戰(zhàn)榮榮，沐萬(wàn)孟，李赟高，張 濤，江 波*

（江南大學(xué)，食品科學(xué)與技術(shù)國(guó)家重點(diǎn)實(shí)驗(yàn)室，江蘇 無(wú)錫 214122）

菊粉的酶法生物轉(zhuǎn)化在食品中應(yīng)用的研究進(jìn)展

戰(zhàn)榮榮，沐萬(wàn)孟，李赟高，張 濤，江 波*

（江南大學(xué)，食品科學(xué)與技術(shù)國(guó)家重點(diǎn)實(shí)驗(yàn)室，江蘇 無(wú)錫 214122）

菊粉是我國(guó)分布廣泛、含量豐富的重要農(nóng)副產(chǎn)品，利用菊粉酶對(duì)其進(jìn)行生物深加工可高效實(shí)現(xiàn)菊粉在食品領(lǐng)域的低成本、多方向、高附加值利用。本文綜述國(guó)內(nèi)外菊粉應(yīng)用于食品的酶法研究開發(fā)現(xiàn)狀，對(duì)不同類型菊粉酶生物轉(zhuǎn)化產(chǎn)品的性質(zhì)及食品應(yīng)用、酶的微生物來(lái)源及菊粉酶的最新研究進(jìn)展進(jìn)行闡述。

內(nèi)切型菊粉酶；外切型菊粉酶；菊糖果糖轉(zhuǎn)移酶；低聚果糖；高果糖漿；雙果糖酐

菊粉，又稱菊糖，是D-呋喃果糖以β-2,1-糖苷鍵連結(jié)而成的多聚果糖，其還原端連接一個(gè)葡萄糖基，呈直鏈結(jié)構(gòu)，聚合度一般在30左右。菊粉是我國(guó)種植廣泛的重要農(nóng)副產(chǎn)品，廣泛存在于菊芋、菊苣、牛蒡、大麗花等多種植物的根莖中[1]。菊粉作為一種膳食纖維，由于具有類似脂肪的質(zhì)構(gòu)與口感，被長(zhǎng)期作為簡(jiǎn)單的食品配料應(yīng)用于食品加工[2]。然而，菊粉存在甜度低、水溶性較差等問(wèn)題，進(jìn)行更深層次、高附加值的生物產(chǎn)品開發(fā)成為充分利用我國(guó)豐富菊粉資源的新方向，也逐漸成為食品領(lǐng)域研究的熱點(diǎn)。

利用微生物發(fā)酵或酶解等現(xiàn)代生物轉(zhuǎn)化方法，菊粉有望實(shí)現(xiàn)多種形勢(shì)的高附加值產(chǎn)品轉(zhuǎn)化[3]。菊粉通過(guò)發(fā)酵策略可獲得單細(xì)胞蛋白[4]，檸檬酸[5]，生物燃料如生物乙醇[6]、單細(xì)胞油[7]，化學(xué)產(chǎn)品如丁二醇[8]、乳酸[9]及糖醇[10]等。菊粉高附加值酶法利用主要應(yīng)用于食品領(lǐng)域，是借助生物菊粉酶生產(chǎn)食品行業(yè)重要的糖類或功能性糖類，其產(chǎn)物屬于天然產(chǎn)品，具有適合現(xiàn)代人們對(duì)食品安全性、健康性及功能性等需求的特點(diǎn)，逐漸成為菊粉生物轉(zhuǎn)化應(yīng)用極其重要的方向。菊粉酶是菊粉酶法生物轉(zhuǎn)化的媒介，為糖酐水解酶32家族（glycoside hydrolase family 32，GH32）酶類，按其水解菊粉的方式分為內(nèi)切型菊粉酶、外切型菊粉酶及菊糖果糖轉(zhuǎn)移酶3種類型[11]，它們是一類作用于菊糖β-2,1-糖苷鍵的水解酶，可將菊糖水解為不同的單糖或低聚糖?，F(xiàn)階段發(fā)現(xiàn)的微生物菊粉酶多來(lái)源于霉菌、酵母菌和部分細(xì)菌[12]，分布于胞外、胞內(nèi)及胞壁中，各種微生物的產(chǎn)酶類型、酶活性及性質(zhì)存在較大差異，且以產(chǎn)外切型菊粉酶微生物最多[13]，更有微生物可以同時(shí)產(chǎn)多種類型菊粉酶。菊粉酶通常以I/S的大小來(lái)區(qū)分內(nèi)切型菊粉酶和外切型菊粉酶，I、S分別是以菊粉、蔗糖作為底物時(shí)的酶活力，一般認(rèn)為當(dāng)I/S＜10時(shí)為外切菊粉酶，I/S≥10時(shí)為內(nèi)切菊粉酶[14]，而菊糖果糖轉(zhuǎn)移酶通常利用高效液相色譜對(duì)酶反應(yīng)產(chǎn)物雙果糖酐進(jìn)行檢測(cè)鑒定[11]。菊粉酶的純化手段一般結(jié)合超濾、硫酸銨分段沉淀、離子交換和Sephadex凝膠過(guò)濾等，并利用HPLC進(jìn)行產(chǎn)物分析。對(duì)于內(nèi)、外切混合酶的分離一般采用NaCl線性梯度洗脫進(jìn)行[15]。利用不同類型的菊粉作底物，菊糖內(nèi)切型、外切型及轉(zhuǎn)移酶的主要轉(zhuǎn)化產(chǎn)物分別為低聚果糖、果糖和雙果糖酐，并逐漸成為這些糖工業(yè)化生產(chǎn)的重要方式。需要指出的是，新型菊粉酶作為利用菊粉進(jìn)行功能性糖——雙果糖酐Ⅲ生產(chǎn)的重要方式，為菊粉在食品領(lǐng)域的應(yīng)用提供最新研究方向，也為菊粉的酶學(xué)生物轉(zhuǎn)化提供更廣闊的開發(fā)空間，如下將以不同類型菊粉酶對(duì)菊粉在國(guó)內(nèi)外食品領(lǐng)域的酶法研究及應(yīng)用進(jìn)展進(jìn)行綜述。

1 內(nèi)切型菊粉酶——低聚果糖

內(nèi)切型菊粉酶可隨機(jī)斷開菊粉鏈內(nèi)部的β-1,2-糖苷鍵，主要水解產(chǎn)物為低聚果糖，是菊粉食品應(yīng)用中已商業(yè)化的重要開發(fā)形式，成為低聚果糖工業(yè)化生產(chǎn)的重要工藝。從20世紀(jì)80年代初日本及歐美等發(fā)達(dá)國(guó)家開始利用微生物菊粉酶水解菊芋制取果糖，到目前對(duì)菊粉低聚糖生產(chǎn)的研究仍然十分火熱。

1.1 低聚果糖的性質(zhì)及食品應(yīng)用

低聚果糖（inulooligosaccharides，IOS）為由2～10個(gè)果糖組成的低聚化合物，又稱果寡糖。IOS具有抗腫瘤和刺激雙歧桿菌生長(zhǎng)的作用，也可作為膳食纖維用于防治糖尿病和減肥，還可用于改善腸胃功能降低血脂和預(yù)防高膽固醇等。在食品中具有廣泛應(yīng)用，常被用于糖果、水果制品、牛奶制品、酸奶、鮮奶酪、烘焙食品、冰激凌和調(diào)味料等[16]。

1.2 IOS生產(chǎn)現(xiàn)狀及內(nèi)切型菊粉酶生產(chǎn)IOS

利用微生物產(chǎn)內(nèi)切型菊粉酶作用于菊粉可獲得較高純度的IOS，其副產(chǎn)物僅有少量果糖，逐漸取代以蔗糖為原料利用果糖基轉(zhuǎn)移酶酶解制備低聚果糖（fructose oligosaccharides，F(xiàn)OS）的工藝，并成為現(xiàn)階段IOS工業(yè)化生產(chǎn)的重要工藝。菊粉IOS的優(yōu)良酶法生產(chǎn)工藝首先對(duì)微生物具有較高要求，是微生物產(chǎn)內(nèi)切型菊粉酶在無(wú)外切酶及轉(zhuǎn)移酶存在時(shí)對(duì)菊粉進(jìn)行的轉(zhuǎn)化方式，因此優(yōu)良的內(nèi)切型菊粉酶微生物資源對(duì)IOS的生產(chǎn)具有重要意義。能產(chǎn)生內(nèi)切型菊粉酶的微生物有真菌如青霉屬、曲霉屬，酵母如克魯威屬、念珠菌屬，細(xì)菌如桿菌屬、假單胞菌屬等[17]，如表1所示。目前為止不同來(lái)源的菊粉資源，利用內(nèi)切型菊粉酶固定化及現(xiàn)代分離純化手段可實(shí)現(xiàn)70%～95%的IOS轉(zhuǎn)化率。Nguyen等[18]通過(guò)殼聚糖固定化菊粉內(nèi)切酶實(shí)現(xiàn)了菊芋汁固定床反應(yīng)器的持續(xù)性發(fā)酵，其固定化酶的半衰期達(dá)48 d，IOS產(chǎn)率為66%。此外，菊粉底物的反應(yīng)形式對(duì)IOS產(chǎn)量也有較為重要的影響。Jin Zhenyu等[28]發(fā)現(xiàn)，A. ficuum菌株所產(chǎn)菊粉內(nèi)切酶利用菊芋汁的能力優(yōu)于菊芋粉，可使IOS產(chǎn)量由50%增至80%。

表1 生產(chǎn)IOS的內(nèi)切菊粉酶微生物來(lái)源Table 1 Biochemical properties of endo-inulinase derived from various microorganniissmmss

1.3 內(nèi)切型菊粉酶的分子水平研究現(xiàn)狀

圖1 A.ficuum菌株菊粉內(nèi)切酶INU2的晶體結(jié)構(gòu)[[3344Fig.1 The crystal structure of endo-inulinase INU2 from A. fi cuum[34]

野生型微生物菊粉內(nèi)切酶產(chǎn)量及酶活力等都相對(duì)較低，單一產(chǎn)內(nèi)切型菊粉酶的微生物較少，使得酶制劑的純化工藝較難、生產(chǎn)成本較高，借助現(xiàn)代分子生物學(xué)技術(shù)構(gòu)建因工程菌株、通過(guò)深入了解酶的結(jié)構(gòu)及作用機(jī)制進(jìn)行分子水平的基因改造等成為解決這一問(wèn)題的有效途徑，也成為現(xiàn)在內(nèi)切型菊粉酶的研究重心和熱點(diǎn)。Yun等[32]將Pseudomonas sp.內(nèi)切菊粉酶基因進(jìn)行原核表達(dá)使IOS的產(chǎn)率為78%，此后利用聚苯乙烯固定化及生物反應(yīng)器等技術(shù)使菊粉內(nèi)切酶50℃穩(wěn)定工作17 d，IOS的產(chǎn)量達(dá)150 g/（L·h）。Kim等[33]先后探索了Arthrobacter sp.S37菌株菊粉內(nèi)切酶基因EnIA的作用機(jī)制，E323、E519及D460是活性中心的關(guān)鍵氨基酸，E323及E519為親核基團(tuán)，而D460為酶催化的必需基團(tuán)，并嚴(yán)重影響該酶的pH值特性。2012年P(guān)ouyez[34]首次將內(nèi)切型菊粉酶INU2（Aspergillus ficuum）晶體結(jié)構(gòu)成功解析，如圖1所示，其晶體結(jié)構(gòu)的催化中心比其他GH32水解酶類多一個(gè)催化反應(yīng)口袋，這個(gè)口袋有兩個(gè)loop環(huán)和一段W-M（I）-N-D（E）-P-N-G保守序列構(gòu)成，這個(gè)額外的反應(yīng)口袋可能是形成內(nèi)切菊粉酶活性的關(guān)鍵，也可有效解釋Trp40對(duì)其酶活具有重要作用及菊粉底物裂解機(jī)制。

2 外切型菊粉酶——高果糖漿

外切菊粉酶可作用于菊粉鏈非還原性末端的糖苷鍵，逐一水解釋放出果糖，是工業(yè)化生產(chǎn)高果糖漿的重要生產(chǎn)方式；高果糖漿（high-fructose syrup，HFS）是果糖含量高達(dá)90%的第3代果葡糖漿產(chǎn)品，目前在美、日等國(guó)，果葡糖漿已成為最重要的甜味劑之一，并且其生產(chǎn)發(fā)展勢(shì)頭強(qiáng)勁，已越來(lái)越受到國(guó)內(nèi)外的重視與歡迎[35]。

2.1 HFS的性質(zhì)及食品應(yīng)用

果糖是一種天然營(yíng)養(yǎng)甜味劑，甜度為蔗糖的1.8倍，為山梨醇的1.5倍，其甜度高、熱值低、具有類似蜂蜜的良好風(fēng)味、結(jié)構(gòu)組成等類似于蔗糖等特性使其成為優(yōu)秀的果汁甜味劑和低熱值食品原料。果葡糖漿是工業(yè)化生成果糖時(shí)的混合產(chǎn)物，因生產(chǎn)原料及工藝方法不同果糖含量在40%～90%，此外還含有葡萄糖及低聚糖等成分。HFS作為高濃度果糖類強(qiáng)化甜味劑，具有高滲透壓、強(qiáng)保濕性、高發(fā)酵性、低冰點(diǎn)、甜味純正和保健性等多種特性，被廣泛應(yīng)用于食品焙烤、飲料制品和食品配料等食品領(lǐng)域，成為蔗糖極具潛力的替代性糖產(chǎn)品之一。

2.2 HFS生產(chǎn)現(xiàn)狀及外切型菊粉酶生產(chǎn)HFS

HFS的制備策略較多，主要包括以菊糖、玉米淀粉、葡萄糖和蔗糖為原料的酶法和化學(xué)法。美國(guó)、日本和我國(guó)HFS的傳統(tǒng)工業(yè)化制備工藝是以玉米淀粉為原料利用異構(gòu)酶酶法制備，然而該生產(chǎn)工藝復(fù)雜、生產(chǎn)成本也較高，成為HFS工業(yè)化生產(chǎn)及食品應(yīng)用的瓶頸問(wèn)題。20世紀(jì)70年代以后，各國(guó)開始關(guān)注以菊粉為原料，酸法和酶法水解制備果糖和高果糖漿的生產(chǎn)工藝。其中酸法制備高果糖漿果糖純度較高，但副產(chǎn)物多、色素重、分離精制難，依然不能滿足HFS的食品應(yīng)用需求。而以菊粉為原料的酶法生產(chǎn)工藝為HFS的工業(yè)化生產(chǎn)及食品應(yīng)用帶來(lái)曙光。依靠外切型菊粉酶的HFS生產(chǎn)被稱為一步法催化反應(yīng)，它以其獨(dú)特的工藝簡(jiǎn)單、成本低、得率高而備受青睞，逐漸成為國(guó)外HFS的主流生產(chǎn)工藝，外切型菊粉酶微生物來(lái)源如表2所示。Sirisansaneeyakul等[51]利用A. niger TISTR 3570（可產(chǎn)內(nèi)切型及外切型菊粉酶）和Candida guilliermondii TISTR 5844菌株（僅產(chǎn)外切型菊粉酶）的菊粉酶混合物降解菊粉，25 h獲得18.2 g/L的果糖產(chǎn)品。Singh等[36]利用K. marxianus YS-1分泌的菊粉內(nèi)切酶固定化于杜奧萊A568后利用菊粉生產(chǎn)HFS，結(jié)果表明菊粉的純度對(duì)HFS的產(chǎn)量影響不大，4 h產(chǎn)量達(dá)40 g/L左右。

表2 生產(chǎn)HPS的外切菊粉酶微生物來(lái)源Table 2 Biochemical properties of exo-inulinase derived from various microorganniissmmss

2.3 外切型菊粉酶的分子水平研究現(xiàn)狀

隨著菊粉外切酶的工業(yè)化發(fā)酵工藝日趨成熟，逐漸迎來(lái)了菊粉外切酶分子及原子水平的研究及機(jī)理研究的高潮。菊粉外切酶的基因INU1首次于1991年由Laloux等[55]從Kluyveromyces marxianus中克隆獲得，此后Gao Wei[53]、Liu Bin等[56]又從Bacillus smithii T7中成功克隆INU1基因，并利用化學(xué)修飾研究證實(shí)其酶的活性中心必定包含2個(gè)Trp和一個(gè)His殘基，組氨酸是酶與底物結(jié)合的關(guān)鍵位點(diǎn)，而色氨酸對(duì)其熱穩(wěn)定性有重要的作用。此后Cao Tianshu等[54]又克隆獲得Cryptococcus aureus HYA的INU1基因，并利用pPICZαA載體在畢赤酵母X-33中成功表達(dá)。Emanuele等[52]利用動(dòng)力學(xué)對(duì)其作用機(jī)制進(jìn)行了研究，以工業(yè)化需求對(duì)進(jìn)行動(dòng)力學(xué)模型的溫度及底物濃度進(jìn)行定義（分別為40～60℃和3～60 g/L），獲得產(chǎn)物果糖和菊糖底物的化學(xué)計(jì)量關(guān)系。目前，外切菊粉酶晶體結(jié)構(gòu)已獲得解析，如Aspergillus awamori[57]及Geobacillus srearothermophilus SK1289p[47]。Aspergillus awamori菊粉外切酶是一種可連接5個(gè)寡糖的糖蛋白酶，如圖2所示，其催化區(qū)三級(jí)結(jié)構(gòu)折疊為兩個(gè)區(qū)域：獨(dú)特的5片β-propeller折疊構(gòu)成的N端催化區(qū)和折疊為β-sandwich結(jié)構(gòu)的C端催化區(qū)，從催化中心側(cè)鏈殘基距離推測(cè)該酶遵循雙位移機(jī)制，Asp41及Glu241分別作為親核基團(tuán)和催化基團(tuán)，而Asp189通過(guò)氫鍵與底物作用，是重要的底物識(shí)別基團(tuán)。

圖2 Aspergillus awamori 菊粉外切酶二級(jí)晶體結(jié)構(gòu)[[5577Fig.2 The secondary structure elements of exo-inulinase from Aspergillus awamori[57]

3 菊糖果糖轉(zhuǎn)移酶——雙果糖酐Ⅲ

菊糖果糖轉(zhuǎn)移酶（inulin fructotransferase，IFTase）又稱新型菊糖酶，是一種新型的菊粉水解酶類，可從菊糖非還原末端以相鄰2個(gè)果糖基為單位水解菊糖糖苷鍵，同時(shí)伴隨分子內(nèi)轉(zhuǎn)果糖基反應(yīng)產(chǎn)生雙果糖酐。利用IFTase生產(chǎn)雙果糖酐的研究主要集中在日本和韓國(guó)，作為菊粉在食品應(yīng)用的新型轉(zhuǎn)化方式，現(xiàn)階段主要應(yīng)用于生物轉(zhuǎn)化開發(fā)雙果糖酐Ⅲ（difructose anhydride Ⅲ，DFAⅢ），目前已有日本甜菜制糖株式會(huì)社2009年采用北海道大學(xué)Arthrobacter sp. H65-7發(fā)酵產(chǎn)生IFTase[58]，已實(shí)現(xiàn)工業(yè)化生產(chǎn)DFAⅢ。然而，目前國(guó)內(nèi)的研究?jī)H本課題組趙萌[11]篩選獲得高產(chǎn)新菌株，并實(shí)現(xiàn)高效原核表達(dá)。隨后，杭華[59-61]通過(guò)中試擴(kuò)大化酶膜反應(yīng)器利用IFTase進(jìn)行DFAⅢ的應(yīng)用性生產(chǎn)研究，實(shí)現(xiàn)400 g/L DFAⅢ的超高濃度生產(chǎn)，為利用菊粉工業(yè)化生產(chǎn)DFAⅢ提供了極為重要的探索性研究。

3.1 DFAⅢ性質(zhì)、食品應(yīng)用及IFTase酶法生產(chǎn)

雙果糖酐又稱二果糖二酐，是由兩個(gè)果糖基組成的一類環(huán)狀二糖，其中兩個(gè)殘基的還原性末端和相對(duì)殘基的非還原性羥基連接，存在16種同分異構(gòu)體[62]。通過(guò)微生物菊粉酶中的菊糖果糖轉(zhuǎn)移酶轉(zhuǎn)化菊糖可以合成雙果糖酐Ⅰ[63]、Ⅲ，而其他同分異構(gòu)體至今仍難以獲得。到目前為止，發(fā)現(xiàn)產(chǎn)雙果糖酐I的菌株仍然較少，國(guó)際產(chǎn)IFTase的菌株主要集中在日本和韓國(guó)。日本食品國(guó)家研究所Haraguchi先后發(fā)現(xiàn)4株產(chǎn)IFTase菌株可用于生產(chǎn)DFAⅢ[80,82,84]，而現(xiàn)階段最高酶活力是由江南大學(xué)Zhao Meng等[64]發(fā)現(xiàn)的A. aurescens SK8.001菌株經(jīng)原核表達(dá)實(shí)現(xiàn)，該菌株可實(shí)現(xiàn)胞內(nèi)胞外同時(shí)產(chǎn)酶，最高胞內(nèi)酶活力達(dá)119 U/mL，胞外酶活力也高達(dá)81 U/mL，這一研究成果有望為國(guó)內(nèi)外深化利用IFTase進(jìn)行菊糖的DFAIII工業(yè)化生產(chǎn)提供可能。

DFAⅢ是近年來(lái)人們發(fā)現(xiàn)的一種新型天然功能性甜味劑，具有促進(jìn)Ca、Fe、Mg、Zn、Cu等礦物質(zhì)元素的吸收、增進(jìn)骨骼生長(zhǎng)[65]、利于排尿、改善便秘[66]、預(yù)防結(jié)腸直腸癌[67]及抑制蛀牙[68]等功能，同時(shí)其甜度較高、能量值低，具有良好的耐熱、耐酸等理化特性，具有成為傳統(tǒng)甜味劑蔗糖替代品的潛能。作為無(wú)糖或低糖食品的關(guān)鍵配料，適用于焙烤食品、飲料、糖果等食品領(lǐng)域，促使其逐漸成為菊粉資源食品開發(fā)的重點(diǎn)和熱點(diǎn)方向。

3.2 IFTase生產(chǎn)DFAⅢ的微生物來(lái)源及分子水平研究現(xiàn)狀

利用菊粉酶法合成DFAⅢ是利用微生物IFTase水解菊粉獲得以DFAⅢ為主要產(chǎn)物的酶解策略，符合現(xiàn)代人們對(duì)食品領(lǐng)域天然產(chǎn)品的追求，也可有效實(shí)現(xiàn)菊粉的高附加值生物轉(zhuǎn)化。此外，利用菊粉還可以通過(guò)化學(xué)合成實(shí)現(xiàn)DFAⅢ的制備（或利用果糖、低聚果糖、果糖多糖原料等[69-70]），然而化學(xué)合成過(guò)程會(huì)產(chǎn)生多種雙果糖酐，且組分復(fù)雜，使DFAⅢ的分離純化困難，產(chǎn)量較低。而酶法合成DFAⅢ的副產(chǎn)物只有少量低聚果糖，可全面有效的解決化學(xué)法合成DFAⅢ所存在的問(wèn)題，成為利用資源豐富的菊粉資源進(jìn)行DFAⅢ工業(yè)化生產(chǎn)的最佳途徑，同時(shí)也對(duì)食品行業(yè)新糖源的開發(fā)具有重要意義。

酶解菊粉生產(chǎn)DFAⅢ的轉(zhuǎn)化效率與IFTase的酶學(xué)性質(zhì)有直接關(guān)系。不同菌株來(lái)源的IFTase的基因序列具有高度相似性，但酶活力、最適作用條件、分子質(zhì)量等具有較大差異，表3給出了迄今為止發(fā)現(xiàn)的所有可產(chǎn)DFAⅢ的微生物IFTase性質(zhì)。自1972年Tanaka[54]首次于產(chǎn)脲節(jié)桿菌中發(fā)酵生產(chǎn)新型菊粉酶可將菊糖降解為DFAⅢ和少量的低聚果糖以來(lái)，此后陸續(xù)有13種產(chǎn)DFAⅢ的野生型菌株被報(bào)道，至今只有Jung等[71]解析了IFTase三聚體晶體結(jié)構(gòu)，如圖3所示，并提出了分子內(nèi)果糖基轉(zhuǎn)移反應(yīng)機(jī)制，如圖4所示，IFTase三聚體結(jié)構(gòu)形成僅可容納一個(gè)二糖的底物結(jié)合口袋，為酶催化的前提條件，其活性位點(diǎn)分布在單體間的界面，并有6個(gè)直接的作用點(diǎn)，4個(gè)位于F1，2個(gè)位于F2，果糖基F1上的O-1’分別與Arg292、Ser133側(cè)鏈結(jié)合，O-3’與Glu244羧基結(jié)合，O-’與Pro291結(jié)合；Asp233*位于另一個(gè)亞基上，Asp233*同時(shí)與果糖基F2上的O-3、O-4結(jié)合；Arg174*胍基與Asp233*羰基相互作用，可穩(wěn)定Asp233*的定位；另外果糖基F1 O-4'及果糖基F2 O-6分別通過(guò)溶劑介導(dǎo)的氫鍵與Tyr197、Gln313結(jié)合。末端果糖基F1及相鄰果糖基F2在催化過(guò)程中分別作為電子供體、電子受體；Glu244的羧基進(jìn)攻F1上的3-OH使其去質(zhì)子化，從而激活受體果糖基F2；Asp233通過(guò)氫鍵為F2定位，使得去質(zhì)子化的F1 3-O更有效地親核進(jìn)攻F2 2-C，最終使得果二糖從菊糖上脫離。而其他可能存在的作用機(jī)理如活性位點(diǎn)殘基的鑒定及作用機(jī)制仍不夠清晰，這表明IFTase利用菊粉進(jìn)行DFAⅢ的研究仍任重道遠(yuǎn)，許多基礎(chǔ)性研究仍亟待開展。

表3 利用菊粉生產(chǎn)DFAⅢ的IFTase的性質(zhì)及微生物來(lái)源Table 3 Biochemical and enzymatic properties of inulin fructotransferases (producing DFAⅢ）from various microorganisms

圖3 Bacillus sp. 菊糖果糖轉(zhuǎn)移酶的亞基結(jié)構(gòu)（A）與晶體結(jié)構(gòu)（B）Fig.3 Subunit structure (A) and crystal structure (B) of inulin fructotransferase from Bacillus sp

圖4 4Bacillus sp. 菊糖果糖轉(zhuǎn)移酶分子內(nèi)果糖基轉(zhuǎn)移反應(yīng)示意Fig.4 Proposed catalytic mechanisms of inulin fructotransferase from Bacillus sp

此外，一些微生物IFTase具有降解菊糖為DFAI的活性，如Arthrobacter golobiformis S14-3所產(chǎn)IFTase（可產(chǎn)DFAIII）為胞外酶，且以單聚體形式存在，與產(chǎn)DFAⅢ的IFTase具有較高同源性[72]。DFAⅠ作為非還原性糖，甜度為蔗糖的一半，是一種低熱量糖，現(xiàn)階段研究較少，也可作為食品領(lǐng)域菊粉酶法轉(zhuǎn)化的應(yīng)用方向。

4 展 望

作為可再生資源的菊粉在食品應(yīng)用中的高附加值開發(fā)應(yīng)用研究仍處于初級(jí)階段。隨著不同微生物菊粉酶的開發(fā)，在食品中具有重要功能的IOS益生元和HFS甜味劑產(chǎn)品的研究已有較好基礎(chǔ)，工業(yè)化應(yīng)用也日趨成熟，新型菊粉酶應(yīng)用于功能性甜味劑DFAⅢ的生產(chǎn)作為新的菊粉開發(fā)形式，進(jìn)一步拓寬了菊粉在食品領(lǐng)域的應(yīng)用，這也有望成為食品研究的新熱點(diǎn)。此外，為實(shí)現(xiàn)菊粉在食品中的酶法高效轉(zhuǎn)化，除卻各種菊粉酶菌株的繼續(xù)發(fā)掘外，以提高酶學(xué)性質(zhì)及產(chǎn)量為目的的各種現(xiàn)有野生型菌株的菊粉酶結(jié)構(gòu)、作用機(jī)理及分子水平的基因改造成為新的研究熱點(diǎn)，這也必將為IOS、HFS及DFAⅢ等食品糖類的規(guī)?；a(chǎn)提供更深入的理論支撐。而利用超濾或納濾等現(xiàn)代酶膜反應(yīng)器[85]進(jìn)行新的酶底物反應(yīng)模式研究，以及固定化技術(shù)、現(xiàn)代純化手段等可以有效增進(jìn)產(chǎn)物轉(zhuǎn)化效率、降低能耗，也有望成為酶制劑的重要發(fā)展方向，這也必將對(duì)食品領(lǐng)域菊粉的酶法生物轉(zhuǎn)化提供更為廣闊的前景和發(fā)展空間。

[1] PANDEY A, SOCCOL C R, SELVAKUMAR P, et al. Recent developments in microbial inulinases, its production, properties and industrial applications[J]. Applied Biochemistry and Biotechnology, 1999, 81: 35-52.

[2] 王珊珊, 孫愛(ài)東, 何洪巨. 菊粉的功能性作用及開發(fā)利用[J]. 中國(guó)食物與營(yíng)養(yǎng), 2009, 15(11): 57-59.

[3] CHI Zhenming, ZHANG Tong, CAO Tianshu, et al. Biotechnological potential of inulin for bioprocesses[J]. Bioresource Technology, 2011, 102: 4295-4303.

[4] ZHAO Chunhai, CHI Zhe, ZHANG Fang, et al. Direct conversion of inulin and extract of tubers of jerusalem artichoke into single cell oil by co-cultures of Rhodotorula mucilaginosa TJY15a and immobilized inulinase-producing yeast cells[J]. Bioresource Technology, 2011, 102(10): 6128-6133.

[5] LIU Xiaoyan, CHI Zhe, LIU Guanglei, et al. Inulin hydrolysis and citric acid production from inulin using the surface-engineered Yarrowia lipolytica displaying inulinase[J]. Metabolic Engineering, 2010, 12(5): 469-476.

[6] HU Nan, YUAN Bo, SUN Juan, et al. Thermotolerant Kluyveromyces marxianus and Saccharomyces cerevisiae strains representing protentials for bioethanol production from Jerusalem artichoke by consolidated bioprocessing[J]. Applied Microbiology and Biotechnology, 2012, 95(5): 1359-1368.

[7] ZHAO Chunhai, CUI Wei, LIU Xiaoyan, et al. Expression of inulinase gene in the oleaginous yeast Yarrowia lipolytica and single cell oil production from inulin-containing materials[J]. Metabolic Engineering, 2010, 12(6): 510-517.

[8] GAO Jian, XU Hong, LI Qiujie, et al. Optimization of medium for one-step fermentation of inulin extract from Jerusalem artichoke tubers using Paenibacillus polymyxa ZJ-9 to produce R,R-2,3-butanediol[J]. Bioresource Technology, 2010, 101(18): 7076-7082.

[9] CHOI H Y, RYU H K, PARK K M, et al. Direct lactic acid fermentation of Jerusalem artichoke tuber extract using Lactobacillus paracasei without acidic or enzymatic inulin hydrolysis[J]. Bioresource Technology, 2012, 114: 745-747.

[10] SAHA B C. Production of mannitol from inulin by simultaneous enzymatic saccharification and fermentation with Lactobacillus intermedius NRRL B-3693[J]. Enzyme Microbial Technology, 2006, 39: 991-995.

[11] 趙萌. 菊糖果糖轉(zhuǎn)移酶的菌種篩選、誘導(dǎo)合成、分離純化及克隆表達(dá)[D]. 無(wú)錫: 江南大學(xué), 2011.

[12] KANGO N, JAIN S C. Production and properties of microbial inulinases: recent advances[J]. Food Biotechnology, 2011, 25: 165-212.

[13] CABEZAS M J C, BRAVO R S, SHENE C. Inulin and sugar contents in Helianthus tuberosus and Cichorium intybus tubers: effect of postharvest storage temperature[J]. Journal of Food Science, 2002, 67: 2860-3865.

[14] 彭英云. Aspergillus fi cuum SK004產(chǎn)外切菊粉酶及其酶解菊粉制備高果糖漿的研究[D]. 無(wú)錫: 江南大學(xué), 2005.

[15] 華成偉, 王建華, 滕達(dá). 菊粉化學(xué)和微生物菊粉內(nèi)切酶研究進(jìn)展[J].中國(guó)食品學(xué)報(bào), 2004, 4(4): 103-108.

[16] KAUR N, GUPTA A K. Applications of inulin and oligofructose in health and nutrition[J]. Journal of Biosciences, 2002, 27: 703-714.

[17] CHI Zhengming, CHI Zhe, ZHANG Tong, et al. Inulinase expressing microorganisms and applications of inulinases[J]. Applied Microbiology and Biotechnology, 2009, 82(2): 211-220.

[18] NGUYEN Q D, REZESSY-SZABO J M, CZUKOR B. Continuous production of oligofructose syrup from Jerusalem artichoke juice by immobilized endo-inulinase[J]. Process Biochemistry, 2011, 46(1): 298-303.

[19] 白春陽(yáng), 蘇文金. 土曲霉金色變種AT8951菊粉酶的純化和性質(zhì)的研究[J]. 真菌學(xué)報(bào), 1994, 13(4): 282-289.

[20] YOKOTA A, TAMAUCHI O. Production of inulotriose from inulin by inulin-degrading enzyme from Strepttomyyces rochei E87[J]. Letters in Applied Microbiology, 1995, 21: 330-333.

[21] NAKAMURA T, OGATA Y, SHITARA A, et al. Continuous production of fructose syrups from inulin by immol/Lobi-lized inulinase from Aspergillus nigermutant 817[J]. Journal of Fermentation and Bioengineering, 1995, 80: 164-169.

[22] ONODERA S, MURAKAMI T, ITO H, et al. Molecular cloningand nucleotide sequences of cDNA and gene encoding endo-inulinuase from Penicillium purpurogenum[J]. Bioscience Biotechnology and Biochemistry, 1996, 60: 1780-1785.

[23] YUN J W, KIM D H, KIM B W, et al. Production of inulooligosaccharides from inulin by immobilized endoinulinase from Pseudomonas sp.[J]. Journal of Fermentation and Bioengineering, 1997, 84: 369-371.

[24] NAKAMURA T, SHITARA A, MATSUDA S. Production, purification and properties of an endo-inulinase of Penicillium sp. TN-88 that liberates inulinase[J]. Journal of Fermentation and Bioengineering, 1997, 84(4): 313-318.

[25] PARK J P, BAE J T, YOU D J, et al. Production of inulooligosaccharides from inulin by a novel endoinulinase from Xanthomonas sp.[J]. Biotechnology Letter, 1999, 21: 1043-1046.

[26] CHO Y J, SINHA J, PARK J P, et al. Production of inulooligosaccharides from chicory extract by endoinulinase from Xanthomonas oryzae No. 5[J]. Enzyme and Microbial Technology, 2001, 28: 439-445.

[27] JEON S J, YOU D J, KWON H J. Cloning and characterization of cycloinulooligosaccharide fructanotransferase (CFTase) from Bacillus polymyxa MGL21[J]. Journal of Microbiology and Biotechnology, 2002, 12(6): 921-928.

[28] JIN Zhengyu, WANG Jing, JIANG Bo, et al. Production of inulooligosaccharides by endoinulinases from Aspergillus fi cuum[J]. Food Research International, 2005, 38(3): 301-308.

[29] CHEN Hanqing, CHEN Xiaoming, LI Yin. Purification and characterization of exo-and endo-inulinase from Aapergillus ficuum JNSP5-06[J]. Food Chemistry, 2009, 115(4): 1206-1212.

[30] WANG L, HUANG Y, LONG X. Cloning of exoinulinase gene from Penicillium janthinellum strain B01 and its high-level expression in Pichia pastoris[J]. Journal of Applied Microbiology, 2011, 111(6): 1371-1380.

[31] YUAN Bo, HU Nan, SUN Juan. Purification and characterization of a novel extracellular inulinase from a new yeast species Candida kutaonensis sp. nov KRF1(T)[J]. Applied Microbiology and Biotechnology, 2012, 96(6): 1517-1526.

[32] YUN J W, CHOI Y J, SONG C H, et al. Microbial production of inulooligosaccharides by an endoinulinase from Pseudomonas sp. expressed in Escherichia coli[J]. Journal of Bioscience and Bioengineering, 1999, 81: 291-295.

[33] KIM K Y, NASCIMENTO A S, GOLUBEV A. Catalytic mechanism of inulinase from Arthrobacter sp. S37[J]. Biochemical and Biophysical Research Communications, 2008, 371(4): 600-605.

[34] POUYEZ J, MAYARD A, VANDAMME A M, et al. First crystal structure of an endo-inulinase, INU2, from Aspergillus ficuum: discovery of an extra-pocket in the catalytic domain responsible for its endo-activity[J]. Biochimie, 2012, 94: 2423-2430.

[35] 王建華, 徐長(zhǎng)警. 菊粉果糖的研究與開發(fā)[J]. 中國(guó)甜菜糖業(yè), 2004(4): 10-14.

[36] SINGH R S, DHALIWAL R, PURI M. Production of high fructose syrup from Asparagus inulin using immobilized exoinulinase from Kluyveromyces marxianus YS-1[J]. Journal of Industrial Microbiology and Biotechnology, 2007, 34: 649-655.

[37] GUIRAUD J P. Inulin hydrolysis by an immol/Lobilized yeast-cell reactor[J]. Enzyme and Microbial Technology, 1983, 5: 185-190.

[38] MUKHERJEE K, SENGUPTA S. Purification and properties of a nonspecific belta-fructofuranosidase (inulinase) from the mushroom Panaeolus papillonaceus[J]. Canadian Journal of Microbiology, 1987, 33: 520-524.

[39] ROUWENHHORST R J, HENSING M, VERBAKEL J, et al. Structure and properties of the extracellular inulinase of Kluyveromyces marxianus CBS6556[J]. Applied Environmental Microbiology, 1990, 56: 3337-3345.

[40] AZHARI R, ALADA M S, EHUD I, et al. Purification and characterization of endo and exo-inulinase[J]. Biotechnology and Applied Biochemistry, 1989, 11: 105-117.

[41] KIM C H, RHEEh S K. Fructose production from Jerusalem artichoke by inulinase immobilized on chitin[J]. Biotechnology Letters, 1989, 11: 201-206.

[42] ETTALIBI M, BARATTI J C. Molecular and kinetic properties of Aspergillus ficuuminulinases[J]. Agricultural and Biological Chemistry, 1990, 54(1): 61-68

[43] ONODERA S, SHIOMI. Purification and subsite affinities of exoinulinase from Penicillium trebinskii[J]. Bioscience Biotechnology and Biochemistry, 1992, 56: 1443-1447.

[44] NAKAMURA T, OGATA Y, SHITARA A, et al. Continuous production of fructose syrups from inulin by immobilized inulinase from Aspergillus niger mutant 817[J]. Journal of Fermentation and Bioengineering, 1995, 80: 164-169.

[45] SCHORR G S, FONTANA A GUIRAUD JP. Fructose syrups and ethanol production by selective fermentation of inulin[J]. Current Microbiology, 1995, 30: 325-330.

[46] 魏文鈴, 鄭中輝, 鄭志成, 等. 克魯維酵母Y-85合成菊粉酶最適條件的研究[J]. 微生物學(xué)報(bào), 1998, 38(3): 208-212.

[47] TSUJIMOTO Y, WATANABE A, NAKANO K. Gene cloning, expression, and crystallization of thermostable exo-inulinase from Geobacillus stearothermophilus KP1289[J]. Applied Microbiology and Biotechnology, 2003, 62(2/3): 180-185.

[48] KIM K Y, KOO B S, JO D. Cloning, expression, and purification of exoinulinase from Bacillus sp. sun-7[J]. Journal of Microbiology and Biotechnology, 2004, 14(2): 344-349.

[49] GILL P K, MANHAS R K, SINGH P. Comparative analysis of thermostability of extracellular inulinase activity from Aspergillus fumigatus with commercially available (Novozyme) inulinase[J]. Bioresource Technology, 2006, 97: 355-358.

[50] SINGH R S, DHALIWAL R, PURI M. Partial purification and characterization of exoinulinase from Kluyveromyces marxianus YS-1 for preparation of highfructose syrup[J]. Journal of Microbiology and Biotechnology, 2007, 17: 733-738.

[51] SIRISANSANEEYAKUL S, WORAWUTHIYANAN N, VANICHSRIRATANA W, et al. Production of fructose from inulin using mixed inulinases from Aspergillus niger and Candida guilliermondii[J]. World Journal of Microbiology and Biotechnology, 2007, 23: 543-552.

[52] EMANUELE R, VINCENZA C, STEFANO C. Fructose production by chicory inulin enzymatic hydrolysis: a kinetic study and reaction mechanism[J]. Process Biochemistry, 2009, 44: 466-470.

[53] GAO Wei, BAO Yongming, LIU Yang. Characterization of thermostable endoinulinase from a new strain Bacillus smthii T7[J]. Applied Biochemistry and Biotechnology, 2009, 157(3): 498-506.

[54] CAO Tianshu, WANG Guangyuan, CHI Zhen, et al. Cloning, characterization and heterelogous expression of the INU1 gene from Cryptococcus aureus HYA[J]. Gene, 2013, 516: 255-262.

[55] LALOUX O, CASSART J P, DELOUR J, et al. Cloning and sequencing of the inulinuase gene of Kluyveromyces marxianusvar. Marxianus ATCC 12424[J]. FEBS Letters, 1991, 289: 64-68.

[56] LIU Bin, WANG Jingyun, BAO Yongming. Characterization of key amino acid residues in active site of inulinase from Bacillus smithii T7 by chemical modification[J]. Chinese Journal of Catalysis, 2009, 30(7): 673-678.

[57] NAGEM R A P. ROJAS A L, GOLUBEV A M. Crystal structure of exo-inulinase from Aspergillus awamori: the enzyme fold and structural determinants of substrate recognition[J]. Journal of Molecular Biology, 2004, 344(2): 471-480.

[58] KIKUCHI H, INOUE M, SAITO, et al. Industrial production of difructose anhydride III (DFA III) from crude inulin extracted from chicory roots using Arthrobacter sp. H65-7 fructosyltransferase[J]. Journal of Bioscience and Bioengineering, 2009, 107(3): 262-265.

[59] 杭華. 菊糖果糖轉(zhuǎn)移酶-模耦合反應(yīng)器制備雙果糖酐III的研究[D].無(wú)錫: 江南大學(xué), 2012.

[60] HANG Hua, MU Wanmeng, JIANG Bo. Enzymatic hydrolysis of inulin in a bioreactor coupled with an ultrafiltration membrane[J]. Desalination, 2012, 284: 309-315.

[61] HANG Hua, MU Wanmeng, JIANG Bo. DFAIII production from inulin with inulin fructotransferase in ultrafiltration membrane bioreactor[J]. Journal of Bioscience and Bioengineering, 2012, 113(1): 55-57.

[62] MELLET C, FERNANDEZ J. Difructose dianhydrides (DFAs) and DFA-enriched products as functional foods[J]. Topics in Current Chemistry, 2010, 294: 49-77.

[63] UEDA M, SASHIDA R, MORIMOTO Y, et al. Purification of inulin fructotransferase(DFA I-producing) from Arthrobacter sp. MCI2493 and production of DFAI from inulin by the enzyme[J]. Bioscience Biotechnology and Biochemistry, 1994, 58(3): 574-575.

[64] ZHAO Meng, MU Wanmeng, JIANG Bo, et al. Purification and characterization of inulin fructotransferase (DFA III-forming) from Arthrobacter aurescens SK 8.001[J]. Bioresource Technology, 2011, 102: 1757-1764.

[65] TOMITA K, SHIOMI T, OKUHARA Y, et al. Ingestion of difructose anhydride III enhances absorption and retention of calcium in healthy men[J]. Bioscience, Biotechnology and Biochemistry, 2007, 71(3): 681-687.

[66] MINAMIDA K, SHIGA K, SUJAYA I N, et al. Effects of difructose anhydride III (DFA III) administration on rat intestinal microbiota[J]. Journal of Bioscience and Bioengineering, 2005, 99(3): 230-236.

[67] MINAMIDA K, OHASHI M, HARA H, et al. Effects of ingestion of difructose anhydride III (DFA III) and the DFA III-assimilating bacterium Ruminococcus productus on rat intestine[J]. Bioscience, Biotechnology and Biochemistry, 2006, 70(2): 332-339.

[68] KIKUCHHI H, NAGURA T, INOUE M, et al. Physical, chemical and physiological properties of difructose anhydride III produced from inulin by enzymatic reaction[J]. Journal of Applied Glycoscience, 2004, 51: 291-296.

[69] KIKUCHI H, NAGURA T, INOUE M, et al. Physical, chemical and physiological properties of difructose anhydride III produced from inulin by enzymatic reaction[J]. Journal of Applied Glycoscience, 2004, 51: 291-296.

[70] RUBIO E M, GARCIA MORENO M I, BALBUENA P, et al. Spacer-mediated synthesis of contra-thermodynamic spiroacetals: stereoselective synthesis of C 2-symmetric difructose dianhydrides[J]. Journal of Organic Chemistry, 2006, 71: 2257-2266.

[71] JUNG W S, HONG K, LEE S, et al. Structural and functional insights into intramolecular fructosyl transfer by inulin fructotransferase[J]. Journal of Biological Chemistry, 2007, 282: 8414-8423.

[72] KAZUTOMO H. Two types of inulin fructotransferases[J]. Materials, 2011, 4: 1543-1547.

[73] TANAKA K, UCHIYAMA T. Formation of difructofuranose 1,2’: 2,3’dianhydride from inulin by an extracellular inulinase of Arthrobacter ureafaciens[J]. Biochimimica et Biophysica Acta, 1972, 284: 248-256.

[74] KAWANMURA M, TAKAHASHI S, UCHIYAMA T. Purification and some properties of inulin fructotransferase (depolymerizing) from Arthrobacter ilicis[J]. Agricultural and Biological Chemistry, 1988, 52: 3209-3210.

[75] HARAGUCHI K, KISHHIMOTO M, SEKI K, et al. Purification and properties of inulin fructotransferase (depolymerizing) from Arthrobacter globiformis C11-1[J]. Agricultural and Biological Chemistry, 1988, 52: 291-292.

[76] YOKOTA A, HIRAYAMA S, ENMOTO K, et al. Production of inulin fructotransferase (depolymerizing) by Arthrobacter sp. H65-7 and preparation of DFA III from inulin by the enzyme[J]. Journal of Fermentation and Bioengineering, 1991, 72: 258-261.

[77] PARK J B, CHOI Y J. Purification and characterization of inulin fructotransferase (depolymerizing) from Arthrobacter sp. A-6[J]. Journal of Microbiology and Biotechnology, 1996, 6(6): 402-406.

[78] CHO C, LIM Y, KANG S, et al. Production of inulin fructotransferase (depolymerizing) Flavobacterium sp. LC-413[J]. Journal of Food Science and Nutrition, 1996, 1(1): 121-126.

[79] KANG S, KIM W, CHANG Y, et al. Purification and properties of inulin fructotransferase (DFA III-producing) from Bacillus sp. snu-7[J]. Bioscience, Biotechnology, and Biochemistry, 1998, 62(4): 628-631.

[80] HARAGUCHI K, YAMANAKA T, OHTSUBO K. Purification and properties of a heat stable inulin fructotransferase (DFA III-producing) from Arthrobacter pascens T13-2[J]. Carbohydrate Polymers, 2002, 50(2): 117-121.

[81] JAHNZ U. Screening-automation auf basis hohlkugelverkapselter zellen und enzymatische bildung von difructoseanhydrid III aus inulin unter thermophilen bedingungen (screening, charakterisierung, immobilisierung)[D]. Braunschweig: Technical University of Braunschweig, 2001.

[82] HARAGUCHI K, YOSHIDA M, OHTSUBO K. Thermostable inulin fructotransferase (DFA III-producing) from Arthrobacter sp. L68-1[J]. Carbohydrate Polymers, 2005, 59(4): 411-416.

[83] HARAGUCHI K, YOSHIHDA M, OHTSUBO K. Inulin fructotransferase (DFA-III producing) from Leifsonia sp. T88-4[J]. Carbohydrate Polymers, 2006, 66(1): 75-80.

[84] HARAGUCHI K. Inulin fructotransferase (DFAIII-Producing) from Arthrobacter ureafaciens D13-3[J]. Carbohydrate Polymers, 2010, 82: 742-746.

[85] HANG Hua, MU Wanmeng, JIANG Bo, et al. Enzymatic hydrolysis of inulin in a bioreactor coupled with an ultrafiltration membrane[J]. Desalination, 2012, 284: 309-315.

Enzymatic Biotransformation of Inulin for Application in Foods

ZHAN Rong-rong, MU Wan-meng, LI Yun-gao, ZHANG Tao, JIANG Bo*
(State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China)

Inulin, which is widely distributed and abundant in China, is an important agricultural and sideline product. Biotechnological processing of inulin with inulinase can provide an efficient approach for low-cost, multi-directions, and high-value-added development and utilization of inulin in the field of food. This paper reviews the current situation of research on enzymatic biotransformation of inulin for application in foods. Additionally, the properties of inulinasecatalyzed bio-transformation products and their applications in the food industry as well as the microbial sources and the latest progress of different types of inullinase are elaborated in depth.

endo-inulinase; exo-inulinase; inulin fructotransferase; inulooligosaccharide; high-fructose syrup; difructose anhydride

Q539；TS210.1

A

1002-6630（2014）03-0226-08

10.7506/spkx1002-6630-201403046

2013-03-12

國(guó)家自然科學(xué)基金項(xiàng)目（21276001；31171705）；江蘇省科技支撐項(xiàng)目（BE2011622；BE2011766；BE2010678；BE2010626）

戰(zhàn)榮榮（1984—），女，博士研究生，研究方向?yàn)槭称飞锛夹g(shù)。E-mail：newlife.zrr@163.com

*通信作者：江波（1962—），男，教授，博士，研究方向?yàn)槊冈谑称飞镏圃熘械膽?yīng)用。E-mail：bjiang@jiangnan.edu.cn