高效液相色譜法測定19個(gè)鷹嘴豆品種中寡糖的含量Abstract

Theextractionprocedureofoligosaccharidesfromchickpeaseedswasoptimizedandsufficientextractionwasachievedwith50%ofethanol-waterinaratioof10:1forsolventtodefattedchickpeamealat50°Cfor30min.Undertheoptimalextractionconditions,thecontentsofoligosaccharidesin19cultivarsofchickpeaseedsweredeterminedbyHPLC.Theresultsshowedthatthemainα-galactooligosaccharides(α-GOS)inchickpeaseedswereraffinose,stachyose,verbascoseandanunknownglycoside,whichwasisolated,purified,andidentifiedasciceritol.Ciceritol,themainsugarinallthechickpeasamples,accountedforabout50%ofthetotalα-GOS.

Therewereconsiderablevariationsinthelevelsofα-GOSandsucrosebetweenspecies,especiallyverbascosewhichcouldonlybedetectedin7samples.Withthehighestamountofα-GOSandalowamountofsucrose,the171wasthebestchoiceforobtainingα-GOSforuseasaprebioticinfunctionalfoods.Keywords:Chickpea(CicerarietinumL)seed;α-Galactooligosaccharide;Sucrose;Optimization;Extractionmethod;HPLC通過實(shí)驗(yàn)對鷹嘴豆種子中寡糖的提取進(jìn)行優(yōu)化，最終確定了寡糖的提取條件為石油醚脫脂，料液比1：10，提取溶劑為50%乙醇，50℃水浴加熱30分鐘，并運(yùn)用HPLC對19個(gè)鷹嘴豆品種中低聚糖的含量進(jìn)行了測定。結(jié)果表明：鷹嘴豆種子中主要含有的α-低聚半乳糖有棉子糖（三糖）,水蘇糖（四糖），毛蕊花糖（五糖），和一個(gè)未知的糖，并對其進(jìn)行了分離，鑒定，確定為Ciceritol。并且Ciceritol是鷹嘴豆中含量最多的低聚糖，約占α-GOS的50%。α-GOS和蔗糖（二糖）的含量在不同品種間存在較大差異，尤其是毛蕊花糖，只在7個(gè)品種中檢測到。通過含量測定，找到了19個(gè)鷹嘴豆品種中，171鷹嘴豆中α-低聚半乳糖的含量最高，同時(shí)蔗糖含量相對較低，是制作功能性食品的最佳選擇。Abstract

EthanolconcentrationTempera-tureExtractiontimeRatio(m/v)10%20℃15min1：5230%50℃30min1：10350%70℃60min1：15470%90℃1：20580%

Opitimizationofextractionconditionsforoligosachharides寡糖的提取與制備1.先將種子在85℃下干燥2小時(shí)，粉碎過6號篩。2.取500g干粉，用2L石油醚脫脂，得到脫脂鷹嘴豆。3.取脫脂鷹嘴豆，一式三份，每份1g，用10ml50%乙醇50℃溫浸3次，每次30min，合并上清，減壓濃縮成浸膏。4.浸膏復(fù)溶于5.0ml的流動相(乙腈:水=75:25)Fig.1Chromatographicprofileoffreesugarsinextractionofchickpeaseeds.1,Ribose;2,Fructose;3,Sucrose;4,Unknowncompound;5,Maltose;6,Raffinose;7,Ciceritol;8,Stachyose;9,Vebascose.Agilent1100seriesSugar-DColum(4.6*250mm)Ace:Water=75:25Flowrate=1ml/minInjvolume=20ul核糖果糖蔗糖(二糖）未知麥芽糖Ciceritol（三糖）水蘇糖（四糖）毛蕊花糖（五糖）棉子糖（三糖）ExtractionandIdentificationofCiceritol

Theextractofchickpeaseedswasloadedontoacolumn(3×50cm)ofcharcoal–Celite(1:1,w/w)andsuccessivelyelutedwithwaterandincreasingconcentrationsofethanol(5–30%).ThefractionswerescreenedbyreactionwithnaphthoresorcinolandthosewithsugarswerefurtherdetectedbyHPLC.Thefractionscontainingciceritolwerecombined,concentratedinvacuo,andpurifiedbygelpermeationonacolumn(1.5×90cm)ofBiogelP-2.Degassedwaterwasusedastheeluentataflowrateof20ml/h.Thefractionscontainingpureciceritol,analyzedbyHPLC,werecombined,concentrated,andfreeze-dried.ThestructurewasconfirmedbyESI-TOF-MS(AppliedBiosystemsmassspectrometer)andNMR(BrukerDRX500)withD2Oasthesolvent.該級分通過間二萘酚反應(yīng)進(jìn)行篩選并用HPLC進(jìn)一步檢測。將含ciceritol的餾分進(jìn)行合并，減壓濃縮，并通過BiogelP-2凝膠色譜柱（1.5×90厘米）進(jìn)行純化，并以20ml/h的流速用脫氣水進(jìn)行洗脫。通過HPLC分析該級分，合并，濃縮，冷凍干燥得到純的Ciceritol。其結(jié)構(gòu)經(jīng)ESI-TOF-MS儀（AppliedBiosystems質(zhì)譜儀）和NMR（Bruker公司DRX500）進(jìn)行鑒定。將鷹嘴豆種子的提取物裝載到木炭硅藻土柱（3×50厘米）上，并依次用水及濃度不斷增加的乙醇（5-30％）進(jìn)行梯度洗脫。Fig.2Thechemicalstructureofciceritol.α-D-吡喃半乳糖基–(1→6)?-α-D-吡喃半乳糖基–(1→2)-4-O-甲基-手性肌醇O-α-

D-galactopyranosyl-(1→6)-O-α-D-galatopyranosyl-(1→2)-1D-4-O-

methyl-chiro-inositol123456123456123456被甲基化的肌醇（環(huán)己六醇）M/Z

541[M+的Na]

+=518α-1,6糖苷鍵Fig.3Theeffectsofextractionconditionsonthecontentsofα-GOSandsucroseinachickpeaseeds.Concentrationsof