| Normally,the oxidative capacity and antioxidant capacity of the body are in a dynamic balance.However,a series of non-specific defense reactions will occur to resist and adapt to these stress after being stimulated by various external stresses.When the level of free radicals produced in the body's cells is higher than its antioxidant defense ability,excessive free radicals exist in tissues or cells,which will cause the imbalance of redox state,induce oxidative stress and lead to oxidative damage.Oxidative stress is one of the main causes of a wide range of human syndromes.Intestinal tract is the organ with the largest contact area with the outside world.It inhabits a large number of microorganisms and carries the main functions of food digestion,absorption and metabolism.Therefore,there is a very active metabolism of substances and energy in the intestine,which is vulnerable to the attack of oxygen free radicals.More seriously,oxidative stress slowly causes very serious intestinal damage.Therefore,maintaining redox balance is very important to maintain the balance of intestinal environment.It has been reported that probiotics can change the microbial structure in the intestine,thereby changing the environment conducive to anti-inflammatory,reducing the production of pro-inflammatory bacterial products,and improving the integrity of the intestinal barrier.Even some probiotics have been proved to reduce intestinal oxidative stress.Urine-based detection and analysis has become an attractive alternative method,because urine collection is simple and convenient,and is the most commonly used biological sample for non-invasive detection.Metabolites of intestinal flora can be excreted into urine and accumulated in the bladder after absorption,reflecting the final changes of the body,and can be collected within a set period of time.Therefore,urine is the "concentration camp" of body metabolism and the "barometer" of body diseases.Urinary metabonomics has been successfully applied to the study of intestinal microbial-related diseases,including tuberculosis,autism,and metabolic syndrome.Therefore,the method of detecting oxidative stress markers and representative intestinal microbial biomarkers in human urine samples was established by using nanofibers solid phase extraction technology created by our research group.A population study paradigm with different stress modes was designed to detect oxidative stress biomarkers in human non-invasive samples and co-metabolites in intestinal microbial community hosts.To study the effect of oxidative stress on intestinal flora metabolism.In addition,new composite nanofibers were prepared by electrospinning technology as encapsulation materials of lactic acid bacteria,and their protective effects on viable bacteria were investigated in vitro.The details are as follows:1.Establishment of methods for detecting oxidative stress markers and intestinal microbial metabolites(1)Polystyrene-nano-copper particles(PS-CuNPs)composite nanofibers were prepared by electrospinning technology.The selective extraction of glutathione from urine samples before derivatization was realized based on the Cu-S covalent bond between nano-copper on the fiber and –SH on the glutathione molecule.Under optimized conditions,the method has a 1.1 ng/mL detection limit and a response that is linear in the 10-1000 ng/mL GSH concentration range.The recoveries of GSH from artificial urine spiked at three levels(80,400 and 800 ng/mL)are in the range of 94.6-98.6% with relative standard deviations(RSD)of < 4.5%(n = 5).The established method was applied to the detection of glutathione in urine of high-risk infants and normal infants.The average level of glutathione in normal children urine is 823 ± 220 ng/mL,and that of high-risk infants is 443 ± 150 ng/mL.The results showed that the content of glutathione in urine of high-risk infants is significantly lower than that of normal infants(P < 0.05),indicating that the body of high-risk infants is in a high state of oxidative stress,indicating that the established method system could be applied to population studies.(2)Based on the principle that potassium permanganate oxidizes mercaptan amino acids in acidic solution to produce weak chemiluminescence,and formaldehyde can greatly enhance the luminescence intensity of potassium permanganate-mercaptan amino acids oxidizing luminescence system,a method for the determination of glutathione and cysteine in human milk by high performance liquid chromatography combined with chemiluminescence was established.This method has the characteristics of high separation efficiency and sensitive detection,and realizes the purpose of direct detection without derivation of mercaptan amino acids.The performance of the method was evaluated under optimal detection conditions.The linear ranges are 0.02-10 ?mol/mL for Cys and 0.05-10 ?mol/mL for GSH,respectively.The detection limits,defined as signal-to-noise ratio of 3.0,are in the range from 0.02 ?mol/mL to 0.05 ?mol/mL for all analytes.The recovery values are up to 96% for low,medium and high concentrations of artificial urine samples.The intra-day precision(n = 5)and the inter-day precision(n = 5)are <10%,which met the testing requirements of biological samples.The new method was used to detect glutathione and cysteine in breast milk.The average level of GSH and Cys in breast milk from high-polluted urban are 0.63 ± 0.05 ?g/mL and 0.52 ± 0.02 ?g/mL,and that of high-polluted urban are 1.71 ± 0.34 ?g/mL and 1.12 ± 0.04?g/mL.The data of breast milk samples from the two areas are analyzed by comparing the air total metals(lead,arsenic,cadmium and mercury).It wasfound that the contents of glutathione and cysteine in breast milk from urban areas with serious self-polluting environment are lower than those of the control group(P < 0.05).It seems that differences in pollution level can lead to different levels of oxidative stress in lactating women.(3)A method based on solid phase extraction with nanofibers and high performance liquid chromatography-fluorescence detection for indoleamine,the intestinal microflora cometabolism with the host in urine,was established.This method can omit the usual steps of rotary evaporation for concentration of indoles,avoid the loss of indoleamine and improve the sensitivity and accuracy of the method.The linearity of the method is confirmed over the concentration range of 2.0-200 ng/mL for melatonin,5.0-400 ng/mL for MEL,IPA,IND and SKT,respectively.The correlation coefficients for all indoleamines are greater than 0.991,which shows a good linearity correlation between concentration and peak areas.The new method has been successfully applied to the determinaction of indoles in students' urine before and after physical exercise.The average level of MEL,IPA,IND and SKT in urine from before physical exercise are 54.2 ± 5.10 ng/mL,14.4 ± 1.30 ng/mL,250.8 ± 14.1 ng/mL and 9.43 ± 1.07 ng/mL,and that of after physical exercise are 84.0 ± 9.69 ng/mL,25.9 ± 3.39 ng/mL,343.7 ± 36.8 ng/mL and 14.6 ± 1.36 ng/mL.The analysis results show that the concentration of indoles in urine after exercise is significantly higher than that before exercise(P < 0.05),indicating that short-term exercise stress will induce the changes in intestinal flora metabolites.2.Effects of oxidative stress caused by environmental pollution on intestinal flora metabolites in childrenIn different urban areas of the same city(the level of environmental pollution index is slightly different),we choose two kindergarten which were basically matched in the geographical environment and management mode,and collect urine samples of two kindergartens' children.The levels of glutathione and indoles in urine were determinated by the above-mentioned proposed method for oxidative stress markers and intestinal flora metabolites.The average level of GSH in high-polluted urban kindergartens is 36.3 ± 2.25 ng/mL,and that of low-polluted urban kindergartens is 49.4 ± 5.27 ng/mL.The result shows that the urinary glutathione level of children in high-polluted urban kindergartens is significantly lower than those in low-polluted urban kindergartens(P < 0.05),indicating that the level of oxidative stress in children in high-polluted urban areas was increased.The average level of MEL,IPA and IND in urine from high-polluted urban kindergartens are 40.8 ± 3.42 ng/mL,195.0 ± 22.4 ng/mL and 181.3 ± 15.0 ng/mL,and that of low-polluted urban kindergartens are 54.0 ± 5.02 ng/mL,113.9 ± 22.3 ng/mL and 243.7 ± 23.4 ng/mL.Indole-3-propionic acid,the secondary metabolite of indole in urine of children,also increased correspondingly,but the concentration of indole and melatonin showed the opposite trend(P < 0.05).It can be speculated that oxidative stress in children increases,and simultaneously affects the co-metabolism of in intestinal microflora with hosts.These results indicate that even minor environmental changes can have a significant impact on human health,especially on children's health.The establishment of a sensitive and accurate detection system is an important means to carry out environmental and human health research.3.Study on immobilization of probiotics based on polyvinyl alcohol/silk fibroin electrospinning technologyPolyvinyl alcohol/silk fibroin/probiotics composite bio-nanofibers embedding active bacteria were prepared by electrospinning technology by adjusting the proportion of each component.After simulated gastric juice stress for 2.0 hours,the biomass of composite bionanofibers in fresh liquid medium was not affected;on the contrary,pure probiotics did not grow in fresh liquid medium after simulated gastric juice stress.The comparative experiments showed that the polyvinyl alcohol/silk fibroin/probiotic cell composite bionanofibers exhibited outstanding advantages in resisting gastric juice stress.This technology has made a useful exploration for the research of new active probiotics preparation. |
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