High-fat and high-alcohol feed for constructing animal model with liver diseases and preparation method of high-fat and high-alcohol feed

Abstract

The invention belongs to the technical field of animal feeds, and particularly relates to a high-fat and high-alcohol feed for constructing a hepatopathy animal model and a preparation method of the high-fat and high-alcohol feed. The high-fat and high-alcohol feed comprises 60-75% of a basal feed, 5-10% of cholesterol and 20-30% of a fatty alcohol premix, the fatty alcohol premix is microspheres of a shell-core structure, a shell layer is glycidyl methacrylate modified silk fibroin, and a core is an ethanol-in-oil emulsion. The novel high-fat and high-alcohol feed disclosed by the invention has good stability, effectively reduces the alcohol odor and better conforms to the olfactory preference of mice, meanwhile, a solid-phase packaging form can effectively ensure the solid form of food and is closer to the occlusion habit of the mice, the intake proportion of alcohol and fat is increased while the normal dietary habit of the mice is maintained, and the health of the mice is improved. The forming process of the food-borne metabolic fatty liver is simulated.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal feeds, and particularly relates to a high-fat and high-alcohol feed for constructing a liver disease animal model and a preparation method thereof. Background Art

[0002] Liver diseases pose a major threat to human health. There is no effective treatment for end-stage liver diseases and the fatality rate is high. Primary liver cancer is the seventh most common malignant tumor in the world, ranking third in terms of the number of deaths, causing approximately 700,000 deaths annually; in addition, severe liver cirrhosis causes more than 1 million deaths annually. In recent years, the number of patients with metabolic liver diseases represented by alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD), also known as metabolic associated fatty liver disease (MAFLD), has increased rapidly. Therefore, it is of great significance to attach importance to the basic and translational research of liver diseases such as ALD and NAFLD to meet the health needs. In this process, accurate and effective animal models are indispensable powerful tools.

[0003] Both ALD and NAFLD are metabolic syndromes caused by the long-term action of harmful factors on the liver. Such liver diseases include a long course of disease and similar evolution stages. Hepatic steatosis is a common early lesion in both ALD and NAFLD. With the chronic change of the microenvironment in the liver, the intrahepatic immune-inflammatory system is over-activated in some patients, resulting in alcoholic hepatitis (AH) or non-alcoholic steatohepatitis (NASH). Continuous inflammation can cause a large number of hepatocytes to be damaged and induce liver fibrosis. If not effectively diagnosed and treated, ultimately a small number of patients will develop into end-stage liver diseases such as advanced liver cirrhosis or primary liver cancer, and the prognosis is poor at this time.

[0004] Metabolic liver diseases represented by ALD and NAFLD have a continuous and complex course of disease evolution, which poses high requirements for related animal models. An ideal animal model should be able to simulate all pathological stages of the occurrence and development of chronic liver diseases. Therefore, in-depth analysis of the advantages and limitations of existing methods will point the way for a new generation of animal diet models.

[0005] The Lieber-DeCarli (LD) diet, first introduced by Lieber et al. in 1963, is the most successful ALD animal model to date. Many important research results on ALD are based on the LD diet model. In view of the habit of rodents to dislike alcohol, this model uses a semi-liquid diet containing alcohol as the only available dietary source, inducing experimental animals to consume sufficient alcohol through eating. The NAFLD diet animal model simulates the high-fat, high-cholesterol diet of humans by increasing the fat energy supply ratio in the feed. Representative high-fat diet models include the simple high-fat diet model (High fat diet, HFD) and the Western diet model (Western diet, WD), and its stable modeling effect has been highly recognized by scientific researchers.

[0006] As the mainstream ALD model, the LD diet has the advantages of low cost, easy implementation, and stable effect. However, it is currently recognized that hepatic steatosis is the main pathological change mediated by the LD model. The inability of the LD diet alone to induce advanced liver lesions including fatty hepatitis and liver fibrosis is its main functional deficiency. Compared with the LD diet, the HFD and WD models only need to replace normal feed, with lower operation and maintenance costs and high safety. HFD and WD feeding can affect the systemic physiological condition of experimental animals, including significant increase in animal weight and hyperlipidemia. There are subtle differences in the effects of HFD and WD diets on the liver: it is generally believed that HFD mediates more severe intrahepatic fat accumulation, while the WD diet, although causing slightly milder hepatic steatosis, can be observed to have a significant inflammatory response. However, HFD or WD diet alone also cannot cause advanced liver disease changes such as liver fibrosis and liver tumors.

[0007] In addition, although the above animal diet models have played a prominent role in the study of metabolic liver disease, they still cannot fully reflect the harmfulness of bad habits to the human population. One of the important reasons is that potential patients often face multiple risk factors at the same time, and the final onset and outcome are often the result of the superposition, interweaving and promotion of multiple factors. For example, a large number of people have both long-term alcoholism and excessive high-fat diet intake. Therefore, it is of great scientific significance to develop a solid diet with a unit energy close to that of conventional mice, a high alcohol content, and a high fat content to accurately simulate the progressive metabolic liver disease caused by bad eating habits in the human population, thereby constructing an animal diet model with the ability to reflect complex dietary risks. Summary of the invention

[0008] In order to solve the above problems, in the first aspect, the present invention provides a high-fat and high-alcohol feed for constructing a liver disease animal model, which comprises, by mass percentage: 60-75% of a basic feed, 5-10% of cholesterol, and 20-30% of a fat-ethanol premix;

[0009] The fatty alcohol premix is a microsphere with a core-shell structure, where the shell is glycidyl methacrylate-modified silk fibroin and the core is an ethanol-in-oil emulsion.

[0010] Furthermore, the ethanol-in-oil emulsion is composed of an aqueous ethanol solution, an edible oil, and an emulsifier.

[0011] Furthermore, the mass ratio of the aqueous ethanol solution, the edible oil, and the emulsifier is 75-100:150-200:1.2-4.5.

[0012] Furthermore, the volume percentage of ethanol in the aqueous ethanol solution is 40-65%.

[0013] Furthermore, the edible oil includes edible vegetable oil or edible animal oil. The edible vegetable oil includes soybean oil, rapeseed oil, peanut oil, sesame oil, olive oil, corn oil, sunflower oil, and edible vegetable blended oil; the edible animal oil includes lard, beef tallow, and fish oil.

[0014] Furthermore, the emulsifier includes one or more of glycerol monostearate, citric acid, sucrose fatty acid ester, sorbitan monooleate, and polyoxyethylene sorbitan monostearate.

[0015] Furthermore, the emulsifier is compounded from glycerol monostearate, citric acid, sucrose fatty acid ester, sorbitan monooleate, and polyoxyethylene sorbitan monostearate at a mass ratio of 5-7:1-2:1-2:3-8:0.5-1.5.

[0016] In a second aspect, the present invention provides a method for preparing a high-fat and high-alcohol feed as described herein, which includes the following steps:

[0017] (1) Thoroughly mix the edible oil and the emulsifier, add the aqueous ethanol to the mixed solution of the oil and the emulsifier, and after thorough mixing, obtain a crude emulsion. Homogenize the crude emulsion to obtain a stable water-in-oil emulsion;

[0018] (2) Dissolve glycidyl methacrylate-modified silk fibroin in water, add a photoinitiator, and drop the resulting solution into the water-in-oil emulsion obtained in step (1) under stirring conditions, and stir and mix thoroughly;

[0019] (3) Perform a light treatment on the emulsion obtained in step (2) to promote cross-linking and curing, centrifuge to collect the microspheres, and after washing and drying, obtain the fatty alcohol premix as the core-shell structure microspheres;

[0020] (4) Thoroughly mix the required amounts of the basal feed, cholesterol, and the fatty alcohol premix, and press them into pellets or blocks to obtain a high-fat and high-alcohol feed for constructing a liver disease animal model.

[0021] Further, the photoinitiator is lithium phenyl(2,4,6-trimethylbenzoyl)phosphate.

[0022] Further, the light irradiation is carried out with ultraviolet light of 365-405 nm.

[0023] As used herein, the basal diet is a common diet for the daily feeding of non-human primates, which can ensure the energy required for the growth and activities of primates, such as the non-human primate diet that can be purchased on the market. In a non-limiting example, the basal diet may contain the following components (1000 g): casein 140 g, L-cystine 1.8 g, corn starch 495.692 g, maltodextrin 125 g, sucrose 100 g, cellulose 50 g, soybean oil 40 g, tert-butylhydroquinone 0.008 g, mixed minerals 35 g, mixed vitamins 10 g, choline bitartrate 2.5 g.

[0024] Advantages of the present invention

[0025] The novel high-fat and high-alcohol diet of the present invention has good stability, effectively reduces the smell of alcohol, is more in line with the olfactory preference of mice. At the same time, the solid-phase encapsulation form can effectively ensure the solid form of the food, which is closer to the biting habit of mice. While maintaining the normal eating habits of mice, it increases the intake ratio of alcohol and fat, and simulates the formation process of diet-induced metabolic fatty liver.

[0026] Since rodents do not like the taste of ethanol, the present invention innovatively uses the oil-in-alcohol method to prepare the high-fat and high-alcohol diet, and by exploring the oil-alcohol ratio, when the oil, alcohol and other components are fixed in total energy, the feed can be formed and the fat can wrap the alcohol, so that the smell of alcohol will not overflow.

[0027] In the mouse modeling experiment, the novel high-fat and high-alcohol diet of the present invention successfully induced the formation of severe fatty liver within 12 weeks. Brief description of the drawings

[0028] Figure 1 Shows the liver H&E staining results of the liver disease mouse model constructed using the novel high-fat and high-alcohol diet.

[0029] Figure 2 Shows the liver oil red staining results of the liver disease mouse model constructed using the novel high-fat and high-alcohol diet. Detailed implementation manners

[0030] The following specific examples are used to further illustrate the present invention, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0031] Example 1: Preparation of glycidyl methacrylate modified silk fibroin

[0032] (1) adjusting the pH of an 8 wt % silk fibroin solution to about 8.5 using PBS buffer;

[0033] (2) adding an appropriate amount of glycidyl methacrylate (GMA) (the molar ratio of GMA to the amino group in SF is 5:1);

[0034] (3) React at room temperature or 37°C for 12 hours;

[0035] (4) Using a dialysis bag with a MWCO of 3500Da, the unreacted GMA and by-products were dialyzed and lyophilized to obtain glycidyl methacrylate-modified silk fibroin.

[0036] Example 2: Preparation of fatty ethanol premix

[0037] (1) uniformly mixing glycerol monofatty acid ester, citric acid, sucrose fatty acid ester, sorbitan monooleate, and polyoxyethylene sorbitan monostearate in a mass ratio of 6:1.5:1.5:5:1 to obtain a composite emulsifier;

[0038] (2) 40 parts of soybean oil, 20 parts of sunflower oil, 60 parts of liquid lard and 60 parts of corn oil are fully mixed with a composite emulsifier, 90 parts of 60% ethanol by volume of aqueous ethanol are added to the mixed solution of oil and emulsifier, and a crude emulsion is obtained by processing with a high-speed disperser, and the crude emulsion is homogenized with a dynamic high-pressure microfluidizer to obtain a stable water-in-oil emulsion; after testing, the water-in-oil emulsion is kept in a temperature range of -10 to 40° C. for more than 30 days without stratification;

[0039] (3) dissolving glycidyl methacrylate modified silk fibroin (SilMA) in water to obtain a 20% (W / V) SilMA solution, adding a photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate at a final concentration of 0.25% (W / V), and adding the obtained solution dropwise to the oil-in-water emulsion obtained in step (2) under stirring conditions, and stirring at 8000 rpm for 30 minutes;

[0040] (4) The emulsion obtained in step (3) is treated with 405 nm ultraviolet light for 10 min to promote cross-linking and curing, and the microspheres are collected by centrifugation at 3000 rpm, washed with distilled water and freeze-dried to obtain a fat ethanol premix as a shell-core structure microsphere.

[0041] Example 3: Preparation of high-fat and high-alcohol feed

[0042] Mix 60% of the basal diet, 10% of cholesterol, and 30% of the fat-ethanol premix thoroughly in a high-speed mixer, and press them into pellets to obtain a high-fat and high-alcohol diet for constructing an animal model of liver disease.

[0043] The composition of the basal diet (1000 g): casein 140 g, L-cystine 1.8 g, corn starch 495.692 g, maltodextrin 125 g, sucrose 100 g, cellulose 50 g, soybean oil 40 g, tert-butylhydroquinone 0.008 g, mixed minerals 35 g, mixed vitamins 10 g, choline bitartrate 2.5 g.

[0044] Example 4: Construction of a severe fatty liver model

[0045] To explore whether the new high-fat and high-alcohol diet can induce severe fatty liver, 8-week-old male C57BL / 6J mice were divided into an experimental group and a control group. The experimental group was fed the new high-fat and high-alcohol diet, and the control group was fed the control diet. Both groups were given ordinary drinking water. Feed and water were freely available, and they were continuously fed for 12 weeks. After the feeding period ended, the mice were euthanized with a lethal dose of isoflurane. After recording the body weight, the liver organs were removed and weighed. The overall condition of the liver was observed by H&E staining and oil red staining, including the degree of hepatocyte steatosis, immune cell infiltration, the formation of liver fibrosis, and apoptosis and necrosis of cells. The results are as Figure 1 and Figure 2 shown. It can be seen that significant fat accumulation was found in the hepatocytes of the experimental group by H&E staining, and a large number of immune cells infiltrated between the hepatocytes. Oil red staining confirmed obvious fat accumulation in the liver of the experimental group, indicating that the new mouse liver disease diet model can induce severe fatty liver.

[0046] Comparative Example 1:

[0047] Prepare the fat-ethanol premix according to Example 2, except that the addition amount of water-containing ethanol in step (2) is 70 parts.

[0048] Prepare the high-fat and high-alcohol diet with the obtained fat-ethanol premix according to Example 3, and construct a severe fatty liver model according to Example 4. Due to the relatively small addition amount of water-containing ethanol in the fat-ethanol premix, the construction time of the severe fatty liver model was prolonged.

[0049] Comparative Example 2:

[0050] Prepare the fat-ethanol premix according to Example 2, except that the addition amount of water-containing ethanol in step (2) is 110 parts.

[0051] The obtained fatty ethanol premix was used to prepare a high-fat and high-alcohol diet according to Example 3, and a severe fatty liver model was constructed according to Example 4. Since the content of water-containing ethanol in the fatty ethanol premix was relatively high, there was a leakage of alcohol smell in the high-fat and high-alcohol diet, resulting in less food intake of mice during the construction of the severe fatty liver model and an extended model construction time.

[0052] It should be noted that the description and drawings of the present invention give preferred embodiments of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments do not serve as additional limitations to the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosed content of the present invention more thorough and comprehensive. Moreover, the above technical features continue to be combined with each other to form various embodiments not listed above, which are all regarded as within the scope described in the specification of the present invention; further, for those of ordinary skill in the art, improvements or transformations can be made according to the above description, and all such improvements and transformations should fall within the protection scope of the appended claims of the present invention.

Claims

1. A high-fat and high-alcohol diet for constructing an animal model of liver disease, characterized in that, Comprising by mass percentage: 60 - 75% of basic feed, 5 - 10% of cholesterol, and 20 - 30% of fatty alcohol premix; The fatty alcohol premix is a microsphere with a core - shell structure, the shell layer is glycidyl methacrylate - modified silk fibroin, and the core is an ethanol - in - oil emulsion.

2. The high-fat and high-alcohol feed according to claim 1, wherein, The ethanol - in - oil emulsion is composed of an aqueous ethanol solution, an edible oil, and an emulsifier.

3. The high-fat and high-alcohol feed according to claim 2, wherein The mass ratio of the aqueous ethanol solution, the edible oil, and the emulsifier is 75 - 100:150 - 200:1.2 - 4.

5.

4. The high-fat and high-alcohol feed according to claim 2, wherein The volume percentage of ethanol in the aqueous ethanol solution is 40 - 65%.

5. The high-fat and high-alcohol feed according to claim 2, wherein The edible oil includes edible vegetable oil or edible animal oil. The edible vegetable oil includes soybean oil, rapeseed oil, peanut oil, sesame oil, olive oil, corn oil, sunflower seed oil, and edible vegetable blended oil; the edible animal oil includes lard, beef tallow, and fish oil.

6. The high-fat and high-alcohol feed according to claim 2, wherein The emulsifier includes one or more of glycerol monostearate, citric acid, sucrose fatty acid ester, sorbitan monooleate, and polyoxyethylene sorbitan monostearate.

7. The high-fat and high-alcohol feed according to claim 6, wherein The emulsifier is compounded from glycerol monostearate, citric acid, sucrose fatty acid ester, sorbitan monooleate, and polyoxyethylene sorbitan monostearate with a mass ratio of 5 - 7:1 - 2:1 - 2:3 - 8:0.5 - 1.

5.

8. A method for preparing a high-fat and high-alcohol feed according to any one of claims 1-7, characterized in that, Including the following steps: (1) Thoroughly mix the edible oil and the emulsifier, add the aqueous ethanol to the mixed solution of the oil and the emulsifier, and after thorough mixing, obtain a crude emulsion. Homogenize the crude emulsion to obtain a stable water - in - oil emulsion; (2) Dissolve glycidyl methacrylate - modified silk fibroin in water, add a photo - initiator, and drop the resulting solution into the water - in - oil emulsion obtained in step (1) under stirring conditions, and stir thoroughly to mix evenly; (3) Perform a light treatment on the emulsion obtained in step (2) to promote cross - linking and curing, centrifuge to collect the microspheres, and after washing and drying, obtain the fatty alcohol premix as a core - shell structure microsphere; (4) Thoroughly mix the required amounts of basic feed, cholesterol, and fatty alcohol premix, and press them into pellets or blocks to obtain a high - fat and high - alcohol feed for constructing a liver disease animal model.

9. The preparation method according to claim 8, wherein The photo - initiator is lithium phenyl(2,4,6 - trimethylbenzoyl)phosphate.

10. The preparation method according to claim 8, characterized in that, The light treatment is carried out using ultraviolet light with a wavelength of 365 - 405 nm.

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