Specific screening medium for Akkermansia muciniphila and preparation method and application thereof

A screening medium and specific technology, applied in the field of microorganisms, can solve problems such as inability to use screening, instability, and difficulty in culturing

Active Publication Date: 2019-02-19
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] However, the above-mentioned separation method has many defects in the actual operation process: first, the above-mentioned separation method requires that all operating processes be carried out under anaerobic, nitrogen-enriched environment and the medium used in the above-mentioned separation method requires gas replacement and deoxygenation in advance and all Oxygen cannot be mixed in the operation process, and most laboratories (especially domestic laboratories) are only equipped with anaerobic workstations, and do not have the device for gas replacement and deoxygenation in the early stage. The method of boiling caused certain difficulties to the cultivation of Akkermansia muciniphila; secondly, the liquid enrichment medium prepared in the above separation method was required to be clear and transparent to judge whether the enrichment tube had grown. However, due to the Willem deVos team and Professor Peng Yongzheng The teams only disclosed the formula of the enrichment medium they used, but did not disclose the preparation method of the enrichment medium. However, the inventor found in actual operation that the enrichment medium prepared according to the conventional solution preparation method was in a turbid state. , cannot be used for the screening of Akkermansia muciniphila at all; again, the enrichment medium used in the above separation method is formulated with NaHCO 3 is a buffer solution, while NaHCO 3 In the process of high-temperature and high-pressure sterilization, it is unstable, and it is easy to make the medium alkaline, and the metal cations in the medium such as Ca 2+ , Mg 2+ Precipitation occurs and a turbid solution is formed; finally, the Noble Agar used in the above separation method is expensive and is not suitable for large-scale separation and screening; and, in order to achieve the purpose of deep anaerobic culture, when the above separation method uses separation medium for separation culture , the pouring method is used, the pouring method will make the colonies growing inside the solid medium small, and there is no typical colony shape, which is very inconvenient for later screening and is not suitable for the screening and isolation of strains

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  • Specific screening medium for Akkermansia muciniphila and preparation method and application thereof
  • Specific screening medium for Akkermansia muciniphila and preparation method and application thereof
  • Specific screening medium for Akkermansia muciniphila and preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0070] Embodiment 1: the configuration of enrichment medium

[0071] Although the Willem de Vos team published the composition of the Akkermansia muciniphila enrichment medium, the configuration method was not mentioned in detail. When the inventors configured the components according to the literature reports and the conventional method, the enrichment medium was turbid. However, the liquid tube enrichment culture finally needs to observe the turbidity of the culture medium to determine whether the target strain is enriched, so the turbidity of the medium will make subsequent experiments impossible. Therefore, the composition and configuration method of the Akkermansiamuciniphila enrichment medium must Make further improvements.

[0072] Due to the complex composition of the medium, the configuration is cumbersome. In order to simplify the configuration of the medium, the different medium components are firstly configured into a high-concentration stock solution, which is con...

Embodiment 2

[0121] Embodiment 2: the configuration of separation medium and the construction of screening environment

[0122] Since the growth of Akkermansia muciniphila requires a lower redox potential and does not grow under aerobic conditions, the Willem de Vos team requires all operations to be carried out in an anaerobic, nitrogen-filled environment and requires the medium used to require gas in advance Replacement deoxygenation and all operations can not be mixed with oxygen, and the separation method adopted by the Willem de Vos team is also the pouring method. The separation principle is that the bacteria can grow in a relatively high anaerobic environment inside the medium by using the deep pouring method However, since most laboratories (especially domestic laboratories) are only equipped with anaerobic workstations, they do not have the device for gas replacement and deoxygenation in the early stage, and the pouring method will make the colonies growing in the solid medium smal...

Embodiment 3

[0132] Example 3: Screening of Akkermansia muciniphila in stool samples

[0133] Specific steps are as follows:

[0134] 1. Source of materials

[0135] 7 feces samples from Haozhou, Anhui. The samples were collected on July 31, 2017. The samples were mixed with sterile 30% glycerol, dispensed into sterile tubes by aseptic operation, and stored at -80°C for later use. The sample numbers were respectively For FAHBZ-9, FAHBZ-12, FAHBZ-19, FAHBZ-26, FAHBZ-28, FAHBZ-30, FAHBZ-31, FAHBZ-39.

[0136] 2. Sample dilution and enrichment culture

[0137] Take the FAHBZ-12 sample as an example: draw the sample in a 200μL glycerol tube, add 4.5mL PBS, and record it as 10 -1 ; diluted to 10 -2 When the turbidity is not visible to the naked eye; continue to dilute 3 gradients downwards and record it as 10 -3 、10 -4 、10 -5 ; from 10 -2 Draw 500 μ L in the dilution tube, add in the rich culture medium that 4.5mL embodiment 1 obtains; Dilute 10 -3 ~10 -5 Inoculate the enrichment medi...

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Abstract

The invention discloses a specific screening medium for Akkermansia muciniphila and a preparation method and application thereof, and belongs to the technical field of microorganism. The medium comprises an enrichment medium, a separation medium and a primary screening medium. The enrichment medium contains mucin as the sole carbon source; the separation medium comprises brain-heart extract brothor brain-heart extract broth and mucin; The primary screening medium comprises brain-heart extract broth or mucin and brain-heart extract broth; by using the specific screening medium for screening the Akkermansia muciniphila in excrement, and the positive rate of the Akkermansia miniphila can be as high as 70%.

Description

technical field [0001] The invention relates to a specific screening medium for Akkermansia and its preparation method and application, belonging to the technical field of microorganisms. Background technique [0002] Akkermansia muciniphila is one of the human intestinal bacteria, and its abundance in the human intestinal tract is low, generally less than 3%. It belongs to the genus Akkermansia, verrucomicrobial phylum, Gram-negative strict anaerobic bacteria, no plasmid; its growth requires a low redox potential, does not grow under aerobic conditions; and it can intestinal goblet cells The secreted mucin serves as the sole carbon source, nitrogen source and energy substance. [0003] At present, studies have shown that the abundance of Akkermansia muciniphila in the intestine is related to various physiological and pathological states, and the abundance of Akkermansia muciniphila in the intestines of people with obesity, diabetes and enteritis will be significantly reduc...

Claims

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Application Information

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IPC IPC(8): C12Q1/04
CPCC12Q1/045
Inventor翟齐啸陈卫冯赛赛田丰伟陆文伟赵建新张灏
OwnerJIANGNAN UNIV