| The total number of cells in the gastrointestinal tract microbes symbiosis with human beings,is about 10 times the number of human cells themselves.These microbial metabolic gene contains a lot of genes for the metabolism of polysaccharides,amino acids,exogenous compounds and formation of methane,regulation of vitamin and biosynthesis of isoprenoid,such as the number of genes encoded by these microbes is about 100 times of the body’s own encoding gene,human and animal body is a superorganism composed by itself and the microbial colonization in vivo.Intestinal flora is the most important micro ecosystem of human and animal body.The quantity is huge and the composition is extremely complex,and intestinal flora has important and irreplaceable functions of metabolism,nutrition,protection,immune regulation,etc.The host and gut microbes interact with external environmental factors,which are closely related to the host health and disease.In the healthy state,the relationship between the bacteria in the intestinal tract is symbiosis or antagonism,maintaining the micro ecological balance.Interference by exogenous environmental factors such as diet,the use of antibiotics and other factors,will lead to changes in the structure of the intestinal flora,intestinal flora once disordered,can cause the intestinal or non intestinal diseases of the host.The correct understanding of the symbiotic relationship between the intestinal micro ecosystem and the host will help us to understand the human being.Black tea in the history is the main supply for China’s northwest and southwest border minority areas.Border areas dominated by high protein foods,lack of fruit and vegetable diet.Dark tea can help digest greasy and digestion,and provides essential vitamins and minerals,is the necessaries of life for the frontier minority areas.In recent years,Yunnan Pu’er Tea and Hunan Fuzhuan Tea are popular with the public in the mainland market,production and sales continued to grow,became a common drink for many families.To investigate the relationship between drinking dark tea and health has become a research hotspot in the field of tea science.Therefore,the research of dark tea for the regulation of intestinal flora has a very important significance.The study selected Yunnan Pu-erh raw tea,Pu-erh ripe tea and Hunan Fuzhuan tea as the research objects,and detected the basic biochemical composition of three kinds of dark tea.Biochemical composition analysis results of the tested dark tea samples showed that the content of water extract,tea polyphenols and total free amino acids of Pu-erh raw tea was the highest.The content of soluble sugar,gallic acid and caffeine of Pu-erh ripe tea were the highest.The content of the basic biochemical composition of Fuzhuan Tea was opposite less,may be closely related to the coarse raw materials and the longer aging time.The content of water extract of Pu-erh raw tea was the highest(47.11±0.02%),Pu-erh ripe tea came second(31.94±0.01%),while Fuzhuan tea was the lowest(20.06±0.01%).The content of theabrownins(TBs)of Pu-erh ripe tea was the highest(9.62±0.02%),Fuzhuan tea came second(6.53±0.01%),while Pu-erh raw tea was the lowest(1.60±0.01%),and the content of thearubigins(TFs)of Pu-erh raw tea was the highest(3.91±0.01%).There is a greate difference in the content of tea polyphenol of the tested tea samples,Pu-erh raw tea was the highest(31.35±0.03%),Pu-erh ripe tea was opposite less(10.71±0.01%),while Fuzhuan tea was the lowest(4.53±0.00%).The study used drug sensitive paper method to test the antibacterial effect of dark tea on intestinal microflora in vitro.Escherichia coli,Staphylococcus aureus,Salmonella typhimurium,Enterococcus faecalis,Lactobacillus acidophilus,Lactococcus lactis was selected as the test strains,and investigated the inhibitory effect of Pu-erh raw tea,Pu-erh ripe tea and Fuzhuan Tea on the test strains.Experimental results show that the test dark tea samples have a significant inhibitory effect on Escherichia coli,Staphylococcus aureus,Salmonella typhimurium,Enterococcus faecalis,and the antibacterial effect of Escherichia coli was the best.There was no significant inhibitory effect on Lactobacillus acidophilus,Lactococcus lactis in the set concentration.It was deduced that the regulation effects of dark tea on the intestinal flora of mice was by inhibiting the growth of Escherichia coli,Salmonella and other conditioned pathogen and promoting probiotics.The strongest antibacterial effect in vitro was Pu-erh raw tea,and Pu-erh ripe tea became second,Fuzhuan tea was relatively weak.The main reason may be related to the content of tea polyphenols,caffeine,gallic acid and other active ingredients with the antibacterial function.The experimental results showed that the higher of the content of tea polyphenol,the stronger of the antimicrobial effect.The method of making mouse flora imbalance model is using lincomycin hydrochloride gastrointestinal tract.The results of PCR-DGGE method combined with tapping sequencing technology showed that the dominant bacteria in the intestinal tract of the model group were Enterobacteriales Enterobacteriaceae,and significantly reduced the ecological diversity of the intestinal flora in mice.The results of real time fluorescence quantitative PCR method showed that the number of Escherichia coli in the intestinal tract of mice increased significantly(P<0.05),while the population of Enterococcus,Lactobacillus and Bifidobacterium were significantly decreased(P<0.05).It showed that the conditioned pathogen in the intestinal tract grew rapidly,while the probiotics were inhibited.Intragastric administration of lincomycin hydrochloride has broken the balance of intestinal flora of mice,changed the structure.The research applied lincomycin hydrochloride in mice successfully constructed mouse flora imbalance model.In this study,we set a low dose(0.83 g/kg·bw),medium dose(1.67 g/kg·bw)and high dose(3.33 g/kg·bw)to intervene the flora imbalance mouse.Denaturing gradient gel electrophoresis(DGGE)of 16 S r RNA V3 region and specific PCR-DGGE of Clostridium leptum group,Bacteroides and Lactic acid bacteria to investigate intestinal microflora,objectively reflected the changes of intestinal flora caused by dark tea.Fuzhuan Tea(medium dose)had the best effect,made a remarkable recovery of intestinal dominant bacteria in the short time.The best regulating effect of intestinal dominant bacteria was Fuzhuan Tea,Pu-erh ripe tea became second,Pu-erh raw tea was relatively weak.Pu-erh raw tea(low dose)and Fuzhuan tea(high dose)of the regulating effect of Clostridium leptum group were incredibly effective.The bast regulating effect of Clostridium leptum group was Pu-erh raw tea,Fuzhuan tea became second,Pu-erh raw tea was relatively weak.Fuzhuan tea(medium dose)of the regulating effect of Bacteroides was incredibly effective.The best regulating effect of Bacteroides was Fuzhuan tea,Pu-erh raw tea became second,Pu-erh raw tea was relatively weak.Pu-erh ripe tea(high dose)of the regulating effect of Lactic acid bacteria was incredibly effective.The best regulating effect of Lactic acid bacteria was Pu-erh ripe tea,Pu-erh raw tea became second,Fuzhuan tea was relatively weak.In order to further verify the regulating effect of dark tea on the intestinal flora,the study used real-time fluorescence quantitative PCR technology to detecte the population of Escherichia coli,Enterococcus,Lactobacillus and Bifidobacterium.Results showed the population of Escherichia colithe test dark tea treatment group significantly decreased(P<0.05),which needing further research to proof the effect adjusted by dark tea or relying on own self-healing functions in mice.Under a certain dose of administration,Pu-erh raw tea and Fuzhuan tea contributed to recovery of the population of Enterococcus in mice,while there was no significant effect of Pu-erh ripe tea.Pu-erh raw tea,Pu-erh ripe tea and Fuzhuan tea undering a certain dose of administration contributed to recovery of the population of Lactobacillus and Bifidobacterium.The largest number of Enterococcus and Lactobacillus in the intestinal tract of mice was Pu-erh raw tea(medium dose)group,respectively as 7.84±0.14 and 8.38±0.02.The largest number of Lactobacillus in the intestinal tract of mice was Fuzhuan tea(medium dose)group(8.61±0.07).In summary,there was a significantly antibacterial effect on Escherichia coli,Staphylococcus aureus,Salmonella typhimurium,Enterococcus faecalis in vitro,especially for Escherichia coli,while there was no significantly antibacterial effect on Lactobacillus acidophilus and Lactococcus lactis.The results of antibacterial test in vitro confirmed that the strongest antibacterial effect was Pu-erh raw tea,followed by Pu-erh ripe tea,while Fuzhuan tea was relatively weak.In the test of regulation of dark tea on the intestinal flora of mice in vivo,the test dark tea samples could improve the flora structure of dominant bacteria,Clostridium leptum group,Bacteroides and Lactic acid bacteria and increase the the population of Enterococcus,Lactobacillus and Bifidobacterium.The strongest regulating effect on dominant bacteria and Bacteroides was Fuzhuan tea,and the strongest regulating effect on Clostridium leptum group was Pu-erh raw tea,while the strongest regulating effect on Lactic acid bacteria was Pu-erh ripe tea.Pu-erh raw tea and Fuzhuan tea could help to recover the number of Enterococcus,while there was no significant effect of Pu-erh ripe tea.Pu-erh raw tea,Pu-erh ripe tea and Fuzhuan tea undering a certain dose of administration contributed to recovery of the population of Lactobacillus and Bifidobacterium.Three kinds of the test dark tea in the inhibitory effect of Escherichia coli had no significant difference.The best regulating effect on Enterococcus and Bifidobacterium was Pu-erh raw tea,while the best regulating effect on Lactobacillus was Fuzhuan tea.Results showed that dark tea could increase the population of Enterococcus,Lactobacillus,Bifidobacterium and other probiotic and decrease the population of Escherichia coli and other conditioned pathogen,optimize the structure of intestinal flora,regulate the structure of the flora in the intestinal tract tending to diversity,make the intestinal micro ecosystem more healthy and stable,to maintain the health of the body.There are some difference between the antibacterial effect of dark tea in vitro and in vivo,which is speculated a certain relationship with the growth state of microorganisms.Innovativeness:1、Tea and health is a hot research topic in tea science field for many years,but there is rarely reports about the regulation effect of dark tea on intestinal flora in vivo.The study used a combination of antibacterial effect in vitro and in vitro to comprehensive evaluate of dark tea on regulation of intestinal flora.It’s greatly significant to provid some theoretical guidance for tea and health.2、The study selected Yunnan Pu-erh raw tea,Pu-erh ripe tea and Hunan Fuzhuan tea as the research objects,which was the popular varieties of dark tea and have a great market impact,to investigate the regulation of intestinal microbes in vitro and in vivo,comprehensive evaluation of dark tea on regulation of intestinal flora.3、The study successful applied the microbial molecular ecology to detecte the regulating effect of black tea on intestinal flora,such as PCR-DGGE and real time quantitative PCR technology.It can objectively reflect the regulation effect of the dark tea on the intestinal flora by overcoming some technology difficulties,such as the results obtained undered artificial conditionsin different from the behavior in the natural ecological environment and strictly anaerobic bacteria difficult to cultivate.can objectively reflect the black tea on the intestinal flora regulation of the real situation. |
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