【作者】 高莉莉；

【导师】 张柏林； Piotr Walczak；

【作者基本信息】 北京林业大学 ， 植物学， 2009， 博士

【摘要】 肠膜明串珠菌在有受体存在的条件下,能够利用葡聚糖蔗糖酶的转糖苷作用合成不同聚合度的低聚葡萄糖。这样的低聚糖能够抵御肠道内多种消化酶的降解,在不被人体消化和吸收的情况下,选择性的刺激肠道内有益微生物的生长和繁殖,被称之为非消化性低聚糖（non-digestible oligosaccharide）或功能性低聚糖,可以作为益生素而应用到多种功能性食品当中。目前,低聚糖生产主要通过淀粉水解来制得,产品中通常含有大量的单糖、二糖或者是麦芽糊精,它们都属于能够被人体消化吸收的碳水化合物,不仅不能选择性的刺激肠道有益微生物的生长和活力,而且还增加了食物产品的热能值,在很大程度上降低了功能性低聚糖的利用效率。本研究利用肠膜明串珠菌菌株6055、DM1-2、PC13和L4葡聚糖蔗糖酶转糖苷作用合成低聚糖的能力,从菌株筛选、培养基选择、合成低聚糖关键酶特性、发酵过程中底物和产物的变化情况以及产物纯化工艺等多个方面对肠膜明串珠菌发酵生成低聚糖的工艺开展了研究,旨在建立以明串珠菌发酵生产功能性低聚糖的新工艺技术,主要研究结果如下:从来源不同的基质材料中分离到生成葡聚糖和代谢柠檬酸的阳性菌株6株,能够将蔗糖转化为葡聚糖的菌株8株,柠檬酸代谢阳性菌株6株。形态学、生理和生化特征测试结果显示,其中15株被鉴定为肠膜明串珠菌（Leuconostoc mesemteroides）。基于葡聚糖生成量、生成速度以及葡聚糖蔗糖酶活力的初步测定结果,确定了葡聚糖蔗糖酶活力相对较高的4个菌株,即DM1-2、PC13和L4以及6055作为研究出发菌株,16s rRNA基因序列的测定结果进一步证实了这四个菌株的分类学地位。通过检测各种营养成分以及发酵条件对肠膜明串珠菌细胞生长的影响,以蔗糖作为碳源,以大豆蛋白胨和酵母膏作为氮源,在发酵过程中进行pH控制和震荡加氧将会在很大程度上提高肠膜明串珠菌的增殖速度和细胞生物量。尽管Mg²⁺和Mn²⁺对细胞的增殖效果不明显,但MnSO₄、NaCl和FeSO₄可显著提高肠膜明串珠菌葡聚糖蔗糖酶产量。Tween80和Ca²⁺能明显改善葡聚糖蔗糖酶的活力稳定性。K₂HPO₄能高度缓冲培养介质的pH值变化,麦芽糖是低聚糖生成过程中必不可少的的受体分子。因此,确定了MS培养基是肠膜明串珠菌低聚糖优化发酵介质,其产酶的优化发酵条件是28℃,100r/min下震荡培养。经SDS-PAGE分离后,采用原位染色和PAS染色方法在电泳胶片上获得葡聚糖蔗糖酶的特异性反应条带。结果显示,菌株6055可产生分子量分别为151kDa、142kDa和117kDa的三种葡聚糖蔗糖酶,其中前两者同时存在于上清液中和细胞表面,而后者只存在于上清液当中;菌株DM1-2可以产生两种葡聚糖蔗糖酶,分子量分别为183kDa和142kDa,前者同时存在于上清液中和细胞表面,而后者仅能存在于细胞表面;菌株PC13可以产生分子量分别为148 kDa、138 kDa和115kDa的三种葡聚糖蔗糖酶,前两者属于胞外酶,而后者与细胞相连;与菌株6055相似,菌株L4可以产生两种同时存在于上清液和细胞表面的葡聚糖蔗糖酶,分子量分别为145 kDa和136kDa,而分子量为115kDa的葡聚糖蔗糖酶只存在于上清液中。酶活力测试表明,菌株6055、DM1-2、PC13和L4的全细胞发酵液和上清液中葡聚糖蔗糖酶活力分别为5.204 U/mL和4.710U/mL、1.638 U/mL和1.352U/mL、3.244U/mL和1.118U/mL、3.250 U/mL和1.006U/mL;而胞外酶所占比例分别为90.5%、82.5%、34.5%和31%。采用硫酸铵沉淀-离心分离-透析-聚乙二醇浓缩-冻干的方法对上清液中的葡聚糖蔗糖酶进行分离和浓缩,按细胞干重计,菌株6055、DM1-2、PC13和L4粗酶粉中葡聚糖蔗糖酶活力分别为10.7 U/mg、2.7 U/mg、2.8 U/mg和2.5U/mg。按蛋白含量测算,4个菌株的酶活力分别为24.4 U/mg、6.5 U/mg、4.2 U/mg和4.0U/mg。各菌株酶活力的回收率分别为32%、32%、44%和43%。在—18℃贮藏2周后,4个菌株的粗酶粉活力分别下降了31.6%、48.2%、41.2%和37.2%。采用低温超滤（100kDa截留分子量）对上清液中葡聚糖蔗糖酶进行提纯并获得浓缩的酶液,从500mL发酵液中获得菌株6055、DM1-2、PC13和L4的浓缩酶力分别是75.8 U/mL、18.7 U/mL、15.8 U/mL和13.9 U/mL,葡聚糖蔗糖酶活力回收率分别为76%、74%、69%和73%。在-18℃贮藏2周后,浓缩酶活力分别下降了6.6%、11.5%、9.1%和6.4%。当蔗糖和麦芽糖（S/M）比例为2:1时,对肠膜明串珠菌发酵生成低聚糖时的底物和产物监测显示,在对数生长期,蔗糖和麦芽糖快速消耗,同时有大量的低聚糖生成,发酵终点时4个菌株均能将蔗糖完全利用,但有少量麦芽糖残余。发酵结束后,菌株6055、DM1-2、PC13和L4的细胞生物量在1.7g/L左右,它们的低聚糖产量分别为69.8g/L、38.2g/L、48.4g/L和54.7g/L,所获低聚糖分别为理论产量的75%、41%、51%和58%。将蔗糖和麦芽糖的比例从2:1提高至7:1,发现在S/M（蔗糖/麦芽糖）为7的培养基中,除菌株PC13生成三种低聚糖之外,其他菌株均生成两种低聚糖。存S/M为2的培养基中,除菌株6055能生成四种低聚糖之外,其他菌株均可生成两种类型的低聚糖。提高蔗糖和麦芽糖的比例有助于肠膜明串珠菌发酵生成聚合度相对较高的低聚糖。在S/M为7的培养基中,菌株6055、DM1-2、PC13和L4的低聚糖产量分别为45.4g/L、35.5g/L、57.3g/L和48.8g/L。相对于蔗糖和麦芽糖比例为7的情况,除菌株PC13之外,其他3个菌株均在蔗糖和麦芽糖比例为2的情况下获得相对较高的低聚糖产量。设计出包含活性炭和硅藻土脱色、离子交换除盐、冷冻结晶除甘露醇和酵母选择性除盐这四个主要步骤的低聚糖纯化工艺路线。利用酿酒酵母选择性发酵碳水化合物的特性对肠膜明串珠菌代谢生成的低聚糖产物进行纯化发现,菌株6055和DM1-2发酵液中的果糖、麦芽糖和潘糖在接入酵母后10h和12h被完全清除。菌株PC13和L4的发酵液中麦芽糖和果糖均在接种后10h内被酵母菌发酵完毕,因这两个菌株生成潘糖含量较高,接种酿酒酵母14h后才被完全清除。菌株6055、DM1-2、PC13和L4生成的潘糖分别为低聚糖总产量的7.6%、22%、32%和30%;整个发酵过程中,DP＞3的低聚糖含量无明显变化,说明DP＞3的低聚糖能够抵御酿酒酵母各种代谢酶的降解,因此可以作为功能性低聚糖来源。菌株6055、DM1-2、PC13和L4的发酵液经酿酒酵母选择性除糖后得到的低聚糖含量分别是124g/L、58g/L、66g/L和83g/L,纯度分别为94.6%、88.7%、89.5%和91.9%。核磁共振检测可知,4株肠膜明串珠菌生成的低聚糖是由α-1,6糖苷键连接的主链,伴有α-1,4糖苷键连接的分枝结构,并且α-1,4糖苷键位于低聚糖的还原型末端。更多还原

【Abstract】 Leuconostoc mesemteroides,widely used in cabbage,butter and cheese fermentation, is able to use saccharides or maltose as acceptors to biosynthesize glucooligosaccharides via transglycosidation of dextransucrase.The glucooligosaccharides show strong resistance to various digestive enzymes existing in the gastro-intestinal system.They favor selectively the growth and proliferation of the beneficial microorganisms in intestinal tract due to not being digested and absorbed by body.These oligosaccharides,defined as non-digestible oligosaccharide,play as probiotics in functional foods.However,the oligosacchafides formed via starch hydrolysation contain generally large amount of monosaccharides,disaccharides or maltodextrin which are the carbohydrates liable to digestive enzymes.They only give energy to food,but do not promote the growth and activation of probiotic bacteria in intestines and thus have little functionality.Therefore,the objectives of this study were to:（1） examine the ability of L.mesemteroides strains 6055,DM1-2,PC13 and L4 isolated from traditional Chinese foods to produce oligosaccharides;（2） optimize medium components available for L.mesemteroides to synthesize oligosaccharides;（3） deal with the properties of key enzymes related to oligosaccharides synthesize;（4） investigate the change between the substrates and the end-products throughout glucooligosaccharides fermentation;and（5） establish a new way to produce functional oligosaccharides via L.mesemteroides.The following are main results:（1） 15 strains derived from different origins were proved to produce glucan and metabolize citrate.Additionally,8 strains isolated were able to transform sucrose into glucan,and 6 strains showed citrate-metabolizing ability.The 15 strains were identified as L.mesemteroides according to their morphological,physiological and biochemical features and 16s rRNA sequence.Among these strains used in this study, 4 strains,named as L.mesemteroides DM1-2,PC13,L4 and 6055 were chosen for further study in terms of their high dextransucrase activity,glucan mass,and the synthesis rate of glucan.（2） Nutrients and fermentation conditions affected the growth of L. mesemteroides cells.Controlled-pH and aeration by shaking culture greatly increased the propagation and biomass of L.mesemteroides cell when sucrose was used as carbon source,and soybean peptone and yeast extract as nitrogen source.The effect of Mg²⁺ and Mn²⁺ on the cell propagation was not remarkable,but MnSO₄,NaCl and FeSO₄ notably promoted the level of dextransucrase by L.mesemteroides.Tween80 and Ca²⁺ significantly improved the stability of dextransucrase activity.K₂HPO₄,due to efficiently buff the pH of media,was the essential ingredient of fermentation media in producing dextransucrase as if maltose played an essential role as acceptor in the process of generating oligosaccharides.So,the medium MS was confirmed to be the optimal fermentation medium for producing oligosacchaddes by L.mesemteroides. The optimized conditions for generating enzyme were 28℃plus shaking at the speed of 100r/min.（3） SDS-PAGE,together with double staining and PAS staining proved the existence of specific reaction bands which indicated dextransucrase.It was seen that strain 6055 generated three kinds of dextransucrases whose molecular weights were 151 kDa,142kDa and 117kDa,respectively.Among them,the former two ones were found both in supematants and cell wall,while the later was only found in the fermentation liquor.Strain DM 1-2 synthesized two kinds of dextransucrases,and their molecular weights were 183kDa and 142kDa,respectively.The former existed both in cell wall and out of cell,but the later only was located on cell surface.Strain PC 13 produced three kinds of dextransucrases,and their molecular weights were 148kDa, 138kDa and 115kDa,respectively.The former two ones were ectoenzyme while the later was linked to cell.Resembled to strain 6055,strain L4 produced two types of dextransucrases coexisting in supernatants and cell wall with molecular weights of 145 kDa and 136kDa,respectively,but the dextransucrase of 115kDa was only found in free-cells supernatants.（4） The activities of dextransucrases from strains 6055,DM1-2,PC13 and L4 in fermentation liquor containing complete cells were 5.204 U/mL,1.638 U/mL,3.244 U/mL and 3.250 U/mL,respectively;while Those in the supematants were 4.710U/mL,1.352U/mL,1.118U/mL and 1.006U/mL,respectively.The proportion held by ectoenzymes was 90.5%,82.5%,34.5%and 31%respectively.The enzyme dextransucrase in cell-free supernatants was purified by saturated NH₄SO₄,centrifugal separation,dialysis-PEG concentration and lyophilization.The activities of dextransucrases yielded from strains 6055,DM1-2,PC13 and L4,calculated in cell dry weight,were 10.7 U/mg,2.7 U/mg,2.8 U/mg and 2.5U/mg,respectively.The activities of dextransucrases,in terms of contents of protein,were 24.4 U/mg for strain 6055,6.5 U/mg for strain DM1-2,4.2 U/mg for strain PC13 and 4.0U/mg for strain L4.The recoveries pertinent to the strains were 32%,32%,44%and 43%, respectively.After stored at -18℃for 2 weeks,the activities of dextransucrases decreased by 31.6%for strain 6055,48.2%for strain DM1-2,41.2%for strain PC13 and 37.2%for strain L4.Ultra-filtration at low temperature（Molecular Weight Cut-Off,100kDa,-4℃） was forwarded to concentrate dextransucrase from the supernatants.The activities of concentrated dextransucrase from 500 mL fermenting liquor were 75.8 U/mL for strain 6055,18.7 U/mL for strain DM1-2,15.8 U/mL for strain PC13 and 13.9 U/mL for strain L4.The recoveries of the enzyme for strains 6055,DM1-2,PC13 and L4 were 76%,74%,69%and 73%,respectively. Conservation for 2 weeks at -18℃decreased the activities of the concentrated dextransucrase by 6.6%for strain 6055,11.5%for strain DM1-2,9.1%for strain PC13 and 6.4%for strain L4.（5） When the ratio of sucrose to maltose was 2 to 1,the monitoring of the substrates and end-products by L.mesemteroides indicated that sucrose and maltose were consumed rapidly in log phase and there was great deal of oligosaccharides to be generated.At the terminal stage of fermentation,all the strains metabolized sucrose except for a little of maltose residue.After fermentation stopped,the biomass of the strains 6055,DM1-2,PC13 and L4 averaged about 1.7g/L,and the yields of corresponding oligosaccharides were 69.8g/L,38.2g/L,48.4g/L and 54.7g/L, respectively.Theoretically,the oligosaccharides harvested were 75%for strain 6055, 41%for strain DM1-2,51%for strain PC13 and 58%for strain L4.（6） When the ratio of sucrose to maltose（S/M） was evaluated to 7:1,strain PC13 was found to generate three kinds of oligosaecharides whilst other 3 strains to generate two kinds of oligosaccharides.In the media in which S/M was 2,strain 6055 produced 4 kinds of oligosaccharides,but other strains produced two kinds of oligosaccharides.Increasing the ratio of sucrose to maltose was conducive to generating the oligosaccharides with relatively high degree of polymerization.In the media in which S/M was 7,the oligosaccharides yields from strain 6055,DM1-2, PC13 and L4 were 45.4g/L,35.5g/L,57.3g/L and 48.8g/L,respectively.Compared to the medium whose ratio of sucrose to maltose was 7:1,other 3 strains got the relatively higher oligosaccharides yields in the medium whose ratio of sucrose to maltose was 2:1 except for strain PC13.（7） A purification scheme including decoloration by active carbon and diatomite, ion-exchange demineralization,mannitol removal by crystallization,and yeast fermentation was conduced to collecting oligosacchadde formed by L.mesemteroides strains.One of advantages of this scheme was that Saccharomyces cerevisiae fermented selectively sugars except for the oligosaccharides generated by L. mesemteroides.It was found that the fructose,maltose and panose in media formed by strains 6055 and DM1-2 were scavenged completely after yeast inoculation for 10h to 12h.Fructose and maltose in media produced by strains PC13 and L4 were completely consumed by yeast cell after 10h incubation.The tri-maltose sugars formed by the two strains were eaten completely after 14h inoculation of yeast cells. The percentage of panose produced by strains 6055,DM1-2,PC13 and L4 in the total oligosaccharides yields was 7.6%,22%,32%and 30%,respectively.Throughout the whole fermentation,no significant change in the oligosaccharides whose DP was greater than 3（DP＞3） occurred.It indicated that the oligosaccharides whose DP was greater than 3（DP＞3） had good resistance to various metabolizing enzymes,and so they were the reliable source of functional oligosaccharides.Finally,the gluco-oligosaccharides’ yields by strains 6055,DM1-2,PC13 and L4,especially after the selective removal of sugars from fermenting liquors by S.cerevisiae,were124g/L, 58g/L,66g/L and 83g/L,respectively.Data from nuclear magnetic resonance illustrated that the oligosaccharide product were linked byα-1,6 linkages in the backbone with several branching structures linked byα-1,4 linkages,and theα-1,4 linkages locad at the reducing end of oligosaccharides.更多还原