CN115287233A

CN115287233A - Ackermanella composite culture medium, and powder prepared from same and application of Ackermanella composite culture medium

Info

Publication number: CN115287233A
Application number: CN202210959021.0A
Authority: CN
Inventors: 贾世奇; 马启明; 余国兴
Original assignee: First Affiliated Hospital of Jinan University
Current assignee: First Affiliated Hospital of Jinan University
Priority date: 2022-08-10
Filing date: 2022-08-10
Publication date: 2022-11-04

Abstract

The invention provides a composite culture medium of Ackermanella, which comprises the following components in percentage by mass: soybean peptone 0.5% -3%; 0.2 to 1 percent of tryptone; 1-3% of brain and heart leaching powder; 0.1 to 3 percent of glucose; 0.2 to 0.3 percent of sodium chloride; 0.1 to 0.2 percent of disodium hydrogen phosphate; 0.4% -0.7% of N-acetyl glucosamine; 0.3 to 0.5 percent of L-threonine; 0.004-0.006% of L-cysteine salt. The invention provides an Ackermanella composite culture medium and powder thereof, which can well support the growth of the Ackermanella and can be produced and popularized in a large scale. The powder is favorable for storage and transportation, the preparation procedure at the use end is simplified, the powder can be directly used for culturing Ackermanella after being filtered by only adding ultrapure water, the complicated preparation process and high-pressure sterilization are not needed, the experimental efficiency is improved, and the popularization range is enlarged. The invention also provides a simple, quick and efficient culture medium preparation method for large-scale culture of Ackermanella.

Description

Ackermanella composite culture medium, and powder prepared from same and application of Ackermanella composite culture medium

Technical Field

The invention relates to the field of culture media, in particular to a composite culture medium of Ackermansia and a preparation method and application of powder thereof.

Background

Obesity and metabolic syndrome have become serious problems threatening the quality of a person's healthy life.

Ackermansia is the new generation of intestinal beneficial bacterium of star with the most development potential. Recently, the research results published in the journal of PNAS, nature Medicine and the like show that Ackermanum or Pasteurella multocida have the effects of remarkably improving glycolipid metabolism and treating metabolic syndromes such as obesity, diabetes, dyslipidemia and the like. As a potential probiotic, akkermansia feeds on mucin, and has a good growth rate and condition in a mucin-containing medium. However, a number of ethical issues may be involved in the promotion of akkermansia from mucin cultures. Therefore, the German research team develops a set of culture medium without using mucin aiming at the characteristics of Ackermanella, which is called Ackermanella synthetic culture medium, the culture medium is prepared by more than ten chemical substances, and relates to a plurality of components with complex component ratios, which brings a plurality of obstacles for the popularization and the commercial application of the Ackermanella.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide a preparation method and application of a composite culture medium of Ackermanella and powder thereof, which can be directly used for culturing the Ackermanella and is particularly suitable for large-scale Ackermanella culture.

The purpose of the invention is realized by the following technical scheme:

one of the purposes of the invention is to provide a compound culture medium of Ackermanella, which comprises the following components by mass percent:

soybean peptone 0.5-3%

Tryptone 0.2-1%

1 to 3 percent of the brain and heart leaching powder

0.1 to 3 percent of glucose

0.2 to 0.3 percent of sodium chloride

0.1 to 0.2 percent of disodium hydrogen phosphate

0.4 to 0.7 percent of N-acetyl glucosamine

0.3 to 0.5 percent of L-threonine

0.004-0.006% of L-cysteine salt.

Another object of the present invention is to provide a powder prepared from the complex culture medium of Acermanium species as described above.

The invention also aims to provide a compound culture medium of Ackermansia and application of the prepared powder thereof in culturing the Ackermansia.

The invention also aims to provide a compound culture medium of Ackermansia and application of the prepared powder in prebiotic preparations or components.

The invention also aims to provide the application of the composite culture medium of Ackermanella and the prepared powder thereof in products or components for beautifying, losing weight or preventing and treating metabolic diseases.

The invention also aims to provide a composite culture medium of Ackermansia and application of the prepared powder in intestinal flora conditioning products or components.

The invention also aims to provide a using method of the powder prepared from the Ackermansia composite culture medium, wherein the powder is added with ultrapure water and is filtered and sterilized to obtain the Ackermansia composite culture medium.

Further, the pore diameter of the filtration pore is 0.22-0.45 micron.

The invention has the outstanding effects that:

the invention provides an Ackermanella composite culture medium and powder thereof, which can well support the growth of the Ackermanella and can be produced and popularized in a large scale. The powder is favorable for storage and transportation, the preparation procedure at the use end is simplified, the powder can be directly used for culturing Ackermanella directly after being added with pure water and filtered, the complicated preparation process and high-pressure sterilization are not needed, the experimental efficiency is improved, and the popularization range is also enlarged. The invention also provides a simple, quick and efficient culture medium preparation method for large-scale culture of Ackermanella.

The following embodiments are provided to further explain the present invention in order to make the technical solutions of the present invention easier to understand and grasp.

Drawings

FIG. 1 is a graph showing the efficiency of culture of Ackermanella in the culture media of examples of the present invention and comparative examples;

FIG. 2 is a graph showing the effects of Ackermanella cultured in a medium according to an embodiment of the present invention;

FIG. 3 is a diagram of the results of the comparison of NCBI websites according to the embodiment of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.

Examples

1. Preparation of composite culture medium of Acermanium

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 put into 1L of ultrapure water, simply shaken and uniformly mixed until no visible substances exist, and then filtered by using a 0.22 micron sterilization filter screen to obtain the compound culture medium which can be directly used for culturing Acermann bacteria, wherein the solution is clear and free of impurities and belongs to a clean sterile solution.

2. The high efficiency of the composite culture medium powder is proved by the culture of Acermanium

2ml of Ackermanella in logarithmic phase of propagation (OD 600= 0.8) were added to 48ml of the mucin medium and 48ml of the medium of this example (filtered), respectively, and cultured under the same conditions in an anaerobic chamber for 48 hours. In addition, two media were provided with corresponding controls (mucin media was controlled with 50ml of blank media; this example was controlled with 50ml of media). The OD600 absorbance values in the culture medium were measured at 12h, 24h, 36h and 48h, respectively. Three biological replicates were set for each group above.

As a result:

setting the OD600 light absorption detection value of the mucin blank culture medium as the blank OD-0h, OD-12h, OD-24h, OD-36h and OD-48h; similarly, the OD600 absorbances of the mucin culture medium are respectively viscosity OD-0h, OD-12h, OD-24h, OD-36h and OD-48h; the culture medium of the embodiment is OD-0h, OD-12h, OD-24h, OD-36h and OD-48h; the culture medium of the embodiment is OD-0h, OD-12h, OD-24h, OD-36h and OD-48h; the results are interpreted as the presence of bacteria at the same time minus the blank medium, expressed as d values. The above results show that: for the growth rate of bacteria, the growth rate of the medium of this example was better than that of the mucin medium at 12h, 24h, 36h and 48h after the transfer of the same amount of bacteria (see Table 1). The time was taken as the abscissa and the OD difference (d) as the ordinate to perform two-dimensional graph analysis, and the results are shown in fig. 1.

TABLE 1 comparison of growth rates of mucin Medium versus Medium of this example

Time	OD (mucin-mucinous blank)	OD (this example-blank)	P(T-Test)
				0h	0.011±0.003	0.009±0.002	0.277
12h	0.193±0.017	0.288±0.015	0.002
				24h	0.416±0.019	0.494±0.009	0.003
36h	0.593±0.041	0.680±0.013	0.024
				48h	0.653±0.040	0.803±0.015	0.003

3. The purity of the medium of this example was confirmed by the purity of the bacterial culture

The culture medium powder of the embodiment is prepared, ultrapure water is added into the culture medium powder, the culture medium powder is balanced into 1L, 25ml of Ackermanella stock solution is added after the culture medium powder is filtered by a 0.22 micron sieve, and after 48 hours, part of the bacterial culture solution is taken out and diluted to 10^ -5 for solid culture dish plate coating counting. The results show that: ackermanella anatipestifer 1ml after 48h culture contained 4X 10^8 bacteria and was free of undesired bacteria (see FIG. 2). 16s second generation sequencing was performed by extracting bacteria from the dishes (27F primer sequence 5'-3': AGA GTT TGA TCC TGG CTC AG;1492R primer sequence 5'-3': TAC GGC TAC CTT GTT ACG ACTT; length was about 1400 bp), and the alignment was performed by NCBI website, wherein the alignment rate was 100% with Ackermanella tabescens 835 sub-strain, and the results confirmed that all bacteria in the dishes were Ackermanella (see Table 2, FIG. 3). The culture medium powder of the embodiment can be used for subsequent bacterial culture only by simple filtration, does not need high-pressure sterilization, is simple and convenient to operate, and is convenient and fast to use by researchers without high-pressure equipment.

TABLE 2.16S sequencing bands results

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered as the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.

Claims

1. A composite culture medium of Ackermanella is characterized in that: the composite material comprises the following components in percentage by mass:

soybean peptone 0.5-3%

Tryptone 0.2-1%

1 to 3 percent of the brain heart leaching powder

0.1 to 3 percent of glucose

0.2 to 0.3 percent of sodium chloride

0.1 to 0.2 percent of disodium hydrogen phosphate

N-acetylglucosamine 0.4% -0.7%

0.3 to 0.5 percent of L-threonine

0.004-0.006% of L-cysteine salt.

2. A powder made from the complex medium of akkermansia species of claim 1.

3. A complex culture medium of akkermansia as claimed in claims 1-2 and the use of the prepared powder for culturing akkermansia.

4. Use of a complex culture medium of akkermansia sp as defined in claims 1-2 and the powders prepared thereof in prebiotic formulations or compositions.

5. Use of the complex culture medium of akkermansia sp as defined in claims 1-2 and the powders prepared therefrom in cosmetic, weight-loss or metabolic disease control products or compositions.

6. A complex culture medium of akkermansia sp as claimed in claims 1-2 and the use of the powder prepared thereof in an intestinal flora conditioning preparation or component.

7. A method of using a powder made of the complex medium of akkermansia according to claim 2, characterized in that: adding ultrapure water into the powder, and filtering and sterilizing to obtain the compound culture medium of the Acermanium bacteria.

8. The method of using a powder made of a complex medium of akkermansia as claimed in claim 7, characterized in that: the aperture of the filtration pore is 0.22-0.45 micron.