CN114569638A

CN114569638A - Ackermanella muciniphila microecological preparation and application thereof to laying hens

Info

Publication number: CN114569638A
Application number: CN202210208096.5A
Authority: CN
Inventors: 刘丹; 胡永飞; 魏福晓; 金晓露
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2022-03-03
Filing date: 2022-03-03
Publication date: 2022-06-03
Anticipated expiration: 2042-03-03
Also published as: CN114569638B

Abstract

The invention discloses an akkermansia muciniphila microecological preparation and application thereof to laying hens. According to the invention, three microecological bactericides of akkermansia muciniphila, including live bactericides, pasteurella inactivated bactericides and freeze-dried bactericides are added into the feed and irrigated by the Hailan brown laying hens, and the result shows that the bactericides of the akmansia muciniphila can reduce the increase of the blood fat and the liver fat level caused by high-fat diet of the Hailan brown laying hens, reduce fat deposition in vivo and improve the quality of eggs. The Ackermanella muciniphila micro-ecological preparation (microbial inoculum) provided by the invention has comprehensive functions, is non-toxic and harmless, has the same action effect in the aspects of preventing/relieving fatty liver of laying hens and improving egg quality by using a pasteurella inactivated microbial inoculum, a freeze-dried microbial inoculum and a live microbial inoculum, and has greater advantages in the aspects of storage and addition modes. The invention provides a basis for taking the akkermansia muciniphila as a novel microecological preparation in the laying hen breeding industry.

Description

Ackermanella muciniphila microecological preparation and application thereof to laying hens

Technical Field

The invention relates to the technical field of biology, in particular to an akkermansia muciniphila microecological preparation and application thereof to laying hens.

Background

The fatty liver syndrome is a common and frequent nutritional metabolic disease of cage-rearing laying hens, and is mainly caused by endocrine dyscrasia and metabolic disorder in vivo caused by laying hen dyscrasia, so that excessive fat deposition is caused. Sick chickens like to lie on the bed, the abdomen of the sick chickens is expanded and drooped, the sick chickens touch the soft abdomen, and have a large amount of fat accumulation, lethargy and lassitude. The egg yield of sick chickens is influenced, the production performance and the egg quality are reduced, particularly, the quality of eggshells of old laying hens is reduced, the number of broken eggs, soft-shelled eggs and eggshell eggs is increased, and huge economic loss is caused to the laying hen industry.

Eggs are high cholesterol food, the serum cholesterol level of a human body cannot be influenced when the eggs are normally taken, but for people with high blood fat and high cholesterol, the intake of the eggs still needs to be strictly controlled, so that the development of the low cholesterol eggs has a great application value.

At present, the fatty liver is relieved mainly by adding substances such as fat (such as linoleic acid), choline, carnitine and the like which are rich in unsaturated fatty acid into the feed; the traditional Chinese medicine is also used for conditioning and preventing fatty liver; in addition, antibiotics are added to change the structure of intestinal flora, inhibit the proliferation of harmful bacteria, reduce the damage to the liver and relieve the fatty liver. However, the abuse of antibiotics can cause the drug resistance of poultry, the veterinary drug residue in poultry products is more and more serious, and the resistance reduction and non-resistance cultivation become the development trend of the poultry industry along with the proposal of the 'banning' oral size. At present, the substitute antibiotic product mainly comprises fermented feed, an enzyme preparation, antibacterial peptide, an acidifier, Chinese herbal medicines, a microecological preparation and the like, wherein the microecological preparation is widely valued and used as a pollution-free green additive in the animal husbandry industry due to the advantages of safety, no medicine residue and the like. Common probiotics used as microecologics comprise lactic acid bacteria, bacillus, bifidobacteria and the like, although the probiotics are various, the effects are different, so that the development of a safe, non-toxic and harmless microecologics for preventing/relieving the fatty liver of the laying hens and improving the egg quality is urgently needed in the field.

Disclosure of Invention

The technical problem to be solved by the invention is how to improve the quality of poultry eggs and/or improve the protein height of the poultry eggs and/or how to reduce the fat accumulation in the abdomen of the poultry and/or how to reduce the blood fat level and the liver fat level of the poultry and/or how to obtain the new function of akkermansia muciniphila.

In order to solve the technical problem, the invention firstly provides at least one of the following applications of akkermansia muciniphila:

A1) the application of the akkermansia muciniphila in preparing products for treating or relieving the metabolic related diseases of poultry.

A2) The application of the akkermansia muciniphila in improving the quality of poultry eggs or preparing products for improving the quality of the poultry eggs.

A3) The application of the akkermansia muciniphila in improving the protein height and the Hardgrove unit level of poultry eggs.

A4) Use of akkermansia muciniphila for reducing the accumulation of abdominal fat in poultry and/or reducing the weight of abdominal fat in poultry and/or for the manufacture of a product for reducing the weight of abdominal fat in poultry.

A5) The application of the akkermansia muciniphila in relieving poultry liver pathological changes or preparing products for relieving poultry liver pathological changes.

A6) The application of the akkermansia muciniphila in maintaining the structural integrity and the morphological integrity of poultry liver and/or relieving poultry liver steatosis and/or preparing products for relieving poultry liver steatosis.

A7) The application of the akkermansia muciniphila in reducing the blood fat level of poultry or preparing products for reducing the blood fat of poultry.

A8) The application of the akkermansia muciniphila in reducing cholesterol and triglyceride in poultry serum and/or reducing the level of poultry liver fat and/or preparing products for reducing poultry liver fat.

A9) The application of the akkermansia muciniphila in reducing the cholesterol content in the egg yolk of poultry and/or preparing products for reducing the cholesterol content in the egg yolk of poultry.

The Akkermansia muciniphila strain described above may be Akkermansia muciniphila (Akkermansia muciniphila) having the strain number ATCC BAA-835 at the american Type Culture collection ATCC (american Type Culture collection).

In the above-mentioned application, the poultry may be layer chicken, and the metabolic-related disease may be layer chicken fatty liver syndrome.

In order to solve the technical problems, the invention also provides a microbial inoculum. The microbial inoculum contains the akkermansia muciniphila described above or/and the metabolites of akkermansia muciniphila described above or/and the culture of akkermansia muciniphila described above. The microbial inoculum has at least one of the following characteristics:

B1) treating or relieving poultry metabolic related diseases.

B2) Improving the quality of poultry eggs.

B3) Improving the protein height and the harderian unit level of the poultry eggs.

B4) Reducing the accumulation of abdominal fat in poultry and/or reducing the abdominal fat weight of poultry.

B5) Relieving poultry liver pathological changes.

B6) Maintaining structural and morphological integrity of poultry liver and/or alleviating poultry liver steatosis.

B7) Reducing the blood lipid level of poultry.

B8) Reducing the levels of cholesterol and triglycerides in the serum of poultry and/or reducing the liver lipid levels of poultry.

B9) Reducing cholesterol content in poultry egg yolk.

The culture of akkermansia muciniphila as described above is a substance obtained by culturing akkermansia muciniphila in a microbial culture medium (e.g., a fermentation broth containing akkermansia muciniphila and a substance secreted into a liquid medium, or a solid medium containing akkermansia muciniphila and a substance secreted into a solid medium).

The active ingredients of the microbial inoculum can be akkermansia muciniphila or/and metabolites of akkermansia muciniphila, the active ingredients of the microbial inoculum can also contain other biological ingredients or non-biological ingredients, and other active ingredients of the microbial inoculum can be determined by a person skilled in the art according to the effect of the microbial inoculum.

The microbial inoculum may also include a carrier. The carrier may be a solid carrier or a liquid carrier. The solid carrier is a mineral material or a biological material; the mineral material may be at least one of grass peat, clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the biological material is at least one of straws, pine shells, rice straws, peanut shells, corn flour, bean flour, starch, grass peat and animal manure of various crops; the liquid carrier can be water; in the microbial inoculum, the akkermansia muciniphila or/and the metabolite of the akkermansia muciniphila can exist in the form of cultured living cells, fermentation liquor of the living cells, filtrate of cell culture or mixture of the cells and the filtrate. The preparation formulation of the microbial inoculum can be various preparation formulations, such as liquid, emulsion, suspending agent, powder, granules, wettable powder or water dispersible granules.

According to the needs, the microbial inoculum can also be added with a surfactant (such as Tween 20, Tween 80 and the like), a binder, a stabilizer (such as an antioxidant), a pH regulator and the like.

As described above, the metabolite of Akkermansia muciniphila may be a fermentation broth of Akkermansia muciniphila. The fermentation liquor of akkermansia muciniphila can be prepared according to the following method: culturing Ackermanella muciniphila in a liquid fermentation culture medium, and collecting a fermentation liquid (containing the Ackermanella muciniphila and substances secreted into the liquid culture medium), wherein the fermentation liquid is a metabolite of the Ackermanella muciniphila.

The Akkermansia muciniphila strain described above may be Akkermansia muciniphila (Akkermansia muciniphila) having the strain number ATCC BAA-835 at the american Type Culture collection ATCC (american Type Culture collection). The poultry can be laying hens, and the metabolic-related disease can be laying hen fatty liver syndrome.

The above-mentioned microbial inoculum can be live microbial inoculum, pasteur inactivated microbial inoculum or freeze-dried powder microbial inoculum.

The effective bacteria concentration of the viscous protein akkermansia in the live bacteria agent and/or the pasteur inactivated bacteria agent can be more than or equal to 1 x 10⁸CFU/mL。

In the microbial inoculum, the preparation process of the freeze-dried powder can also comprise adding a cryoprotectant into the culture for vacuum freeze-drying. The cryoprotectant may be skim milk powder. The skimmed milk powder can be added in an amount of 10%.

In order to solve the technical problems, the invention also provides at least one application of the microbial inoculum, which is described above, as follows:

C1) the application of the microbial inoculum in preparing products for treating or relieving poultry metabolic related diseases.

C2) The microbial inoculum is applied to improving the quality of poultry eggs or preparing products for improving the quality of the poultry eggs.

C3) The microbial inoculum is applied to improving the protein height and the Hardgrove unit level of poultry eggs.

C4) The application of the microbial inoculum in reducing the accumulation of poultry abdominal fat and/or reducing the weight of poultry abdominal fat and/or preparing products for reducing the weight of poultry abdominal fat.

C5) The microbial inoculum is applied to relieving poultry liver pathological changes or preparing products for relieving poultry liver pathological changes.

C6) The microbial inoculum is applied to maintaining the integrity of the poultry liver structure and shape and/or relieving poultry liver steatosis and/or preparing products for relieving poultry liver steatosis.

C7) The microbial inoculum is applied to reducing the blood fat level of poultry or preparing products for reducing the blood fat of poultry.

C8) The microbial inoculum is applied to reducing cholesterol and triglyceride in poultry serum and/or reducing the level of poultry liver fat and/or preparing products for reducing poultry liver fat.

C9) The microbial inoculum is applied to reducing the cholesterol content in the egg yolk of poultry and/or preparing products for reducing the cholesterol content in the egg yolk of poultry.

In the microbial inoculum or the application, the laying hens can be 28-week-old laying hens and/or 64-week-old laying hens.

In the above application, the laying hen may be a harland brown laying hen.

The product described above may be a feed, a feed additive and/or a medicament.

According to the invention, three micro-ecological preparations of the akkermansia muciniphila are added into the feed and are irrigated to the helan brown laying hens, wherein the micro-ecological preparations comprise live bacteria, pasteur inactivation bacteria and freeze-dried powder bacteria. The akkermansia muciniphila microecological preparation (microbial agent) provided by the invention has comprehensive functions, is non-toxic and harmless, and has the same action effect in the aspects of preventing/relieving the fatty liver of laying hens and improving the egg quality as the pasteurella inactivated microbial agent, the freeze-dried microbial agent and the viable microbial agent, and has greater advantages in the aspects of storage and addition modes. The invention provides a basis for taking the akkermansia muciniphila as a novel microecological preparation in the laying hen breeding industry.

Compared with the prior art, the invention has the following advantages:

(1) the invention reports that the akkermansia muciniphila can prevent/relieve fatty liver of laying hens for the first time, improves the quality of eggs, determines a novel probiotic micro-ecological preparation, can be used as a feed additive, has the characteristics of safety, no toxicity and no harm, and has wide market application prospect.

(2) The invention provides three preparation methods of a viscous protein akkermansia microecological preparation.

(3) The adhesive protein akkermansia occulta pasteur inactivated bacterial agent, the freeze-dried powder bacterial agent and the viable bacterial agent have the same effect in the aspects of preventing/relieving the fatty liver of laying hens and improving the egg quality, and have greater advantages in the aspects of storage and addition modes.

Drawings

Fig. 1 is a liver anatomy diagram.

Fig. 2 is the liver weight and liver index.

Fig. 3 is a diagram of pathological section of liver. The length of the scale is 200 μm.

FIG. 4 shows the effect of the live and inactivated bacterial agents of akkermansia muciniphila on the cholesterol content of egg yolk.

FIG. 5 is a diagram showing the anatomy of liver and abdominal fat.

FIG. 6 shows the effect of Akkermansia muciniphila lyophilized powder on the content of cholesterol in egg yolk of old-aged laying hens.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The Akkermansia muciniphila (Akkermansia muciniphila) BAA-835 in the following examples is derived from American Type Culture Collection (ATCC) and has the strain number of ATCC BAA-835, hereinafter referred to as BAA-835.

The experimental data in the examples of the invention were statistically analyzed using SPSS 22.0 software, with one-way anova, P <0.05 being a difference of statistical significance.

Example 1 preparation of Ackermanella muciniphila Microecopreparations

1. Preparation of Ackermanella muciniphila BAA-835 viable bacteria agent

Under aseptic condition, resuspending Akkermansia muciniphila (Akkermansia muciniphila) strain BAA-835 freeze-dried powder with sterile water, sucking bacterial suspension, pumping all into 1-2 Columbia blood plates with 200 μ L/plate, uniformly coating, placing the plates in an Anaerobic incubator (Whitley DG250 Anaerobic Workstation, Bailai Puhui (Beijing) science and technology Co., Ltd.), culturing at 37 deg.C for 5-7d, wherein the Anaerobic gas component is N₂:CO₂:H₂80:10:10, then selecting a single colony to be activated for two generations in a modified GAM liquid culture medium (15 g/L of peptone, 10g/L of tryptone, 3g/L of soytone, 5g/L of yeast extract, 2g/L of beef powder, 13.5g/L of digested serum powder, 1.2g/L of beef liver extract, 3g/L of glucose, 2.5g/L of monopotassium phosphate, 3g/L of sodium chloride, 0.3g/L of soluble starch, 0.3g/L of L-cysteine, 0.15g/L of sodium thioglycolate and pH 7.2). The activated BAA-835 is inoculated in an anaerobic improved GAM liquid culture medium according to the inoculation amount of 5 percent, and is placed in a biochemical incubator (LRH-250F, Shanghai-Hengyue scientific instruments, Co., Ltd.) at 37 ℃ for culturing for 48 hours to obtain a BAA-835 thallus culture solution, and the phenomenon that the liquid culture medium becomes turbid and precipitates appear at the bottom can be obviously observed. Centrifuging the thallus culture solution at 8000rpm for 30min, discarding supernatant, resuspending the precipitate with sterile anaerobic PBS to obtain bacterial suspension, counting GAM plate, and adjusting the concentration of bacterial suspension to make BAA-835 content 3.5 × 10⁸CFU/mL was stored in a refrigerator at-80 ℃ and used in the following examples as a viable bacterial agent of Ackermanella muciniphila BAA-835.

Wherein:

(1) sterile anaerobic PBS was applied directly to PBS (1.54g Na)₂HPO₄·12H₂O、0.24g KH₂PO₄8g NaCl and 0.2g KCl are dissolved and the volume is fixed to 1000mL), glycerol is diluted to the concentration of 20%, the glycerol is filled into an anaerobic bottle, an automatic air pumping and exchanging device (Beijing Esche instruments Co., Ltd.) is used for introducing nitrogen into the anaerobic bottle for air replacement, the glycerol is sterilized in an autoclave at the temperature of 121 ℃ for 15min after oxygen is exhausted, the glycerol is cooled and then is subpackaged into 5mL centrifuge tubes with 2mL per tube to obtain sterile anaerobic PBS, and the sterile anaerobic PBS is frozen and stored in a refrigerator with the temperature of 80 ℃ below zero for subsequent use.

(2) The GAM plate is prepared by adding agar powder at a ratio of 15g/L on the basis of improved GAM liquid culture medium.

2. Preparation of akkermansia muciniphila BAA-835 pasteurella inactivated bacteria agent

The Ackermanella muciniphila BAA-835 viable bacteria agent in the step 1 (the BAA-835 content of the bacteria agent is 3.5 multiplied by 10)⁸CFU/mL) was inactivated in a water bath at 70 ℃ for 30min, and used in the following examples as a pasteurisation inoculum of akkermansia muciniphila BAA-835.

3. Preparation of akkermansia muciniphila BAA-835 lyophilized powder microbial inoculum

Obtaining BAA-835 thallus culture solution according to the culture method of the step 1, centrifuging the BAA-835 thallus culture solution at 8000rpm for 30min, removing supernatant to obtain BAA-835 thallus precipitate, adding 10% of skimmed milk powder into the BAA-835 thallus precipitate, mixing well, and performing vacuum freeze drying (24h) by using a freeze dryer (Beijing Bo Yi kang laboratory instruments Co., Ltd.), wherein the obtained freeze-dried powder is stored at-20 ℃. The concentration of the fermentation liquid before the BAA-835 freeze-dried powder is 1 x 10 as the Ackermanella mucronatum BAA-835 freeze-dried powder microbial inoculum⁸CFU/mL (since both live and Pasteurella viscolytica BAA-835 bacteria can function, the loss rate during lyophilization is not considered) was used in the following examples.

Example 2 application of Ackermanella muciniphila BAA-835 live bacteria and inactivated bacteria agent in relieving fatty liver of laying hens

1. The experimental method comprises the following steps:

the layer model selected in this embodiment is: high energy low protein diet (HFD) induced fatty liver model. 80 kalanchoe brown laying hens (28095; bonnong poultry limited, city, n.) with 28 weeks of age and similar body weights (2.010 ± 0.10kg) were randomly divided into 4 groups: a control group (ND), a high fat model group (HFD), an akkermansia muciniphila viable bacteria-buffered group (HFD _ LA), and an akkermansia muciniphila inactivated bacteria-buffered group (HFD _ PA), 20 per group. The site of this example was at poultry test base of the university of agriculture in china (28095; state, north river) and included a1 week prefeed period and an 18 week gavage period. All groups were fed basal diets during the pre-feeding period.

Control group (ND): basal diet was fed during the positive feeding period, and each chicken was gavaged with 5mL of sterile PBS per day.

High fat model group (HFD): feeding high-energy low-protein daily ration in a positive feeding period, and feeding 5mL of sterile PBS to each chicken every day. The formula and nutritional levels of the basal diet and the high energy low protein diet are shown in table 1.

TABLE 1 daily ration formulas and nutritional levels

Note: providing a Provided per kilogram of ration of Provided per kilogram of ration: mn,60 mg; fe,80 mg; cu,8 mg; i,1.2 mg; se,0.3mg. VA,12,500 IU; VD3,32,500 IU; VE,18.75 mg; VK3,2.65 mg; VB1,2 mg; VB2,6 mg; VB12,0.025 mg; pantothenic acid,12 mg; niacin,50 mg; folic acid,1.25 mg; biotin,0.325 mg.

Akkermansia muciniphila viable bacteria-releasing group (HFD _ LA): feeding the high-energy low-protein daily ration in a positive feeding period, and feeding 5mL of Ackermanella mucronatum BAA-835 viable bacteria preparation (the effective bacteria concentration of the Ackermanella mucronatum viable bacteria preparation is 3.5 multiplied by 10) to each chicken per day⁸CFU/mL)。

Akkermansia muciniphila-inactivated bacteria-delayed group (HFD _ PA): the high-energy low-protein daily ration is fed in a positive feeding period, and each chicken is gavaged with 5mL of the akkermansia muciniphila BAA-835 pasteurella inoculum of example 1 every day.

At the end of 18 weeks (day 126 of the positive feeding period), all the layers were starved for 8h, weighed and blood was collected from the pterygoid vein, the blood was centrifuged at 3000rpm for 10min at 4 ℃ and serum was collected and stored in a refrigerator at-20 ℃. After blood collection, the jugular vein of the laying hen is exsanguinated to death, and the abdominal cavity is immediately cut open to extract the liver and abdominal fat. Photographing and weighing the liver, and storing the liver tissue in 4% paraformaldehyde fixing solution; weighing abdominal fat; rapidly collecting liver tissue samples, quickly freezing in liquid nitrogen, and storing in an ultra-low temperature refrigerator at-80 ℃.

Method for measuring lipid in liver:

a) thawing the frozen liver sample at 4 ℃ to 80 ℃, weighing about 50mg in a 2mL centrifuge tube, and recording the liver moisture content;

b) adding 1mL chloroform/methanol (2:1), homogenizing on ice, and standing at 4 deg.C overnight;

c) adding 0.3mL of distilled water, centrifuging at 3000rpm for 10 min;

d) taking 0.2mL of lower organic phase, and drying by nitrogen;

e) adding 0.2mL of 5% Triton (X-100) PBS for redissolving;

f) the lipid assay was performed on the reconstituted solution using a cholesterol (TC) assay kit (beijing zhongsheng bei chow) and a Triglyceride (TG) assay kit (beijing zhongsheng bei chow).

Method for measuring lipid in serum:

serum was taken and sent to the Lancet Biotechnology Ltd to detect the levels of TC and TG using a fully automated biochemical detector.

2. The experimental results are as follows:

2.1 Ackermansia muciniphila BAA-835 can reduce abdominal fat deposition of laying hens

The results in table 2 show that the abdominal fat weight and abdominal fat rate of the HFD group laying hens are significantly higher than those of the ND control group; the abdominal fat weight and abdominal fat rate of the HFD _ LA and HFD _ PA groups were significantly lower than those of the HFD group; the results show that both the live bacterium agent and the pasteurella multocida agent of the gavage muciniphila can inhibit fat deposition of laying hens induced by high-fat diet, and have the fat reducing effect.

TABLE 2 Effect of Akkermansia muciniphila on abdominal fat

Note: SEM represents standard error of mean; p <0.05 is the criterion for significance of difference, and P <0.01 is the criterion for very significance of difference.

2.2 Ackermansia muciniphila BAA-835 can reduce the blood fat and liver fat level of laying hens

The results in table 3 show that the serum cholesterol (TC) and Triglyceride (TG) contents and the liver TG contents of the layers in HFD _ LA and HFD _ PA groups are significantly lower than those in HFD groups, and the liver TC contents of the HFD _ LA and HFD _ PA groups tend to decrease, indicating that both the akstringy protein akkermansia BAA-835 live bacteria and the pasteurella bacteria have the effect of reducing blood fat.

TABLE 3 Effect of Ackermanella muciniphila on blood lipid and liver lipid content

2.3 Ackermanella muciniphila BAA-835 live bacteria can reduce liver lipid accumulation of laying hens and relieve liver steatosis

The anatomical results show that the liver of the layer chicken in the HFD group has larger volume than that of the layer chicken in the ND group (figure 1), the color is obviously lighter and more yellow, and the non-alcoholic fatty liver caused by obesity is formed. The liver surface smooth color volume of the laying hens in the HFD _ LA and HFD _ PA groups is similar to that of the laying hens in the ND control group, and no obvious pathological change appears; compared with the ND control group, high-fat diet causes the increase of the liver weight of the laying hens (represented by HFD in figure 2), and the liver weight of the HFD _ LA and HFD _ PA groups is reduced to a certain extent (figure 2), which indicates that the akkermansia gastricicola BAA-835 can improve the liver enlargement of the laying hens and has the effect of preventing fatty liver.

Liver tissue blocks fixed in 4% paraformaldehyde solution were cut to appropriate size, embedded in paraffin, and stained with hematoxylin-eosin (HE). The results of light microscope observation show that the HFD group has disordered arrangement of liver cells, partial karyolysis and fat vacuoles with different sizes in the cytoplasm of the liver cells and obviously increased number compared with the ND group, which indicates that high fat diet indeed causes liver tissue damage and causes steatosis. Compared with the HFD group, the HFD _ LA and HFD _ PA groups have the advantages that the number of fat vacuoles in liver cells is obviously reduced, and the vacuole volume is obviously reduced, so that the live bacterium agent and the pasteurella killed bacterium agent of the Ackermansia gastrinosa BAA-835 have the effects of relieving liver injury caused by the fatty degeneration of laying hens and protecting normal tissue morphology of the liver.

2.4 Ackermansia muciniphila can improve egg quality

At the end of 18 weeks of the positive feeding period, eggs of laying hens of each treatment group are collected, 10 eggs in each group are continuously measured for three days, and the quality of the eggs is measured within 12 hours of the collected eggs. Measuring egg weight, eggshell strength, egg white height, Ha's unit, and yolk color with egg quality measuring instrument (DET-6000, NABELCo., Ltd. Japan); measuring the thickness of the eggshell using an eggshell thickness tester (TI-PVX, Orka Technology Ltd. Israel); the egg yolk was separated and weighed by an egg separator, the specific gravity of the egg yolk was calculated, and the egg yolk sample was stored in a refrigerator at-20 ℃. The results of the experiments are shown in Table 4, and compared with the HFD group, the HFD _ LA and HFD _ PA groups have the tendency of increasing Ha's unit, which indicates that both the Ackermanella mucronatum BAA-835 live bacteria and the Pasteurella pasteurianum bacteria have the effect of improving the quality of the eggs.

TABLE 4 Effect of Ackermansia muciniphila BAA-835 on egg quality

2.5 Ackermanella muciniphila can reduce cholesterol content in egg yolk

Thawing the frozen yolk sample in a refrigerator at-20 deg.C at 4 deg.C, and stirring with a glass rod. Weighing 100mg of the uniformly mixed sample, adding 4mL of isopropanol, and uniformly mixing by vortex; centrifuging at 3000rpm for 10min, collecting supernatant, and measuring cholesterol content in egg yolk. The results in FIG. 4 show that the egg yolk cholesterol content of the egg chicken in HFD group is significantly higher than that of the egg yolk cholesterol content in ND group, and the egg yolk cholesterol content of HFD _ LA and HFD _ PA groups is significantly lower than that of the egg yolk cholesterol content in HFD group, which indicates that the live bacteria of Ackermanella viscosus BAA-835 and the pasteurella can reduce the cholesterol content in the egg yolk.

Example 3 application of akkermansia muciniphila BAA-835 lyophilized powder to old laying hens

1. Design of experiments

In this example, 200 healthy helan brown laying hens aged 64 weeks were selected, blood was collected by fasting for 8h of the hind wing vein, and serum was separated and the contents of TC and TG in serum were measured (beijing kimchikoku corner biotechnology development ltd). According to the content of TC and TG in serum and the laying rate, 140 chickens are selected and randomly divided into two groups, each group is divided into 7 times, each group is divided into 10 times, and the initial weight (a control group: 2.17 +/-0.12 kg; a test group: 2.16 +/-0.12 kg), the laying rate (a control group: 89.6 +/-5.67%; a test group: 89.6 +/-5.48%), the TC (a control group: 3.71 +/-0.42 mmol/L; a test group: 3.71 +/-0.40 mmol/L) and the TG (a control group: 8.59 +/-2.80 mmol/L; and a test group: 8.59 +/-2.74 mmol/L) of the serum are ensured to be consistent. The site of this example was in poultry testing base of the university of agriculture in china (28095; state, north river), 2 weeks during the pre-feeding period and 12 weeks during the forward feeding period.

The Control group (Control) was fed a basal diet, the basal diet composition and nutrient levels are shown in table 5.

Table 5 basal diet composition and nutritional levels (%)

Trial group (Treatment) in basal diet as 10⁹The Ackermanella muciniphila BAA-835 lyophilized powder microbial inoculum prepared in example 1 was added to CFU in a daily amount, and the concentration of the fermentation solution before vacuum freeze-drying of BAA-835 lyophilized powder was 1X 10⁸CFU/mL。

At the end of 12 weeks of the positive feeding period (day 84 of the positive feeding period), all the layers were starved for 8 hours and weighed as a single layer. All test chicken wings were subjected to venous blood collection, centrifuged at 3000rpm at 4 ℃ for 10min, serum was separated, and the TC and TG contents in the serum were measured (Beijing Kinghaike Biotech development Co., Ltd.). And selecting 1 laying hen slaughter sample close to the average blood lipid level repeatedly. And (4) bleeding and killing jugular vein, immediately cutting open the abdominal cavity, and picking liver and abdominal fat. Photographing and weighing the liver; weighing abdominal fat; rapidly collecting liver tissue samples, quickly freezing in liquid nitrogen, and storing in an ultra-low temperature refrigerator at-80 ℃.

2. The experimental results are as follows:

2.1 Ackermansia muciniphila BAA-835 lyophilized powder for reducing abdominal fat deposition of laying hens

The anatomical result shows (figure 5), the liver of the laying hen of the control group (the left figure in figure 5) is slightly yellow, the texture is soft and fragile, the surface of the liver of the laying hen of the akkermansia muciniphila BAA-835 freeze-dried powder addition group (the right figure in figure 5) is smooth and compact, the liver color is normally dark red, and no bleeding point exists on the surface of the liver, so that the liver is basically recovered to be normal; and the abdominal fat of the laying hens is obviously reduced by adding the akkermansia muciniphila BAA-835 freeze-dried powder (Table 6). As can be seen from table 6, the akkermansia muciniphila BAA-835 lyophilized powder addition group (Treatment) significantly reduced the abdominal fat weight and abdominal fat rate compared to the Control group (Control), consistent with the observed anatomical results.

TABLE 6 influence of Akkermansia muciniphila BAA-835 lyophilized powder on fat deposition of old layer chicken

Note: results are expressed as "mean ± standard deviation"; p <0.05 is the criterion for significance of difference, and P <0.01 is the criterion for very significance of difference.

2.2 Ackermansia muciniphila BAA-835 lyophilized powder for reducing serum and liver cholesterol content of old layer chicken

As is clear from Table 7, the Ackermanella muciniphila BAA-835 lyophilized powder addition group (Treatment) tended to lower the serum total cholesterol content as compared with the Control group (Control).

TABLE 7 influence of Akkermansia muciniphila BAA-835 lyophilized powder on blood lipid and liver lipid content of old layer chicken

Note: results are expressed as "mean ± standard deviation"; p <0.05 is the standard of significance of difference, P <0.01 is the standard of significance of difference, and P <0.05 <0.10 is the trend of difference.

2.3 Ackermansia muciniphila BAA-835 freeze-dried powder can improve the quality of eggs laid by old people

TABLE 8 Effect of Akkermansia muciniphila BAA-835 Freeze-dried powder on the quality of the eggs of the aged

Note: results are expressed as "mean ± standard deviation"; p <0.05 is the standard of significance of difference, P <0.01 is the standard of significance of difference, and 0.05< P <0.10 is the trend of difference.

As can be seen from Table 8, compared with the Control group (Control), the addition of the akkermansia muciniphila BAA-835 lyophilized powder has the tendency of increasing the Ha's unit, and the eggshell thickness is extremely remarkably increased, which indicates that the addition of the akkermansia muciniphila BAA-835 lyophilized powder has the effect of improving the quality of the aged eggs.

2.4 Ackermanella muciniphila BAA-835 lyophilized powder capable of reducing cholesterol content of egg yolk of old laying hens

Thawing the frozen yolk sample in a refrigerator at-20 deg.C at 4 deg.C, and stirring with a glass rod. Weighing 100mg of the uniformly mixed sample, adding 4mL of isopropanol, and uniformly mixing by vortex; centrifuging at 3000rpm for 10min, and collecting supernatant to determine cholesterol content in egg yolk. The results in FIG. 6 show that the addition of Ackermansia muciniphila BAA-835 lyophilized powder (shown in Treatment in FIG. 6) can significantly reduce the cholesterol content in egg yolk compared to the Control (shown in Control in FIG. 6).

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Claims

1. At least one of the following uses of akkermansia muciniphila:

A1) the application of the akkermansia muciniphila in preparing products for treating or relieving the metabolic related diseases of poultry;

A2) the application of the akkermansia muciniphila in improving the quality of poultry eggs or preparing products for improving the quality of the poultry eggs;

A3) the application of the akkermansia muciniphila in improving the protein height and the Hardgrove unit level of poultry eggs;

A4) the use of akkermansia muciniphila for reducing the accumulation of poultry abdominal fat and/or reducing poultry abdominal fat weight and/or for the manufacture of a product for reducing poultry abdominal fat weight;

A5) the application of the akkermansia muciniphila in relieving poultry liver lesion or preparing a product for relieving poultry liver lesion;

A6) the application of the akkermansia muciniphila in maintaining the structural integrity and the morphological integrity of poultry liver and/or relieving poultry liver steatosis and/or preparing products for relieving poultry liver steatosis;

A7) the application of the akkermansia muciniphila in reducing the blood fat level of poultry or preparing products for reducing the blood fat of poultry;

A8) the application of the akkermansia muciniphila in reducing cholesterol and triglyceride in poultry serum and/or reducing the level of poultry liver fat and/or preparing products for reducing poultry liver fat;

2. Use according to claim 1, characterized in that: the Ackermanella muciniphila is Akkermanella muciniphila (Akkermansia muciniphila), which has a strain number of ATCC BAA-835 at the American Type Culture Collection.

3. Use according to claim 1 or 2, characterized in that: the poultry is laying hens, and the metabolic related diseases are laying hen fatty liver syndrome.

4. A microbial inoculum, which is characterized in that: the microbial agent contains akkermansia muciniphila as described in claim 1 or/and a metabolite of akkermansia muciniphila as described in claim 1 or/and a culture of akkermansia muciniphila as described in claim 1;

the microbial inoculum has at least one of the following characteristics:

B1) treating or ameliorating metabolic-related disorders in poultry;

B2) improving the quality of the poultry eggs;

B3) improving the protein height and the harderian unit level of the poultry eggs;

B4) reducing the accumulation of abdominal fat in poultry and/or reducing the abdominal fat weight of poultry;

B5) relieving liver lesions of poultry;

B6) maintaining structural and morphological integrity and/or mitigating liver steatosis in poultry;

B7) reducing the blood lipid level of poultry;

B8) reducing the levels of cholesterol and triglycerides in the serum of poultry and/or reducing the liver lipid levels of poultry;

B9) reducing cholesterol content in poultry egg yolk.

5. The microbial inoculum according to claim 4, characterized in that: the poultry is laying hens, and the metabolic related diseases are laying hen fatty liver syndrome.

6. Use of the inoculant of claim 4 or 5 for at least one of:

C1) the application of the microbial inoculum in preparing products for treating or relieving poultry metabolic related diseases;

C2) the microbial inoculum is applied to improving the quality of poultry eggs or preparing products for improving the quality of the poultry eggs;

C3) the application of the microbial inoculum in improving the protein height and the Haugh unit level of poultry eggs;

C4) the application of the microbial inoculum in reducing the accumulation of poultry abdominal fat and/or reducing the weight of poultry abdominal fat and/or preparing products for reducing the weight of poultry abdominal fat;

C5) the microbial inoculum is applied to relieving poultry liver pathological changes or preparing products for relieving poultry liver pathological changes;

C6) the microbial inoculum is applied to maintaining the integrity of the poultry liver structure and shape and/or relieving the poultry liver steatosis and/or preparing a product for relieving the poultry liver steatosis;

C7) the microbial inoculum is applied to reducing the blood fat level of poultry or preparing products for reducing the blood fat level of poultry;

C8) the application of the microbial inoculum in reducing cholesterol and triglyceride in poultry serum and/or reducing the level of poultry liver fat and/or preparing products for reducing poultry liver fat;

C9) the microbial inoculum is applied to reducing the cholesterol content in poultry egg yolk and/or preparing products for reducing the cholesterol content in poultry egg yolk.

7. Use according to claim 6, characterized in that: the poultry is laying hens, and the metabolic related diseases are laying hen fatty liver syndrome.