CN-120866154-B - Fermentation method and application of human intestinal flora

CN120866154BCN 120866154 BCN120866154 BCN 120866154BCN-120866154-B

Abstract

The invention relates to a fermentation method and application of human intestinal flora, and relates to the field of microbial fermentation, comprising the following steps of screening a donor, namely, a 12-14 year old boy, healthy, free of major disease history, free of obvious gastrointestinal symptoms, free of any antibiotics within at least 6 months and free of living rules; the method comprises the steps of collecting and processing a sample, quickly transferring fresh feces of a donor to an anaerobic workstation, weighing, adding pre-reduced sterile normal saline according to a proportion, fully and uniformly vortex, filtering, collecting a feces suspension, fermenting, transferring the feces suspension to a culture medium, transferring the culture medium into a multi-layer movable coaxial cylindrical ring anaerobic fermentation tank, sealing and fermenting, and simulating physical structure, chemical environment and physiological activity of human intestinal tracts by constructing a dynamic bionic fermentation system, so that the in-vitro planting efficiency, metabolic activity and yield and stability of functional products of intestinal flora are obviously improved.

Inventors

ZHOU SHUHAN
GOU JUNBO
ZHANG YANG
LIU ZHIYI
Qin Lingxi
ZENG YAXIN

Assignees

湖北中医药大学
湖北时珍实验室

Dates

Publication Date: 20260512
Application Date: 20250801

Claims (3)

1. A method of fermenting a human intestinal flora, comprising the steps of: a. Screening the donor, namely, 12-14 years old boys, healthy body, no history of major diseases, no obvious gastrointestinal symptoms, no antibiotics taken within at least 6 months and no living rule; b. Sample collection and treatment, namely collecting fresh feces of a donor, rapidly transferring to an anaerobic workstation, weighing, adding pre-reduced sterile normal saline according to a proportion, fully and uniformly vortex mixing, filtering, and collecting feces suspension; c. Fermentation, namely transferring the fecal suspension to a culture medium, transferring the culture medium into a multilayer movable coaxial cylindrical ring anaerobic fermentation tank, and sealing and fermenting; The fermentation tank is made of stainless steel, the diameter of the tank is 300 mm, a plurality of layers of coaxial cylindrical rings are arranged in the tank, the ring gap is 15mm, the surface of each cylindrical ring is provided with micro-ravines with the depth of 200-300 mu m and the width of 50 mu m, and the density of the ravines is 35 strips/mm < 2 >; the multilayer movable coaxial cylindrical ring is capable of displacement, including radial displacement, axial displacement and rotation, The radial displacement, the radial displacement generated by the telescopic cylinders A to D moving to the telescopic cylinders A to D of the fixed frame, the offset amplitude of 10-20 mu m, the frequency of 0.05 Hz, the axial displacement, the amplitude of 50-100 mu m, the frequency of 0.033 Hz, the rotating speed of 5-10 rpm, the rotating direction of 30 seconds forward/30 seconds reverse are in bidirectional alternation; Performing displacement operation according to preset time in the fermentation process, wherein the displacement operation comprises 0-20 seconds of radial displacement, 20-60 seconds of axial displacement, 60-180 seconds of bidirectional alternate rotation, and repeating the displacement operation when the concentration of butyric acid is detected to be less than 20 mM; the method also comprises a refreshing period operation, wherein every 72 hours or when the synthesis rate of the butyric acid continuously increases for 4 hours by <5%, the bidirectional alternate rotation of 25+/-1 rpm is started for 90 seconds, and the axial displacement with the amplitude of 120 mu m and the frequency of 0.1Hz is carried out simultaneously; performing displacement in stages after the update period is over: the activation period of the streamer is 0-30 minutes, namely radial displacement, amplitude is 15 mu m, and frequency is 0.05Hz; the nutrition conveying period is 30-90 minutes, namely, bidirectional alternate rotation is carried out, radial displacement is carried out, the rotation speed is 15+/-0.5 rpm, the amplitude of axial displacement is 80 mu m, and the frequency is 0.033Hz; the synergistic period of the gully bacteria is 90-180 minutes, the axial synergistic radial displacement is 100 mu m, the frequency is 0.1Hz, the radial displacement is 20 mu m, and the frequency is 0.05Hz.
2. The method of claim 1, wherein the medium in fermentation comprises peptone 10.0g/L, glucose 5.0g/L, disodium hydrogen phosphate 8.0g/L, potassium dihydrogen phosphate 2.0g/L, bovine bile salt 20.0g/L, brilliant green 0.015g/L, and prebiotics 1.0-2.0g/L; Wherein the prebiotic is at least one of inulin, fructo-oligosaccharide and beta-glucan.
3. The method for fermenting human intestinal flora according to claim 1, wherein the fermentation conditions are that the temperature is 37+/-0.5℃, pH 5.8.8-6.8, and the volume ratio of N 2 80%、CO 2 % to H 2 % is maintained by introducing gas.

Description

Fermentation method and application of human intestinal flora Technical Field The invention relates to the field of microbial fermentation, in particular to a fermentation method and application of human intestinal flora. Background In recent years, the role of intestinal microecology in human health and diseases has been widely studied and accepted, and particularly has important regulatory potential in metabolic syndrome, obesity, inflammatory bowel disease, diabetes, neuropsychiatric diseases and the like. Simulating human intestinal microenvironment and developing in vitro artificial intestinal system fermentation has become an important means for exploring microorganism metabolism mechanism, prebiotic function evaluation and personalized nutrition strategy development. However, the existing system for in-vitro external human intestinal flora fermentation has the problems of unstable anaerobic control, poor flora structure repeatability, low metabolite collection efficiency and the like, and is difficult to truly reflect the in-vivo flora ecological structure and the conversion process of functional products. Therefore, an anaerobic fermentation system with high stability and high repeatability, which can accurately regulate and control key parameters (such as temperature, pH and anaerobic degree) is developed so as to rebuild and dynamically maintain the micro-ecological environment similar to the intestinal tract of a human body, and has important practical significance and industrialization value for improving the research efficiency and accuracy of related functional products of the intestinal flora. Disclosure of Invention Aiming at the technical problems existing in the prior art, the invention provides a fermentation method and application of human intestinal flora, solves the problems of insufficient traditional fermentation microenvironment and unstable flora structure, simulates the physical structure, chemical environment and physiological activity of human intestinal by constructing a dynamic bionic fermentation system, improves the in-vitro field planting efficiency, metabolic activity and yield and stability of functional products of the intestinal flora, and can be widely used for prebiotic screening, microecological function evaluation, nutrition group study and personalized intervention product development. The technical scheme for solving the technical problems is as follows, the fermentation method of the human intestinal flora comprises the following steps: a. Screening the donor, namely, 12-14 years old boys, healthy body, no history of major diseases, no obvious gastrointestinal symptoms, no antibiotics taken within at least 6 months and no living rule; b. Sample collection and treatment, namely collecting fresh feces of a donor, rapidly transferring to an anaerobic workstation, weighing, adding pre-reduced sterile normal saline according to a proportion, fully and uniformly vortex mixing, filtering, and collecting feces suspension; c. And (3) fermenting, namely transferring the fecal suspension to a culture medium, and then transferring the culture medium into a multilayer movable coaxial cylindrical ring anaerobic fermentation tank for sealed fermentation. Further, the culture medium in fermentation comprises 10.0g/L of peptone, 5.0g/L of glucose, 8.0g/L of disodium hydrogen phosphate, 2.0g/L of monopotassium phosphate, 20.0g/L of ox gall salt, 0.015g/L of brilliant green and 1.0-2.0g/L of prebiotics; Wherein the prebiotic is at least one of inulin, fructo-oligosaccharide and beta-glucan. Further, the fermentation condition is that the temperature is 37+/-0.5℃, pH 5.8.8-6.8, and the volume proportion of N 280%、CO215%、H2% is maintained by introducing gas. Further, the tank body of the fermentation tank is made of stainless steel, the diameter of the tank body is 300 mm, a plurality of layers of coaxial cylindrical rings are arranged in the tank body, the ring gap is 15mm, the surface of each cylindrical ring is provided with micro-ravines with the depth of 200-300 mu m and the width of 50 mu m, and the density of the ravines is 35 strips/mm < 2 >. Further, the multi-layer movable coaxial cylindrical ring is capable of displacement, including radial displacement, axial displacement and rotation, Wherein, radial displacement is generated when the telescopic cylinder moves to telescopic cylinders A to D of the fixed frame, the offset amplitude is 10-20 mu m, and the frequency is 0.05 Hz; axial displacement, amplitude of 50-100 μm and frequency of 0.033 Hz; The rotation speed is 5-10 rpm, the rotation direction is bidirectional alternation, and 30 seconds is clockwise/30 seconds is anticlockwise. Further, the displacement operation is carried out according to the preset time in the fermentation process, wherein the radial displacement is 0-20 seconds; axial displacement for 20-60 seconds; 60-180 seconds, namely bidirectional alternate rotation; When the butyric acid concentration was