【作者】 徐宁宁；

【导师】 刘建新； 俞培强；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2018， 博士

【摘要】 反刍动物营养研究多关注粗饲料的利用,对于精饲料(淀粉)的研究相对较少,并且多集中在不同用量和比例对瘤胃健康和生产性能的影响,对于淀粉降解机制缺少系统研究。为此,本研究旨在从胚乳物理结构和化学键组成两个层面探究玉米内在结构差异,并结合瘤胃降解参数,系统解析玉米瘤胃降解机制。在比较玉米和小麦(淀粉)瘤胃降解特性的基础上,选用玉米为材料,研究了不同品种玉米内在结构、蒸汽预处理改变玉米内在结构等对瘤胃降解特性的影响,并从玉米淀粉瘤胃发酵过程中微生物粘附、降解参数等方面分析了作用机制。1玉米和小麦淀粉的瘤胃发酵特征以玉米、小麦作为精料,稻草为粗料。按照3个精粗比(35:65、50:50、65:35)、4个小麦与玉米比(20:80、40:60、60:40、80:20)进行体外模拟瘤胃发酵试验,测定产气量和发酵参数,并计算氮利用效率(ENU):ENU(%)=微生物N(g)/可利用N(g)×100。结果发现,随着底物中小麦比例的提高,产气量、挥发性脂肪酸和氨态氮浓度线性提高,微生物蛋白合成、干物质降解率和ENU降低。随着精料含量的提高,挥发性脂肪酸浓度和微生物蛋白合成线性增加,ENU线性降低。利用扫描电镜和共聚焦显微镜观察玉米和小麦胚乳结构(starch-protein matrix)发现,玉米胚乳中大部分淀粉颗粒被蛋白质包被,小麦淀粉颗粒却相对散布于胚乳中。结果显示,玉米和小麦胚乳结构明显差异,这些差异可能导致了它们在瘤胃中发酵特征的不同。2不同品种玉米内在结构差异对瘤胃降解特性的影响选用三个玉米品种(LM01,LM10和LD999),用4头瘘管泌乳荷斯坦奶牛(DIM = 250±20d),按照尼龙袋法测定其瘤胃降解参数;同时利用傅里叶红外光谱仪测定它们的化学结构,用扫描电镜和小角度散射技术测定了各品种玉米的胚乳和淀粉颗粒形态学结构;分析了玉米内在理化结构与瘤胃降解参数的关联。2.1胚乳形态结构LD999胚乳中存在较大面积的层状蛋白膜片,淀粉颗粒呈规则且表面光滑的圆球形,但LM10和LM01淀粉颗粒表面存在较大的碎片状蛋白颗粒;小角度散射技术分析结果,玉米淀粉颗粒晶型和颗粒结晶度在三种品种间无显著差异。2.2化学键组成碳水化合物相关化学键(C=O)的红外吸收峰(ca.1187-950 cm-1)峰强在LM01中高于LM10和LD999,表明三种玉米对应的碳水化合物内在化学结构和物质总量存在差异。非结构性碳水化合物对应的化学键及官能团吸收峰(ca.950-820cm-1)在三种玉米品种间差异显著;LD999的蛋白(-CO-NH-)相关吸收峰(AmideI和Amide Ⅱ)及次级结构(α-helix和β-sheet)吸收峰峰强均高于LM10和LM01,表明LD999中含有更多的蛋白质。2.3瘤胃降解参数瘤胃可降解干物质(%RDDM)、瘤胃可降解淀粉(%RDSt)在LM01中最高,但淀粉降解速率(Kd)的品种间差异不显著;LM01的瘤胃可降解蛋白(%RDP)高于LM10和LD999,由于蛋白含量有差异,LD999的瘤胃可降解蛋白总量(RDP,g/kg DM)最大。通过分析这些参数与胚乳形态、化学键的关系发现,玉米DM和淀粉的瘤胃降解率同碳水化合物相关化学键和原子基团吸收峰峰强成正相关,蛋白降解率同肽键含量成正相关,并且受到胚乳结构和淀粉颗粒形态的影响。3蒸汽压片处理改变玉米内在结构对瘤胃降解参数的影响对两种玉米原料处理前(RC-I和RC-II)和处理后(SFC-I和SFC-II)的样品进行胚乳内在结构、化学键和原子基团组成及瘤胃降解参数测定,分析玉米内在理化结构与瘤胃降解参数的关系。3.1胚乳形态结构SFC中淀粉颗粒与纤维、蛋白间的铰链作用受到严重破坏,醇溶蛋白γ-zein的两种单体16kDa和27kDa浓度在蒸汽压片玉米(SFC)中显著低于未处理玉米(RC)。对淀粉颗粒进行扫描电镜和粒径分布分析发现,SFC胚乳中淀粉颗粒出现明显塌陷,由规则的球状变成不规则的圆饼状,且淀粉颗粒粒径变大;SFC的淀粉颗粒晶型与RC无显著差异,但是颗粒结晶度却显著降低,表明蒸汽压片处理显著改变了玉米胚乳物理形态结构。3.2化学键和原子基团组成总碳水化合物(ca.1188-950cm-1)和非结构性碳水化合物(ca.950-820 cm-1)相关的化学键(C=O)和官能团相对应的红外吸收峰峰强在SFC中显著高于RC,表明SFC含有更高的可利用碳水化合物。SFC的蛋白相关化学键(-CO-NH-)吸收峰低于RC,且二次结构a-helix和β-sheet显著低于RC,说明蒸汽压片处理破坏了蛋白质结构,降低了蛋白质含量。3.3瘤胃降解参数SFC的干物质和淀粉瘤胃降解速率(Kd)及降解率显著高于RC,瘤胃可溶性部分显著降低;SFC的蛋白降解率(%RDP)显著低于RC,但蛋白降解速率两者间无显著差异。相关性分析表明,碳水化合物化学键组成与玉米DM、淀粉降解率成正相关,蛋白化学基团相关吸收峰同蛋白降解率成正相关。通过分析这些参数与胚乳形态、化学键的关系发现,蒸汽压片处理改变了玉米胚乳和淀粉颗粒形态结构,并破坏了玉米碳水化合物和蛋白相关化学键和原子基团振动模式,进而改善了蒸汽压片玉米的瘤胃降解。4玉米内在结构影响瘤胃降解的分子机制选取蒸汽压片处理前后的玉米(RC和SFC),通过测定其瘤胃发酵过程中的化学键组成,并结合荧光标记和原位荧光杂交(FISH)技术,从化学键组成和细菌粘附两个角度探究玉米瘤胃降解机制。4.1瘤胃发酵过程中化学结构变化随着瘤胃发酵时间延长,RC和SFC发酵残余物中的非结构性碳水化合物相关化学键和基团(ca.950-820 cm-1)逐渐减少至0,总碳水化合物相关化学键吸收峰(ca.1188-950cm-1)逐渐降低,但蛋白相关化学键吸收峰(ca.1720-1480cm-1)呈升高趋势。主成分分析显示,瘤胃发酵0、2、4、8和12 h残余物的化学键和原子基团组成在RC与SFC间无显著差异,但前12 h与24 h、48 h发酵的残余物化学键组成差异显著。RC和SFC发酵残余物中的淀粉含量随发酵时间延长而降低,在前12h发酵过程中,RC的淀粉含量无显著降低,但SFC的淀粉含量呈线性降低。4.2 淀粉降解菌(starch hydrolyzing bacteria,SHB)的粘附作用在RC样品中,SHB丰度在4 h发酵时间点达到峰值,然后逐渐降低;Ruminococcaceae、phylum Firmicutes 丰度(R_SHB)存在相似趋势。在 SFC 发酵过程中,SHB和R_SHB丰度在4h和24h两个时间点出现峰值,并且以24h丰度最高。结合RC和SFC样品中化学键组成与SHB丰度分析发现,SHB丰度与化学键组成无显著相关性,但是SHB丰度在RC和SFC之间存在显著差异,说明淀粉颗粒的物理结构很大程度上影响了 SHB对淀粉颗粒的粘附。综上所述,玉米瘤胃降解过程受到胚乳物理结构和化学键组成的影响。在物理水平上,胚乳中的starch-protein matrix结构、淀粉颗粒形态、粒径大小、结晶度,醇溶蛋白含量等均会对玉米干物质和淀粉瘤胃降解产生影响;在化学水平上,玉米的官能团组成和化学键强度影响其瘤胃降解。淀粉降解菌在不同结构的玉米表面粘附程度有差异,这种差异会显著影响玉米的瘤胃降解。结果提示,通过改变玉米的内在理化结构,可以改善玉米的瘤胃降解特性。更多还原

【Abstract】 In ruminant nutrition,much attention has been paid on forage utilization,but few study focus on the concentrate including starch.Most of researches on concentrate are related to the effects of concentrate level on rumen health and milk yield,but little information is available on the mechanism of starch degradation in the rumen.Therefore,the objectives of current study were to investigate endosperm physical structures and chemical bonds in corn,along with rumen degradation characteristics,and to elucidate the mechanism of rumen degradation.Based on comparison of rumen fermentation characteristics between wheat and corn grain,we intended to to clarify the effect of the intrinsic molecular structures of different varieties of corn as well as untreated and steam-flaked corn on rumen fermentation characteristics.The adhesion of rumen microbes to corn granules during the degradation was also investigated to clarify the mechanism with which corn was degraded in the rumen1.Rumen fermentation characteristics of corn and wheat starchThree ratios of concentrate to forage(35:65,50:50,and 65:35)and 4 ratios of wheat to corn(20:80,40:60,60:40,and 80:20)were applied to in vitro gas test to evaluate rumen fermentation characteristics and nitrogen(N)utilization,and calculated efficiency of N utilization(ENU),expressed as ratio of microbial N to available N.With the increasing ratio of wheat,gas production,total volatile fatty acids and ammonia N increased linearly,but microbial protein synthesis,dry matter degradation and ENU decreased linearly.With the increasing ratio of concentrate-to-forage,volatile fatty acids and microbial protein increased linearly,but ENU degreased linearly.The endosperm structures(starch-protein matrix)were detected by scanning electronic microscopy and confocal laser scanning microscopic.Starch granules were embedded in the phosphorus-associated protein matrix in corn,but more granules were separated from the matrix in wheat endosperm.It is suggested that corn and wheat exhibited different fermentation characteristics with various endosperm structures.2.Influence of intrinsic structures differing among corn varieties on rumen degradabilityThree corn varieties(LM10,LM01 and LD999)were allocated into four cannulated mid-lactation dairy cows to detect rumen degradation characteristics in situ.Endosperm physical structures were detected by scanning electron microscopy and small angle X-ray scattering technique(SAXS).Chemical bonds and functional groups vibritions were detected by Fourier transform Vibrational Molecular Spectroscopy.The correction was analyzed between rumen degradation and intrinsic structure or chemical bonds of corn varieties.2.1 Endopserm morphologyMuch more protein fractions were detected in LD999 than other two corn.Starch granules were sphericity and smooth in LD999,but covered by more protein in LM01and LM10.The SAXS results indicated that no difference was observed in crystal form and crystallinity of starch granules among corn lines.2.2 Chemical bonds and functional groupsAbsorption peak(ca.1188-950 cm-1)of chemical bonds(C=O)related to carbohydrate and non-structural carbohydrate was greater in LM01 than that in LM10 and LD999,suggesting more fermentable carbohydrate in LM01.Structural carbohydrate associated chemical bonds intensities were greatest in LD999.Protein chemical bonds(-CO-NH-)and secondary structure α-helix and β-sheet was greatest in LD999,indicating high protein content in LD999.2.3 Rumen degradation characteristicsThe LM01 had greatest%RDDM and%RDSt.No difference was observed in rate of starch degradation among three corn lines.The LM01 had greater%RDP,but LD999 had greatest value of rumen degradable protein(RDP,g/kg DM).Combined with degradation characteristics and intrinsic structures,it is suggested that carbohydrate related functional groups vibrations were positively associated to%RDDM and%RDSt,but protein molecular structures were negatively correlated to%RDDM and%RDP.Endosperm physical structures played an important role in determining rumen degradation.3.Rumen degradation characteristics of corn influenced by alteration of intrinsic structures by steam flakingDifferent raw(RC-1 and RC-II)and steam flaked corn(SFC-Ⅰ and SFC-Ⅱ)were allocated into ruminant cannulated dairy cows to test rumwn degradation characteristics,endosperm physical structures and chemical bonds.The correlation was analyzed between rumen degradation and intrinsic structure or chemical bonds of untreated and steam-treated corn.3.1 Endosperm morphologySteam flaking process destroyed y-zein that determined starch-protein matrix.Both 16-kDa and 27-kDa y-zein were significantly lower in SFC than in RC.Starchgranules were swollen and gelatinized during steam flaking,and median particle size increased from 17.8 to 30.8 or 26.0 μm for RC-I or RC-II,respectively.Granule crystallinity of RC-I and RC-II decreased from 22.0 and 25.0%to 9.9 and 16.9%,respectively,but no influence was observed on crystal form.Thus,steam flaking process altered endosperm and starch granules physical structures.3.2 Chemical bonds and functional groupsQuantity and intensities of functional groups and chemical bonds related to carbohydrate(ca.1188-950 cm-1)and non-structural carbohydrate(ca.950-820 cm-1)were much greater in SFC than in RC,but functional groups of structural carbohydrate was lower in SFC,suggesting that steam flaking process destroyed fiber and increased fermentable carbohydrate.Steam flaking destroyed protein related chemical bonds(Amide I and Amide II),inducing lower degradable protein in SFC.Along with multivariate analysis,steam flaking process changed carbohydrate and protein chemical bonds significantly.3.3 Rumen degradation kineticsRate and extent of dry matter and starch rumen degradation in SFC were significantly greater than RC.No difference was observed between RC and SFC as to rate of protein degradation,but%RDP was lower in SFC.Correlation analysis showed that chemical bonds of carbohydrate were positively associated with rate and extend of DM and starch degradation.Chemical bonds of protein were positively associated with%RDP.In combination with physical structures and degradation characteristics,it is suggested that steam flaking changed endosperm physical structures and chemical bonds,resulting in increased rumen degradation of the carbohydrate fractions.4.Mechanism of corn carbohydrate degradation in rumenRaw(RC)and steam flaked corn(SFC)were used to detect the chemical bonds in fermented residues during rumen fermentation.The fluorescence probe and fluorescence in situ hybridization(FISH)technique were employed to investigate starch rumen degradation process based on breakdown of chemical bonds and bacteria colonization on corn granule.4.1 Alteration of carbohydrate chemical bonds during rumen fermentationWith increasing incubation in the rumen,quantities of chemical bonds related to carbohydrate(ca.1188-950 cm-1)and non-structural carbohydrate(ca.950-820 cm-1,)decreased linearly in both RC and SFC,but protein(ca.1720-1480 cm-1)related chemical bonds tended to increase.Molecular structures of the residues after 0,2,4,8 and 12 h fermentation could not be separated by principal component analysis(PCA),but the difference was totally different between the residues for 24 h and 48 h fermentation.4.2 Effect of starch hydrolyzing bacteria(SHB)on starch degradationAbundance of the SHB in RC were greatest after 4 h fermentation,then decreased gradually.The quantity of Ruminococcaceae and phylum Firmicutes(R_SHB)exhibited similar trends as the SHB.Both SHB and R_SHB exhibited two peaks at 4 h and 24 h rumen fermentation of SFC.The SHB abundance were totally different between RC and SFC,but no correlation was observed between colonized bacteria and molecular structures,indicating that granules physical structures played an important role in determining SHB attachment.In summary,starch-protein matrix,endosperm and granule morphology,starch granule crystallinity exert great influences on rumen degradation characteristics of DM and starch of corn.As to chemical structures,the functional groups and chemical bonds in corn could result in different rumen degradation kinetics.Intrinsic molecular structures influenced SHB abundance,resulting in different degradation characteristics.It is suggested that rumen fermentation characteristics of corn can be improved through altering its intrinsic structure and chemical bonds.更多还原