体外消化实验.doc

体外消化实验.doc

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体外模拟消化（口腔、胃、小肠）：

1、参考Gawlik-Dziki等（2009）方法（TheeffectofsimulateddigestioninvitroonbioactivityofwheatbreadwithTartarybuckwheatflavonesaddition）

2.4.1.Preparationofsimulatedsaliva,gastricandintestinalfluidsSimulatedsalivasolutionwaspreparedbydissolveof2.38g

Na2HPO4,0.19gKH2PO4and8gNaClin1lofdistilledwater.ThesolutionwasadjustedtopH6.75anda-amylase（EC3.2.1.1.）wasaddedtoobtain200Uofenzymeactivity.Forthegastricdigestion,0.32%pepsin（fromporcinestomachmucosa,pepsinA,EC3.4.23.1）dilutionin0.03MNaCl,pH1.2wasprepared.Simulatedintestinal.juicewaspreparedbydilutionof0.05gofpancreatinand0.3gofbileextractin35ml0.1MNaHCO3.

2.4.1.Preparationofsimulatedsaliva,gastricandintestinalfluids

Simulatedsalivasolutionwaspreparedbydissolveof2.38gNa2HPO4,0.19gKH2PO4and8gNaClin1lofdistilledwater.ThesolutionwasadjustedtopH6.75andα-amylase（EC3.2.1.1.）wasaddedtoobtain200Uofenzymeactivity.Forthegastricdigestion,0.32%pepsin（fromporcinestomachmucosa,pepsinA,EC3.4.23.1）dilutionin0.03MNaCl,pH1.2wasprepared.Simulatedintestinaljuicewaspreparedbydilutionof0.05gofpancreatinand0.3gofbileextractin35ml0.1MNaHCO3.

2、参考Versantvoort等（2005）（Applicabilityofaninvitrodigestionmodelinassessingthebioaccessibilityofmycotoxinsfromfood）和Hur等（2009）的方法（Influenceofinitialemulsifiertypeonmicrostructuralchangesoccurringinemulsifiedlipidsduringinvitrodigestion）

Table1

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

9mlCaCl2·2H2O22.2g/l

10mlCaCl2·2H2O22.2g/l

15mguricacid

2.5gpepsin

1gBSA

1.8gBSA

25mgmucin

3gmucin

9gpancreatin

30gbile

1.5glipase

pH

6.8±0.2

1.30±0.02

8.1±0.2

8.2±0.2

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）at60ºCinashakerwaterbathfor30min.ThesampledispersionwasadjustedtopH4.4–4.6byaddingHClandthenhydrolyzedwithamyloglucosidase（300U,Sigmachemical,Cat.No.A9913）at60ºCinashakerwater-bathfor30min.Thedigestedsamplewasfilteredthroughacelitelayerinacrucibleandwashedtwicewith15mlof78%ethanol,twicewith15mlof100%ethanolandrinsedwith15mlofacetone.Theremainingsamplewasdriedinanovenat100ºCovernight.Theresistantstarchcontentwascalculatedusingtheequation:

（%）RScontent=Remainingsampleweight（g,dsb）/initialsampleweight（g,dsb）×100%

ForRSdeterminedusingEnglyst’smethod（1992）,Starch（1.000g,db）in20mLofsodiumacetatebuffer（0.1M,pH5.2）wascookedinaboilingwater-bathfor30min.Thestarchdispersionwascooleddownto37ºC,mixedwithanenzymesolution（5mL）consistingofpancreatinextractandamyloglucosidase,andincubatedinawater-bathat37ºC.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）