Development Of The In-vitro Human Gastrointestinal Microbial Ecosystem And Its Stability Evaluation

In recent years, the food and drug safety, nutrients digestion and absorption were greatly concerned by public. In-vitro digestion model was largely used in food nutrients absorption,drug metabolism, biological pollutants accessibility analysis and food intake. However, the current in-vitro digestion systems have the limitation of using only stomach and small intestine,and using animal model such as mice and rats, which cannot mimic the human gut micrtoflora,and with high cost, low reproduction rate, long life cycle and ethical issues. Therefore it is necessary to establish and validate a set of flexible, accurate and repeatable in-vitro digestion model which adapt oriental eating habits and gastrointestinal micro-ecology. This system will be used in nutrition, toxicology, microbiology, and other research area. This study aimed to develop a human gastrointestinal tract microecological digestive simulation system(SmartGut,SG) and the stability of the system was evaluated:1. Development and design of the SG system. SG system model was established based on the human intestinal micro ecology system simulator(SHIME) designed by the University of Ghent in Belgium, combined with oriental eating habits and auto-controller. SG system model mainly include: system controller, simulation of the digestive system, mixing system, time controller, speed control system, pneumatic control system, temperature control system, pH feedback regulation system, material liquid reagent and digestive juices reagent bottles.2. The SG system has the function to control: system time, speed and other parameters setting, engraftment inoculation fluid preparation, intestinal microecological engraftment,digestive juices, nutrient solution preparation, digestion simulation as well as the determination of the stability parameters.3. The feasibility and stability of SG system were assessed, mainly includes 4 parts of the system parameters. Results are as follows:(1) Using two aged 20 to 35 year-old healthy adults’ feces to prepare inoculation fluids using shearing and vortex methods followed by-80oC storage. Bacterial culture counting and denaturing gel electrophoresis(PCR-DGGE) technology were used to assess the microflora changes during the storage. Results showed that bacteria such as lactic acid bacteria, the totalaerobic bacteria count,the total anaerobic bacteria count and DGGE profiles are stable during the storage time, indicating that prepared inoculation fluids are in a stable state in the process of-80℃ storage. Among them, adultⅠhas higher amounts of microbial yield which is suitable for SG system’s inoculation fluids. Moreover, inoculation fluid prepared by shear method was better than using vortex method.(2) During the SG system running cycle, bactrial counting was determined in AC, TC and DC at 6:00 and 12:00 every day. The microflora in AC, TC and DC fluctuated until 7d. Since 7d,the number of each bacteria in the different stages of the large intestine maintained a stable state,indicating that SG system need at least 7 days to get a standard microflora.(3) PCR-DGGE was used to assess the microflora stability in AC, TC and DC at 6:00 and12:00 every day during the SG system running cycle and get the following conclusion: The microflora deiversity in AC, TC and DC fluctuated until 7d. From 7d it goes into the plateau, the most stable microecological structure which proved that the system achieve stability. Among 3different colon distances, the descending colon has more complex microbial diversity,comparing with transverse colon and ascending colon.(4) By using gas chromatography-hydrogen flame ionization detection(GC-FID)technology, the short chain fatty acid(SCFAs) in AC, TC and DC were analyzed, and the conclusions are:SG system constantly produced SCFAs, inculding acetic acid, propionic acid, butyric acid and valeric acid. Among them, acetic acid content was the highest(86.40 μg/mL), followed by propionic acid and butyric acid, and pentanoic acid content is the lowest(10.21 μg/mL).In system operation cycle, SCFAs content in AC, TC and DC fluctuated until 7d. Since 7d,total SCFA and basic content of each short chain fatty acids were in a stable state, slightly fluctuations within a certain range.At the different samLing time(6:00 or 12:00), total SCFA and each short chain fatty acids detected at 12:00 in the AC, TC, DC were significantly higher than 6:00 samples. It indicated that after overnight operation without nutrient feeding, the microflora in the system has metabolic consumpted the SCFAs, therefore at 6:00 the total SCFA and short chain fatty acidswas obviously lower than that of at 12:00.In summary, this study has developed the human gastrointestinal tract microecological digestion system(SmartGut, SG) and evaluated its feasibility and stability. SG system can mimic the oriental eating habits and simulate human gut microecological digestive system.Therefore it can provide preliminary theoretical basis and has apply value in gut microflora research.