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A method for constructing an in vitro simulation model of Prevot enterotype

A technology of Prevo intestinal and in vitro simulation, applied in bacteria and other directions, can solve the problems of unclear physiological and biochemical conditions, difficult to determine Prevo intestinal simulation conditions, and inability to determine the decisive role of growth.

Active Publication Date: 2021-09-21
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the simulation of Prevotella enterotype has been in a bottleneck period. There are two key unknown factors in the simulation of Prevotia enterotype: one is that it is impossible to determine which key nutrients are effective for Prevotella in in vitro simulation. Second, the specific physiological and biochemical conditions are not clear when simulated in vitro
[0005] A literature search of the prior art found that in the literature (FanBetal,) the simulation of the rod-shaped enterotype by the VI medium is better, but it is difficult to determine the simulation conditions for the Prevot enterotype

Method used

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  • A method for constructing an in vitro simulation model of Prevot enterotype
  • A method for constructing an in vitro simulation model of Prevot enterotype
  • A method for constructing an in vitro simulation model of Prevot enterotype

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Experimental program
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Effect test

Embodiment 1

[0025] (1) Preparation of culture medium

[0026] The composition of the improved VI (VI-IMO) medium: isomaltooligosaccharide (IMO, polymerization degree 2-5, molecular weight 342-823) 8g / L, tryptone 3g / L, peptone 3g / L, yeast extract 4.5g / L, mucin 0.5g / L, 3# bile salt 0.4g / L, L-cysteine ​​hydrochloride 0.8g / L, heme 0.05g / L, 1mL / L Tween 80, NaCl 4.5g / L, KCl 2.5g / L, MgCl 2 ·6H 2 O 4.5g / L, CaCl 2 ·6H 2 O 0.2g / L, KH 2 PO 4 0.4g / L, trace elements 2ml / L, the solvent is distilled water, the pH value is 6.3-6.8, after sterilization, put the medium on the continuous fermentation device, turn on the magnetic stirring and let in nitrogen.

[0027] The concentration of trace elements is: MgSO 4 ·7H 2 O 3.0g / L, CaCl 2 2H 2 O 0.1g / L, MnCl 2 4H 2 O0.32g / L, FeSO 4 ·7H 2 O 0.1g / L, CoSO 4 ·7H 2O 0.18g / L, ZnSO 4 ·7H 2 O 0.18g / L, CuSO 4 ·5H 2 O0.01g / L, NiCl 2 ·6H 2 O 0.092g / L, the solvent is distilled water.

[0028] (2) Automatic outdoor simulation model device of intesti...

Embodiment 2

[0042] Same as Example 1, the fresh feces of YYS Prevot enteric volunteers were used as samples, and the flow rate of the culture medium was set to 330ml / 12h ( Figure 5 Medium yys-12h-d10), 330ml / 24h ( Figure 5 Medium yys-24h-d10), 330ml / 36h ( Figure 5 Medium yys-36h-d10), 330ml / 48h ( Figure 5 Middle yys-48h-d10), pH is 6.5, other is with embodiment 1, the result sees Figure 5 shown.

[0043] like Figure 5 As shown, the proportion of Prevotella in the 16S sequencing results of the feces stock solution yys-10% reached more than 80%, and IMO was used as the main sugar source, when the pH was set to 6.5, the flow speed was changed, and after 11 days of fermentation, 4 species The proportion of Prevotella in the simulation results of flow acceleration is very low, that is, Enterotype Prevotti cannot be simulated. It can also be seen from the simulation results of 12h wherein that when the IMO is used as the sugar source and the flow velocity is 12h, the Przewalski's ent...

Embodiment 3

[0045] Same as Example 1, using the fresh feces of CHH and YYS Prevot enteric volunteers, the medium flow rate is set to 330ml / 12h, when pH5.5, replace IMO with fructooligosaccharide (FOS) as sugar source, and ferment to On the 11th day, the fermentation broth was taken for 16SrRNA sequencing, and the results of bacterial flora sequencing can be found in Figure 6 shown.

[0046] Depend on Figure 6 It can be seen that when other conditions remain unchanged, when IMO is replaced by FOS, the proportion of Prevotella in the fermentation broth is low, and the simulation effect on Enteroplasma pratziformis is poor.

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Abstract

The invention discloses a method for constructing an in vitro simulation model of Prevot enterotype. The fresh feces of healthy volunteers with Prevot enterotype are diluted with PBS into a feces suspension by using a fully automatic in vitro simulation model device for intestinal flora. Then the feces suspension was inoculated into the improved VI medium, fermented and cultured at 37°C, pH 5.2-5.8, magnetic stirring speed 150rpm, and supplemented with medium at a rate of 330ml / 12h, to obtain Prevot enterotype in vitro Simulation model; the present invention finds the important nutritional conditions for simulating Prevot enterotype in vitro, and explores the physical and chemical conditions in the continuous fermentation process, which can increase the ratio of Prevotella in the fermentation broth to 40%-80%, At the same time, the correlation coefficient between the microbiota in the fermentation broth and the original stool sample reached about 80%, so that the Prevot enterotype could be stably simulated in vitro.

Description

(1) Technical field [0001] The invention relates to an in vitro method for simulating Prevot enterotype in human intestine, in particular to a method for constructing an in vitro simulation model of Prevot enterotype. (2) Background technology [0002] The human body contains tens of thousands of microbiota, and the microbiota that settle in the gut is regarded as an additional organ of the human body. They can produce some important factors necessary for the human body, and can also form a special metabolic pathway with the host. The small intestine is equivalent to a powerful anaerobic bioreactor. The utilization of fermentation substrates by microorganisms and intestinal epithelial cells and the exchange of materials and information between them are carried out in an orderly manner in this reactor. Microbes obtain energy by fermenting indigestible carbohydrates in the diet, and can also metabolize essential nutrients such as amino acids and vitamins for the host, and can ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20
CPCC12N1/20
Inventor 陈军奎王欣刘伟朱立颖皮雄娥费笛波李正鹏杨云生
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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