真菌發(fā)酵胞外多糖的研究進展

楊同香，吳孔陽，陳俊亮，唐浩國，康懷彬（.河南科技大學(xué)食品與生物工程學(xué)院，河南 洛陽 4703；.洛陽師范學(xué)院生命科學(xué)學(xué)院，河南 洛陽 470）

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真菌發(fā)酵胞外多糖的研究進展

楊同香1，吳孔陽2，陳俊亮1，唐浩國1，康懷彬1
（1.河南科技大學(xué)食品與生物工程學(xué)院，河南 洛陽 471023；2.洛陽師范學(xué)院生命科學(xué)學(xué)院，河南 洛陽 471022）

摘 要：真菌胞外多糖具有高黏性、抗氧化、抗病毒和抗腫瘤等多種生物學(xué)活性，在食品和醫(yī)藥工業(yè)等領(lǐng)域應(yīng)用廣泛。本文結(jié)合國內(nèi)外最新研究成果，綜述了近年來有關(guān)產(chǎn)胞外多糖的真菌、影響真菌胞外多糖產(chǎn)量的因素、產(chǎn)胞外多糖的菌株的選育策略以及真菌胞外多糖的功能研究，以期為真菌發(fā)酵胞外多糖的研究和應(yīng)用提供參考。

關(guān)鍵詞：真菌胞外多糖；產(chǎn)量；發(fā)酵；功能

引文格式：

楊同香,吳孔陽,陳俊亮,等.真菌發(fā)酵胞外多糖的研究進展[J].食品科學(xué),2016,37(5):265-270.DOI:10.7506/spkx1002-6630-201605046.http://www.spkx.net.cn

YANG Tongxiang,WU Kongyang,CHEN Junliang,et al.Recent advances in fungal exopolysaccharide fermentation[J].Food Science,2016,37(5):265-270.(in Chinese with English abstract)DOI:10.7506/spkx1002-6630-201605046.http://www.spkx.net.cn

多糖在自然界分布極其廣泛，植物、動物和微生物體內(nèi)均有存在，胞外多糖（exopolysaccharide，EPS）通常指微生物在生長代謝過程中分泌到細(xì)胞外的一類多糖[1-3]。由于微生物EPS具有多元化的多糖結(jié)構(gòu)和不同的流變學(xué)特性，在生物技術(shù)領(lǐng)域應(yīng)用前景十分廣闊[4]。近年來，研究人員對微生物EPS開展了廣泛的研究，主要包括菌株的分離和鑒定、產(chǎn)EPS菌株選育、EPS生物合成途徑、工程菌構(gòu)建、發(fā)酵條件的優(yōu)化、EPS的分離、純化、結(jié)構(gòu)鑒定及其生物學(xué)活性的研究等，尤其是對細(xì)菌EPS的研究進行了較全面的歸納和總結(jié)[4-7]，而單就對真菌發(fā)酵EPS的研究概述則相對較少。另外，如何獲得高產(chǎn)EPS產(chǎn)生菌以及如何提高EPS產(chǎn)量，也是研究人員一直以來關(guān)心的問題。因此，本課題組結(jié)合自己的研究工作，從EPS產(chǎn)生菌、影響真菌EPS產(chǎn)量的因素、產(chǎn)EPS的菌株的選育策略等方面對真菌發(fā)酵EPS的研究做一簡要概述。

1　真菌EPSS產(chǎn)生菌

目前公開報道產(chǎn)EPS的真菌至少有上百種[8]。以往研究人員主要從陸地（包括極地環(huán)境）采樣并分離產(chǎn)EPS的真菌，近些年發(fā)現(xiàn)海洋蘊藏著數(shù)量豐富且能產(chǎn)生結(jié)構(gòu)新穎EPS的真菌[9]。本文將近5 a有關(guān)產(chǎn)EPS真菌的文獻進行歸納，見表1。大多數(shù)研究者選擇馬鈴薯葡萄糖瓊脂培養(yǎng)基（potato dextrose agar，PDA）對產(chǎn)EPS真菌的培養(yǎng)和分離。由于不同真菌生長特性差異，培養(yǎng)溫度一般在18～30 ℃。所產(chǎn)EPS的結(jié)構(gòu)也因菌株種屬差異而種類繁多，通常發(fā)現(xiàn)的真菌EPS以雜多糖居多，當(dāng)然也有關(guān)于同多糖的報道，如Mukhopadhyay等[10]鑒定了一株產(chǎn)EPS的南極土壤嗜冷真菌Thelebolus sp.IITKGP-BT12，進一步研究發(fā)現(xiàn)該EPS為葡聚糖，且在基礎(chǔ)鹽培養(yǎng)基中，多糖產(chǎn)量高達1.94 g/L。

表1　近5 a報道產(chǎn)EPS的真菌Table 1 Exopolysaccharide production by fungi reported in last five years

2　影響真菌EPS產(chǎn)量的因素

研究發(fā)現(xiàn)EPS的生物合成是在細(xì)胞內(nèi)進行，然后分泌到細(xì)胞外。而這一過程通常受到諸多因素影響，其中對EPS產(chǎn)量的影響主要受到真菌類型、培養(yǎng)基組成以及發(fā)酵條件等因素。

2.1真菌類型

對于未研究的和未開發(fā)的真菌，不在統(tǒng)計范圍之類，從公開報道的真菌發(fā)酵EPS水平來看，擔(dān)子菌通常比絲狀真菌和酵母菌EPS的產(chǎn)量要高[8]。本文對近年來真菌發(fā)酵EPS的文獻進行歸納（表2），從表2中可以基本了解不同真菌發(fā)酵產(chǎn)EPS的能力。

表2　國內(nèi)外部分真菌EPSS產(chǎn)量Table 2 Overseas studies on the production of fungal exopolysaccharide

2.2培養(yǎng)基組成

研究表明培養(yǎng)基組成對EPS產(chǎn)量的影響至關(guān)重要。微生物發(fā)酵EPS，對碳源選擇具有一定的偏好性，細(xì)菌通常利用蔗糖發(fā)酵EPS[32]，真菌則通常利用葡萄糖發(fā)酵EPS（表2），并且在該條件下可以促進菌絲生長和獲得較多的多糖。對于氮源而言，多數(shù)真菌發(fā)酵EPS，會考慮使用酵母粉[33]，有些會輔以脫脂牛奶作為氮源[34]。不過對于工業(yè)化發(fā)酵生產(chǎn)EPS，最好還要考慮到原料的價格，Sharma等[35]對A.pullulans RBF 4A3利用5 種不同的農(nóng)副產(chǎn)品作為營養(yǎng)來源發(fā)酵生產(chǎn)普魯蘭多糖進行研究，結(jié)果發(fā)現(xiàn)添加有玉米漿和葡萄糖的培養(yǎng)基，菌株產(chǎn)多糖水平上升，而且經(jīng)過單因素條件優(yōu)化后，普魯蘭多糖的產(chǎn)量提高了14%，質(zhì)量濃度達到88.59 g/L。相關(guān)研究結(jié)果表明，磷酸鹽對于真菌發(fā)酵EPS這一過程非常重要，其中磷酸二氫鉀和磷酸氫二鉀被認(rèn)為是最為有效的磷源補充劑，而對于其他的一些無機鹽，硫酸鎂則被認(rèn)為對真菌發(fā)酵EPS有很好促進作用[8]。另外，在真菌發(fā)酵EPS過程中，添加某些因子也可以提高真菌發(fā)酵EPS水平。這些因子包括植物油、脂肪酸、表面活性劑、核苷酸和維生素等。比如Sheng Long等[36]研究了尿嘧啶對Aureobasidium pullulans CGMCC1234發(fā)酵多糖的影響，當(dāng)菌株發(fā)酵48 h時，向培養(yǎng)基中添加5 mmol/L的尿嘧啶，結(jié)果發(fā)現(xiàn)與不加尿嘧啶的對照組相比，Aureobasidium pullulans CGMCC1234普魯蘭多糖質(zhì)量濃度達到49.07 g/L，提高了30%。Sheng Long等[37]發(fā)現(xiàn)在培養(yǎng)基中加入體積分?jǐn)?shù)為0.5%的吐溫-80可以顯著提高Aureobasidium pullulans CGMCC1234普魯蘭多糖的產(chǎn)量。事實上，其他研究結(jié)果也表明吐溫-80能夠促進對多種真菌EPS的合成，并從蛋白質(zhì)組學(xué)角度去闡述吐溫-80的作用機制[38-39]。

培養(yǎng)基的組分可以直接影響菌體的生長狀況，菌體生長狀況與EPS產(chǎn)量是否有關(guān)聯(lián)，尚無統(tǒng)一的論證。培養(yǎng)基中的某些成分盡管能夠促進菌體生長，但會減少EPS產(chǎn)量。Wu Shengjun等[25]研究發(fā)現(xiàn)來源于Aureobasidium pullulans CJ001所產(chǎn)的普魯蘭多糖，與細(xì)胞生物量沒有相關(guān)性，而這與某些細(xì)菌EPS產(chǎn)量與生物量相關(guān)的結(jié)論不一致[40]，這有助于研究人員更好地認(rèn)識菌株類型和培養(yǎng)基差異對真菌發(fā)酵EPS的影響。

2.3發(fā)酵條件

菌株深層培養(yǎng)技術(shù)在商業(yè)上被認(rèn)為是真菌發(fā)酵多糖十分有效的方法，且未出現(xiàn)顯著的污染問題[46]。大量研究表明真菌發(fā)酵EPS需要在好氧條件下完成，溶氧限制將影響EPS產(chǎn)量。Ruperez等[47]通過靜息培養(yǎng)和深層培養(yǎng)兩種方式比較了Aspergillus parasiticus發(fā)酵EPS的能力，結(jié)果發(fā)現(xiàn)深層培養(yǎng)條件下所得到的EPS產(chǎn)量是靜息培養(yǎng)條件下的2.3 倍。目前有關(guān)真菌發(fā)酵EPS條件研究，多集中于對發(fā)酵培養(yǎng)基和發(fā)酵產(chǎn)EPS條件的優(yōu)化，所涉及的實驗方法采用常規(guī)的統(tǒng)計方法，包括單因素試驗、正交試驗、部分因子試驗以及中心組合試驗等優(yōu)化EPS產(chǎn)量[23,41,44]。不同于細(xì)菌生長周期快，真菌發(fā)酵EPS通常需要較長的時間，一般需要1 周左右，多的甚至需要發(fā)酵20 d（表2）。

3 EPS菌株的選育策略

獲得高產(chǎn)EPS的菌株是發(fā)酵優(yōu)化的前提。對于真核微生物而言，當(dāng)傳統(tǒng)微生物育種技術(shù)難以對某些特定菌株的產(chǎn)多糖水平有較大提高時，有必要開發(fā)新的菌種選育技術(shù)。當(dāng)然，研究人員需要根據(jù)菌株自身特性及實驗室條件來設(shè)計合理的選育路線。通常情況下，可以在傳統(tǒng)育種技術(shù)基礎(chǔ)上，通過改良或引入新開發(fā)的高效微生物進化育種技術(shù)獲得高產(chǎn)EPS突變株。比如Liu Bin等[48]采用常溫常壓等離子體技術(shù)對海洋Crypthecodinium cohnii進行突變選育，獲得一株高產(chǎn)EPS突變子M7，EPS產(chǎn)量達到1.02 g/L，較出發(fā)菌株提高了33.85%。DNA改組技術(shù)也可獲得高產(chǎn)EPS的突變株，Kang Jianxiong等[49]通過在紫外/甲基磺酸乙酯誘變基礎(chǔ)上，通過DNA改組技術(shù)獲得Aureobasidium pullulans突變株F3-2，EPS產(chǎn)量較野生型菌株提高了168%，極大地提高了EPS產(chǎn)量。另外通過控制菌株生長的外部環(huán)境，可以在不同程度上提高真菌發(fā)酵EPS的產(chǎn)量，比如限制氮源種類和添加量，不僅可以減少投入成本，還可以極大促進EPS的合成[50]。

4 真菌EPS的功能研究

由表1可知，不同真菌所產(chǎn)EPS具有多樣性的結(jié)構(gòu)類型，因其結(jié)構(gòu)的獨特性，使得真菌EPS具有豐富的生物學(xué)功能和極其廣泛的工業(yè)應(yīng)用價值[51]。

4.1抗腫瘤及免疫調(diào)節(jié)活性

活性真菌多糖通常被描述為生物反應(yīng)調(diào)節(jié)劑，主要是基于它們能夠觸發(fā)免疫系統(tǒng)針對癌細(xì)胞的非特異性反應(yīng)[21,52]。Ikekawa[53]通過小鼠喂養(yǎng)真姬菇實驗發(fā)現(xiàn)，實驗組小鼠患癌比例（3/36）遠(yuǎn)小于對照組（21/36），并認(rèn)為抑制腫瘤的機制由于免疫增強作用所致，Wasser[54]也認(rèn)同這一觀點，認(rèn)為食用一些大型真菌可以預(yù)防腫瘤的發(fā)生和轉(zhuǎn)移。事實上，對于癌癥的預(yù)防和治療，從臨床實踐中發(fā)現(xiàn)，大型真菌EPS與化學(xué)療法、外科手術(shù)結(jié)合起來被認(rèn)為是非常有效的方法。在動物模型和人類臨床實踐中，香菇多糖的研究最為透徹，在化學(xué)療法治療之前，通過給患癌病人注射香菇多糖，可以很好地改善治療效果[54]。最新研究發(fā)現(xiàn)，翅鱗傘菌株（Pholiota squarrosa Quel.）AS5.245 水溶性胞外多糖（water-soluble exopolysaccharide，PEPS）-1對植入小鼠Heps肝癌細(xì)胞具有抗腫瘤活性，并猜測這種特性很可能是PEPS-1通過刺激宿主的免疫應(yīng)答反應(yīng)實現(xiàn)，但是PEPS-1的結(jié)構(gòu)與功能的關(guān)系以及分子作用機制還需進一步研究[11]。除此之外，Aureobasidium pullulans所產(chǎn)的普魯蘭多糖及其各種衍生物也成為研究人員用于治療肝、肺、腦、脾等相關(guān)腫瘤疾病治療的潛在藥物[55-56]。比如Li Huanan等[57]通過合成普魯蘭多糖衍生物（普魯蘭多糖-多柔比星結(jié)合物），研究該物質(zhì)對人肝癌細(xì)胞的影響，并認(rèn)為該結(jié)合物有望成為靶向藥物載體安全用于給藥體系。

4.2抗炎癥及抗菌作用

大型真菌所產(chǎn)的一些多糖，其治療效果已得公認(rèn)，比如鳳尾菇EPS具有鎮(zhèn)痛和抗炎作用[13]。Smiderle等[58]從Pleurotus pulmonarius中分離出一種3-O-甲基-半乳甘露聚糖，發(fā)現(xiàn)該多糖對醋酸誘發(fā)小鼠扭體反應(yīng)具有鎮(zhèn)痛作用，盡管與吲哚美辛鎮(zhèn)痛作用相似，但地塞米松和吲哚美辛在一定程度上抑制炎癥應(yīng)答反應(yīng)，而3-O-甲基-半乳甘露聚糖則不存在這種現(xiàn)象，不過由3-O-甲基-半乳甘露聚糖引起的鎮(zhèn)痛作用機制仍然不清楚。Silveira等[13]首次報道了來源于Pleurotus sajor-caju的甲基化EPS可以減輕角叉菜所致小鼠足腫脹，表明這種EPS很可能成為一種有效的鎮(zhèn)痛和抗炎癥藥劑。此外，研究發(fā)現(xiàn)真菌EPS具有抗細(xì)菌和抗病毒作用[52,59-60]，比如香菇EPS輔助治療抗藥性的肺結(jié)核，可以提高中性粒細(xì)胞對結(jié)核菌的吞噬作用，而葡聚糖和靈芝多糖分別對風(fēng)疹病毒和乙型肝炎病毒具有一定的抗性。

4.3抗氧化活性

天然抗氧化劑因在疾病預(yù)防與治療、延緩衰老過程中發(fā)揮重要作用而廣為人知，在這些天然抗氧化劑中，多糖通常被認(rèn)為是抗氧化性最強的一種[61]。有研究表明真菌EPS具有抗氧化功能，可以清除自由基，比如金針菇EPS[12]、血紅密孔菌EPS[62]和被毛孢屬真菌EPS[14]。多糖的抗氧化活性主要取決其結(jié)構(gòu)特征，包括分子質(zhì)量大小、單糖組成及糖苷鍵連接方式等，這往往是多種因素共同作用的結(jié)果[63]。即使對于同一來源的真菌EPS，分離得到不同分子質(zhì)量及單糖組成的EPS，其抗氧化性也有一定的差別。Zheng Jianqiang等[61]純化了來源于Boletus aereus的EPS，獲得3 種EPS，分別命名為Fr-I、Fr-II和Fr-III，通過體外抗氧化活性實驗，發(fā)現(xiàn)Fr-I是其中抗氧化活性最高的EPS。

4.4益生功能

開發(fā)促進腸道有益菌群增殖，抑制有害微生物生長的益生元備受研究人員青睞，目前已經(jīng)上市的各種功能性低聚糖種類繁多，包括低聚木糖、低聚半乳糖、低聚果糖等[64-65]，該類益生元因進入末端結(jié)腸保留時間因素限制其應(yīng)有功效[66-67]。而研究發(fā)現(xiàn)真菌EPS在人腸道不易被消化分解，具有防止細(xì)胞脫水、吞噬、減少血液中葡萄糖釋放速率和膽固醇的積累[23,52]。另外，還有研究表明來源于釀酒酵母的可溶性和不可溶性葡聚糖具有顯著降血脂的功效[52]。

5　結(jié) 語

開發(fā)能夠應(yīng)用于工業(yè)上的新型EPS，歷來會引起科研人員的興趣，隨著真菌EPS研究的深入以及EPS高通量篩選平臺的建立[2,68]，勢必會有更多新型的EPS被發(fā)現(xiàn)，其相應(yīng)的生理學(xué)功能也將會被闡述。盡管近年來真菌EPS研究取得了豐碩的成果，但是有關(guān)真菌EPS生物合成途徑和相關(guān)酶的基因調(diào)控和表達研究還相對較少，而這對于提高EPS產(chǎn)量研究具有很強的理論指導(dǎo)意義。另外EPS工業(yè)規(guī)?；a(chǎn)過程中，如果菌株所利用的碳源單一，勢必會增加EPS的生產(chǎn)成本、缺乏市場競爭力，進一步開發(fā)利用工農(nóng)業(yè)廢棄物，如漁業(yè)殼多糖水解物、甘蔗糖蜜等有可能提高真菌發(fā)酵EPS水平。本課題組已著手此方面研究，以期獲得這方面特性的菌株以及具有特殊生物學(xué)活性的EPS。

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Recent Advances in Fungal Exopolysaccharide Fermentation

YANG Tongxiang1,WU Kongyang2,CHEN Junliang1,TANG Haoguo1,KANG Huaibin1
(1.College of Food and Bioengineering,Henan University of Science and Technology,Luoyang 471023,China; 2.College of Life Science,Luoyang Normal University,Luoyang 471022,China)

Abstract:Fungal exopolysaccharide possesses high viscosity,and antioxidant,antiviral and antitumor activity,which is widely used in the food and pharmaceutical industries.In this review,we summarizes the latest progress made in China and abroad in studying exopolysaccharide-producing strains,factors influencing the production of fungal exopolysaccharide,breeding strategies for exopolysaccharide production and the function of fungal exopolysaccharide,aiming to provide some references for further investigation and application of fungal exopolysaccharide.

Key words:fungal exopolysaccharide; production; fermentation; function

中圖分類號：Q939.9

文獻標(biāo)志碼：A

文章編號：1002-6630（2016）05-0265-06

DOI:10.7506/spkx1002-6630-201605046

作者簡介：楊同香（1983—），女，講師，博士，研究方向為乳品科學(xué)與工程。E-mail：txyamy@163.com

基金項目：河南省高等學(xué)校重點科研基金項目（15A180050）；河南科技大學(xué)博士科研啟動基金項目（13480043）；河南科技大學(xué)青年科學(xué)基金項目（2015QN038）

收稿日期：2015-06-12