阅读说明：本技术 一种提高抗氧化功能的蛋鸡饲料添加剂及其应用 (Laying hen feed additive capable of improving antioxidant function and application thereof ) 是由 张晓彤 勇强 黄曹兴 陶昱恒 陈雨姝 廖吉丽 董昊路 李鑫 赖晨欢 于 2021-01-29 设计创作，主要内容包括：本发明公开了一种提高抗氧化功能的蛋鸡饲料添加剂及其应用,属于动物饲料添加剂领域。本发明通过将豆科种子中提取的半乳甘露聚糖使用内切甘露聚糖酶进行酶解,过滤除去单糖,得到一种半乳甘露聚糖不完全降解产物,将其添加到蛋鸡饲料中饲喂蛋鸡进行试验,发现本发明的半乳甘露聚糖不完全降解产物作为饲料添加剂可以提高动物体内抗氧化酶的活力,降低脂质过氧化物丙二醛的含量。由于本发明得到的半乳甘露聚糖不完全降解产物是从豆科种子中提取,是一种能够提高动物抗氧化能力的绿色饲料添加剂。(The invention discloses a laying hen feed additive for improving an antioxidant function and application thereof, and belongs to the field of animal feed additives. The galactomannan is extracted from leguminous seeds and is subjected to enzymolysis by using endo-mannanase, monosaccharide is removed by filtering, a galactomannan incomplete degradation product is obtained, and the galactomannan incomplete degradation product is added into laying hen feed to feed laying hens for tests. The galactomannan incompletely degraded product is extracted from leguminous seeds, and is a green feed additive capable of improving the antioxidant capacity of animals.)

1. A layer feed additive for improving the antioxidation function is characterized in that leguminous seeds containing galactomannan are used as raw materials, and a galactomannan incomplete degradation product is obtained by chemical or enzymatic degradation.

2. The laying hen feed additive for improving antioxidant function according to claim 1, wherein the leguminous seeds are selected from one or more of sesbania seeds, guar beans, locust beans and cucurbit pepo.

3. The layer feed additive for improving antioxidant function as claimed in claim 1, wherein the molecular weight of the galactomannan incomplete degradation product is between 342-15000 Da.

4. The layer chicken feed additive for improving antioxidant function according to claim 1, wherein the enzyme in the enzymolysis method is endo-mannanase.

5. The laying hen feed additive for improving the antioxidant function according to claim 1, wherein the enzymatic degradation to obtain the incomplete degradation product of galactomannan comprises the steps of:

1) mechanically pulverizing leguminous seeds containing galactomannan, adding distilled water, extracting and centrifuging to obtain supernatant, adding anhydrous ethanol into the supernatant to obtain precipitate, and drying the precipitate to obtain galactomannan powder solid;

2) weighing galactomannan powder solid, adding distilled water, endo-mannase and citric acid buffer solution, fully and uniformly mixing, keeping enzyme hydrolysis reaction conditions until the reaction is finished, and centrifuging to obtain an enzymolysis liquid supernatant containing galactomannan incomplete degradation products;

3) taking the supernatant of the enzymolysis liquid containing the galactomannan incomplete degradation product, filtering to remove monosaccharide to obtain trapped fluid, concentrating and drying to obtain the galactomannan incomplete degradation product.

6. The laying hen feed additive for improving the antioxidant function according to claim 5, wherein the step 1) is specifically as follows: mechanically pulverizing leguminous seeds containing galactomannan, adding distilled water at a solid-to-liquid ratio of 1: 50, extracting and centrifuging to obtain supernatant, adding anhydrous ethanol into the supernatant to obtain precipitate, and drying the precipitate to obtain galactomannan powder solid.

7. The laying hen feed additive for improving the antioxidant function according to claim 5, wherein the step 2) is specifically as follows: weighing galactomannan powder solid, adding distilled water, endo-mannase and citric acid buffer solution, fully and uniformly mixing, reacting for 24h at the temperature of 50 ℃ with the galactomannan substrate concentration of 2 percent and the enzyme addition of 20U/g galactomannan, the pH value of 4.8, inactivating the endo-mannase after the enzymatic hydrolysis reaction is finished, and centrifuging to obtain the enzymolysis liquid supernatant containing galactomannan incomplete degradation products.

8. The laying hen feed additive for improving the antioxidant function according to claim 5, wherein the step 3) is specifically as follows: and (3) taking the supernatant of the enzymolysis liquid containing the galactomannan incomplete degradation product, filtering by using a nanofiltration membrane with the molecular weight of 200Da to remove monosaccharide to obtain trapped fluid, and concentrating and drying the trapped fluid to obtain the galactomannan incomplete degradation product.

9. A layer feed containing the layer feed additive for improving antioxidant function according to claim 1.

10. The layer feed containing the layer feed additive for improving antioxidant function according to claim 9, wherein: the addition amount of galactomannan incompletely degraded product in the feed for laying hens is 0.01-0.05%.

Technical Field

The invention belongs to the technical field of animal feed additives, and particularly relates to a laying hen feed additive for improving an antioxidant function and application thereof.

Background

A large amount of free radicals are generated in the normal physiological metabolism process of livestock and poultry, but because an antioxidant system exists in vivo, organism tissues and cells can be protected from being damaged by excessive free radicals, namely the generation and the elimination of oxygen radicals are in a dynamic balance. However, with the continuous development of intensive culture, various stress factors exist in the culture process, so that the oxidation resistance of an oxidation resistance system in a livestock body is easily reduced, oxygen free radicals exceed the reasonable level required in the animal body, cells are damaged, the functions of tissues and organs are reduced, diseases are caused, even the livestock and poultry die, and huge economic loss is brought. Thus, oxidative stress has become a significant problem that compromises intensive farming.

However, the current methods for solving the problem of oxidative damage in the feed industry are limited, and the prior art mainly adds a chemical antioxidant such as ethoxyquinoline in the feed, but the latest research finds that the chemical antioxidant has potential risks to human health. Therefore, it is very important to find safe and reliable natural antioxidants. In recent years, many natural polysaccharides and their degradation products have received increasing attention due to their effect of improving the antioxidant capacity of animals. Whether the galactomannan, an important natural polysaccharide, also has the function of improving the antioxidant capacity of animals needs to be fully researched and utilized.

By incomplete galactomannan degradation products is meant natural galactomannan degradation products having a degree of polymerization greater than 2 (molecular weight 342), excluding monosaccharides from the degradation products. The incomplete galactomannan degradation product is prepared from seeds of leguminous plants rich in galactomannan, such as sesbania, guar bean, locust bean, etc., by acid hydrolysis or enzyme hydrolysis. The acid hydrolysis method has high reaction speed, but has the problems of difficult control, more byproducts, complex separation and purification and the like. The biological enzyme method is a preferred method for selectively degrading galactomannan by B-mannase, and is gradually produced in large scale due to the advantages of mild reaction conditions, high selectivity, small load on environmental pollution and the like.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide the laying hen feed for improving the anti-oxidation function. The invention also aims to provide the application of the laying hen feed for improving the anti-oxidation function in improving the anti-oxidation function of laying hens.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a laying hen feed additive for improving antioxidation function is a galactomannan incomplete degradation product obtained by degrading leguminous seeds containing galactomannan by chemical method or enzymatic method.

Further, the leguminous seeds are selected from one or more of sesbania seeds, guar beans, locust beans and cucurbit seeds.

Further, the molecular weight of the incomplete degradation product of galactomannan is between 342-15000 Da.

Further, the enzyme used in the enzymatic method is an endo-mannanase.

Further, the enzymatic degradation step to obtain the incomplete degradation product of galactomannan is:

1) mechanically pulverizing leguminous seeds containing galactomannan, adding distilled water, extracting and centrifuging to obtain supernatant, adding anhydrous ethanol into the supernatant to obtain precipitate, and drying the precipitate to obtain galactomannan powder solid;

2) weighing galactomannan powder solid, adding distilled water, endo-mannase and citric acid buffer solution, fully and uniformly mixing, keeping enzyme hydrolysis reaction conditions until the reaction is finished, and centrifuging to obtain an enzymolysis liquid supernatant containing galactomannan incomplete degradation products;

3) taking the supernatant of the enzymolysis liquid containing the galactomannan incomplete degradation product, filtering to remove monosaccharide to obtain trapped fluid, concentrating and drying to obtain the galactomannan incomplete degradation product.

Further, the step 1) is specifically as follows: mechanically pulverizing leguminous seeds containing galactomannan, adding distilled water at a solid-to-liquid ratio of 1: 50, extracting and centrifuging to obtain supernatant, adding anhydrous ethanol into the supernatant to obtain precipitate, and drying the precipitate to obtain galactomannan powder solid.

Further, the step 2) is specifically as follows: weighing galactomannan powder solid, adding distilled water, endo-mannase and citric acid buffer solution, fully and uniformly mixing, reacting for 24h at the temperature of 50 ℃ with the galactomannan substrate concentration of 2 percent and the enzyme addition of 20U/g galactomannan, the pH value of 4.8, inactivating the endo-mannase after the enzymatic hydrolysis reaction is finished, and centrifuging to obtain the enzymolysis liquid supernatant containing galactomannan incomplete degradation products.

Further, the step 3) is specifically as follows: and (3) taking the supernatant of the enzymolysis liquid containing the galactomannan incomplete degradation product, filtering by using a nanofiltration membrane with the molecular weight of 200Da to remove monosaccharide to obtain trapped fluid, and concentrating and drying the trapped fluid to obtain the galactomannan incomplete degradation product.

A layer feed containing the layer feed additive for improving the antioxidation function.

Furthermore, in the layer feed containing the layer feed additive for improving the antioxidation function, the addition amount of the galactomannan incomplete degradation product in the layer feed is 0.01-0.05%.

Compared with the prior art, the invention has the beneficial effects that:

the galactomannan incomplete degradation product provided by the invention is added into daily ration of laying hens as a feed additive, so that the content of antioxidants (glutathione peroxidase, superoxide dismutase and total antioxidant capacity) in the laying hens can be increased, the antioxidant capacity of the laying hens is improved, and the antioxidant capacity of the laying hens is improved by reducing the content of malonaldehyde in the laying hens. This shows that the galactomannan incomplete degradation product provided by the invention is an effective feed additive capable of improving the antioxidant capacity of animals.

Detailed Description

The invention is further described with reference to specific examples. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

In the following examples, the molecular weight distribution of galactomannans, incomplete degradation products of galactomannans, was determined by Gel Permeation Chromatography (GPC). The chromatographic conditions were as follows: chromatograph: agilent high performance liquid chromatograph 1260, chromatographic column: waters Ultrahydrogel TM 2000 (7.8X 300mm), Waters Ultrahydrogel TM 250 (7.8X 300mm) and Waters Ultrahydrogel^TMThree columns 120 (7.8X 300mm) are connected in series in sequence, and the column is protected: waters Ultrahydrogel^TMGuard Column (6 × 40mm), detector: differential detector, mobile phase: water, mobile phase flow rate: 0.60mL/min, column temperature: 65 ℃, sample injection volume: 10.0. mu.L, molecular weight determination was performed using polyethylene glycol as a standard sample.

Example 1: preparation of galactomannan incomplete degradation product

The preparation method of the galactomannan incomplete degradation product comprises the following steps:

(1) targeted enzymatic hydrolysis of galactomannans

Mechanically pulverizing the seeds of Leguminosae family (sesbania) containing galactomannan to 20-100 mesh, adding distilled water at a solid-to-liquid ratio of 1: 50, extracting at 50 deg.C for 24 hr, centrifuging at 10000 rpm for 10min to obtain supernatant, adding anhydrous ethanol into the supernatant to obtain precipitate, and freeze drying the precipitate to obtain galactomannan powder solid.

Weighing 20.0g of the galactomannan, placing the galactomannan in a 2L enzyme reaction tank, adding distilled water, endo-mannase and 1mol/L citric acid buffer solution to ensure that the volume of reaction liquid is 1000mL, fully and uniformly mixing, reacting for 24h under the conditions of substrate concentration of 2%, enzyme addition of 20U/g galactomannan, pH value of 4.8 and 50 ℃, after the enzymatic hydrolysis reaction is finished, placing enzymatic hydrolysate at 100 ℃ for 10min to inactivate the endo-mannase, centrifuging for 10min under the condition of 10000 r/min, and obtaining supernatant, namely the enzymatic hydrolysate containing the incomplete degradation product of the galactomannan.

(2) Preparation of galactomannan incomplete degradation product

And (2) taking 1000mL of the supernatant of the enzymolysis liquid containing the incomplete degradation product of the galactomannan in the step (1), adding absolute ethyl alcohol under the stirring condition to ensure that the concentration of the ethyl alcohol in the system is 30% (v/v), and centrifuging for 10min under the condition of 10000 r/min to obtain the supernatant and a precipitate. Filtering the supernatant with nanofiltration membrane with molecular weight of 200Da to remove monosaccharide, and collecting the retentate. Concentrating and drying the trapped fluid to obtain galactomannan incomplete degradation product.

The results show that the molecular weight range of the galactomannan incomplete degradation products was determined by GPC as 342-13500 Da.

Example 2: preparation of compound feed containing galactomannan incompletely degraded product

The preparation method of the compound feed containing the galactomannan incomplete degradation product comprises the following steps:

(1) the compound feed for laying hens can be conventional commercial feed for laying hens and can also be feed prepared according to a conventional formula, and the embodiment takes corn-soybean meal type basic feed as an example for explanation

The compound feed for laying hens (corn-soybean meal type basic feed) comprises the following components: every 100kg of compound feed contains: 62kg of corn, 25kg of soybean meal, 8kg of stone powder and 5kg of premix (purchased from Wuhan Huamu biotechnology limited). And fully and uniformly mixing the corn, the soybean meal, the stone powder and the premix to obtain the compound feed for the laying hens.

(2) Preparation of laying hen feed containing galactomannan incomplete degradation product

And (3) respectively taking 22g, 54g and 109g of galactomannan incomplete degradation products to be uniformly mixed with 1kg of the laying hen compound feed in the step (1), and then adding the rest 99kg of laying hen compound feed to be fully mixed to obtain the laying hen feed respectively containing 220 g/ton, 540 g/ton and 1090 g/ton of galactomannan incomplete degradation products.

Example 3: laying hen feed feeding test of mannan incomplete degradation product

Test protocol: 288 Hailan brown laying hens which are 476 days old and have similar and healthy production performance are selected for the test and randomly divided into four groups of I, II, III and IV, each group is divided into 6 repetitions, and each repetition is 12 chickens. II. The III and IV groups are test groups, and 100kg of laying hen feed contains 22g, 54g and 109g of galactomannan incomplete degradation products respectively. Group I was a blank control group fed only corn-soybean meal type basal diet without galactomannan incomplete degradation product additive. Laying hens are raised in a three-layer ladder cage and are raised in an open chicken house. In order to reduce the influence of temperature, light and other non-human factors on results to the maximum extent, all groups of chickens are distributed in two rows of hencoops in the south and the north. The nipple drinker was free to drink water, 16 hours a day. Feeding twice a day (6: 00 am, 3:00 pm), and regularly stirring the feed in the homogenizing groove to ensure that the laying hens fully eat the feed. The feeding amount of the day is properly adjusted according to the feeding condition of the day, so that the material groove is basically free of residual materials at night. Two weeks after the pilot test, the test period was 8 weeks.

One chicken, 24 in total, was randomly selected from each replicate at the end of the experimental period. Blood samples were collected from the pterygoid vein using a sterile needle and syringe, the samples were centrifuged at 3,000 rpm for 15 minutes at 4 ℃ to separate the serum, the obtained serum was collected in a new tube and stored at-20 ℃. The hens were then euthanized by cervical dislocation, the entire gastrointestinal tract was quickly removed and placed on a refrigerated stainless steel tray, the jejunum (from the end of the pancreatic annulus to the Meckel diverticulum) and ileum (from the Meckel diverticulum to the ileocecal junction), mesenteric membranes were separated, the jejunum and ileum were opened longitudinally and rinsed with cold phosphate buffer, the jejunum and ileum mucosa were carefully scraped using a sterile glass microscope slide, collected with a cryovial, placed in liquid nitrogen for quick freezing, and stored at-80 ℃ for further analysis.

Using a homogenizer, approximately 0.3g of the jejunal or ileal mucosa is homogenized with cold 0.86% sodium chloride solution (1: 9, wt/vol) and centrifuged at 3,000 rpm for 15 minutes at 4 ℃ and the supernatant is stored at-20 ℃ for subsequent analysis. Total protein concentration was determined according to the Bradford method using bovine serum albumin as a standard protein. The levels of superoxide dismutase, malondialdehyde, glutathione peroxidase and total antioxidant capacity in serum, jejunum and ileum were determined using an enzyme-linked immunosorbent assay kit provided by the institute of bioengineering, buhui, Nanjing.

The influence of adding galactomannan incomplete degradation products into daily ration of the laying hens on the oxidation resistance index of the laying hens is shown in table 1, and the result shows that the supplement of the galactomannan incomplete degradation products in basic diet of the laying hens can improve the levels of superoxide dismutase, glutathione peroxidase and total oxidation resistance. In evaluating the antioxidant capacity of any animal, the total antioxidant capacity, superoxide dismutase and glutathione peroxidase activities are important indicators for evaluating the antioxidant capacity. Of these, superoxide dismutase and glutathione peroxidase are considered as the major elements of the first level of antioxidant defense in cells, as they form the main protective system against oxidative damage. In addition, total antioxidant capacity is also a useful proxy for overall levels of enzymatic and non-enzymatic antioxidants. In the present invention, the effect of increasing antioxidant levels is different in four tissues or organs (serum, liver, jejunum and ileum), and more pronounced in the ileum. Meanwhile, the results in table 1 also show that the addition of galactomannan incompletely degraded products in the basal diet of laying hens can reduce the content of malondialdehyde. Malondialdehyde content may reflect the degree of lipid peroxidation and indirectly the degree of cell damage. From the above, it is known that incomplete degradation products of galactomannan improve the antioxidant capacity mainly by increasing the antioxidant content (glutathione peroxidase, superoxide dismutase and total antioxidant capacity) and secondly by decreasing the malondialdehyde content. This shows that the galactomannan incomplete degradation product provided by the invention is an effective feed additive capable of improving the antioxidant capacity of animals.

TABLE 1 influence of galactomannan incomplete degradation products on the antioxidant index of egg-laying hens