体外消化实验文档格式.doc
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体外消化实验文档格式.doc
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Constituentsandconcentrationsofthevarioussyntheticjuicesoftheinvitrodigestionmodelrepresentingfedconditions
Saliva唾液
Gastricjuice胃液
Duodenaljuice十二指肠液
Bilejuice胆汁
Inorganicsolution
10mlKCl89.6g/l
15.7mlNaCl175.3g/l
40mlNaCl175.3g/l
30mlNaCl175.3g/l
10mlKSCN20g/l
3.0mlNaH2PO488.8g/l
40mlNaHCO384.7g/l
68.3mlNaHCO384.7g/l
10mlNaH2PO488.8g/l
9.2mlKCl89.6g/l
10mlKH2PO48g/l
4.2mlKCl89.6g/l
10mlNaSO457g/l
18mlCaCl2·
2H2O22.2g/l
6.3mlKCl89.6g/l
150μlHCl37%g/g
1.7mlNaCl175.3g/l
10mlNH4Cl30.6g/l
10mlMgCl25g/l
20mlNaHCO384.7g/l
6.5mlHCl37%g/g
180μlHCl37%g/g
Organicsolution
8mlurea25g/l
10mlglucose65g/l
4mlurea25g/l
10mlurea25g/l
10mlglucuronicacid(葡萄糖醛酸)2g/l
3.4mlurea25g/l
10mlglucosaminehydrochloride(盐酸氨基葡萄糖)33g/l
Addtomixtureorganic
+inorganicsolution
290mgα-amylase
1gBSA
9mlCaCl2·
10mlCaCl2·
15mguricacid
2.5gpepsin
1.8gBSA
25mgmucin
3gmucin
9gpancreatin
30gbile
1.5glipase
pH
6.8±
0.2
1.30±
0.02
8.1±
8.2±
Theinorganicandorganicsolutionsareaugmentedto500mlwithdistilledwater.Aftermixingoftheinorganicandorganicsolutions,somefurtherconstituentsareaddedanddissolved.Ifnecessary,thepHofthejuicesisadjustedtotheappropriateinterval.
另外,
抗性淀粉含量测定:
1、参考Li等(2008)方法(Characterizationofmaizeamylose-extender(ae)mutantstarches.PartI:
Relationshipbetweenresistantstarchcontentsandmolecularstructures)
2.3.Resistantstarch(RS)content
RScontentsoftheae-mutantstarchsamplesweredeterminedusingtheAOACMethod991.43fortotaldietaryfiber(AOAC,2003)andEnglyst’smethod(1992)forcomparison.
FortheAOAC991.43method,starch(1.0g,dry-starchbasis,dsb)wassuspendedinaMes-trisbuffersolution(0.05M,40ml)andhydrolyzedwith500uofa-amylasefromBacilluslicheniformis(SigmaChemical,Cat.No.A3403)inaboilingwater-bathfor30minwithstir.ThesamplewasthendigestedwithproteasefromBacilluslicheniformis(5mg,SigmaChemical,Cat.No.P3910)at60º
Cinashakerwaterbathfor30min.ThesampledispersionwasadjustedtopH4.4–4.6byaddingHClandthenhydrolyzedwithamyloglucosidase(300U,Sigmachemical,Cat.No.A9913)at60º
Cinashakerwater-bathfor30min.Thedigestedsamplewasfilteredthroughacelitelayerinacrucibleandwashedtwicewith15mlof78%ethanol,twicewith15mlof100%ethanolandrinsedwith15mlofacetone.Theremainingsamplewasdriedinanovenat100º
Covernight.Theresistantstarchcontentwascalculatedusingtheequation:
(%)RScontent=Remainingsampleweight(g,dsb)/initialsampleweight(g,dsb)×
100%
ForRSdeterminedusingEnglyst’smethod(1992),Starch(1.000g,db)in20mLofsodiumacetatebuffer(0.1M,pH5.2)wascookedinaboilingwater-bathfor30min.Thestarchdispersionwascooleddownto37º
C,mixedwithanenzymesolution(5mL)consistingofpancreatinextractandamyloglucosidase,andincubatedinawater-bathat37º
C.Thepancreatinextractwaspreparedasfollows;
3.0gofpancreatin(Sigma,Cat.No.P7545)wassuspendedin20mLdeionizedwater,stirredfor10minatroomtemperature,andcentrifugedat1500gfor10min.Theenzymesolutionwaspreparedbymixing13.5mlsupernatantofpancreatinextract,210Uamyloglucosidase(Sigma,Cat.No.A7095),and1.0mLdeionizedwater.Therapiddigestiblestarch(RDS)wasdefinedasthetotalstarchdigestedwithinthefirst20min,andtheslowlydigestiblestarch(SDS)wasthestarchdigestedbetween20and120min(Englystetal.,1992).Theresistantstarchcontentwascalculatedasfollows:
(%)RScontent=100%×
(totalstarch–RDS-SDS)(g,dsb)/totalstarch(g,dsb)
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- 体外 消化 实验
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