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Excrement flora in-vitro culture method and culture medium

A technology for in vitro culture and fecal flora, applied in biochemical equipment and methods, microorganisms, microorganisms, etc., can solve problems such as Clostridium difficile infection, affect the reliability of results, and complex operations, and achieve the speed of bacterial flora reproduction. Fast, precise control of operating conditions, reproducible results

Active Publication Date: 2016-08-03
济南万泉生物技术有限公司
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

At present, fecal microbiota transplantation has been regarded as a special treatment method for Clostridium difficile infection, inflammatory bowel disease, irritable bowel syndrome, autoimmune enteropathy, intestinal food allergy and other diseases, However, due to the extremely strict screening of feces donors, the feces need to be collected and used immediately, which greatly limits the application of this technology.
The Chinese invention patent with the application number 201510619640.5 discloses the application of artificially cultured fecal flora transplantation in the treatment of intestinal diseases. The invention isolates a single intestinal flora, and through fecal bacteria transplantation, it realizes the adjustment and restoration of human intestinal tract. The purpose of the environment and the composition of the flora, but the flora only achieves the isolation and cultivation of a single flora, and there is no research on other beneficial flora to the human body in the feces; the Chinese invention patent application number 200810048065.8 discloses a human intestine System and method for continuous cultivation of Tao flora. This invention focuses on the changes of dominant flora and metabolites (mainly short-chain fatty acids) before and after cultivation. However, from the published instructions and claims, the process is relatively rough. , complex operation, medium and fermentation conditions are only suitable for the growth of several specific dominant bacteria
Although it also analyzed the flora structure before and after fermentation, due to the limitation of technical methods at that time, the inventor only used the traditional method of counting bacterial cultures to compare the limited number of cultivable dominant bacteria in the intestinal tract. More than 80% of the bacteria in the tract can not be purely cultured in vitro so far, which affects the reliability of the results to a certain extent

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  • Excrement flora in-vitro culture method and culture medium

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Embodiment 1

[0030] In the in vitro medium of the present embodiment, every 1000 milliliters of medium contains 2 grams of peptone, 1 gram of yeast powder, 2 grams of beef extract, 0.5 grams of glucose, 0.5 grams of cysteine, 0.5 grams of bile salts, and 1 gram of cellobiose. gram, fructose 1 gram, magnesium sulfate 0.02 gram, potassium dihydrogen phosphate 2 gram, vitamin K 0.002 gram, ferrous sulfate 0.001 gram, heme 0.003 gram, 1MCaCl 2 The solution is 0.3 ml, and the balance is distilled water; the pH of the medium is adjusted to 6 with 6M sodium hydroxide solution or hydrochloric acid.

[0031] The in vitro culture method of this specific embodiment comprises the following steps:

[0032] 1. Preparation of original fecal flora:

[0033] (1) Weigh 100g of normal saline, add 0.05g of cysteine, after the cysteine ​​is completely dissolved, filter through a 0.22μm filter membrane, and the filtrate is the eluent;

[0034] (2) Weigh 10g of fresh feces from healthy people, add 20ml of elue...

Embodiment 2

[0041] In the in vitro medium of the present embodiment, every 1000 milliliters of medium contains 3 grams of peptone, 2 grams of yeast powder, 4 grams of beef extract, 1 gram of glucose, 1 gram of cysteine, 2 grams of bile salts, and 2 grams of cellobiose. gram, fructose 2 grams, magnesium sulfate 0.04 grams, potassium dihydrogen phosphate 3 grams, vitamin K 0.004 grams, ferrous sulfate 0.002 grams, heme 0.005 grams, 1MCaCl2 The solution is 0.5 ml, and the balance is water; the pH of the medium is adjusted to 8 with 6M sodium hydroxide solution or hydrochloric acid.

[0042] The in vitro culture method of this specific embodiment comprises the following steps:

[0043] 1. Preparation of original fecal flora:

[0044] (1) Weigh 100g of normal saline, add 0.1g of sodium hydrosulfite, after the sodium hydrosulfite is completely dissolved, filter through a 0.22μm filter membrane, and the filtrate is the eluent;

[0045] (2) Weigh 10g of fresh feces from healthy people, add 100ml...

Embodiment 3

[0052] In the in vitro medium of the present embodiment, every 1000 milliliters of medium contains 2.5 grams of peptone, 1.5 grams of yeast powder, 3 grams of beef extract, 0.8 grams of glucose, 0.8 grams of cysteine, 1.2 grams of bile salts, and 1.5 grams of cellobiose. grams, fructose 1.5 grams, magnesium sulfate 0.03 grams, potassium dihydrogen phosphate 2.5 grams, vitamin K 0.003 grams, ferrous sulfate 0.001 grams, heme 0.004 grams, 1MCaCl 2 The solution is 0.4 ml, and the balance is distilled water; the pH of the medium is adjusted to 8 with 6M sodium hydroxide solution or hydrochloric acid.

[0053] The in vitro culture method of this specific embodiment comprises the following steps:

[0054] 1. Preparation of original fecal flora:

[0055] (1) Weigh 100g of normal saline, add 0.08g of cysteine, after the cysteine ​​is completely dissolved, filter through a 0.22μm filter membrane, and the filtrate is the eluent;

[0056] (2) Weigh 10g of fresh feces from healthy peopl...

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Abstract

The invention belongs to the technical field of excrement flora transplantation, and particularly relates to an excrement flora in-vitro culture method and a culture medium .The pH of the culture medium is 6.0-8.0 .The culture medium is prepared from peptone, yeast powder, a beef extract, glucose, cysteine, cholate, cellose, fructose, magnesium sulfate, monopotassium phosphate, vitamin K, ferrous sulfate, heme, a 1M CaCl2 solution and distilled water .The composition and the contents of the components of the culture medium determine that the content of probiotics, the proportion of bacillus and cocci, the proportion of gram-positive and negative bacteria and the like in flora obtained through culture are consistent with those of original flora, and by adopting the culture medium with the components and the ratio, flora consistent with fresh excrement flora in proportion can be well cultured .The adopted excrement flora in-vitro culture method is simple, the operation conditions can be accurately controlled, and the excrement flora prepared through the method is stable in proportion, good in repeatability, and suitable for industrial production .

Description

technical field [0001] The invention belongs to the technical field of fecal bacteria transplantation, and in particular relates to an external culture method and culture medium of fecal bacteria. Background technique [0002] There are about 1000 to 1050 kinds of microorganisms living in the human gastrointestinal tract, with a quantity of 10 trillion. These microorganisms are called intestinal flora. The normal intestinal microorganisms of the human body can synthesize a variety of vitamins necessary for human growth and development, participate in the growth and differentiation of the intestinal epithelium and the decomposition of toxic and harmful substances, form a defense barrier and participate in the immune process. Diet structure, stress and antibiotic use and other factors that change the internal environment of the human body can lead to an imbalance of intestinal flora, which in turn can cause a variety of diseases, including a variety of gastrointestinal disease...

Claims

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Application Information

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IPC IPC(8): C12N1/00
CPCC12N1/00
Inventor 张海燕李理想戴晓宇李栋
Owner 济南万泉生物技术有限公司
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