实验方法与实验动物 EXPERIMENTAL METHOD AND ANIMAL

2种纤维源饲粮细胞壁多糖的化学结构及其在猪消化道降解规律的核磁共振研究

  • 杨亚敏 ,
  • 庄苏 ,
  • 张志静 ,
  • 张子珩 ,
  • 刘强
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  • 南京农业大学动物科技学院, 南京 210095
杨亚敏(1994-),女,河南商丘人,硕士研究生,研究方向为饲料资源开发与利用。E-mail:Yaaajoshua@outlook.com

收稿日期: 2020-01-15

  网络出版日期: 2020-07-15

基金资助

国家自然科学基金项目(31172237)

A Study of Chemical Structure of Cell Wall Polysaccharides from Two Fibrous Diets and Their Degradation Rule in Digestive Tract of Pigs Using Nuclear Magnetic Resonance Method

  • YANG Yamin ,
  • ZHUANG Su ,
  • ZHANG Zhijing ,
  • ZHANG Ziheng ,
  • LIU Qiang
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  • College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China

Received date: 2020-01-15

  Online published: 2020-07-15

摘要

采用二维异核单量子相干核磁共振(2D HSQC NMR)技术对由小麦麸和西兰花茎叶粉为单一纤维来源的饲粮以及对应饲粮的猪回肠末端食糜和粪便样品中细胞壁多糖进行定性解析和量化表达,并验证该方法用于饲料细胞壁多糖化学结构解析和定量分析的潜力,为细胞壁相关的饲料营养研究提供新实验技术。经脱脂、去淀粉和去蛋白质处理的饲粮、食糜和粪便样品经球磨处理后,用氘代二甲基亚砜和吡啶混合(DP)溶液(4:1,体积比)溶胀制成胶态样品,在配有5 mm低温探头的600 MHz核磁共振(NMR)谱仪上采集其异核单量子相干(HSQC)图谱,并用Bruker TopSpin 3.6.1软件对多糖异头区的共振信号进行定性和定量分析,同时用气相色谱(GC)法测定样品中非淀粉多糖的糖苷组成和含量。结果表明:参照现有文献数据可对大多数链中和还原端糖苷的共振峰进行准确归属。小麦麸饲粮细胞壁多糖中阿拉伯呋喃糖苷(Araf)种类较多,且多以多糖侧链的形式存在;而西兰花饲粮细胞壁多糖则相对简单,主要以阿拉伯聚糖的形式存在。小麦麸饲粮细胞壁多糖中木吡喃糖苷(Xylp)不带侧链的->4)-β-D-Xylp-(1->相对积分强度显著高于西兰花饲粮细胞壁多糖(P<0.05);西兰花饲粮细胞壁多糖中半乳糖醛酸吡喃糖苷(GalpA)和糖醛酸苷(UA)相对积分强度显著高于小麦麸饲粮细胞壁多糖(P<0.05);二者的葡吡喃糖苷(Glcp)相对积分强度均较高,而且均以->4)-β-D-Glcp-(1->为主,但西兰花饲粮细胞壁多糖中->3)-β-D-Glcp-(1->的相对积分强度显著高于小麦麸饲粮细胞壁多糖(P<0.05)。小麦麸饲粮细胞壁多糖主要在猪大肠内降解,而西兰花饲粮细胞壁多糖在猪回肠末端之前就有明显降解。西兰花饲粮细胞壁多糖中Araf和Galp的降解速度显著快于小麦麸饲粮细胞壁多糖(P<0.05),但后者的->4)-β-D-Xylp-(1->在猪大肠中的降解速度显著快于前者(P<0.05),二者的Glcp均不易降解,以侧链形式存在的Araf和有侧链修饰基团的多糖主链(木聚糖和半乳糖醛酸聚糖)在猪消化道中也均不易被降解。饲粮、食糜和粪便样品中大多数糖苷通过核磁共振(NMR)法测得的相对积分强度与通过GC法测得的摩尔百分比变化趋势一致,对于含量较高的糖苷,二者具有显著的回归关系(P<0.05)。综上,2D HSQC NMR技术在解析细胞壁多糖精细化学结构方面具有独特优势,而且还具有一定的定量分析潜力,但用于饲料营养研究尚需进一步完善。

本文引用格式

杨亚敏 , 庄苏 , 张志静 , 张子珩 , 刘强 . 2种纤维源饲粮细胞壁多糖的化学结构及其在猪消化道降解规律的核磁共振研究[J]. 动物营养学报, 2020 , 32(7) : 3343 -3357 . DOI: 10.3969/j.issn.1006-267x.2020.07.044

Abstract

The objective of this study was to evaluate the feasibility of the application of 2 dimensional heteronuclear single quantum coherence nuclear magnetic resonance (2D HSQC NMR) technique in analyzing the cell wall polysaccharides from trial pig diet (using wheat bran or broccoli leaf and stem meal as single fiber source), terminal chyme of ileum and fecal samples both qualitatively and quantitatively, and to verify its potential in analyzing chemical structure and quantitative analysis and to provide new experimental techniques for cell wall related feed nutrition research. After removing lipid, starch and protein, the cell wall polysaccharide extracts of the diet, ileal digesta and fecal samples were ball milled and dissolved in dimethyl sulfoxide (DMSO)-d6/pyridine-d5 (4:1, V/V) to form gel mixture. The HSQC spectra of the gel samples were collected on a 600 MHz nuclear magnetic resonance (NMR) spectrometer equipped with a cryogenically cooled 5-mm TXI gradient probe. The assignment and volume integration of the signals in the anomeric zoon of polysaccharides were performed by using Bruker TopSpin 3.6 with reference to the published data of chemical shift of polysaccharides. The glycosides content and composition of the cell wall polysaccharide extracts were analyzed using gas chromatography (GC) method simultaneously. The results showed that the most of signals in anomeric zoon of spectra could be assigned to corresponding glycoside in polysaccharides referring to the existing references. The arabinfuranoside (Araf) in the cell wall polysaccharide of wheat bran-based diet (WB) were complicated and most of them existed as the form of side chains of polysaccharides. That of broccoli leaf and stem meal-based diet (BR) was relatively simpler and formed arabinan mainly. The relative integral strength of xylopyranoside (Xylp) without side chain ->4)-β-D-Xylp-(1-> in the cell wall polysaccharide of WB was higher significantly than that of BR (P<0.05). The relative integral strengths of galacturonopyranoside (GalpA) and uronic acids glycoside (UA) in the cell wall polysaccharide of BR were significantly higher than those of WB (P<0.05). The relative integral strength of glucopyranosides (Glcp) was higher and mainly ->4)-β-D-Glcp-(1-> in the cell wall polysaccharide of WB and BR, but in BR cell wall polysaccharides, the relative integral strength of ->3)-β-D-Glcp-(1-> was significantly higher than that of WB (P<0.05). The cell wall polysaccharide of WB was mainly degraded in the large intestine of pigs, while that of BR was obviously degraded before entering the terminal ileum. The degrading rates of Araf and Galp in the cell wall polysaccharide of BR was faster than those of WB (P<0.05), but the ->4)-β-D-Xylp-(1-> in the cell wall polysaccharide of WB was more easily degraded in large intestine of pigs than that of WB (P<0.05). The Glcp in the two diets were the same difficult to degrade. It also demonstrated that Araf presented as side chain and the main chain of xylan and galacturonan with side chains were harder to be degraded in pig digestive tract. The relative integral strength of glycosides measured by the NMR method shared the same trend from diets, chyme to fecal samples with their molar percentages determined by GC method. There were significant regressive relationships between above two methods in glycosides with high content (P<0.05). It can be concluded that the 2D HSQC NMR technique illustrated an obvious advantage not only in resolving the detail structures of cell wall polysaccharides but also possess high potential in quantitative analysis. The 2D HSQC NMR method using feed nutrition research still need to be further improved.

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