A feed for inducing type 2 diabetes and application thereof in establishing a type 2 diabetes animal model

Abstract

The application relates to an induced type 2 diabetes mellitus feed and application thereof in establishing a type 2 diabetes mellitus animal model, and relates to the field of experimental animal models, and particularly relates to an induced type 2 diabetes mellitus feed and application thereof in establishing a type 2 diabetes mellitus animal model. The application aims at solving the problems of long modeling time, instability and easy change into a type 1 diabetes mellitus model of the existing type 2 diabetes mellitus animal model establishing method. The feed comprises desulfurization vibrio powder, symbiotic clostridium powder and high-fat high-sugar feed. The method comprises the following steps: drying desulfurization vibrio liquid and symbiotic clostridium liquid into powder, uniformly mixing the powder and the high-fat high-sugar feed, and extruding into a column shape by using an extrusion molding machine. The application significantly shortens the establishment time of the type 2 diabetes mellitus animal model, does not need to inject a high-dose STZ, and does not cause the type 1 diabetes mellitus model of absolute insulin deficiency of rats in the case of using a higher concentration of STZ induction. The induced type 2 diabetes mellitus feed is used for establishing the type 2 diabetes mellitus animal model.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of experimental animal models, and particularly relates to a type 2 diabetes inducing feed and application thereof in establishing a type 2 diabetes animal model. BACKGROUND

[0002] Diabetes is a group of clinical syndromes caused by the interaction of genetic and environmental factors. Due to absolute or relative deficiency of insulin secretion and decreased sensitivity of target tissue cells to insulin, a series of metabolic disorders such as sugar, protein, fat, water and electrolyte are caused, the prevalence rate increases year by year, and diabetes has become the most prevalent disease among global chronic non-communicable diseases. Among the diabetes patients, type 2 diabetes patients account for more than 90%. In order to conquer these major and frequently-occurring difficult diseases, life scientists and pharmacologists in various countries in the world have carried out a large amount of innovation and research, but the curative treatment result of diabetes still stays at the level of ten years ago. Up to now, an effective drug or method has not been found to cure diabetes from the etiology. Type 2 diabetes is an endocrine and metabolic disease with insulin resistance and insufficient insulin secretion as the pathophysiological basis. The causes of type 2 diabetes include susceptible genes, living habits and various reasons.

[0003] The causes and pathogenesis of type 2 diabetes are various, and the causes thereof have not been completely elucidated at present. Researches in recent years show that intestinal flora is closely related to obesity and insulin resistance, and plays an important role in the occurrence and development of type 2 diabetes.

[0004] A large number of microorganisms in the human digestive tract become intestinal flora, forming a human microbial system. According to the relationship with the host, the intestinal flora has physiological microorganisms (accounting for 99% of the total number of microorganisms, such as Bifidobacterium and Lactobacillus) that symbiotically coexist with the host, pathogenic bacteria (such as Enterococcus and Enterobacter) that symbiotically coexist with the host, and harmful pathogenic bacteria. Intestinal flora participates in energy metabolism of the host, provides various vitamins, amino acids, antibiotics and polypeptides, decomposes harmful substances in the body, participates in the growth, differentiation and inflammatory response of the human intestinal epithelium. Intestinal flora imbalance not only causes a variety of gastrointestinal diseases such as diarrhea, constipation, enteritis, but also induces chronic diseases such as obesity, cardiovascular disease, diabetes and metabolic syndrome.

[0005] Microecology research finds that the number of intestinal flora has a certain relationship with the occurrence and development of diabetes. Researches show that unbalanced intestinal flora increases body weight, produces and absorbs more LPS, activates low-grade chronic inflammation, promotes insulin resistance and impaired beta cell secretion function, causes metabolic disorders of cholesterol and triglyceride in the body, and further causes or promotes the occurrence and development of type 2 diabetes. Further researches show that type 2 diabetes patients all have changes such as significant intestinal flora structure imbalance, increase of harmful bacteria such as Escherichia coli and significant decrease of Bifidobacterium.

[0006] At present, the main methods for establishing animal models of type 2 diabetes include high-sugar high-fat feed induction, chemical drug induction, high-sugar high-fat combined with chemical drug induction, spontaneous diabetes animal model, and transgenic animal, etc. Although the high-sugar high-fat feed method is very close to the conventional pathogenesis of the human population, it needs a long time and high cost in the animal induction process, and is easy to reverse. The type 2 diabetes model induced by chemical drugs generally uses streptozotocin (STZ) intraperitoneal injection. In the past articles, the dosage of STZ is 35-150 mg / kg, but it is found that the dosage of STZ is difficult to control. If the dosage of STZ is less than 20 mg / kg of body weight, the rats may recover naturally, or the rats are fed with high-fat feed for a long time after STZ injection to maintain the insulin resistance state, which is long in modeling time and unstable. STZ is a nitroso-containing compound, which can selectively damage pancreatic beta cells, and the damaged islet is reconstructed and then fibrosis. If the dosage of STZ is higher than or equal to 35 mg / kg of body weight, the concentration of coenzyme I in the beta cells decreases, which leads to the direct death of the beta cells, thereby becoming type 1 diabetes with absolute lack of insulin. The animals fed with high-fat high-sugar diet are particularly prone to death after injection of STZ, which affects the development of research. SUMMARY

[0007] The present application aims to solve the problems of long modeling time, instability and easy change into type 1 diabetes of the existing method for establishing animal models of type 2 diabetes, and provides a type 2 diabetes inducing feed and its application in establishing animal models of type 2 diabetes.

[0008] The type 2 diabetes inducing feed of the present application comprises Desulfovibrio bacteria powder, Clostridium symbiosum bacteria powder and high-fat high-sugar feed.

[0009] Further, the live bacteria concentration of Desulfovibrio in the type 2 diabetes inducing feed is 1×10 8 cfu / g-1.2×10 8 cfu / g.

[0010] Further, the live bacteria concentration of Clostridium symbiosum in the type 2 diabetes inducing feed is 1×10 8 cfu / g-1.2×10 8 cfu / g.

[0011] The preparation method of the type 2 diabetes inducing feed of the present application comprises the following steps:

[0012] The Desulfovibrio desulfuricans bacterial liquid and the symbiotic Clostridium bacteria liquid are dried into bacterial powder at low temperature respectively, the Desulfovibrio desulfuricans bacterial powder, the symbiotic Clostridium bacteria powder and high-fat high-sugar feed are mixed uniformly, and then are extruded into a column shape by an extrusion molding machine.

[0013] The application further provides application of the feed for inducing type 2 diabetes in establishing a type 2 diabetes animal model.

[0014] Further, a specific method for establishing a type 2 diabetes animal model by using the feed for inducing type 2 diabetes comprises the following steps:

[0015] I. The Desulfovibrio desulfuricans bacterial liquid and the symbiotic Clostridium bacteria liquid are respectively prepared into bacterial liquid with a concentration of 4*10 9 cfu / mL by using sterile PBS, then the two kinds of bacterial liquid are mixed according to a volume ratio of 1:1 to obtain mixed bacterial liquid;

[0016] II. Select rats with a weight of 300-350g, and make each rat fast for 12 hours, first, use the mixed bacterial liquid to perform gavage on each rat with 2mL, then use the feed for inducing type 2 diabetes to feed each rat every day, after continuous feeding for 4 weeks, make the rats fast for 12 hours, and perform intraperitoneal injection of STZ 25mg / kg in a fasting state, and then continue to use the high-fat high-sugar feed to feed the rats for 1 week, so that a type 2 diabetes animal model is obtained.

[0017] Further, the preparation method of the Desulfovibrio desulfuricans bacterial liquid in step I is as follows:

[0018] The freeze-dried powder of Desulfovibrio desulfuricans is placed into ATCC 260 liquid medium to perform sterile rehydration, so as to obtain activated bacterial liquid, the bacterial liquid is sterilely transferred into a container containing ATCC 260 liquid medium to perform anaerobic primary culture, and the culture condition is 37℃ for 24 hours, then ATCC 260 liquid medium is used again to perform anaerobic subculture and expansion culture, and the culture condition is 37℃ for 24 hours.

[0019] Further, the preparation method of the symbiotic Clostridium bacteria liquid in step I is as follows:

[0020] The freeze-dried powder of symbiotic Clostridium is placed into ATCC 260 liquid medium to perform sterile rehydration, so as to obtain activated bacterial liquid, the bacterial liquid is sterilely transferred into a container containing ATCC 260 liquid medium to perform anaerobic primary culture, and the culture condition is 37℃ for 24 hours, then ATCC 260 liquid medium is used again to perform anaerobic subculture and expansion culture, and the culture condition is 37℃ for 24 hours.

[0021] After the feed for inducing type 2 diabetes is prepared, it can be stored at 4℃, and needs to be restored to room temperature before use.

[0022] The application has the following beneficial effects:

[0023] 1. The type 2 diabetes induction diet of the present invention can induce rapid weight gain in rats after feeding. After 4 weeks of feeding, the rats have reached the weight of rats fed a normal high-sugar and high-fat diet after 6 weeks. Therefore, STZ injection can be performed two weeks earlier, which significantly shortens the establishment time of the type 2 diabetes animal model.

[0024] 2. The type 2 diabetes induction diet of this invention requires only one STZ injection after feeding to rats, and all rats in the experimental group successfully modeled fasting blood glucose levels in the first week after injection. In contrast, some rats in the group fed with ordinary high-sugar and high-fat diets required two STZ injections to achieve successful modeling in the third week. Therefore, this step is completed two weeks earlier.

[0025] 3. It does not require the use of high-dose STZ injections, and will not result in a type 1 diabetes model in rats that suffers from absolute insulin deficiency due to the use of high-concentration STZ induction.

[0026] 4. The feed of this invention will not cause dysfunction of other organs in rats.

[0027] 5. The type 2 diabetic rat model established by this invention has a long maintenance time, and the fasting blood glucose stability of the rats after modeling is significant. The blood glucose synchronicity of the rats in the experimental group is good, and the utilization rate of the rats is high. If this model is used for treatment research, not only can more specific data be obtained, but the time for future experiments using model animals can also be extended to within 4 weeks, which is convenient for carrying out scientific research. Attached Figure Description

[0028] Figure 1 The changes in body weight of rats in each group after feeding; where ● represents group A, ■ represents group B, and ▲ represents group C.

[0029] Figure 2 The changes in fasting blood glucose in rats of each group after STZ injection are shown; where ● represents group A, ■ represents group B, and ▲ represents group C.

[0030] Figure 3 The changes in body weight of rats in each group after STZ injection are shown; where ■ represents group A, ▲ represents group B, and ▼ represents group C.

[0031] Figure 4 The changes in food intake of rats in each group after STZ injection are shown; ● represents group A, ■ represents group B, and ▲ represents group C.

[0032] Figure 5 The changes in water intake of rats in each group after STZ injection are shown; where ■ represents group A, ▲ represents group B, and ▼ represents group C. Detailed Implementation

[0033] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

[0034] Specific embodiment one: the type 2 diabetes inducing feed comprises Desulfovibrio powder, Clostridium symbiosum powder and high-fat high-sugar feed.

[0035] The Desulfovibrio is purchased, and the number is ATCC29098. The Clostridium symbiosum is purchased, and the number is ATCC14940.

[0036] The high-fat high-sugar feed is a type 2 diabetes model feed, which is purchased from Beijing Keao Cooperation, and the product number is 1016711983920930816.

[0037] The type 2 diabetes inducing feed of the present embodiment can make rats gain weight in a short time after feeding, and the body weight has reached that of rats fed with ordinary high-sugar high-fat feed for 6 weeks after feeding for 4 weeks. Therefore, STZ injection can be performed two weeks in advance, and the establishment time of the type 2 diabetes animal model is significantly shortened.

[0038] Specific embodiment two: the difference between the present embodiment and specific embodiment one is that the viable bacterial concentration of Desulfovibrio in the type 2 diabetes inducing feed is 1×10 8 cfu / g~1.2×10 8 cfu / g. The others are the same as specific embodiment one.

[0039] Specific embodiment three: the difference between the present embodiment and specific embodiment one is that the viable bacterial concentration of Clostridium symbiosum in the type 2 diabetes inducing feed is 1×10 8 cfu / g~1.2×10 8 cfu / g. The others are the same as specific embodiment one.

[0040] Specific embodiment four: the preparation method of the type 2 diabetes inducing feed of the present embodiment comprises the following steps:

[0041] The Desulfovibrio bacterial liquid and the Clostridium symbiosum bacterial liquid are dried at low temperature into bacterial powder respectively, and the Desulfovibrio bacterial powder, the Clostridium symbiosum bacterial powder and the high-fat high-sugar feed are mixed uniformly and then extruded into a column shape by an extrusion molding machine.

[0042] Specific embodiment five: the difference between the present embodiment and specific embodiment four is that the temperature of the low-temperature drying is -60°C. The others are the same as specific embodiment four.

[0043] Specific embodiment six: the application of the type 2 diabetes inducing feed of the present embodiment in the establishment of a type 2 diabetes animal model.

[0044] Specific embodiment seven: the difference between this embodiment and specific embodiment six is that the specific method for establishing a type 2 diabetes animal model by using the type 2 diabetes inducing feed comprises the following steps:

[0045] I. The desulfurication Vibrio bacterial solution and the symbiotic Clostridium bacterial solution are respectively prepared into bacterial solutions with a concentration of 4x10 9 cfu / mL by using sterile PBS, and then the two bacterial solutions are mixed according to a volume ratio of 1:1 to obtain a mixed bacterial solution;

[0046] II. Select rats with a weight of 300-350 grams, and fast the rats for 12 hours. First, use the mixed bacterial solution to gavage each rat with 2 mL, and then use the type 2 diabetes inducing feed to feed the rats every day. After continuous feeding for 4 weeks, fast the rats for 12 hours, and then inject STZ at 25 mg / kg by intraperitoneal injection. After the injection, continue to feed the rats with the high-fat and high-sugar feed for 1 week to obtain a type 2 diabetes animal model. The other steps are the same as those in specific embodiment six.

[0047] After the rats are fed with the type 2 diabetes inducing feed in this embodiment, only one injection of STZ is needed, and all the rats in the experimental group successfully model the type 2 diabetes after the first week of injection. However, for the group fed with the ordinary high-sugar and high-fat feed, some rats need to be injected with STZ twice to successfully model the type 2 diabetes in the third week, so this step is 2 weeks earlier.

[0048] Without using the method of injecting a high dose of STZ, the type 1 diabetes model of absolute insulin deficiency caused by using a high concentration of STZ is avoided.

[0049] Specific embodiment eight: the difference between this embodiment and specific embodiment seven is that the preparation method of the desulfurication Vibrio bacterial solution in step one is as follows:

[0050] The desulfurication Vibrio freeze-dried powder is placed in ATCC 260 liquid medium for sterile rehydration to obtain an activated bacterial solution. The bacterial solution is sterilely transferred to a container containing ATCC 260 liquid medium for anaerobic primary culture at 37°C for 24 hours. Then, the ATCC 260 liquid medium is used again for anaerobic subculture and expansion culture at 37°C for 24 hours. The other steps are the same as those in specific embodiment seven.

[0051] The desulfurication Vibrio is in the form of freeze-dried powder when purchased. Sterile rehydration is to add the freeze-dried powder of the bacterial body to the ATCC 260 liquid medium at room temperature. After the freeze-dried powder of the bacterial body gradually absorbs water, the bacterial body returns to an activated state.

[0052] Specific embodiment nine: the difference between this embodiment and specific embodiment seven is that the preparation method of the symbiotic Clostridium bacterial solution in step one is as follows:

[0053] The symbiotic Clostridium freeze-dried powder is put into ATCC 260 liquid medium for sterile rehydration to obtain the activated bacteria liquid, and the bacteria liquid is sterilely transferred to a container containing ATCC 260 liquid culture for anaerobic primary culture, and the culture condition is 37℃ for 24 hours, and then ATCC 260 liquid medium is used again for anaerobic subculture and expansion culture, and the culture condition is 37℃ for 24 hours. The rest is the same as embodiment seven.

[0054] The symbiotic Clostridium is in the form of freeze-dried powder when purchased, and sterile rehydration is to add the bacteria freeze-dried powder into ATCC 260 liquid medium at room temperature, and the bacteria freeze-dried powder gradually absorbs water to restore to the activated state.

[0055] Embodiment ten: different from one of embodiments seven to nine is that the type 2 diabetes inducing feed is stored at 4℃ after preparation, and needs to be restored to room temperature before use. The rest is the same as one of embodiments seven to nine.

[0056] The following embodiments of the application are described in detail, and the following embodiments are implemented on the premise of the technical scheme of the application, and detailed implementation schemes and specific operation processes are given, but the protection scope of the application is not limited to the following embodiments.

[0057] Embodiment 1:

[0058] I. Preparation of Desulfovibrio bacteria liquid

[0059] The purchased Desulfovibrio ATCC29098 is sterilely rehydrated with about 0.5mL ATCC 260 medium. The whole content is sterilely transferred to a 5mL tube containing ATCC 260 liquid medium broth for 37℃ anaerobic primary culture for 24 hours, and then ATCC 260 liquid medium is used again for 37℃ anaerobic subculture and expansion culture for 24 hours.

[0060] II. Preparation of symbiotic Clostridium bacteria liquid

[0061] The purchased symbiotic Clostridium ATCC14940 is sterilely rehydrated with about 0.5mL ATCC 260 medium. The whole content is sterilely transferred to a 5mL tube containing ATCC 260 liquid medium broth for 37℃ anaerobic primary culture for 24 hours, and then ATCC 260 liquid medium is used again for 37℃ anaerobic subculture and expansion culture for 24 hours.

[0062] III. The two bacterial liquids in step one and two are dried into viable bacteria powder at -60℃, and the viable bacteria number in the powder is determined by gradient dilution method on ATCC 260 agar solid medium at 37℃ for 48 hours. According to the viable bacteria number, the feed is prepared. The feed ratio: 100g of conventional high-fat and high-sugar feed (Beijing Keao Cooperation II type diabetes model feed, commodity number: 1016711983920930816 feed) is low-temperature pulverized, and each of the two bacterial liquids with a bacteria number of 1x10 10 cfu is mixed with the conventional high-fat and high-sugar feed uniformly, and then the new feed is extruded into a column shape by an extrusion molding machine to become the feed of the present application, which contains 1x10 8 cfu / g of Desulfovibrio vulgaris and 1x10 8 cfu / g of Clostridium symbiosum. The total amount of the feed is prepared according to the number of the modeling animals, and it is prepared once a week and stored in a refrigerator at 4℃.

[0063] IV. The two bacterial liquids in step one and two are determined for viable bacteria number by gradient dilution method on ATCC 260 agar solid medium at 37℃ for 48 hours, and then 4x10 9 cfu / mL of bacterial suspension is prepared with sterile PBS according to the viable bacteria number. Then 1mL of each of the two bacterial liquids is taken and mixed to become 2mL of bacterial liquid with a total bacteria number of 8x10 9 cfu.

[0064] V. Establishment of a type 2 diabetes model in rats

[0065] 300-350g male Wister rats are taken, each of which is fasted for 12 hours, and then 2ml of the bacterial liquid with a bacteria number of 8x10 9 cfu prepared in step IV is used for a single gavage, and then the feed prepared in step III is used for continuous feeding for 4 weeks after being warmed to room temperature in advance. The rats grow rapidly. Then the rats are fasted for 12 hours, and STZ 25mg / kg is injected intraperitoneally under fasting. After the injection, the rats are fed with high-fat and high-sugar feed (Beijing Keao Cooperation feed, commodity number: 1016711983920930816) for 1 week, and the rats can stably achieve fasting blood glucose≥7.0mmol / L.

[0066] Example 2: Change of rat body weight after 6 weeks of feed feeding

[0067] After the rats are fed for a period of time, if the rat body weight increases rapidly and significantly, and obesity occurs, injection of STZ can cause type 2 diabetes, so in the process of establishing a type 2 diabetes model, rapid obesity of the rats is an important link to save the whole type 2 diabetes rat model, and the change of body weight is detected to understand when to inject STZ.

[0068] Take 300 grams of male Wister rats, first in the laboratory with ordinary maintenance type feed (Beijing Kaohe cooperation feed Co., Ltd., commodity number: 1016706714625204224) feeding adaptation for 7 days, record the amount of food, water, change once every 1-2 days litter, ensure the hygiene of the cage, the environment of the breeding room is suitable. After the rats go through the adaptation period, start the normal experiment.

[0069] The rats were randomly divided into three groups, group A was 10 rats, always using ordinary maintenance type feed (Beijing Kaohe cooperation feed Co., Ltd., commodity number: 1016706714625204224) feeding, group B was 15 rats ordinary high-fat high-sugar type feed (Beijing Kaohe cooperation feed Co., Ltd., commodity number: 1016711983920930816), group C was 15 rats prepared by example 1 of the application. On the first day of the experiment, A and B groups of animals were fasted once with sterile PBS, 2mL per animal; C group of animals at the same time fasted once with mixed bacteria liquid, 2mL per animal. In order to find out the change of rat body weight, the animals in this example were fed for 6 weeks, free drinking water, the rat body weight, food intake, water intake and fasting blood glucose change were observed every week.

[0070] Fasting body weight determination: the rats were measured for fasting body weight every 7 days. The rats were fasted for 12 hours before the body weight was measured, and the water was normal;

[0071] Fasting blood glucose determination: the rats were measured for fasting blood glucose every 7 days. After the rats were fasted for 12 hours, the tail vein blood was drawn to measure the fasting blood glucose;

[0072] 24-hour food intake and water intake determination: the rats were recorded for food intake and water intake every 7 days. The weight of the feed and the water bottle was weighed at the beginning of feeding, and the remaining feed and water bottle weight was weighed again after 24 hours, the difference was the food intake and water intake of the rats in 24 hours.

[0073] The body weight change of the three groups of rats after feeding is shown in Table 1 Figure 1 Figure 1 ​The results show that the body weight of the rats in each group increases with time, but the body weight of the rats fed in the B group and the C group increases much higher than that of the rats fed in the A group. The body weight of the rats fed in the C group has significantly exceeded that of the rats fed in the B group since the third week. Therefore, the feed of the present application shows a trend of rapidly promoting the weight gain of the rats. Other physical examination results show that after the rats are fed by the method of the present application, the color and temperature of the rats are not found to change; the respiratory obstruction and the motor function are not found to change; the nervous system is not found to have involuntary contraction or convulsive contraction reaction; the reflex, salivation, skin, hair, muscle tension, cardiovascular and the like are normal; and no diarrhea, dysentery and the like are found.

[0074] Example 3: Inducing a rat to form a type 2 diabetes model

[0075] In order to stably form a type 2 diabetes rat, after the rats have been obese, STZ is injected once or twice, so that the rats have obvious symptoms of diabetes such as polyphagia, polydipsia, polyuria, hyperglycemia and the like. After the injection of STZ is completed, the diet amount, the water amount and the blood glucose value are detected every week, and if the fasting blood glucose is ≥7.0 mmol / L, it is considered that the modeling is initially successful.

[0076] After the change of the body weight of the rats is observed in Example 2, the following experiments are continued:

[0077] (1) The rats in the A group are continuously fed with the ordinary maintenance type feed (Beijing Keao Cooperation Feed Co., Ltd., commodity number: 1016706714625204224) for 4 weeks.

[0078] (2) The rats in the B group are fasted for 12 hours, and then STZ is injected intraperitoneally at 25 mg / kg. After the injection, the rats are continuously fed with the ordinary high-fat high-sugar feed (Beijing Keao Cooperation Feed Co., Ltd., commodity number: 1016711983920930816) for 1 week, and then the fasting blood glucose or the random blood glucose is detected. If the fasting blood glucose is ≥7.0 mmol / L, it is considered that the modeling is initially successful. If the blood glucose does not reach the modeling standard, the rats can be continuously waited for one week. If the blood glucose is still not more than 7.0 mmol / L, the rats are injected with STZ at 25 mg / kg again, and then the blood glucose is detected after the rats are continuously fed with the high-fat high-sugar feed for 1 week. Generally, the blood glucose can reach the modeling standard after the injection. If the modeling is not successful, the rats are discarded.

[0079] (3) Group C rats were fasted for 12 hours and injected with STZ 25 mg / kg intraperitoneally. After injection, the rats were fed with the feed of the application for 1 week, and then the fasting blood glucose or random blood glucose was detected. If the fasting blood glucose was equal to or greater than 7.0 mmol / L, it was considered that the modeling was successful. If the blood glucose did not reach the modeling standard, the rats were continued to be injected with STZ 25 mg / kg once again, and then the blood glucose was detected after the rats were fed with the high-fat and high-sugar feed for 1 week. If the blood glucose was still less than 7.0 mmol / L, the rats were abandoned.

[0080] After the modeling was successful, the ordinary feed was replaced, and the animals were allowed to drink freely. The state of the animals was observed, the blood glucose and the body weight were detected every week, the monitoring was performed for 4 weeks, and the stability of the model was observed. The bedding was replaced every 1-2 days to ensure the hygiene of the cages and the environment of the breeding room was suitable.

[0081] The changes of the fasting blood glucose of the rats in each group after the injection of STZ once are shown in Table 1, and the fasting blood glucose of the rats in group B after the injection of STZ once is shown in Table 2. As can be seen from the data in Tables 1 and 2, the fasting blood glucose of the rats in group A was below the normal value. The rats in group C were in a hyperglycemic state after the injection of STZ for the first week, and the fasting blood glucose of all the rats was greater than 7.0 mmol / L, and continued to increase in the second week, indicating that the modeling of group C was successful after the injection of STZ for the first week. The average fasting blood glucose of the rats in group B was 6.79±1.70 mmol / L after the injection for the first week, and only 9 of the 15 rats were greater than 7.0 mmol / L. After the rats were fed with the high-fat and high-sugar feed for 1 week, the average fasting blood glucose increased to 7.61±2.13 mmol / L, but the fasting blood glucose of the animals in the group was quite different, and only 10 of the 15 animals were greater than 7.0 mmol / L. Therefore, the remaining animals in group B were injected with STZ twice after the fasting blood glucose was detected in the second week. The animals in the three groups were continuously observed for 2 weeks. The results are shown in Table 3. The animals in group A were still normal in fasting blood glucose, the animals in group C were still in the 2-type diabetes onset stage with the fasting blood glucose being greater than 7.0 mmol / L after the injection of STZ once, and the remaining animals in group B were all in the 2-type diabetes onset stage with the fasting blood glucose being greater than 7.0 mmol / L after the injection of STZ twice. The changes of the fasting blood glucose of the animals in the three groups after the injection of STZ can also be seen from the curve. Figure 2 The change trend of the body weight of the rats can be seen from Figure 3 Table 4. The body weight of the rats in group A increased gradually, which showed the trend that the body weight of the normal rats increased with the increase of the age, while the body weight of the rats in groups B and C decreased obviously after the injection of STZ. Figure 4 , Figure 5The change of polydipsia, polyphagia is observed. Meanwhile, the dampness of the rat bedding is observed, and the rat bedding of the group B and the group C is very damp, and the rats generally show the state of polyphagia, polydipsia and polyuria. It is shown that compared with the rats fed with the common high-sugar high-fat food, the rats of the present application only need to be injected with low-dose STZ once, and the rats can complete the establishment of the type 2 diabetes model 2 weeks in advance.

[0082] Table 1 Change of fasting blood glucose of rats in each group after injection of STZ once (mmol / L)

[0083] Group Group A (not injected) Group B (after injection once) Group C (after injection once) 1w 4.77±0.78 6.79±1.70 10.23±1.01 2w 5.44±1.02 7.61±2.13 17.87±0.88

[0084] Table 2 Condition that the fasting blood glucose of rats in the group B exceeds 7.0 mmol / L after injection of STZ once

[0085] Time Number of rats over 7.0 mmol / L 1st week 9 2nd week 10

[0086] Table 3 Change of fasting blood glucose of rats in each group after injection of STZ (mmol / L)

[0087] Group Group A (not injected) Group B (after injection twice) Group C (after injection once) 3rd week 5.88±1.11 11.06±1.51 18.12±1.67 4th week 5.25±0.85 17.55±1.09 20.45±0.75

[0088] Example 4: Stability of the type 2 diabetes rat model

[0089] Because the model of type 2 diabetes is established, the influence of any drug or treatment on the type 2 diabetes rats needs to be studied under the condition of non-high-sugar high-fat food feeding. This is also the simulation of the human body condition. When the patient is diagnosed as type 2 diabetes, the patient generally controls the dietary intake according to the doctor's advice. Therefore, in order to determine that the type 2 diabetes model established by the method of the application is stable and does not self-recover to the normal state due to the change of food, 10 male rats of about 350g with fasting blood glucose in the range of 17-20mmol / L are selected, and the change of blood glucose after changing to ordinary maintenance type rat feed is observed. During the period, the rats are free to drink water, the animal state is observed, the blood glucose, body weight, intravenous insulin and C peptide changes are detected every week, and the stability of the model is monitored for 4 weeks. Table 4 can be seen that after changing to the maintenance type ordinary feed, the type 2 diabetes rats in group B all have a tendency of automatic blood glucose decrease with time, especially after 1 time of STZ injection, 3 rats have blood glucose lower than 7.0mmol / L in the third week, and 5 rats have self-blood glucose restored to 7.0mmol / L in the fourth week, and the fasting blood glucose of the rats in the group differs greatly. Like this, the rats with fasting blood glucose lower than 7.0mmol / L cannot be used for further experiments, which reduces the experimental efficiency and increases the experimental cost. The rats in group B injected with 2 times of STZ have this phenomenon in the fourth week. The fasting blood glucose of the rats in group C of the application is relatively stable within 4 weeks after changing to ordinary feed. It can be seen that after the rats are fed by the feed of the application, the stability of the fasting blood glucose of the rats is significantly improved, the blood glucose of the rats in the group is synchronous, and the model maintenance time is long. If this kind of model is used for treatment research, more clear data can be obtained.

[0090] Table 4 Stability of fasting blood glucose of type 2 diabetes rats (mmol / L)

[0091]

[0092] Example 5: Comparison of the stability of type 2 diabetes rats with different starting feeding times

[0093] In general, in the establishment of a type 2 diabetes model, high-sugar high-fat food feed is used to feed the rats for 6 weeks after the body weight changes, and then STZ is used for induction. In order to understand whether the method of the application can save the modeling time, this embodiment compares the difference between starting culture for 4 weeks and 6 weeks for modeling success.

[0094] 320g male wister rats were randomly divided into A and B groups, 10 rats in each group. On the first day of the study, each animal was given a mixed bacterial solution by gavage once, 2mL per animal. Then the A group was fed with the feed of the present application for 4 weeks, and then injected with STZ 25mg / kg by intraperitoneal injection, and then continued to be fed with the feed of the present application for 2 weeks, and the fasting blood glucose and 2-hour postprandial blood glucose were detected every week. The B group was fed with the feed of the present application for 6 weeks, and then injected with STZ 25mg / kg by intraperitoneal injection, and then continued to be fed with the feed of the present application for 2 weeks, and the fasting blood glucose and 2-hour postprandial blood glucose were detected every week. As can be seen from Table 5, after the animals were fed for 4 weeks or 6 weeks, STZ induction was performed, and a stable type 2 diabetic rat model was established, and the fasting blood glucose and postprandial blood glucose could reach fasting blood glucose≥7.0mmol / L, and 2h postprandial blood glucose≥11.1mmol / L. Therefore, induction can be performed after 4 weeks, which not only saves the modeling time, but also saves the cost of rat feed.

[0095] Table 5 Stability of type 2 diabetic rats with different initial feeding times (mmol / L)

[0096]

[0097] Example 6: Stability of bacteria in gastric juice and intestinal juice

[0098] The desulfovibrio bacteria solution and the symbiotic clostridium bacteria solution with a concentration of 2×10 9 cfu / ml were centrifuged at 8000×g to obtain bacterial precipitate. And resuspended with artificial gastric juice (Beijing Coolab Technology Co., Ltd. SL6600 artificial gastric juice) and artificial small intestinal juice (Beijing Coolab Technology Co., Ltd. SL6610A artificial small intestinal juice) and cultured anaerobically at 37℃. Gradient dilution and plate counting method was used to determine the viable bacterial count in artificial gastric juice and intestinal juice at 0h, 1.5h and 3h, respectively, to observe the survival of the two strains exposed to gastric juice and intestinal juice within 3h and evaluate their tolerance. The results are shown in Table 6. Although the number of bacteria of the two strains decreased with the extension of time in artificial gastric juice, after 3 hours, the remaining viable bacterial concentration was about 10 6 cfu / mL, but because the rat food stays in the stomach for no more than 1.5 hours, both of the two bacteria can resist the exposure of gastric acid, and still have a remaining viable bacterial concentration of more than 10 8 cfu / mL to reach the small intestine. The tolerance of the two bacteria in small intestinal juice is better, and after 3 hours of exposure, there are still viable bacteria with a concentration of more than 10 8 cfu / mL.

[0099] Table 6 Survival bacterial count exposed to gastric juice and intestinal juice (lg cfu / ml)

[0100]

[0101] Example 7: Influence of changing the feeding method of animals

[0102] In order to understand the influence of gavage and adding two kinds of bacteria in feed, this example carries out comparative study in multiple ways.

[0103] Male wister rats with a body weight of 350g were randomly divided into 3 groups, 16 rats in each group, and each group was further divided into two subgroups. Group A was not gavaged, only continuously fed with the feed of the present application, 8 rats were fed for 4 weeks and 8 rats were fed for 6 weeks; Group B was gavaged with mixed bacteria solution once a day on an empty stomach, 2mL per rat, and then fed with ordinary high-sugar high-fat feed every day, 8 rats were fed for 4 weeks and 8 rats were fed for 6 weeks; Group C was gavaged with mixed bacteria solution once on the first day of the study, 2mL per rat. Then continue to feed with the feed of the present application, 8 rats were fed for 4 weeks and 8 rats were fed for 6 weeks. The animals in the three groups were injected with STZ 25mg / kg once on an empty stomach after 4 weeks or 6 weeks, and then fed with the feed of the present application for 2 weeks, and the fasting blood glucose was detected. The comparison results of fasting blood glucose are shown in Table 7. It can be seen that rats need to be fed for 6 weeks after feeding with the feed of the present application alone to complete the model establishment. Although the average fasting blood glucose of rats is more than 7mmol / L, the value is very close to 7mmol / L. For some experiments that need to be studied for a long time and cannot be completed within two weeks, it is possible that rats will spontaneously recover to normal. The fasting blood glucose of both group B and group C can exceed 7mmol / L after 4 weeks of feeding and the fasting blood glucose of rats remains in a diabetic state after STZ injection, without natural recovery. Compared with group B, group C only needs to be gavaged once, and the remaining time can be automatically fed by rats, which is more convenient to operate. Moreover, the total amount of bacteria used by group C is significantly reduced compared with group B, saving cost.

[0104] Table 7 Comparison of fasting blood glucose of animals after changing the feeding method (mmol / L)

[0105]

[0106] Male Wister rats with a body weight of 350 g were randomly divided into 3 groups, 10 rats in each group. Group A was given a mixed bacteria solution by gavage once on the first day of the study, 2 mL per animal, and then was fed with the invented feed for 4 weeks. Group B was given Clostridium symbiosum solution by gavage once, 2 mL per animal, and other steps were the same as those of group A. Group C was given Desulfovibrio desulfuricans solution by gavage once, 2 mL per animal, and other steps were the same as those of group A. After 4 weeks, the rats in each group were given STZ 25 mg / kg by intraperitoneal injection once, and then were fed with the invented feed for 2 weeks, and the fasting blood glucose was detected. As shown in Table 8, if one kind of microorganism is used alone, the average fasting blood glucose of the rats exceeds 7 mmol / L, but among 10 animals, 4 or 6 rats do not reach the fasting blood glucose value, and the success rate of inducing type 2 diabetes is not high, while the fasting blood glucose of the animals in group C is uniform, and the established rat type 2 diabetes model is stable.

[0107] Table 8 Change of fasting blood glucose after gavage of microorganisms

[0108]

[0109] Example 8: Change of intestinal flora diversity of rats

[0110] In order to understand the change of intestinal flora of rats after the animal model is established by the method of the application, the following research was carried out.

[0111] Male Wister rats with a body weight of 320 g were randomly divided into 4 groups, 8 rats in each group, and were fed according to Table 9. Groups A and B are control groups, and groups C and D are type 2 diabetes induction groups. Groups A and C are fed for a total of 6 weeks, and groups B and D are fed for a total of 8 weeks. After the feeding of each group is completed, 1 g of fresh fecal sample is collected from each animal, and is immediately stored at -80℃. The intestinal flora of rats is constructed, sequenced and bioinformatically analyzed by taking 16S rRNA gene V3 and V4 variable regions as targets to understand the change of α-diversity index.

[0112] Table 9 Experimental scheme of each group in intestinal flora change research

[0113] Group Gavage Feeding time STZ injection Continue to feed A 2ml PBS once Common feed for 4 weeks None Common feed for 2 weeks B 2ml PBS once Common feed for 6 weeks None Common feed for 2 weeks C 2ml bacterial solution once Invention feed for 4 weeks Once Invention feed for 2 weeks D 2ml bacterial solution once Invention feed for 6 weeks Once Invention feed for 2 weeks

[0114] The change of intestinal flora diversity of each group is shown in Table 10. As shown in Table 10, the flora diversity of rats fed with ordinary feed (group A, group B) does not change, and the Outs, Chao1, Shannon and Simpson indexes of the two groups of animals have no significant difference. However, the intestinal flora of the type 2 diabetes rats established by the present application changes significantly, the Outs, Chao1 and Shannon indexes decrease significantly, and the Simpson index increases significantly, and there is no significant difference between the 4-week and 6-week initial feeding using the method of the present application, thus proving that the STZ induction can be performed after 4 weeks of feeding using the method of the present application, which saves 2 weeks of time compared with the ordinary high-sugar and high-fat feed.

[0115] Table 10 Change of intestinal flora diversity

[0116]

[0117] The change of intestinal flora relative abundance at the door level is shown in Table 11. As shown in Table 11, the main intestinal flora of rats fed with ordinary feed (group A, group B) does not change significantly at the door level (Bacteroidetes, Firmicutes, Proteobacteria, Actinobacteria). However, the intestinal flora of the type 2 diabetes rats established by the present application (group C, group D) changes significantly at the door level, the abundance of Proteobacteria and Actinobacteria decreases, the abundance of Firmicutes increases, and although there is no difference in the abundance of Bacteroidetes, compared with the increase in the abundance of Firmicutes at the corresponding time, the ratio of Bacteroidetes / Firmicutes decreases compared with that of ordinary rats.

[0118] Table 11 Change of intestinal flora relative abundance at the door level

[0119] Group Bacteroidetes Firmicutes Proteobacteria Actinobacteria A 33.22±11.11 56.35±10.18 2.41±0.36 1.21±0.25 B 34.53±12.09 54.04±11.23 2.28±0.21 1.33±0.29 C 30.13±10.67 66.41±7.02 1.46±0.51 1.04±0.41 D 29.78±9.68 67.22±6.93 1.53±0.22 1.11±0.32

Claims

1. A method for establishing a type 2 diabetes animal model using a type 2 diabetes-inducing diet, characterized in that, Includes the following steps:

1. Prepare 4×10⁻⁶ concentrations of *Vibrio desulfurans* and *Clostridium symbioticans* culture separately using sterile PBS. 9 The bacterial suspension was prepared at a concentration of cfu / mL, and then the two bacterial suspensions were mixed at a volume ratio of 1:1 to obtain a mixed bacterial suspension.

2. Select rats weighing 300-350 grams, fast for 12 hours, first administer 2 mL of mixed bacterial solution by gavage, then feed them with type 2 diabetes induction diet every day for 4 consecutive weeks. After fasting for 12 hours, inject STZ 25 mg / kg into the peritoneum on an empty stomach. Continue to feed them with type 2 diabetes induction diet for 1 week after the injection to obtain a type 2 diabetes animal model. The type 2 diabetes-inducing feed includes desulfurized Vibrio (… Desulfovibrio ) bacterial powder, symbiotic Clostridium ( Clostridium symbiosum Microbial powder and high-fat, high-sugar feed, wherein the viable concentration of desulfurizing Vibrio is 1×10⁻⁶. 8 cfu / g ~1.2×10 8 The cfu / g concentration of the symbiotic Clostridium was 1×10⁻⁶. 8 cfu / g ~1.2×10 8 cfu / g.

2. The method according to claim 1, characterized in that... The method for preparing the desulfurization Vibrio bacterial solution in step one is as follows: The lyophilized powder of *Vibrio desulfurans* was placed in ATCC 260 liquid medium and aseptically rehydrated to obtain an activated bacterial solution. The bacterial solution was then aseptically transferred to a container containing ATCC 260 liquid medium for anaerobic primary culture at 37°C for 24 hours. Then, it was anaerobically passaged and expanded again using ATCC 260 liquid medium at 37°C for 24 hours.

3. The method according to claim 1, characterized in that... The method for preparing the symbiotic Clostridium bacterial solution in step one is as follows: The lyophilized Clostridium symbioticis powder was placed in ATCC 260 liquid medium and aseptically rehydrated to obtain an activated bacterial solution. The bacterial solution was aseptically transferred to a container containing ATCC 260 liquid medium for anaerobic primary culture at 37°C for 24 hours. Then, it was anaerobically passaged and expanded again using ATCC 260 liquid medium at 37°C for 24 hours.

4. The method according to claim 1, characterized in that... After the type 2 diabetes-inducing feed is prepared, it should be stored at 4°C and brought back to room temperature before use.

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