【作者】 Mohanad Osman Adam Bashari；

【导师】 Jin Zhengyu；

【作者基本信息】 江南大学 ， Food Science&Technology， 2014， 博士

【摘要】 在制糖过程中,葡聚糖是最主要的杂质之一。葡聚糖因影响蔗糖结晶而导致蔗糖损失,给蔗糖加工和精制增加了难度,并造成经济损失。因此,近年来很多研究采用微生物降解法降低制糖过程中葡聚糖的分子量,从而使葡聚糖对蔗糖加工影响最小化。到目前为止,葡聚糖酶降解法是糖加工厂和精炼厂用来水解葡聚糖最有效的方法。本论文的主要目的包括：(1)优化蔗糖生产过程中葡聚糖酶的添加条件(2)研究葡聚糖酶降解葡聚糖对蔗糖结晶过程和糖晶体的影响(3)探索改善葡聚糖酶活性以减少工业应用成本的新方法。研究了不同分子量和不同浓度的葡聚糖对过饱和蔗糖溶液的流变和玻璃化转变特性的影响。分别将三种分子量葡聚糖100000g/mol、500000g/mol、2000000g/mol以60%-75%(w/w)添加量加入到浓度为1000-10000mg/kg的蔗糖溶液中,发现表观黏度和动态模量随着葡聚糖浓度增加而增加,且与其分子量呈现强相关性；采用差示扫描量热仪法测量样品玻璃化转变温度(Tg),用Fox模型和扩展的Gordon-Taylor模型对Tg与葡聚糖分子量和浓度之间的相关性进行分析,发现葡聚糖分子量和浓度越大,玻璃化转变温度升高越大,证实了扩展的Gordon-Taylor模型可以有效预测不同葡聚糖-蔗糖混合溶液的玻璃化转变温度。采用1H,13C及二维核磁共振技术(COSY和HMQC)、GC-MS、MALDI-TOF等解析了葡聚糖(SC-Dex)的结构。采用酒精沉淀和凝胶过滤层析的方法,从变质甘蔗中提取葡聚糖。利用总酸水解和酶解的方法确定定分离到葡聚糖的纯度。结果表明,SC-Dex萄聚糖主链是由D-葡萄糖通过α-(1-6)糖苷键连接而成,分支主要为D-葡萄糖通过α-(1-3)糖苷键连接；甲基化分析表明,α-(1-3)分支度为4.37%；MALDI-TOF分析显示分子离子峰的质量差异为162g/mol,表明SC-Dex的重复单元为D-葡萄糖；SC-Dex的表面形态为球形并具有多孔结构；HPSEC-MALLS-RI分析表明,SC-Dex的重均分子量为1.753x106g/mol,多分散指数为1.069。研究了糖加工不同阶段葡聚糖酶的添加对剩余葡聚糖分子量参数和固有黏度的影响。采用分光光度法测定葡聚糖酶的相对活性,并用高效液相色谱测定还原糖含量的方法对其进行验证。为进行对比,分别对葡聚糖酶进行了浓缩和稀释。发现在蔗糖汁中添加葡聚糖酶比在蒸发糖浆中添加能更有效降低剩余葡聚糖分子量,且更有经济价值；在优化条件糖汁pH5.5时添加葡聚糖酶可得到最低特性黏度和最小分子量,、与优化温度为55.0℃时的结果相似,但当糖度超过20°Brix时,酶活性降低；葡聚糖去除率随着葡聚糖酶添加量增加而增大,当葡聚糖酶浓度达到100ppm时,葡聚糖去除率最高,为80.29%；此外,在加工过程中,反应时间越长,剩余葡聚糖分子量越低；为达到满意的水解度,葡聚糖酶的用量需根据因高糖度造成酶活损失来矫正。在优化了萄聚糖酶使用后,研究了不同温度下葡聚糖酶催化萄聚糖水解物对糖生产中蔗糖结晶速率和纯蔗糖溶液中晶体生长速率的影响。为说明萄聚糖水解对蔗糖晶体生长速率的影响,将2,000,000g/mol (T2000)分子量的葡聚糖以1000-10000mg/kg添加量加入到55%-70%(w/w)的蔗糖溶液中,并分别加入3种浓度50、75、100ppm的萄聚糖酶。在加入T2000的糖液酶解后,发现蔗糖结晶速度提高了,并发现结晶表面比报道的仅添加T2000葡聚糖的更好。结果表明,在添加100ppm萄聚糖酶水解T2000葡聚糖后,相比于纯蔗糖液中添加T2000葡聚糖,结晶速率提高了高达73.56%。因此,采用添加萄聚糖酶水解萄聚糖可以提高蔗糖结晶速率,从而降低糖制造成本。研究了提高葡聚糖酶活性的新方法,超声辐射(US)处理对葡聚糖酶催化活性和酶水解动力学参数的影响。发现US能够提高葡聚糖酶的催化动力学活性；在超声频率25kHz、超声功率40W、超声时间15min时,葡聚糖酶活性达最高,比常规50℃加热处理提高了13.43%；动力学研究表明,超声处理后Vmax和KM值均增加,表明超声处理能够促进底物转化。另外,我们还研究使用其它方法结合超声处理来提高酶的活性。研究了US与高静水压(US/HHP)联合处理对葡聚糖酶催化活性和酶反应动力学参数的影响,采用荧光光谱和圆二色谱对处理后葡聚糖酶的结构进行了分析。结果表明,当US功率40W、频率25kHz、处理时间15min,HHP压力400MPa、处理时间25min时,葡聚糖水解度达到最大,比常规50℃加热处理增加了163.79%；Vmax、Km、Kcat均比US、HHP单独处理及常规方法有所提高；与US、HHP和常规方法相比,US/HHP组合处理降低了酶反应的Ea、△G和ΔH,但ΔS略微升高；荧光光谱法和圆二色谱解析表明,US/HHP处理增加了葡聚糖酶表面的色氨酸数量,α折叠增加了19.8%,无规卷曲减少6.94%,这有利于提高酶活。研究了超声波和微波辐射(US/MIS)对葡聚糖酶水解葡聚糖的协同作用。采用US(50W,40kHz)和不同功率的微波辐射(10-140W、2450MHz、20sec/min)对葡聚糖酶进行处理,结果显示,US/MI-S处理后葡聚糖水解度比单独采用US、MI-S以及常规方法明显增大,在US功率50W、MI-S功率60W、频率20sec/min、时间25min时,水解度达到最大,比常规加热方法提高了163.58%；与单独处理相比,组合处理后酶的Vmax和KM增加,Kcat和(Kcak/Km)比常规方法提高；组合处理后酶的Ea、ΔG和ΔH均降低,ΔS略微升高；圆二色谱法显示组合处理和US处理降低了酶分子β折叠和无规卷曲含量,MI-S处理提高了β折叠和无规卷曲含量；然而,荧光光谱显示酶二级结构的降解速率比三级结构降解速率慢,可见US/MI-S处理导致了葡聚糖酶二级结构的重排,有助于提高其活性。综上所述,在糖的工业生产过程中,将US与HHP或MI-S进行联用,可以作为一种新的手段来提高葡聚糖酶的工业应用效率,本研究将为相关领域的研究提供依据。更多还原

【Abstract】 Dextran is one of the most significant impurities present in sugar production process. Thepresence of dextran in the canesugar factories and refineries leads to many technical possessingdifficulties, however, from the processing point of view, the most damaging effects of elevateddextran concentrations in a technical sucrose solution are foreseen in the crystallization process.All these possessing difficulties caused by dextrans lead, in the end, to the sugar losses and theseeconomic losses are continuous throughout the process. Therefore, in recent years many studieshave been undertaken to minimization of dextran effects in the sugar factory by controllingmicroorganisms and reducing its molecular weight during the manufacturing process. To date, theuse of the dextranase enzyme is the most efficient method for hydrolyzing dextrans at canesugarfactories and refineries.The objectives of this research are to (1) optimize the addition and investigate the conditionsof using dextranase in cane sugar manufacturing,(2) characterize the effects of thebiodegradation of dextran using dextranase enzyme on the crystallization process and the qualityof the final sugar crystal and (3) find novel methods to improve dextranases activity to decreasetheir industrial application costs.To begin with, the effects of dextran at differents molecular weight (Mw) and theconcentrations of on the rheological and glass transition properties of supersaturated sucrosesolution were investigated. Three dextrans of various Mw, namely100,000g/mol,500,000g/moland2,000,000g/mol, were admixed in concentrations between (1000-10000ppm) with (60%-75%w/w) sucrose solution. The results indicated that both the apparent viscosity and dynamicmodulus increased with an increase in dextran concentrations and they demonstrated strongdependence on its Mw. Glass transition temperature (Tg) of the samples were measured bydifferential scanning calorimetry, and their dependence on dextran Mw and concentration wasanalyzed by the Fox and expanded Gordon-Taylor models. It was found that, the higher the Mwand concentration of the dextran, the greater the increase in Tg. The expanded Gordon-Taylorequation has proved useful in predicting the Tg of different dextrans and sucrose solutionmixtures.In the further step, dextran extracted from deteriorated sugarcane was characterized using themore recently available techniques such as (1H,13C) and two-dimensional (COSY and HMQC)NMR spectral analysis, methylation GC-MS and MALDI-TOF mass spectrometry, the structureof sugarcane dextran (SC-Dex). dextran was extracted from deteriorated sugarcane by alcoholprecipitation and purified by gel filtration chromatography. Total acid hydrolysis and enzymatic degradation were utilized to confirm the purity of separated polysaccharide. On the basis of allspectra, SC-Dex showed a branched polysaccharide that contained only D-glucose residues in-(1-(13) branches. Methylation analysis-(13) branching levels was4.37%. Several structural fragments wereidentified from MALDI-TOF spectrum with peak-to-peak mass difference of162gmol-1, whichconfirmed that the repeat unit in SC-Dex was D-glucose. The surface morphology of SC-Dex,revealed the spherically shaped and porous structure. Using HPSEC-MALLS-RI system, theaverage molecular weight of SC-Dex was estimated to be1.753x106gmol1with an index ofpolydispersity value of1.069.The influence of dextranase enzyme on the molecular weight (Mw) parameters of remainingdextran and intrinsic viscosity after different enzymatic treatments at different steps during sugarmanufacturing was investigated. A spectrophotometric method was used to determine the relativeactivity of dextranase and the result has been confirmed by measured reducing sugar using HPLCsystem. For comparison, the action patterns of concentrated and diluted enzymes wereadditionally included in the experiments. Additions of dextranase to juice were much moreefficient and economical to reduce the Mw of remaining dextran than adding it to evaporatorsyrups. Addition of dextranase at juice pH5.5showed similar minimum Mwwith the lowestintrinsic viscosity, observed at55.0°C, and the enzymes activity was decreased after20°Brix.The highest dextran removal was observed at dextranase concentration at100ppm/juice whichwas resulted in80.29%removal dextran in the juice, Moreover, the higher the level ofconcentrated dextranase applied to the juice, the more the removal of dextran occurred. Inaddition, the longer the availability of the residence time in the factory, the lower dextran Mw hasbeen observed. To reach a satisfactory level of dextran hydrolysis, it was necessary to correct thedose of dextranase enzyme according to the losses of activity caused by the high°Brix.After optimizing the dextranase application, the Influence of hydrolysis of dextran catalyzedby dextranase enzymes during sugar manufacturing on the rate of sucrose crystallization andgrowth rate of sucrose crystals in pure sucrose solution at different temperatures was investigated.To elucidate the influence of hydrolysis of dextran on the growth rate of sucrose crystals, dextranat Mw, of2,000,000g/mol (T2000), was admixed in concentrations between (1000-10000ppm)with (55%-70%w/w) sucrose solution, however, three different concentrations of dextranaseenzyme (50,75,100ppm) were applied for the emzymatic hydrolysis. After enzymatic hydrolysisof dextran T2000presence in the sucrose solution, the growth rate of the sucrose crystal wasincreased, in addition, more perfect crystal surfaces was observed compared with that reported inthe presence of dextran T2000. From the results it could be shown that an increase of crystallization rate of up to73.56%after hydrolysis of dextran T2000using dextranase enzyme at100ppm,compared to crystallization rate with pure sucrose solution in the presence of dextran T2000. Sucha positive influence of enzymatic hydrolysis of dextran using dextranase enzyme could decreasethe crystallization time in the sugar house and thus decreases the production costs of sugarmanufacturing.Based on the above result, that dextranase could minimize of dextran effects during thecrystallization process, further investigations to find novel methods for improving dextranasesactivity to decrease their industrial application costs were carried out. Sequently, the effect ofultrasound irradiation (US) on the enzymatic activity and enzymatic hydrolysis kinetic parametersof dextran catalysis by dextranase were investigated. US has improved the catalytic kineticsactivity of dextranase at all the reaction conditions studied. The maximum activity of dextranasewas observed when the sample was treated with US at25kHz,40W for15min, under which theenzyme activity increased by13.43%compared the routine thermal incubation at50°C.Experimental Kinetics results, demonstrated that, both the Vmaxand KMvalues of dextranaseincreased with US-treated compared with the incubation at50°C. Likewise, both the catalyticand specificity constants are higher under the effects of US field, indicating that, the substrate isconverted into the product at an increased rate when compared with the incubation at50°C. Inaddition, to date, no research work is available on the combination of other methods with US toimprove the enzymes activities. Therefore, we decided to study the effect of combination of othermethods with the ultrasonic irradiation on the enzymes activities.After the enhancement of dextranase activity by means of US, the effect of combination ofUS and high hydrostatic pressure (US/HHP) on the enzymatic activity and enzymatic hydrolysiskinetic parameters of dextran catalytic by dextranase were investigated. Furthermore, the effectsof US/HHP on the structure of dextranase were also discussed with the aid of fluorescencespectroscopy and circular dichroism (CD) spectroscopy. The maximum hydrolysis of dextran wasobserved under US (40W at25kHz for15min) combined with HHP (400MPa for25min), inwhich the hydrolysis of dextran increased by163.79%compared with the conventional thermalincubation at50°C. Results also showed that, Vmaxand KMvalues, as well as, kcatof dextranaseunder US/HHP treatment were higher than that under US, HHP and thermal incubation at50°C,indicating that, the substrate is converted into the product at an increased rate when comparedwith the conventional thermal incubation at50°C. Compared to the enzymatic reaction under US,HHP, and conventional thermal incubation, dextranase enzymatic reaction under US/HHPtreatIn addition, Fluorescence and CD spectra reflected that US/HHP treatment had increased the num-helix by19.80%and reducedrandom coil by6.94%upon US/HHP-treated dextranase protein compared to the control, whichwere helpful for the improvement of its activity.The synergetic effects of ultrasonic and microwave irradiation sock (US/MIS) on hydrolysis ofdextran by dextranase were finally studied. The US treatments were performed at fixed power of50W/40kHz, and the microwave irradiation shock (MI-S) was applied at different powerconditions, of10-140W at2450MHz at sock rate of20sec/min. The hydrolysis of dextran underUS/MI-S was significantly higher than those performed under US, MI-S and conventionalthermal incubation at all condition studied. The maximum hydrolysis rate was observed whenUS/MI-S (US of50W combined with MI-S of60W at sock rate of20sec/min for25min) wasused in which the dextran hydrolysis increased by163.58%compared with routine conventionalheating. Results also showed that, Vmax and KM values of dextranase under US/MI-S treatmentwere higher than those under US, MI-S and conventional thermal incubation. Higher kcatand(Kcat/Km) values were obtained under US/MI-s in comparison with the values in the respectiveconventional heating. Compared to the enzymatic reaction under US, MI-S, and conventionalheating incubation, dextranase enzymatic reaction under US/MI-S treatment showed decreases inOn the other hand, CD spectrareflected that-sheet and random coil content were decreased by the mean of the US/MI-s andUS treatments and increased under MI-s treatment compared to control. However, fluorescencespectra demonstrate the rate of degradation of its secondary structure was slower than that ofenzyme tertiary structure. Therefore, US/MI-s treatment results in reordered secondary structureof dextranase, which were helpful for the improvement of its activity.These results, therefore, indicated that, the combination of US with HHP or MI-Streatments could be used as a novel technique method for improving the industrial efficiency ofdextranases in many industrial applications including sugar manufacturing processes. This studywill serve as a basis for the future work in this area of research.更多还原