| 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 ?-helix and ?-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-? and SFC-?)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 ?m 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. |
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