| In the past dozens of years of evolutionary process, including the period of the farming civilization when it had begun before ten thousand years'ago, until the period of the modern industrial revolution, high in fiber, high in carbohydrates was the basic features in the human diet. After the completion of the industrial revolution, the structure of human diet rapidly changes into low fiber, high protein and high fat mode. Due to a serious shortage of dietary fiber delivery to the gut, the destruction of the balance between human metabolism and the intestinal microbial metabolism, which has developed for tens of thousands of years, leads to a series of major diseases. A fundamental way to solve the problem of the intestinal flora imbalance under the condition of modern life is to raise the intake level of non-starch polysaccharides - which is also called dietary fiber. Searching for a daily fiber resource which provides a high performance of foodstuff processing is a feasible means to settle the problem of the inadequate intake of dietary fiber of modern people. Xylan, there are enough resources to meet the continuous large-scale need of human society, is the major component of the grasses hemicelluloses which is a renewable resource, and it is also obtained from crop residues. Xylan can be degraded and digested into fermentable monosaccharide which is the necessary premise for the use of intestinal microbial metabolism-human metabolism. Learning about its digest and metabolize characteristics in intestine, figuring out its effect on the change of intestinal flora, illustrating the relationship among the xylan, microbial metabolism and animal metabolism is an important scientific evidence of developing xylan into a new dietary fiber product.In the study, the researcher feeds the SPF SD rats by adding free xylan to interfere their diet, aims to discuss the digestive characteristics of xylan from two aspects which are the features of metabolite and the change of intestinal microflora. By analyzing the index of the animal biochemistry, it reveals the metabolic relationship between xylan microorganisms and animal physiology. The main results of this research include the following aspects:1. A method for marking the digestive features of xylan gastrointestine by using insoluble colored xylan has been established. Crosslinking agent connects gas balaam(a water-soluble blue dye) with xylan, while, it forms an insoluble xylan colored by substance-gas balaam. When the insoluble colored xylan is hydrolyzed by enzyme, it releases the dye and makes the reactants colored. Diluting the active enzyme sample in different times and then hydrolyzing the Bagasse xylan colored by gas balaam proves the good linear relationship between the burst size of gas balaam (OD625nm) and marked xylanase enzyme activity (IU/mL). The release rate of the dye is equivalent to the enzymatic hydrolysis rate, it means that the relative amounts of the dye released are equal to the relative amount of xylan degraded. Therefore, this method not only can be used to quantitatively analyze the intestinal xylanase activity, but also can be used to trace the digestion rate of xylan in different position of digestive tract.2. It proves that xylan can obviously improve the xylanase emzyme activity of tested rats' feces. After the three-day feeding with xylan, the labeling method of colored xylan clearly reveals that the enzyme activity, impressively increased. After the rats having been fed for five weeks continuously, the enzyme activity of the rat feces (3.461IU/g) rose 24 times-fold than the feeding starting point, comparing to enzyme activity of the same phase model group(0.512IU/g) it only improved 6.8 times. After the rats having been fed for 6 weeks continuously, the enzyme activity tended to be steady. Therefore, the relatively reliable results of evaluating the regulating effect of xylan on the intestinal microbiological system can probably be obtained from the tested animals which should be fed continuously with xylan more than one month at least. The regulation of xylan on human microbial systems can only be achieved through long-term dietary intervention.3. It shows the features of xylan degraded in the intestine and proves that the macromolecular xylan can almost be sent to the farthest point of the intestine for the fermentation use of the microorganisms there. The labeling method of colored xylan clearly shows that xylan can be entirely degraded by intestinal microorganisms, and it is less than 10% of the total intake(2.38g/kg·d) in the small intestine. The main intestinal segment of xylan degraded is the large intestine, the rate of degradation in the cecum and colon reached respectively up to 60% and 85%, and about 15% of xylan can be totally degraded in the rectum.4. It is observed that, once generated, the enzymic hydrolysates of xylan in the intestine can be digested by intestinal microorganisms. Adding xylan (2.38g/kg·d) to the feed, the containers enzyme liquid in the rats intestines were kept warm with the xylan, then xylan was converted into xylo-oligosaccharide and xylose. Comparing with previous rate, the amount of xylo-oligosaccharide and xylose increased respectively to 3.5 times and 10.4 times, but carbohydrate cannot be found in the intestinal containers themselves. It is proved that in the intestinal environment, the enzymic hydrolysates of xylan won't be accumulated in the enteric cavity but consumed immediately by intestinal microbial metabolism.5. Through analyzing the organic acid of rats'feces with GC-MS, it is found that xylan is fermented by intestinal microorganisms and mainly generating short-chain fatty acids. The features of yielding short-chain fatty acids shows that xylan is a qualified dietary fiber resources which involves a significant value of commercial development.6. Aiming to justify the healthy safety of the current technique in manufacturing xylan product, xylan is added to high fat and high sugar feed in the dose of 2.38g/kg·d. After the rats having been kept by this feed for two months, the biochemical indexes of growth and development and serum of the rats were measured. It demonstrates that there is a little(limited) effect on reducing body weight but no statistically significant differences on rats'GLU, TG, Tch, HDL and LDL by adding the xylan to their feed, meanwhile, it proves that the current technique of producing xylan is basically safet.7. The study indicates that the rat's fecal microbial community structure is culturable due to the apparently change by feeding xylan. However, there is no relationship between fecal xylanase activity and the number of cultivated enzymic microorganisms. Feces culture treatment finds that, the total number of colonies in model group and xylan group are respectively approximately 1.05×109CFU/g and 1.00×109CFU/g. The average number of the transparent circle in single tablet is 3.67±2.31 and 6.33±2.51. Although the total number of bacteria values is close, the difference of some colony morphology and the proportion between two plates can be distinguished with naked eyes. Although feeding with xylan makes the xylanase activity increase greatly in the rats'feces, the phenomenon of a increasing amount of enzyme-producing microorganisms in fecal culture does not exist. A possible reason is that when xylanase microorganisms are evolved in the rats'intestine, there is a large proportion of it belonging to the uncultivated microorganisms. We should adopt molecular biology methods, for example. DNA polymorphisms, metagenomics and so on to study the effect of xylan on the xylanase microbial flora evolved in intestine. In this way, it can be expected to reveal the internal relationship between xylanase activity and the structure of microbial flora.8. It reveals that after the rats were fed by xylan, the abundance of fecal flora diversity is reduced, with the reduction of species of the cultured bacterium known, but the number of clones is increased, compared with the model group, the ratio is from 5% to 60%. Through analyzing 16S rRNA clone library (capacity 500), it is found that there are 109 species bacteria in the model group library, including 63 species of bacteria, its sequence homology is greater than or equal to 97% with the GenBank database bacteria 16S rDNA which have been cloned, and there are 46 kinds of homology less than 97%. This data in the xylan group library are only 57,40 and 17 species. In the model group library, there are 10 kinds of microorganisms are known, Bacteroides and Firmicutes Clostridium bacteria are dominant bacteria, but in the xylan group library, there are only 6 species, Proteobacteria is the dominant bacteria, the number of clones is up to 60%, Firmicutes Clostridium bacteria is also existed, but the Bacteroides bacteria is almost not to be found. In the library model group, there are 104 species of uncultivated bacterium, accounting for 95.4% of the clones, but in the library xylan group, there are only 55 species, clones accounted for 39.6%. |
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