLASTP with an e-value ≤ 1×10 -5 The results are as follows Figure 7 shown.

[0144] from Figure 7 As can be seen from the results, similar to the composition of the intestinal microbiota, PCoA also showed separate clusters between laying hens in the CK and MF2-MF5 groups (adonis test, p < 0.05, Figure 7 a). In addition, the complete diets described in Examples 1 to 4 also increased the inter-group variation in intestinal microbial function (Tukey's HSD test, p < 0.05, Figure 7 b), similar to the composition of the intestinal microbiota. Based on the KEGG annotation, it can be observed that the abundance of several metabolic pathways in the cecal contents of laying hens was significantly affected by the complete feed described in Examples 1 to 4 (Tukey's HSD test, p < 0.05, Figure 7c). The complete feed described in Examples 1 to 4 reduces the ability to metabolize amino acids and biosynthesize O-glycans, while improving the metabolism of major foods such as fatty acids and several sugars. In addition, the complete feed described in Examples 1 to 4 enhances the biosynthesis of some antibiotics (penicillin and cephalosporin), the degradation of some toxic substances (atrazine), and the metabolism of some vitamins (taurine and ascorbic acid), while restricting other pathways (antibiotics: neomycin, kanamycin, and gentamicin; toxic substances: limonene, acetone, and caprolactam; vitamins: retinol and phosphoinositide). In addition, the present invention uses network analysis to explore the potential relationship between intestinal microbial composition and these different functional pathways. The results of the study indicate that intestinal microbial function is more closely related to bacteria, has limited correlation with intestinal viruses, and has few relationships involving bacteria, viruses, and functional pathways ( Figure 7 d) In particular, the abundance of functional pathways related to enteroviruses, such as taurine and oligotaurine metabolism (map00430), β-alanine metabolism (map00410), and retinol metabolism (map00830), decreased after the addition of the complete feed described in Examples 1 to 4.

[0145] Experimental Example 6 Metabolism of Intestinal Microbiota

[0146] All standards of short-chain fatty acids (SCFAs) and amino acids (AAs) used in this experiment were from Sigma-Aldrich (St. Louis, MO, USA).

[0147] The cecal digests of the laying hens in the CK and MF2-MF4 groups were sent to Lingen Biotech for targeted metabolomics analysis. The specific steps were as follows: the cecal digests of the laying hens in the CK and MF2-MF4 groups were resuspended in liquid nitrogen and then added to water as diluted samples by thorough vortexing; then, 50 μl of the diluted sample was taken and thoroughly vortexed with 200 μl of acetonitrile / methanol (1:1) (containing mixed internal standards) to make it homogenous, placed on ice for 30 minutes, and centrifuged at 12000 rpm for 10 minutes. The ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) system (ExionLC TMQuantification of SCFAs and AAs by ACQUITY UPLC Separation was performed on a BEH amide column (2.1 mm × 100 mm, 1.7 μm) maintained at 50°C. The mobile phase consisted of 0.1% formic acid-5 mM ammonium acetate (solvent A) and 0.1% formic acid-acetonitrile (solvent B), delivered at a flow rate of 0.30 mL / min. The solvent gradient was set as follows: initial 80% solvent B for 0.5 min; 80% to 70% solvent B for 2 min; 70% to 45% solvent B for 4 min; 45% to 80% solvent B for 6.01 min; and 80% solvent B for 9 min. The mass spectrometer was operated in positive multiple reaction mode with the following parameters: ion spray voltage of 5500 V, curtain gas of 35 psi, ion source temperature of 550°C, and ion source gases of 50 and 60 psi for 1 and 2, respectively. Based on the results of metagenomic sequencing, the composition of SCFAs (short-chain fatty acids) and AAs (amino acids) in the cecal contents of laying hens in the CK and MF5 groups was determined using targeted metabolomics. The results are as follows Figure 8 shown.

[0148] The results showed that there were significant differences in the composition of SCFAs between the CK and MF5 groups, but not for AAs ( Figure 8 In addition, the cecal contents of laying hens in the MF5 group were found to contain significantly higher levels of acetic acid and propionic acid than those in the CK group ( Figure 8 b), indicating that the complete feed described in Example 4 promoted the synthesis of SCFAs by intestinal microbiota. In contrast, compared with CK, the contents of ornithine and asparagine in the cecal digestive fluid of laying hens in the MF5 group were significantly increased and decreased, respectively ( Figure 8 d), indicating the effect of the complete feed described in Example 4 on the urea cycle. Subsequently, genes responsible for the biosynthesis of acetate and propionate and key genes in the urea cycle were selected from the metagenomic dataset. The results showed that the effect of the complete feed described in Example 4 on the biosynthesis of SCFAs (short-chain fatty acids) and the urea cycle was mainly related to the enrichment of ACSS1_2 and arg genes ( Figure 8 e).

[0149] Experimental Example 7 Regulatory Mechanism of Complete Feed on Laying Hens

[0150] Partial least squares path modeling (PLS-PM) was used to quantify the potential mechanisms of the effects of the complete diet on liver metabolism and egg quality described in Example 4 ("PLSPM" package).

[0151] PLS-PM was conducted to explore the potential mechanism by which the complete feed described in Example 4 regulates the health status of laying hens and egg quality. Figure 9 shown.

[0152] The results showed that the complete feed of the present invention had positive and negative effects on the diversity and composition of the intestinal viral community in laying hens, respectively, which further modulated the biosynthesis of short-chain fatty acids. Furthermore, the addition of the complete feed of the present invention had a positive impact on the intestinal bacterial composition and liver enzyme activity in laying hens, thereby controlling fatty acids in the liver of laying hens. Furthermore, dietary MF improved egg quality by directly affecting liver enzyme activity and indirectly affecting intestinal bacterial diversity.

[0153] As can be seen from the above embodiments, the present invention provides a premix for improving egg quality, a preparation method and an application thereof. Mulberry branch fiber, flax seeds, a vitamin and mineral mixture, and rice husk are mixed to prepare a premix, and the premix is ​​mixed with corn, soybean meal, soybean oil, stone powder, and calcium hydrogen phosphate to prepare a complete feed. The complete feed can reduce the accumulation of fat in the liver of laying hens, effectively control the risk of laying hens suffering from fatty liver, and can also increase the content of unsaturated fatty acids in egg yolks, reduce the content of saturated fatty acids, improve the intestinal microorganisms of laying hens, and thus improve the quality of eggs.

[0154] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A premix for improving egg quality, characterized in that: The invention comprises the following components in parts by weight: 10 to 55 parts of mulberry branch fiber, 5 to 40 parts of flax seeds, 10 to 20 parts of vitamin and mineral mixture, and 10 to 60 parts of rice husk.

2. The premix according to claim 1, wherein The invention comprises the following components in parts by weight: 20-45 parts of mulberry branch fiber, 15-30 parts of flax seeds, 13-17 parts of vitamin and mineral mixture, and 20-50 parts of rice husk.

3. The premix according to claim 2, wherein The preparation method of the mulberry branch fiber comprises the following steps: drying and crushing the mulberry branch to obtain the mulberry branch fiber; The drying temperature is 50-60° C., the drying time is 20-28 hours, and the particle size of the mulberry branch fiber is 100-500 μm.

4. The premix according to claim 1, wherein The vitamin and mineral mixture comprises the following components at final concentrations: vitamin A 800,000-1,000,000 IU / kg, vitamin D3 220,000-500,000 IU / kg, vitamin E 1300-2300 IU / kg, vitamin K3 140-480 mg / kg, vitamin B1 180-280 mg / kg, vitamin B2 300-500 mg / kg, vitamin B6 300-500 mg / kg, vitamin B12 1-3 mg / kg, nicotinamide 2000-4000 mg / kg, D-calcium pantothenate 1000-3000 mg / kg, folic acid 55-75 mg / kg, D-biotin 15-35 mg / kg, choline chloride 280-480 mg / kg, Fe 5000-7000 mg / kg, Cu 700-900 mg / kg, Mn 5000~7000mg / kg, Zn 5000~7000mg / kg, I 30~40mg / kg, Se 12~48mg / kg, Ca 6000~18000mg / kg.

5. The method for preparing the premix according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: mixing mulberry branch fiber, flax seeds, vitamin and mineral mixture and rice husk to obtain a premix.

6. Use of the premix according to any one of claims 1 to 4 in preparing a diet for improving the metabolic performance of the liver of laying hens, improving the quality of eggs and improving the intestinal microorganisms of laying hens.

7. A complete feed for improving egg quality, characterized in that: The invention comprises the following components in parts by weight: 5 to 10 parts of premix, 55 to 65 parts of corn, 20 to 30 parts of soybean meal, 0.5 to 4 parts of soybean oil, 9 to 10 parts of stone powder, and 0.5 to 1 part of calcium hydrogen phosphate; The premix is ​​the premix according to any one of claims 1 to 4.

8. The method for preparing the complete feed according to claim 7, characterized in that: The method comprises the following steps: mixing premix, corn, soybean meal, soybean oil, stone powder and calcium hydrogen phosphate to prepare complete feed.

9. Use of the complete feed according to claim 7 in improving the metabolic performance of laying hens' livers, improving egg quality, and improving the intestinal microorganisms of laying hens.

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