CN115927109A - Preparation method and application of Acermannium aphanidermatum solid preparation - Google Patents
Preparation method and application of Acermannium aphanidermatum solid preparation Download PDFInfo
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- CN115927109A CN115927109A CN202211637261.5A CN202211637261A CN115927109A CN 115927109 A CN115927109 A CN 115927109A CN 202211637261 A CN202211637261 A CN 202211637261A CN 115927109 A CN115927109 A CN 115927109A
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Abstract
The invention relates to a preparation method and application of an Ackermanella posterior solid preparation, wherein the Ackermanella is a muciniphila Akkermansonia, preferably Akkermansonia ATCCBA-835 and a sub-strain thereof. The solid product prepared by the method has the similar effect of treating the glucose metabolism diseases with the Ackermann metazoan liquid preparation and the Ackermann fresh viable bacteria, and can obviously improve insulin sensitivity, improve metabolic indexes such as glucose tolerance and the like. In addition, the Ackermann metazoan solid preparation is transported in a solid form, is more convenient and economic compared with a liquid preparation, simplifies the transportation mode, simultaneously can not need sterile filling, can still keep the activity and the curative effect of the medicine for a long time, and has the quality guarantee period of at least more than 3 months. The acquired solid preparation of Ackermanella by the method changes the physical state of the Ackermanella, simultaneously reserves the effective components of the Ackermanella, and provides a foundation for commercialization and clinical application of the Ackermanella.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to a preparation method and application of an Ackermansia metazoan solid preparation.
Background
Ackermansia is the new generation of intestinal beneficial bacterium of star with the most development potential. Recently, the research results published in PNAS and Nature medicine and other periodicals show that the Acermanium living bacteria have the effects of remarkably improving glycolipid metabolism and treating metabolic syndromes such as obesity, diabetes, dyslipidemia and the like. However, for market promotion, ackermansia must maintain its basic activity in an anaerobic environment. In addition, ackermansia bacteria are very sensitive to ambient temperature, oxygen and humidity, the main transportation mode is to store liquid Ackermansia culture medium at low temperature and place the Ackermansia culture medium in a special closed container to isolate oxygen, and the special transportation mode seriously hinders the application of the Ackermansia bacteria.
Therefore, scientists at home and abroad carry out a great deal of research on the Ackermann fungi after pasteurization, and as a result, the inactivated Ackermann fungi (metazoan fungi) still have the function of improving glycolipid metabolism and have better effect than live fungi. The research accelerates the clinical transformation application process of Ackermanella. However, the market transformation of the Ackermansia hindustanus has the characteristics of low energy consumption, high effectiveness and the like, most of the existing Ackermansia hindustanus are transported in a liquid state, the transportation cost and the energy consumption are obviously increased by the liquid transportation mode compared with the solid state, and in addition, the liquid preparation is very easy to cause pollution and mildew, so that the long-term storage is difficult. In order to avoid pollution and mildew, aseptic filling technology is adopted to achieve the effect of prolonging the storage life. Although the series of operations can prolong the shelf life of the post-growth of Ackermansia, the preparation cost is further increased, and the aseptic filling technology is required to carry out aseptic filling on the liquid product, and the storage condition is also improved, namely the storage under the low-temperature condition is required.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a preparation method and application of a post-growth solid preparation of Ackermanella so as to solve the problems of liquid transportation, storage and pollution of the Ackermanella.
The purpose of the invention is realized by the following technical scheme:
the invention aims to provide a preparation method of an Ackermanella metaplasia solid preparation, which comprises the following steps:
the method comprises the following steps: inoculating an Ackermanomyces lacticus original bacterial liquid to a culture medium to obtain a liquid A;
step two: placing the solution A in an anaerobic incubator for culture, centrifuging the prepared culture medium containing Ackermanella, removing the supernatant to obtain A bacteria mud, placing the obtained A bacteria mud in a centrifuge tube, adding a PBS solution containing 20% of glycerol, and resuspending into a suspension called as solution B;
step three: inactivating the solution B at 65-70 deg.C for 20-30 min, and cooling to obtain solution C;
step four: adding the cryoprotectant into the solution C, and thoroughly mixing to obtain solution D;
step five: placing the solution D in a culture dish, uniformly shaking, and then placing in a refrigerator for freezing to obtain solution E;
step six: and putting the E liquid into a freeze dryer to obtain the post-growth solid preparation of the Ackermansia.
Further, in the second step, the solution A is placed in an anaerobic incubator for 48 hours and is taken out when OD600 is more than 0.8; setting the rotating speed of a centrifugal machine at 6000 rpm, centrifuging the prepared culture medium containing the Ackermanomyces, and removing the supernatant to obtain A bacteria mud.
Further, in the fourth step, the cryoprotectant is 20% of milk powder, 5% of glycerol and 75% of PBS solution by mass percentage.
Further, in the fifth step, the solution D is put into a refrigerator at-80 ℃ for freezing for 30 minutes to obtain the solution E.
Further, in the sixth step, the freeze dryer is programmed to: the operation is carried out for 48-60 hours at-40 ℃ to-50 ℃ and at a pressure of 1pa to 10pa, and then the temperature is raised to 20 ℃ for 2-5 hours.
Further, the Ackermanella is Akkermansia muciniphila ATCC BAA-835 and/or a sub-strain thereof.
The invention also aims to provide application of the Ackermansia henryi metazoan solid preparation obtained by the preparation method in preparing a medicament or health-care product for preventing or treating metabolic diseases.
Furthermore, the drug or health care product of the Ackermansia metaplasia solid preparation can be prepared into a pharmaceutically acceptable dosage form.
Furthermore, the medicine or health-care product of the Ackermansia metaplasia solid preparation also comprises pharmaceutically acceptable auxiliary materials.
Furthermore, the administration mode of the drug or health-care product of the Acermanium retrogradation solid preparation comprises oral administration, buccal administration, external application and/or smearing.
Compared with the prior art, the invention has the following advantages and effects:
the solid product prepared by the method has the similar effect of treating the glucose metabolism diseases with the Ackermann metazoan liquid preparation and the Ackermann fresh viable bacteria, and can obviously improve insulin sensitivity, improve metabolic indexes such as glucose tolerance and the like. In addition, the Ackermann metazoan solid preparation is transported in a solid form, is more convenient and economic compared with a liquid preparation, simplifies the transportation mode, simultaneously can not need sterile filling, can still keep the activity and the curative effect of the medicine for a long time, and has the quality guarantee period of at least more than 3 months. The acquired solid preparation of Ackermanella by the method changes the physical state of the Ackermanella, simultaneously reserves the effective components of the Ackermanella, and provides a foundation for commercialization and clinical application of the Ackermanella.
Drawings
FIG. 1 is a diagram of a finished product of post-growth solid preparation of Ackermanella of example 1 of the present invention;
FIG. 2 is a graph showing the count of viable Ackermanobacter and post-growth solid preparation of Ackermanobacter in example 1 of the present invention;
FIG. 3 is a graph comparing the body weight parameters of three groups of mice in PBS group/live AKK group/post-AKK solid formulation group of example 2 of the present invention;
fig. 4 is a graph of the results of the glucose tolerance test of three groups of mice in the PBS group/live AKK group/post-AKK prebiotic solid formulation group of example 2 of the invention;
FIG. 5 is a graph showing the results of the insulin sensitivity test of three groups of mice in PBS group/live AKK group/post-AKK solid formulation group according to example 2 of the present invention;
FIG. 6 is a graph of the area under the glucose tolerance test curve of example 2 of the present invention;
FIG. 7 is the area under the curve for the insulin sensitivity test of example 2 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. The experimental procedures, for which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions, conditions described in laboratory manuals or conditions recommended by the manufacturer. The raw materials, reagents and the like used in the present invention are commercially available unless otherwise specified.
EXAMPLE 1 preparation of post-growth solid preparation of Ackermansia
And performing amplification culture on the Ackermanella by using the Ackermanella special culture medium. The components of the culture medium are as follows: 13g of soybean peptone, 5g of tryptone, 17.5g of brain heart leaching powder, 1g of glucose, 2.8g of sodium chloride, 1.4g of disodium hydrogen phosphate, 5.5g of N-acetyl glucosamine, 4g of L-threonine and 0.05g of L-cysteine hydrochloride are dissolved in 1L of ultrapure water, the mixture is simply shaken and uniformly mixed until no visible substances exist, and then the obtained mixture is filtered by using a 0.22 mu m degerming filter screen to obtain the akkermansia special culture medium. Inoculating the strain into 1L of Ackermanella special culture medium called solution A according to the proportion of the original strain solution 1. Setting the rotation speed of a centrifugal machine at 6000 rpm, centrifuging the prepared culture medium containing the Ackermanella, removing a supernatant to obtain A bacterial sludge, putting the A bacterial sludge obtained by 1L of the culture medium into the same centrifugal tube, and adding 4ml of PBS (containing 20% of glycerol) to be re-suspended into a suspension called B liquid. And (3) inactivating the solution B at 65-70 ℃ for 20-30 minutes (pasteurizing inactivation), and cooling the solution to obtain solution C.
To solution C, 8ml of a cryoprotectant (20% milk powder, 5% glycerol, 75% PBS solution) was added and mixed thoroughly, called solution D. The solution D is placed in a 15cm culture dish, uniformly shaken and then placed in a-80 ℃ refrigerator for freezing for 30 minutes to be called solution E. Placing the solution E into a freeze dryer. The freeze dryer was programmed to: performing at-40 deg.C to-50 deg.C under 1Pa to 10Pa for 48-60 hr, heating to 20 deg.C for 2-5 hr to obtain Ackermanella zakii metazoan solid preparation called F powder, and encapsulating to obtain Ackermanella zakii metazoan capsule shown in FIG. 1. The Acermanium postbiotic solid preparation is placed at room temperature for 3 months and is called G powder. The post-growth solid preparation of akkermansia (F powder) was weighed using a precision electronic scale. The above solution B, solution C and powder G were each 0.1% resuspended in 1ml of PBS solution and bacteria were counted at 10^ -5, as shown in FIG. 2.
The results show that: every 1ml of original bacterial liquid contains 3-5 multiplied by 10^8 Ackermanensis viable bacteria, and no viable bacteria are found after pasteurization. The prepared Ackermanella metazoa post-growth solid preparation contains the Ackermanella complex with the amount of 1.6-1.8 multiplied by 10^11CFU/g, and the Ackermanella metazoa post-growth solid preparation still keeps the sterile state after being stored for 3 months at room temperature. In conclusion, the Ackermanella metazoa solid preparation contains the Ackermanella complex with the content of 1.6-1.8 x 10^11CFU/g and the storage period at room temperature is at least 3 months.
Example 2 in vivo experiments to examine the effectiveness of solid preparation of Ackermanella
1. Model building
15C 57/BL6 mice of 6-7 weeks were included as subjects and randomized into three groups (PBS group; fresh AKK group; post-AKK prebiotic solid formulation group). The treatment method comprises the following steps: PBS group (control group), the same way of processing with AKK postbiotic solid preparation, only does not contain Ackermanella and its compound, called P reagent for short; in the fresh AKK group, bacteria in a fresh Ackermanella culture medium are taken every day, centrifuged and bacterial sludge is taken, and 200 mu l of PBS solution containing 20 percent of glycerol is added into the Ackermanella of 2-4 multiplied by 10^8 and is uniformly mixed to be called H solution; weighing 0.002g-0.004g of the Ackermanella posterior solid preparation (containing 2-4 x 10^8 of the Ackermanella compound) prepared in the AKK posterior solid preparation group, using 200 mu l of PBS for resuspension to obtain an Ackermanella posterior oral liquid K, and intragastrically administering 200 mu l of the Ackermanella oral liquid K every day. GTT (glucose tolerance test) or ITT (insulin sensitivity test) was performed once a week, and body weight was weighed weekly for a 7-week period of the experiment.
2 results
2.1 post-growth solid preparation of Ackermanobacter has slight weight loss effect on common diet mice
The mice perfused with the above solutions P, H and K were weighed once a week, and at the end of the experiment, statistical analysis was performed on the weight gain of the mice. As a result, it was found that: compared with the control group (PBS group), the AKK live bacteria group and AKK post-growth solid preparation have slight weight loss effect; the weight loss effect of the live AKK bacteria and the post-AKK prebiotics solid preparation has no statistical significance, and the result is shown in figure 3.
2.2 post-growth solid preparation of Ackermanobacter for improving glucose tolerance of mice
Three groups of mice were subjected to the Glucose Tolerance Test (GTT) weekly: after a 16-hour fasting, the blood glucose levels were measured at 0 min, 15 min, 30 min, 60 min and 120 min by intraperitoneal injection of 20% glucose solution (2 g/kg) per 10g of body weight, and plotted with time as abscissa and blood glucose as ordinate, and the area under the curve was calculated, using a weighted statistical method between the different experimental groups. The results show that: compared with a control group, the fresh live Ackermanella bacteria and the post-growth solid preparation of the Ackermanella bacteria can improve the glucose tolerance of mice, and the difference has statistical significance, but the fresh live Ackermanella bacteria and the post-growth solid preparation of the Ackermanella bacteria have no statistical difference, and the result is shown in figure 4.
2.3 post-growth solid preparation of Ackermanobacter for improving insulin sensitivity of mice
Three groups of mice were tested weekly for insulin sensitivity (ITT): after fasting for 6 hours, 100. Mu.l of insulin solution (0.55U/kg) was intraperitoneally injected per 10g of body weight, blood glucose levels were measured at 0 min, 15 min, 30 min, 60 min and 120 min, respectively, and plotted with time as abscissa and blood glucose level as ordinate, and the area under the curve was calculated, using a weighted statistical method between the different experimental groups. The results show that: compared with a control group, the fresh Ackermanella live bacteria and the post-growth solid preparation of the Ackermanella can improve the insulin sensitivity of mice, and the difference has statistical significance, and the result is shown in figure 5.
2.4 therapeutic effect of post-growth solid preparation of Ackermansia on mice is similar to that of fresh Ackermansia
In the tests of body weight, glucose endurance and insulin sensitivity, the hind-born solid preparation of Ackermanella and the fresh live Ackermanella are found to have no obvious difference in the test of the metabolic indexes of mice, and the metabolic indexes can be improved, as shown in figure 6 and figure 7. In addition, the Ackermann metazoan solid preparation can be stored for at least 3 months under aerobic conditions at room temperature, and no mildew or deterioration is found in the preparation.
It should be understood that the above examples are only for illustrating the technical solutions of the present invention, and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes or modifications based on the present invention may be made by those skilled in the art after reading the present disclosure, and the equivalents of these changes or modifications may fall within the scope of the invention defined by the appended claims.
Claims (10)
1. A preparation method of an Ackermanella metazoa solid preparation is characterized by comprising the following steps:
the method comprises the following steps: inoculating the Ackermanomyces lactis original bacterial liquid to a culture medium to obtain a liquid A;
step two: placing the solution A in an anaerobic incubator for culture, centrifuging the prepared culture medium containing Ackermanella, removing the supernatant to obtain A bacteria mud, placing the obtained A bacteria mud in a centrifuge tube, adding a PBS solution containing 20% of glycerol, and resuspending into a suspension called as solution B;
step three: inactivating the solution B at 65-70 deg.C for 20-30 min, and cooling to obtain solution C;
step four: adding the cryoprotectant into the solution C, and thoroughly mixing to obtain solution D;
step five: placing the solution D in a culture dish, uniformly shaking, and then placing in a refrigerator for freezing to obtain solution E;
step six: and putting the E liquid into a freeze dryer to obtain the post-growth solid preparation of the Ackermansia.
2. The method of claim 1, wherein:
in the second step, the solution A is placed in an anaerobic incubator for 48 hours and is taken out when OD600 is more than 0.8; setting the rotating speed of a centrifugal machine at 6000 rpm, centrifuging the prepared culture medium containing the Ackermanomyces, and removing the supernatant to obtain A bacteria mud.
3. The production method according to claim 1, characterized in that:
in the fourth step, the cryoprotectant is 20% of fat milk powder, 5% of glycerol and 75% of PBS solution by mass percentage.
4. The production method according to claim 1, characterized in that:
and in the fifth step, the solution D is placed into a refrigerator at the temperature of-80 ℃ for freezing for 30 minutes to obtain the solution E.
5. The production method according to claim 1, characterized in that:
in the sixth step, the program of the freeze dryer is set as follows: the operation is carried out for 48-60 hours at-40 ℃ to-50 ℃ and at a pressure of 1pa to 10pa, and then the temperature is raised to 20 ℃ for 2-5 hours.
6. The method of claim 1, wherein: the akkermansia is akkermansia muciniphila atccbaa-835 and/or a sub-strain thereof.
7. The use of the Ackermansia post-growth solid preparation obtained by the preparation method according to claim 1 in the preparation of a medicament or health product for preventing or treating metabolic diseases.
8. The use of claim 7, wherein: the drug or health care product of the Ackermanella anabiotic solid preparation is prepared into a pharmaceutically acceptable dosage form.
9. The use of claim 7, wherein: the drug or health product of the Ackermanella anabiotic solid preparation also comprises pharmaceutically acceptable auxiliary materials.
10. The use of claim 7, wherein: the administration mode of the drug or health product of the Acermanium aphanidermatum solid preparation comprises oral administration, buccal administration, external application and/or smearing.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021203083A2 (en) * | 2020-04-02 | 2021-10-07 | Dupont Nutrition Biosciences Aps | Compositions for metabolic health |
| WO2022096501A1 (en) * | 2020-11-09 | 2022-05-12 | A-Mansia Biotech | Composition comprising akkermansia muciniphila and green tea extract |
| CN114569638A (en) * | 2022-03-03 | 2022-06-03 | 中国农业大学 | Ackermanella muciniphila microecological preparation and application thereof to laying hens |
| CN115463159A (en) * | 2022-09-26 | 2022-12-13 | 暨南大学附属第一医院（广州华侨医院） | Application of Acermanium and Acermanium postbiotic in preparation of medicine or health-care product for preventing or treating hepatic steatosis |
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- 2022-12-16 CN CN202211637261.5A patent/CN115927109A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021203083A2 (en) * | 2020-04-02 | 2021-10-07 | Dupont Nutrition Biosciences Aps | Compositions for metabolic health |
| WO2022096501A1 (en) * | 2020-11-09 | 2022-05-12 | A-Mansia Biotech | Composition comprising akkermansia muciniphila and green tea extract |
| CN114569638A (en) * | 2022-03-03 | 2022-06-03 | 中国农业大学 | Ackermanella muciniphila microecological preparation and application thereof to laying hens |
| CN115463159A (en) * | 2022-09-26 | 2022-12-13 | 暨南大学附属第一医院（广州华侨医院） | Application of Acermanium and Acermanium postbiotic in preparation of medicine or health-care product for preventing or treating hepatic steatosis |
Non-Patent Citations (3)
| Title |
|---|
| EFSA NDA PANEL等: "Safety of pasteurised Akkermansia muciniphila as a novel food pursuant to Regulation (EU) 2015/2283", 《EFSA JOURNAL》, vol. 19, no. 9, pages 217 - 5 * |
| XIAO-QI LIN等: "Akkermansia muciniphila Suppresses High-Fat Diet-Induced Obesity and Related Metabolic Disorders in Beagles", 《MOLECULES》, vol. 27 * |
| 李娜: "预防比格犬肥胖的嗜黏蛋白阿克曼菌微胶囊的研制", 《中国优秀硕士学位论文全文数据库》, no. 08 * |
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