谷物β-葡聚糖結(jié)構(gòu)、功能及其 應(yīng)用研究進(jìn)展

張 寧,艾連中,張 匯

(上海理工大學(xué)醫(yī)療器械與食品學(xué)院,上海食品微生物工程技術(shù)研究中心,上海 200093)

膳食纖維是不被人體胃腸道消化吸收,但可被腸道微生物降解的多糖及木質(zhì)素類的總稱,主要存在于植物細(xì)胞壁中[1-2]。精細(xì)食品在人們飲食中的比例不斷增加,膳食纖維的比例不斷減少,導(dǎo)致我國(guó)糖尿病、心血管疾病等患者逐年增加[3]。谷物β-葡聚糖作為一種可溶性膳食纖維得到廣泛關(guān)注[4],對(duì)人體健康起著不可忽視的作用。谷物β-(1→3,1→4)-葡聚糖(β-葡聚糖)是由不同聚合度的β-(1→4)-寡葡聚糖(纖維片段)組成,這些纖維片段通過(guò)β-(1→3)-鍵連接,形成鏈狀大分子形式的β-葡聚糖。不同谷物來(lái)源β-葡聚糖的精細(xì)結(jié)構(gòu),比如β-(1→4)-和β-(1→3)-鍵比例和DP3∶DP4比例不同,鏈狀的空間構(gòu)象會(huì)發(fā)生相應(yīng)的變化,從而導(dǎo)致其理化性質(zhì)(如溶解性和成膠性)和生理活性的差異[5]。研究表明:β-葡聚糖能有效預(yù)防代謝綜合征和II型糖尿病等慢性疾病[6-7],具有改善脂肪性肥胖[8]、維護(hù)腸道健康[9]、抗腫瘤[10]、抗炎[11]和免疫調(diào)節(jié)[12]等生物活性。

歐洲食品安全局(EFSA)已認(rèn)證每天食用定量的β-葡聚糖能維持并降低血液膽固醇水平和餐后血糖響應(yīng)值[13]。近年來(lái)我國(guó)對(duì)β-葡聚糖的認(rèn)識(shí)也越來(lái)越深入,同時(shí),西藏、青海和內(nèi)蒙古等地區(qū)大量種植青稞和燕麥,為β-葡聚糖的提取和開發(fā)利用提供了廣泛的原料基礎(chǔ)[14]?；诖?本文將對(duì)谷物β-葡聚糖的提取純化方法進(jìn)行總結(jié),對(duì)谷物β-葡聚糖的結(jié)構(gòu)特征、理化性質(zhì)、功能特性等進(jìn)行綜述,對(duì)β-葡聚糖的開發(fā)應(yīng)用進(jìn)行展望。

1 谷物β-葡聚糖的提取純化

糧食作物中大麥的β-葡聚糖含量最高,其次是燕麥和小麥,分別為2.5%～11.3%,2.2%～7.8%和0.2%～1.2%[13,15]。β-葡聚糖主要分布于谷物種子的胚乳層和糊粉層細(xì)胞壁,采用剛果紅染色法對(duì)谷物種子進(jìn)行處理,切片觀察到β-葡聚糖(紅色區(qū)域)的分布情況,如圖1所示[16],約85%燕麥β-葡聚糖分布于胚乳層細(xì)胞壁,并主要集中于亞糊粉層次生壁(圖1a),約70%大麥β-葡聚糖均勻分布于胚乳層細(xì)胞壁(圖1b),然而,小麥β-葡聚糖主要分布于糊粉層細(xì)胞壁(圖1c)。由此可見,β-葡聚糖是細(xì)胞壁的重要組成成分,對(duì)細(xì)胞的結(jié)構(gòu)起著固定和支撐的作用?；诖?選擇合適的方法對(duì)谷物β-葡聚糖進(jìn)行制備。谷物β-葡聚糖的制備一般包括谷物前處理、谷物β-葡聚糖的提取和純化三個(gè)部分[17]。

圖1 剛果紅染色觀察燕麥(a)和大麥(b) 和小麥(c)中的β-葡聚糖分布情況Fig.1 The distribution of β-glucan in oat(a)，barley(b)and wheat(c)by congo red staining

1.1 谷物前處理

Myriam等[18]通過(guò)0.5 mm篩減小谷物粒徑,增大谷物與提取液接觸的機(jī)會(huì),增加β-葡聚糖的得率。谷物中的內(nèi)源性β-葡聚糖酶能降解β-葡聚糖,導(dǎo)致其分子量下降[19],因此常用高壓滅菌法、乙醇回流法和烘箱加熱法等滅活酶,不同滅酶方法結(jié)果差異較大,以乙醇回流法最為常用[20]。

1.2 谷物β-葡聚糖的提取

谷物β-葡聚糖大多利用水提、堿提或酶處理等從谷物中提取可溶性的β-葡聚糖。Izydorczyk等[21]采用熱水法從大麥中提取得到了β-葡聚糖,發(fā)現(xiàn)60 ℃相比于40 ℃水溶液提取的β-葡聚糖具有更小的摩爾質(zhì)量。Cui等[17]以小麥麩皮為原料,用1.0 mol/L的NaOH溶液提取β-葡聚糖,提取率為1.37%,純度為57.13%。酶法提取β-葡聚糖作為一種綠色工藝[22],具有操作簡(jiǎn)單、耗費(fèi)時(shí)間少、環(huán)保無(wú)污染、提取率高等優(yōu)點(diǎn),但由于谷物中含有阿拉伯木聚糖等可溶性膳食纖維使得產(chǎn)品純度相對(duì)較低。此外,Hematian等[23]借助超聲波輔助提取大麥中的β-葡聚糖,有效地縮短了提取時(shí)間,并且β-葡聚糖提取率達(dá)3.87%。不同提取谷物β-葡聚糖方法的優(yōu)缺點(diǎn)如表1所示。

表1 不同提取β-葡聚糖方法比較Table 1 Comparison of the extraction methods for β-glucans

1.3 谷物β-葡聚糖的純化

谷物β-葡聚糖提取物中通常含有少量的淀粉、蛋白質(zhì)和阿拉伯木聚糖等成分,為進(jìn)一步提高產(chǎn)品純度,采用硫酸銨沉淀、乙醇分級(jí)沉淀和柱層析等方法進(jìn)行純化。

β-葡聚糖在較低濃度的硫酸銨溶液(20%～55%)中形成沉淀,而阿拉伯木聚糖需要在較高濃度的硫酸銨溶液(55%～95%)中才能沉淀,因此,可通過(guò)該性質(zhì)將兩者進(jìn)行分離。Cui等[21]經(jīng)硫酸銨分級(jí)小麥麩皮中β-葡聚糖得到91.58%β-葡聚糖產(chǎn)品。乙醇分級(jí)沉淀是基于不同分子量的多糖在不同濃度乙醇溶液中溶解度不同而達(dá)到分離效果,隨著乙醇濃度的增加,分子量從大到小的β-葡聚糖可逐漸被分級(jí)。宋雪梅等[24]用乙醇沉淀莜麥麩皮中的β-葡聚糖,結(jié)果表明乙醇濃度為70%時(shí)分級(jí)效果最好。凝膠柱層析是多糖分離純化最常用的一種方法,基于不同分子量進(jìn)行分級(jí),可得到分子量分布窄,純度高的多糖組分,但純化效率較低,成本較高。對(duì)于谷物β-葡聚糖,一般可采用瓊脂糖凝膠柱層析法進(jìn)行分離純化[24],袁建等[25]用此方法分離純化小麥麩皮的β-葡聚糖提取物,得到產(chǎn)品的純度為97.03%。

綜上,制備谷物β-葡聚糖的一般流程可總結(jié)如下:

谷物(麩皮或顆粒)→粉碎過(guò)篩→脫脂滅酶→溶劑提取→離心取上清(殘?jiān)啥翁崛?→去除淀粉、蛋白→乙醇沉淀→干燥→β-葡聚糖粗品→純化→高純度β-葡聚糖。

以上流程得到β-葡聚糖粗品的純度為33%～87%[26]。在制備谷物β-葡聚糖時(shí),需綜合考慮成本、得率、純度及特性等各方面因素,選擇合適的提取和純化工藝,最大程度地利用谷物β-葡聚糖。

2 谷物β-葡聚糖的結(jié)構(gòu)

谷物β-葡聚糖由β-D-吡喃型葡萄糖基單元通過(guò)β-(1→4)-糖苷鍵重復(fù)連接形成纖維三糖或四糖片段,同時(shí)含有5～14個(gè)葡萄糖殘基的纖維素寡聚物,這些纖維素寡聚物再通過(guò)單一的β-(1→3)-糖苷鍵連接組合,形成線型同聚多糖[27]。谷物β-葡聚糖的化學(xué)結(jié)構(gòu)相對(duì)簡(jiǎn)單,可通過(guò)常規(guī)的甲基化和核磁共振分析對(duì)其化學(xué)結(jié)構(gòu)進(jìn)行解析[27-28]。

此外,還可通過(guò)選擇性酶解的方式釋放寡糖片段,通過(guò)寡糖片段的組合判斷谷物β-葡聚糖的精細(xì)結(jié)構(gòu)。地衣聚糖酶(Licheninase,EC 3.2.1.73)可特異性水解谷物β-葡聚糖中與β-(1→3)-鍵相連的β-(1→4)-糖苷鍵,從而得到不同聚合度的寡糖片段[29],如圖2所示。通過(guò)陰離子交換色譜(HPAEC)或基質(zhì)輔助激光解吸電離飛行時(shí)間質(zhì)譜(MALDI-TOF MS)技術(shù)判斷寡糖片段的聚合度和比例,通過(guò)組合即可進(jìn)一步明確谷物β-葡聚糖的化學(xué)結(jié)構(gòu)。

圖2 谷物β-葡聚糖經(jīng)地衣聚糖酶(EC 3.2.1.73)水解后寡糖產(chǎn)物Fig.2 The structure of β-glucan and hydrolysates after treatment of licheninase

不同來(lái)源谷物β-葡聚糖的精細(xì)結(jié)構(gòu)具有較大的差異,主要體現(xiàn)在纖維三糖和纖維四糖的比例以及β-(1→4)-與β-(1→3)-糖苷鍵的比例[30-32]。如小麥、大麥和燕麥β-葡聚糖里纖維三糖(DP3)的相對(duì)比例分別為67%～72%、52%～69%和53%～61%,而纖維四糖(DP4)的相對(duì)比例分別為21%～24%、25%～33%和34%～41%,DP3和DP4比例分別為3.0%～4.5%、1.8%～3.5%和1.5%～2.3%[29,33],兩者的摩爾比被稱為谷物β-葡聚糖的結(jié)構(gòu)指紋,是結(jié)構(gòu)特征的重要差異,比例越高,纖維三糖的含量就越高[34]。此外,即使同種谷物β-葡聚糖的DP3和DP4比例也有差異,從而β-(1→4)-和β-(1→3)-糖苷鍵的比例也有差異,這可能與谷物品種和生長(zhǎng)環(huán)境的不同有關(guān)[17,35]。常見的谷物的β-葡聚糖含量、分子量、纖維三糖和四糖比例及β-(1→3)-與β-(1→4)-糖苷鍵的比例如表2所示。

表2 谷物β-葡聚糖含量、分子量、纖維三糖/ 纖維四糖比例及β-(1→4)-與β-(1→3)-糖苷鍵的比例Table 2 Content,molecular weight,ratio of trisaccharide and tetrasaccharide and ratio of β-(1→4)- and β-(1→3)-glycosidic linkage of cereal β-glucans

3 谷物β-葡聚糖含量的測(cè)定方法

由于谷物β-葡聚糖的結(jié)構(gòu)比較明確,因此,可通過(guò)特定的方法對(duì)樣品中的β-葡聚糖含量進(jìn)行定性和定量測(cè)定。

3.1 熒光法

β-葡聚糖與熒光增白劑特異性結(jié)合,結(jié)合后Caleofluor熒光強(qiáng)度增強(qiáng),其熒光強(qiáng)度增量與β-葡聚糖含量呈一定的線性關(guān)系[36]。該法簡(jiǎn)便快速、精確度高、實(shí)用性強(qiáng),對(duì)工業(yè)上β-葡聚糖的含量測(cè)定起一定的指導(dǎo)意義。但熒光增白劑對(duì)光敏感,影響測(cè)定結(jié)果,因此,難以在較廣范圍內(nèi)使用。

3.2 剛果紅法

β-葡聚糖與剛果紅染料特異性結(jié)合,吸收強(qiáng)度隨β-葡聚糖的濃度增加而增強(qiáng),可用于β-葡聚糖的定量分析[37]。該方法快速、簡(jiǎn)便,但水不溶的β-葡聚糖無(wú)法被檢測(cè)到,結(jié)果偏低。

3.3 酶法

最常用的就是Mcclear等[38]提出的酶法,經(jīng)過(guò)簡(jiǎn)化以后成為常用分析方法,即AOAC955.16法[39]。β-葡聚糖酶特異性水解與β-(1→3)-鍵相連的β-(1→4)-糖苷鍵,降解為寡糖片段,β-葡聚糖苷酶再將寡糖片段降解為葡萄糖,通過(guò)測(cè)定葡萄糖含量,進(jìn)而換算得到β-葡聚糖含量。該法要求酶的純度高,否則測(cè)定結(jié)果不準(zhǔn)確。該法簡(jiǎn)便,用樣量少,但價(jià)格相對(duì)較高,是目前定量測(cè)定β-葡聚糖廣泛使用的方法。表3總結(jié)了不同測(cè)定β-葡聚糖方法的優(yōu)缺點(diǎn),在實(shí)際應(yīng)用中可選擇合適的方法進(jìn)行定性和定量分析。

表3 不同測(cè)定β-葡聚糖方法比較Table 3 Comparison of the methods of measurement of β-glucans

4 谷物β-葡聚糖的理化性質(zhì)

4.1 溶液流變性質(zhì)

通常情況下,谷物β-葡聚糖為白色粉末,溶于水呈淡黃色,不溶于乙醇、丙酮等有機(jī)試劑,具有吸水溶脹能力,有良好的持水性,Lee等[40]報(bào)道,大麥β-葡聚糖的持水性達(dá)6.02～6.81 g/g。水溶液中β-葡聚糖分子間相互纏繞并發(fā)生氫鍵作用形成聚集體,表現(xiàn)出高黏度和凝膠化的性質(zhì)[3,41-42]。Nicolai等[43]表示隨β-葡聚糖濃度增加,分子鏈間聚集纏繞更緊密;分子量降低,分子鏈短而移動(dòng)性增加,成膠速率增加,濃度增加較分子量降低而言更有利于成膠。Li等[41]表示三糖和四糖比例高的β-葡聚糖分子濃溶液更容易聚集而成膠,這是由于纖維三糖單元易形成網(wǎng)絡(luò)交聯(lián)區(qū)所致。Burkus等[44]研究表明β-葡聚糖的臨界濃度、黏度、黏彈行為和剪切變稀性質(zhì)主要和多糖分子量有關(guān),分子量在100～200 kDa的谷物β-葡聚糖在室溫下很容易成膠(8%～10%,w/v),分子量越大,黏度越大,儲(chǔ)能模量越小。谷物β-葡聚糖的高黏度和易成膠特性使之具有良好生理功能和潛在的商業(yè)應(yīng)用價(jià)值。

4.2 乳化性

β-葡聚糖增加乳狀液的外相黏度,限制了液滴的運(yùn)動(dòng),降低油水界面表面張力[45],有利于乳狀液的穩(wěn)定。在制備和穩(wěn)定乳液的過(guò)程中,大液滴被外力作用后變小,乳化劑吸附到小液滴上,阻止液滴間聚集或絮凝成大液滴,進(jìn)而維持其長(zhǎng)期穩(wěn)定性。當(dāng)乳化劑濃度太低或外力作用較大而乳化劑不能及時(shí)被液滴吸附時(shí),乳化劑在界面連續(xù)相一側(cè)形成的具有粘彈性的界面膜被破壞,液滴就會(huì)聚集或絮凝,乳化穩(wěn)定性下降,如圖3所示[46]。Kontogiorgos等[47]表明低分子量β-葡聚糖分子鏈向水相中伸展,形成網(wǎng)絡(luò)結(jié)構(gòu),產(chǎn)生空間位阻、靜電排斥等作用抑制了微粒的聚集,而高分子量β-葡聚糖通過(guò)提高水相黏度分別表現(xiàn)出良好的乳化穩(wěn)定性。

圖3 制備和穩(wěn)定乳液的主要物化過(guò)程示意圖Fig.3 Illustration of main physico-chemical processes involved in making of emulsions

5 谷物β-葡聚糖的生物活性

谷物β-葡聚糖作為一種水溶性膳食纖維,表現(xiàn)出廣泛的生物活性,如降血脂、降血糖、改善腸道健康等。

5.1 降血脂,預(yù)防心血管疾病

谷物β-葡聚糖增加腸道的黏性,結(jié)合膽汁酸,吸收多余的膽固醇和甘油三酯,從而減少膽固醇的肝腸循環(huán),并促進(jìn)其排出體外[48]。研究表明燕麥β-葡聚糖能顯著降低由高脂飲食引起的小鼠血清總甘油三酯和血清總膽固醇水平[49-50]。此外,Aoki等[51]通過(guò)高脂飲食喂養(yǎng)的小鼠表明β-葡聚糖能增加膽固醇7α-羥化酶基因的表達(dá),加快膽固醇轉(zhuǎn)化成膽酸,體內(nèi)膽固醇水平顯著下降。

5.2 降血糖,預(yù)防糖尿病

β-葡聚糖分子互相交聯(lián)的網(wǎng)狀結(jié)構(gòu)形成物理屏障阻礙食物和消化酶的接觸,在胃腸道中形成的黏液,延長(zhǎng)胃的排空時(shí)間,減緩小腸收縮率,減少胰島素的釋放量,從而降低葡萄糖的吸收率,降低患II型糖尿病的風(fēng)險(xiǎn)[52-54]。Bi?rklund等[55]研究發(fā)現(xiàn)燕麥麩β-葡聚糖分子量越高,黏度越大,降低餐后血糖指數(shù)與胰島素水平效果越顯著。

5.3 改善腸道健康

β-葡聚糖作為一種可溶性膳食纖維不能被消化酶降解,進(jìn)入腸道與腸粘膜,與腸道菌群作用,在大腸中被發(fā)酵產(chǎn)生乙酸、丙酸和丁酸等短鏈脂肪酸,腸道pH降低,病原菌生長(zhǎng)繁殖能力下降,同時(shí)丁酸為大腸細(xì)胞的代謝提供能量[51,56-57]。β-葡聚糖作為新一代益生元,選擇性刺激結(jié)腸中某些細(xì)菌的生長(zhǎng)活性,促進(jìn)有益菌生長(zhǎng),抑制有害菌繁殖,改善腸道菌群結(jié)構(gòu)和多樣性,刺激腸道蠕動(dòng),加速有毒物質(zhì)排出體外[58-59]。Wang等[58]研究表明,高分子量β-葡聚糖的攝入改變腸道菌群組成,增加擬桿菌屬和降低厚壁菌屬數(shù)量,推測(cè)β-葡聚糖對(duì)腸道菌群的影響可能和分子量有關(guān)。燕麥β-葡聚糖能顯著抑制由高脂飲食引起的小鼠腸道菌群中乳桿菌屬和擬桿菌屬細(xì)菌多樣性的降低[49]。

6 谷物β-葡聚糖的應(yīng)用前景

目前,谷物β-葡聚糖主要應(yīng)用在食品和化妝品領(lǐng)域。

6.1 在食品中的應(yīng)用

β-葡聚糖有著增稠性、穩(wěn)定性、乳化性和成膠性等良好物性[60]。研究顯示,冰淇淋中添加燕麥β-葡聚糖能提高其黏度、膨脹率和抗溶性,賦予產(chǎn)品潤(rùn)滑和糯性口感,蛋糕中添加1%～5%燕麥纖維能增加成品體積,改善組織結(jié)構(gòu)并賦予產(chǎn)品良好口感[61-62]。β-葡聚糖良好的耐熱性和高持水性,一定程度上阻礙了面包芯水分的擴(kuò)散,阻礙了淀粉的重結(jié)晶化,淀粉老化速率下降,從而延長(zhǎng)貨架期[63]。β-葡聚糖良好的持水性和凝膠性能使香腸富有彈性,多汁易咀嚼,有良好的色澤和風(fēng)味[64]。牛奶和高分子量燕麥β-葡聚糖的結(jié)合制得一種低熱量和低膽固醇乳品,且β-葡聚糖和酪蛋白顆粒的結(jié)合改善了乳品外觀[65]。

6.2 在化妝品中的應(yīng)用

β-葡聚糖可刺激免疫系統(tǒng),激活巨噬細(xì)胞,產(chǎn)生促進(jìn)傷口愈合的細(xì)胞因子,同時(shí)被免疫細(xì)胞膜上受體識(shí)別激活免疫活性細(xì)胞,促進(jìn)纖維細(xì)胞合成膠原蛋白,促淡化疤痕,祛除皺紋,并對(duì)皮炎、濕疹和牛皮癬等皮膚病的情況均有改善[60,66]。Pillai等[67]發(fā)現(xiàn)燕麥β-葡聚糖通過(guò)細(xì)胞間隙滲透到皮膚表層和深層,提高細(xì)胞滋潤(rùn)度,令皮膚富有彈性,是一種天然的保濕因子。Kanlayavattanakul等[68]在護(hù)膚膏中加入0.04%的β-葡聚糖,受試者28 d后皮膚狀況顯著改善,眼部皺紋變淺,前臂皮膚緊致。

7 結(jié)語(yǔ)

谷物β-葡聚糖是膳食纖維的重要來(lái)源,表現(xiàn)出降血糖、降血脂、提高免疫活性及改善腸道健康等生物活性,在疾病預(yù)防和治療中有著的重要作用,與人類健康息息相關(guān)。本文比較了谷物β-葡聚糖不同提取方式和測(cè)定方法的優(yōu)缺點(diǎn),總結(jié)了其結(jié)構(gòu)和溶液性質(zhì)及生理活性,并概述了β-葡聚糖在食品和化妝品行業(yè)的應(yīng)用。谷物β-葡聚糖的生理活性受其理化性質(zhì)和結(jié)構(gòu)特征影響,過(guò)去研究β-葡聚糖主要通過(guò)提高腸道黏度阻礙葡萄糖和膽固醇等的吸收預(yù)防糖尿病和肥胖等慢性疾病,在腸道中發(fā)酵產(chǎn)生短鏈脂肪酸改善腸道健康。不同結(jié)構(gòu)特征的谷物β-葡聚糖在生理功能中所起的作用有待進(jìn)一步研究,為進(jìn)一步發(fā)揮β-葡聚糖的生理活性具有極大指導(dǎo)意義。

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