Differing Molecular Weights Of Condensed Tannins And Their Effects On In Vitro Ruminal Fermentation

In this experient, Yanbian wild forages was chosen as the materials to separated condensed tannins(CT), then determined the molecular weights and structures, investigated the effects on protein. In in vitro test, added CTs to substrates to research the effects of different molecular weights CTs on ruminal fermentation, then expound the influence on ruminal fermentation from molecular angle.All above study would provid scientific and theoretical basis for application of CT in protecting UDP.Experiment I:Extracting, purification, isolation and analyze of CT in pastureWe have measured the levels of condensed tannin、 soluble protein-binding condensed tannin (PCT) and fiber-binding condensed tannin (FCT) in four species of plants that contains tannin in Yanbian. Then the leaves of two of the four samples (that is Vicia. amoena and Lespedeza hedysaroides Kitag) were further analyzed.Simnply, we optimized the condition of CT extraction process at first followed by a further purification. Secondly, the extracted CT was isolated according to their molecular weight by Gel filtration chromatography. At last, degree of polymerization, molecular structure and average molecular mass of CT was estimated by mass spectrometry. In addition, the protein-binding capacity of CT was also measured at the same time.The results showed that the differences of tannin content among different species were large, with an average content of3.4g/kg to88.6g/kg.When the solid to liquid ratio and extractive liquid ratio in extraction process in V. amoena was adjusted to1:40and2:2(Lespedeza hedysaroides Kitag was1:30and2:3), the time was4hours, respectively, it showed the bestresults. The CT molecular in V. amoena was eventually identified as heptamer with the highest molecular weight of1952Da and in Lespedeza hedysaroides Kitag was eventually identified as Decamer with the highest molecular weight of2850Da. Inferring from the molecular structure, the CT in two plant were not monomer buta complex contained-Gwhich made up of Procyanidin、Prodelphinidin and Propelargonidin. The molecular weight of three components extracted from V.amoena were915.60Da、783.94Da、456.37Da, while in Lespedeza hedysaroides Kitag were1368.57Da、886.32Da、607.14Da, respectively.Experiment II:The effect of different molecular masses ofCTon the parameters of ferment in rumen.We performed an experiment with six groups which designated Control, V1, V2, V3, L1, L2and L3, respectively. Then add3%six kinds of corresponding CTs as described in experiment I into the substrate fermented in vitro.The pH value, gas effectS、NH3-N、 MCP and VFA were measured after cultured for24hours in intro.The pH value in each group were significantly higher compared to control group before24hour culture. The total gas and methane production in treatment groups were significantly lower than control group during the process of the culture except the3-hour period in methane production measurement. With regarded to NH3-N and MCP, their value lower than that in control group at beginning, but the difference decreased gradually with time lasteduntilit disappeared after cultured9hours in V3group(with the lightest molecule weight).Total volatile fatty acidsandacetic acid production were also lower than control group. The group L1, which had the biggestmolecule weight, showed the lowest value in total volatile fatty acidsandacetic acid production followed by V1and L2groups, and also showed the highest value in propionic acid production made it significantly higherthan contron and L3group.It turned out that the larger moleculesof CT had more obvious effects on rumen physiological parameter. With the increasing of the molecular mass, pH value reduced gradually slowly, and all the values of biogas effects, digestibility, methane production, NH3-N, MCP, total volatile fatty acidsandacetic acid productionshowed a decreased tendency except propionic acid production.