CN110960558A - Preparation method of Akk bacteria capsule for animals related to metabolic diseases - Google Patents
Preparation method of Akk bacteria capsule for animals related to metabolic diseases Download PDFInfo
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- CN110960558A CN110960558A CN201911355192.7A CN201911355192A CN110960558A CN 110960558 A CN110960558 A CN 110960558A CN 201911355192 A CN201911355192 A CN 201911355192A CN 110960558 A CN110960558 A CN 110960558A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
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- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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Abstract
The invention belongs to the technical field of capsule preparation, and discloses a preparation method of Akk bacteria animal capsules related to metabolic diseases, which is characterized in that 60 percent of conjugated linoleic acid, 15 percent of L-carnitine and 25 percent of Akk bacteria are weighed and placed in a mixing tank for stirring; grinding in a colloid mill for three times, and sieving with a 100-mesh sieve to obtain a content material; placing the mixture in a vacuum pan to remove gas in the material; weighing gelatin, water and glycerol according to a ratio of 100:40:100, feeding into a gelatin melting tank, heating to 60-75 ℃, after the gelatin is completely melted, decompressing, vacuumizing, and sieving with a 80-mesh sieve to obtain a capsule filtrate; sending the filtrate of the capsule body and the materials of the content into a pill press according to the weight ratio of 1:2, and pressing into soft capsules; washing off oil stain on the surface of the capsule, selecting and removing non-positive pills to obtain positive soft capsule, and refrigerating at low temperature for storage. The oral capsule is used for flora transplantation, the composition of intestinal flora of animals with metabolic diseases is adjusted, and the control and treatment of the metabolic diseases are realized.
Description
Technical Field
The invention belongs to the technical field of capsule preparation, and particularly relates to a preparation method of Akk bacteria animal capsule related to metabolic diseases.
Background
Currently, the closest prior art: the intestinal flora refers to tens of thousands of microorganisms living in our intestines, including prokaryotes, eukaryotes, and archaea. The metagenome is different from the flora, and refers to the presence of more than 10 times the number of human genes in all gut flora. The intestinal flora is closely related to human health, and microorganisms residing in the human intestinal tract are key factors of host metabolism and are considered as potential sources of new therapies. A number of recent papers and reviews cover different aspects of the microbiome and its potential role in human health, including early in life, but also specific diseases such as cardiometabolic disorders, inflammatory bowel diseases, neuropsychiatric diseases and cancer, etc.
Akkermansia muciniphila (Akk bacteria) is a bacterium capable of degrading mucin in human intestinal tracts, and research shows that the Akkermansia muciniphila is negatively related to obesity, diabetes, inflammation and metabolic disorder; the mechanism of the probiotic effect can be to regulate the thickness of mucus in the intestinal tract and maintain the integrity of intestinal tract barrier and other regulation effects on endocrine; especially has good regulating effect on hyperglycemia and obesity; the content of Akk bacteria in intestinal tract of people with hyperglycemia and obesity is less than 1/100 bacteria of common people. There are studies that have shown pathogenic effects of the bacterium in reducing body fat mass, improving glucose homogeneity, reducing adipose tissue in inflammation and increasing gut integrity in mouse studies. In obese and type 2 diabetic mice, the abundance of Akk bacteria was reduced. And feeding with prebiotics normalized Akk germ abundance, which was associated with improvement in metabolites. It was also demonstrated that myxophilus treatment reversed high fat diet-induced metabolic disorders including increased fat mass, metabolic endotoxemia, adipose tissue flow and insulin resistance.
In some researches, A.muciniphila is separated from human excrement for the first time by using porcine intestinal mucin as a selective culture medium, and the A.muciniphila belongs to the Microbacterium verrucosum family by 16S rRNA gene sequencing analysis and is classified into the Akkermansiaceae family. Muciniphila is commonly colonized in human intestines, at least 8 different Akkermansia genera exist in intestinal mucus layers, and the a muciniphila of different genera is likely to be colonized in the same human intestines at the same time.
Akkermansia polyciniphila is a gram-negative strict anaerobic bacterium that colonizes the mucus layer of the gastrointestinal tract and specifically degrades mucin, which is isolated from human feces. Akk bacteria play an important role in the mucosal surface of the inner cavity of a host, can regulate the health of the organism and is gradually the focus of intestinal micro-ecology and probiotic research in recent years. Akk bacteria are among the most abundant members of the human intestinal microbiota, accounting for 1% to 5% of intestinal microorganisms. The diversity of intestinal flora is subject to great differences due to individual differences (age, diet, health, etc.) and is often reflected by the species, number, abundance, etc. of a genus.
The colonization of a. muciniphila also varies somewhat at different age stages: the number of A.muciniphila in the feces of young people is lower than that of the feces of old people, so that the old people probably pay more attention to body health and have more balanced diet; the abundance of a. mucini phila of centenarians in china is significantly weaker than that of people not in centenarians, and the abundance of european adults is higher than that of older people; the abundance and colonization rates of infants are lower than those of adults, but reach the adult level within one year. The correlation analysis of the age and the A.muciniphila needs further research, and the expansion of the sample size and the optimization of the detection technology are the primary problems. Due to different regions, the living environment and dietary structure of people are different, and the influence on the colonization of some flora is possible: the planting rate of Brazil population A. muciniphila is about 79.17%, European population is about 74.70%, and southern Chinese population is about 51.74%; mucini philia is less abundant in the stools of autistic children in australia than normal children, while the flora is more abundant in autistic children in italy and in the united states than normal children. The abundance of A.muciniphila in patients with intestinal inflammation diseases is lower than that of common people, and the quantity and the abundance of A.muciniphila in overweight or obese people and patients with type II diabetes are lower than that of common people. The quantity and the abundance of the A.muciniphila can be increased by controlling diet, taking prebiotics, losing weight surgery and other modes, the diversity of intestinal flora of athletes who exercise frequently is higher, the quantity of the A.muciniphila is obviously higher than that of obese people with abnormal metabolism, the reasonable diet, the work and rest rule and proper exercise can be used for keeping the quantity and the abundance of the A.muciniphila at a higher level, and the A.muciniphila can be indirectly proved to be used as a potential index for measuring the physical health condition.
Many studies on intestinal flora indicate that imbalance of intestinal flora (changes in the structure or functional structure of intestinal flora) can cause various diseases, such as obesity, diabetes, nervous system diseases, allergy, and various inflammations. There are studies showing that Akk bacteria are reduced in numbers during obesity and diabetes and that higher baseline abundance is significantly associated with improved cardiometabolic parameters in obese patients receiving heat limitation. It was found that daily administration of Akk bacteria grown on a mucus-based medium could counteract the development of High Fat Diet (HFD) -induced obesity and intestinal barrier dysfunction. Further, it has been found that Akk bacterium is associated with obesity, type II diabetes, and progressive frostbite, and therefore, Akk bacterium is negatively associated with metabolic diseases such as obesity and type II diabetes, and thus, administration of a drug containing Akk bacterium can provide a good therapeutic effect on the diseases.
Akk bacteria have been separated from human feces for the first time for more than 10 years, but most of the research is only limited to the physiological characteristics of Akk bacteria, and the research on the molecular action mechanism does not appear yet. Meanwhile, the optimization of the separation method is always in a standstill, and how to rapidly separate and classify the materials is always a bottleneck of research.
At present, the operation process of intestinal flora transplantation is mostly to irrigate the flora to be transplanted from the anus of an animal in an enema mode, and a test tube containing the flora can also be implanted into the animal body in an operation mode. Intestinal flora transplantation is becoming more and more common, because the intestinal flora in the animal body is disordered after the animal takes some antibacterial drugs, the state of the intestinal flora can be improved as soon as possible by the intestinal flora transplantation, and the occurrence of secondary reaction is prevented. However, the transplantation of the intestinal flora by means of enema cannot be controlled precisely in quantity and concentration gradient and is cumbersome to handle. Therefore, there is a need to develop a precise, effective and economical intestinal flora drug for metabolic disease animals.
In summary, the problems of the prior art are as follows:
(1) the existing research on Akk bacteria is only limited to the physiological characteristics of Akk bacteria, and the research on molecular action mechanisms does not appear. Meanwhile, the optimization of the separation method is always in a standstill, and how to rapidly separate and classify the materials is always a bottleneck of research. This makes the application of Akk bacteria in metabolic diseases slow.
(2) The existing intestinal flora transplanted in an enema form cannot accurately control the quantity and concentration gradient of the intestinal flora and is complicated to operate.
The difficulty of solving the technical problems is as follows: the method for extracting Akk bacteria with high efficiency and carrying out pure culture, and the method for preparing capsules.
The significance of solving the technical problems is as follows: akk bacteria are subjected to pure culture and prepared into capsules efficiently, and the intestinal flora of animals suffering from metabolic diseases is adjusted in an oral mode, so that the aims of controlling and treating the intestinal flora are fulfilled.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a preparation method of Akk bacteria capsule for animals, which is related to metabolic diseases.
The invention is realized by a preparation method of Akk bacteria capsule for animals related to metabolic diseases, and the preparation method of Akk bacteria capsule for animals comprises the following steps:
step one, weighing 60% of conjugated linoleic acid, 15% of L-carnitine and 25% of Akk bacteria by weight percent, placing the materials into a mixing tank, and uniformly stirring the materials.
Step two, grinding the uniformly stirred conjugated linoleic acid, L-carnitine and Akk bacteria in a colloid mill for three times, and sieving with a 100-mesh sieve to obtain a content material; then the content material is put into a vacuum pan, the vacuum degree is controlled to be-0.06 Mpa, and the gas in the material is removed.
Weighing the gelatin, the water and the glycerol according to the ratio of 100:40:100, feeding the materials into a gelatin melting tank, stirring and heating to 60-75 ℃, reducing pressure and vacuumizing after the gelatin is completely melted, sieving by a 80-mesh sieve to obtain capsule body filtrate, and placing the capsule body filtrate in a heat-preserving container for later use.
And step four, respectively feeding the capsule body filtrate obtained in the step three and the obtained content material into a pelleting press according to the weight ratio of 1:2, and pressing to obtain the soft capsule.
Step five, washing off oil stains on the surface of the capsule by using alcohol with the concentration of more than 90% in the capsule obtained in the step four; selecting the capsule with oil stain on the surface, removing non-positive pill to obtain positive soft capsule, and storing at 2-8 deg.C.
Further, in the fourth step, the temperature of the pelleting machine is controlled to be 24-28 ℃, the humidity is controlled to be 30-40%, and after static drying is carried out for 24 hours, the capsules are produced.
Further, the collection conditions of the Akk bacteria are as follows: collecting fresh feces of healthy people, sealing the bag, and storing for use.
Further, the screening conditions of the Akk strain are as follows: a. diarrhea did not occur in the last half year. b. Antibiotics have not been used in the last year. c. No history of intestinal disease or intestinal surgery. d. Without any infectious disease. e. Without any metabolic disease.
Further, the Akk bacteria extraction and pure culture method comprises the following steps:
(1) fresh fecal samples of healthy people were separately collected in a polyethylene bag and stored in a sealed condition for future use, and 0.5g of feces was diluted into 9ml of sterile anaerobic ringer's solution containing 0.5g of cysteine.
(2) The suspension obtained in step (1) was mixed thoroughly and diluted serially (10-fold) in ringer's solution. Each dilution (1ml) was then inoculated in triplicate into 9ml of bicarbonate buffer.
(3) All compounds except vitamins were autoclaved. To this basal medium 0.7% (v/v) of clear sterile rumen fluid and 0.25% (v/v) of commercial porcine gastric mucin were added and purified by ethanol precipitation. This medium is called mucin medium.
(4) Pure cultures were isolated using soft agar technique: the highest dilution of observed culture growth was serially diluted to 10 in phosphate buffer (pH7)-9Then 10 is put-6-10-9The dilutions were re-inoculated into the same medium containing 0.75% agar, individual target colonies were picked, allowed to grow in mucin medium, and re-inoculated into soft agar mucin medium.
(5) And (5) repeating the step (4) until purification.
Further, in step (2), the basal medium contains: 0.4g of monopotassium phosphate, 0.53g of sodium monohydrogen phosphate, 0.3g of ammonium chloride, 0.3g of sodium chloride, 0.1g of magnesium chloride hexahydrate, 0.11g of calcium chloride, 1ml of alkaline trace element solution, 1ml of acidic trace element solution, 1ml of vitamin solution, 0.5mg of resazurin, 4g of sodium bicarbonate and 0.25g of sodium sulfide nonahydrate.
Further, instead of mucin, soybean peptone at a concentration of 16g/L, threonine at a concentration of 4g/L and mixed sugars (glucose, N-acetylglucosamine) at a concentration of 25mM were used. Cultures were washed and concentrated under strictly anaerobic conditions with 25% (vol/vol) glycerol in anaerobic PBS. In addition, the same number of Akk bacteria grown on synthetic medium were inactivated by pasteurization at 70 ℃ for 30 minutes, after which the culture was immediately frozen and stored at-80 ℃.
The invention also aims to provide a capsule prepared by the preparation method of the Akk bacteria animal capsule related to the metabolic disease. Akk bacteria capsule belongs to a medicine for animals, and the capsule contains: conjugated linoleic acid, L-carnitine and Akk bacteria. The specific application of the capsule is that animals can treat metabolic diseases by taking the capsule orally.
It is another object of the present invention to provide a medicament for treating obesity in animals comprising the capsule.
Another object of the present invention is to provide a medicament for treating type ii diabetes comprising the capsule.
In summary, the advantages and positive effects of the invention are: the invention adopts pure culture of Akk bacteria under multiple strict conditions and makes the pure culture into capsules, and the oral capsules are used for flora transplantation to adjust the composition of intestinal flora of animals with metabolic diseases, thereby realizing the control and treatment of the diseases and achieving the purpose of treating the metabolic diseases of the animals, such as obesity, type II diabetes, and the like. The invention has the advantages of no toxicity, long-term effectiveness and the like.
Drawings
FIG. 1 is a flow chart of a preparation method of Akk bacteria capsule for animals relevant to metabolic diseases, which is provided by the embodiment of the invention.
FIG. 2 is a flow chart of the Akk bacteria extraction and pure culture method provided by the embodiment of the invention.
FIG. 3 is a flow chart of a Akk bacteria preservation method according to an embodiment of the present invention.
Fig. 4 is a flow chart of a preparation method of Akk bacteria capsule content provided by an embodiment of the invention.
Fig. 5 and fig. 6 are flow charts of Akk microcapsule body preparation methods provided by embodiments of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In view of the problems in the prior art, the present invention provides a method for preparing Akk bacteria capsule for animals, which is related to metabolic diseases, and the present invention is described in detail below with reference to the accompanying drawings.
As shown in fig. 1, the preparation method of Akk bacteria capsule for animals related to metabolic diseases provided by the embodiment of the invention comprises the following steps:
s101: weighing 60% of conjugated linoleic acid, 15% of L-carnitine and 25% of Akk bacteria according to the weight percentage, placing the materials into a mixing tank, and uniformly stirring the materials.
S102: grinding the uniformly stirred conjugated linoleic acid, L-carnitine and Akk bacteria obtained in the step S101 in a colloid mill for three times, and sieving with a 100-mesh sieve to obtain a content material; then the content material is put into a vacuum pan, the vacuum degree is controlled to be-0.06 Mpa, and the gas in the material is removed.
S103: weighing gelatin, water and glycerol according to a ratio of 100:40:100, feeding into a gelatin melting tank, stirring and heating to 60-75 ℃, reducing pressure and vacuumizing after the gelatin is completely melted, sieving with a 80-mesh sieve to obtain capsule filtrate, and placing in a heat-insulating bucket for later use.
S104: and (4) respectively feeding the capsule body filtrate obtained in the step (S103) and the obtained content materials into a pelleting press according to the weight ratio of 1:2, and pressing to obtain the soft capsule. Controlling the temperature of the pelleting machine to be 24-28 ℃ and the humidity to be 30-40%, statically drying for 24 hours, and then pelleting the capsules.
S105: washing oil stains on the surface of the capsule by using alcohol with the concentration of more than 90% in the capsule obtained in the step S104; selecting the capsule with oil stain on the surface, removing non-positive pill to obtain positive soft capsule, and storing at 2-8 deg.C.
The technical solution of the present invention is further described with reference to the following examples.
Example 1
The embodiment of the invention provides a preparation method of Akk bacteria capsules for patients with metabolic diseases such as obesity, type II diabetes and the like, which comprises the following steps:
1. akk Collection and screening of bacteria
1.1 acquisition conditions: collecting fresh feces of healthy people, sealing the bag, and storing for use.
1.2 screening conditions: a. diarrhea did not occur in the last half year. b. Antibiotics have not been used in the last year. c. No history of intestinal disease or intestinal surgery. d. Has no infectious diseases, such as hepatitis A, hepatitis B, hepatitis C, etc. e. Has no metabolic diseases, such as diabetes, obesity, hypoglycemia, etc.
2. Akk extraction and pure culture of fungus
2.1 fresh stool samples from healthy persons were separately collected in a polyethylene bag and stored in a sealed condition, and 0.5g of stool was diluted into 9ml of sterile anaerobic ringer's solution containing 0.5g of cysteine. This suspension was mixed well and serially diluted (10-fold) in ringer's solution. Each dilution (1ml) was then inoculated in triplicate into 9ml of bicarbonate buffer. The basic culture medium contains: 0.4g of monopotassium phosphate, 0.53g of sodium monohydrogen phosphate, 0.3g of ammonium chloride, 0.3g of sodium chloride, 0.1g of magnesium chloride hexahydrate, 0.11g of calcium chloride, 1ml of alkaline trace element solution, 1ml of acidic trace element solution, 1ml of vitamin solution, 0.5mg of resazurin, 4g of sodium bicarbonate and 0.25g of sodium sulfide nonahydrate. To this basal medium 0.7% (v/v) of clear sterile rumen fluid and 0.25% (v/v) of commercial porcine gastric mucin were added and purified by ethanol precipitation. This medium is called mucin medium. Pure cultures were isolated using soft agar technique: growing the observed cultureThe highest dilution was serially diluted to 10 in phosphate buffer (pH7)-9Then 10 is put-6-10-9The dilutions were re-inoculated into the same medium containing 0.75% agar, individual target colonies were picked, allowed to grow in mucin medium, and re-inoculated into soft agar mucin medium. This step was repeated until purification (as shown in figure 2).
2.2 replacement of mucin by Soytone at a concentration of 16g/L, threonine at a concentration of 4g/L and mixed sugars of 25mM (glucose, N-acetylglucosamine). Cultures were washed and concentrated under strictly anaerobic conditions with 25% (vol/vol) glycerol in anaerobic PBS. In addition, the same number of Akk bacteria grown on synthetic medium were inactivated by pasteurization at 70 ℃ for 30 minutes, and then the culture was immediately frozen and stored at-80 ℃ (as shown in FIG. 3).
3. Akk preparation method of fungus capsule
3.1 weighing 60% of conjugated linoleic acid, 15% of L-carnitine and 25% of Akk bacteria by weight percent, placing the materials in a mixing tank, and uniformly stirring. Grinding the uniformly stirred conjugated linoleic acid, L-carnitine and Akk bacteria in a colloid mill for three times, sieving with a 100-mesh sieve to obtain a content material, placing the content material in a vacuum pan, controlling the vacuum degree to be-0.06 Mpa, and removing gas from the material (as shown in figure 4).
3.2 weighing the gelatin, the water and the glycerol according to the proportion of 100:40:100, feeding the materials into a gelatin melting tank, stirring and heating to 60-75 ℃, carrying out vacuum pumping under reduced pressure after the gelatin is completely melted, sieving by a 80-mesh sieve to obtain capsule body filtrate, and placing the capsule body filtrate in a heat-preserving barrel for later use (as shown in figure 5).
3.3 the obtained capsule body filtrate and the obtained content materials are respectively sent into a pelleting press according to the weight ratio of 1:2, and soft capsules are obtained by pressing. Controlling the temperature at 24-28 deg.C and humidity at 30-40%, statically drying for 24 hr, and making into pill. The obtained capsule is washed with above 90% ethanol to remove oil stain on the surface of the capsule. Selecting the capsule with oil on the surface, removing non-positive pellet to obtain positive soft capsule, and storing at 2-8 deg.C (shown in figure 6).
Enters the intestinal tract of animals with metabolic diseases by oral administration to adjust and improve the composition of intestinal flora, thereby having good treatment effect on the animals with metabolic diseases.
Example 2
The preparation method of the Akk bacteria capsule for animals related to metabolic diseases provided by the embodiment of the invention comprises the following steps:
1: akk extraction and pure culture of fungus
Fresh fecal samples of healthy people were separately collected in a polyethylene bag and stored in a sealed condition for future use, and 0.5g of feces was diluted into 9ml of sterile anaerobic ringer's solution containing 0.5g of cysteine. This suspension was mixed well and serially diluted (10-fold) in ringer's solution. Each dilution (1ml) was then inoculated in triplicate into 9ml of bicarbonate buffer. The basic culture medium contains: 0.4g of monopotassium phosphate, 0.53g of sodium monohydrogen phosphate, 0.3g of ammonium chloride, 0.3g of sodium chloride, 0.1g of magnesium chloride hexahydrate, 0.11g of calcium chloride, 1ml of alkaline trace element solution, 1ml of acidic trace element solution, 1ml of vitamin solution, 0.5mg of resazurin, 4g of sodium bicarbonate and 0.25g of sodium sulfide nonahydrate. To this basal medium 0.7% (v/v) of clear sterile rumen fluid and 0.25% (v/v) of commercial porcine gastric mucin were added and purified by ethanol precipitation. This medium is called mucin medium. Pure cultures were isolated using soft agar technique: the highest dilution of observed culture growth was serially diluted to 10 in phosphate buffer (pH7)-9Then 10 is put-6-10-9The dilutions were re-inoculated into the same medium containing 0.75% agar, individual target colonies were picked, allowed to grow in mucin medium, and re-inoculated into soft agar mucin medium. This step was repeated until purification (as shown in figure 2).
2: akk preparation method of fungus capsule
Weighing 60% of conjugated linoleic acid, 15% of L-carnitine and 25% of Akk bacteria according to the weight percentage, placing the materials into a mixing tank, and uniformly stirring the materials. Grinding the uniformly stirred conjugated linoleic acid, L-carnitine and Akk bacteria in a colloid mill for three times, sieving with a 100-mesh sieve to obtain a content material, placing the content material in a vacuum pan, controlling the vacuum degree to be-0.06 Mpa, and removing gas from the material (as shown in figure 4). Weighing gelatin, water and glycerol at a ratio of 100:40:100, feeding into a gelatin melting tank, stirring and heating to 60-75 deg.C, melting gelatin completely, vacuum-pumping under reduced pressure, sieving with 80 mesh sieve to obtain capsule filtrate, and placing in a heat-insulating bucket (shown in figure 5). And respectively feeding the obtained capsule body filtrate and the obtained content materials into a pelleting press according to the weight ratio of 1:2, and pressing to obtain the soft capsule. Controlling the temperature at 24-28 deg.C and humidity at 30-40%, statically drying for 24 hr, and making into pill. The obtained capsule is washed with above 90% ethanol to remove oil stain on the surface of the capsule. Selecting the capsule with oil on the surface, removing non-positive pellet to obtain positive soft capsule, and storing at 2-8 deg.C (shown in figure 6).
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A preparation method of Akk bacteria and animal capsule related to metabolic disease is characterized in that the preparation method of Akk bacteria and animal capsule comprises the following steps:
step one, weighing 60% of conjugated linoleic acid, 15% of L-carnitine and 25% of Akk bacteria by weight percent, placing the materials into a mixing tank, and uniformly stirring the materials;
step two, grinding the uniformly stirred conjugated linoleic acid, L-carnitine and Akk bacteria in a colloid mill for three times, and sieving with a 100-mesh sieve to obtain a content material; then placing the content material in a vacuum pan, controlling the vacuum degree to be-0.06 Mpa, and removing gas in the material;
weighing the gelatin, water and glycerol according to the ratio of 100:40:100, feeding the materials into a gelatin melting tank, stirring and heating to 60-75 ℃, performing vacuum pumping under reduced pressure after the gelatin is completely melted, sieving by a 80-mesh sieve to obtain capsule body filtrate, and placing the capsule body filtrate in a heat-preserving container for later use;
step four, respectively feeding the capsule body filtrate obtained in the step three and the obtained content materials into a pelleting press according to the weight ratio of 1:2, and pressing to obtain soft capsules;
step five, washing off oil stains on the surface of the capsule by using alcohol with the concentration of more than 90% in the capsule obtained in the step four; selecting the capsule with oil stain on the surface, and removing the non-positive pill to obtain the positive soft capsule.
2. The method for preparing Akk bacteria-animal capsule associated with metabolic diseases according to claim 1, wherein in the fourth step, the temperature of the pellet press is controlled to 24-28 ℃, the humidity is controlled to 30-40%, and the capsule is dried statically for 24 hours and then is made into pills.
3. The method for preparing Akk bacteria capsule for animals related to metabolic diseases according to claim 1, wherein the collection conditions of Akk bacteria are: collecting fresh feces of healthy people, sealing the bag, and storing for use.
4. The method for preparing Akk bacteria capsule for animals related to metabolic disease as claimed in claim 1, wherein the method for extracting and pure culturing Akk bacteria comprises the following steps:
(1) respectively collecting fresh excrement samples of healthy people in a polyethylene bag for sealed storage for later use, and diluting 0.5g of excrement into 9ml of sterile anaerobic ringer's solution containing 0.5g of cysteine;
(2) fully mixing the suspension obtained in the step (1), continuously diluting the suspension in ringer's solution by 10 times, and inoculating 1ml of each dilution in triplicate into 9ml of bicarbonate buffer solution;
(3) autoclaving all compounds except vitamins; adding 0.7% v/v of sterile rumen fluid and 0.25% v/v of commercial porcine gastric mucin to the basal medium and purifying by ethanol precipitation; the culture medium is a mucin culture medium;
(4) isolating the pure culture using soft agar technology;
(5) and (5) repeating the step (4) until purification.
5. The method of claim 4, wherein the basic culture medium comprises, in step (2): 0.4g of monopotassium phosphate, 0.53g of sodium monohydrogen phosphate, 0.3g of ammonium chloride, 0.3g of sodium chloride, 0.1g of magnesium chloride hexahydrate, 0.11g of calcium chloride, 1ml of alkaline trace element solution, 1ml of acidic trace element solution, 1ml of vitamin solution, 0.5mg of resazurin, 4g of sodium bicarbonate and 0.25g of sodium sulfide nonahydrate.
6. The method for preparing Akk fungus capsule for animals according to metabolic disease as claimed in claim 4, wherein the method for separating pure culture by soft agar technique in step (4) comprises: the highest dilution of observed culture growth was serially diluted to 10 in phosphate buffer pH7-9Then 10 is put-6-10-9The dilutions were re-inoculated into the same medium containing 0.75% agar, individual target colonies were picked, grown in mucin medium, and re-inoculated in soft agar mucin medium.
7. The method of preparing a Akk bacterial animal capsule related to metabolic disease of claim 4, wherein mucin is replaced with soy peptone at a concentration of 16g/L, threonine at a concentration of 4g/L and mixed sugar at a concentration of 25 mM; the culture was washed and concentrated under strictly anaerobic conditions with 25% vol/vol glycerol in anaerobic PBS; the same number of Akk bacteria grown on synthetic medium were inactivated by pasteurization at 70 ℃ for 30 minutes, after which the culture was immediately frozen and stored at-80 ℃.
8. A capsule prepared by the method for preparing Akk bacteria-containing animal capsule for metabolic diseases according to any one of claims 1 to 7.
9. A medicament for treating obesity in an animal comprising the capsule of claim 8.
10. A medicament for the treatment of type ii diabetes comprising the capsule of claim 8.
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