Application of response surface methodology to the modeling of cellulase purification by solvent extraction

Central composite design (CCD)sp. JS14 in a solvent extraction was established with Response surface methodology (RSM). Solvent concentration, pH, temperature and retention time were selected as process variables to evaluate the purification impact factor in solvent precipitation, including the purification fold and % recovery. An experimental space with 13 purification fold and 23 recovery percentage recovery is achieved through the optimized condition based on the model. The molecular weight of the purified enzyme was estimated to be 32.5 KDa. Optimum activity of purified enzyme was at pH and temperature 6.5℃ and 40℃ respectively. Enzyme showed maximum activity with carboxymethyl cellulose as substrate with compare to rice husk, wheat straw and sucrose. The purified cellulase activity was inhibited by Na+, Cl- Mg2+ Tween 80 and EDTA.

Optimization of ultrasound assisted extraction of celastrol from Celastrus monospermus Roxb.using response surface methodology

In the present study, we aimed to investigate the effects of key extraction parameters including extraction time（10–20 min）, extraction temperature（30–60 °C）, ultrasonic power（60–90 W） and solvent-to-solid（S/S） ratio（10–30 m L/g） on yield of celastrol from Celastrus monospermus Roxb. To optimize the conditions, we investigated the effects of parameters on the ultrasound assisted extraction（UAE） with the Box-Behnken Design（BBD）, one widely used form of Response Surface Methodology（RSM）. In all tested solvents, ethanol was the most effective for celastrol extraction, followed by methanol, ethanol, ethyl acetate, n-butanol and water. A second order polynomial model was fitted well to the extraction experimental data with R2 of 0.9928. Extraction yield of 3.116 mg/g was obtained for celastrol under the optimized extraction conditions of extraction time（20 min）, extraction temperature（46 °C）, ultrasonic power（60 W） and S/S ratio（30 m L/g）. Experimental validation was performed, and the experimental values agreed well with the predicted values. The results indicated that the UAE was good extraction material for celastrol from C. monospermus Roxb.

Ultrasound-assisted Extraction of Kamebakaurin from Rabdosia excisa by Response Surface Methodology

For the efficient extraction of kamebakaurin（KA）, the ultrasound-assisted extraction（UAE） of KA from Rabdosia excisa（R, excisa） via response surface methodology（RSM） was investigated with high-performance liquid chromatography（HPLC）. Effects of the experimental parameters such as extraction solvent, ratio of liquid to plant material, extraction time and extraction temperature on the extracting efficiency of KA from R. excisa were evaluated, and the purity of KA in residual was calculated. The optimized conditions were 65.5%（volume fraction） acetone, 35 ℃, time of 24.6 min with ultrasound of 80 W/L, 40 kHz, ratio of liquid to plant material at 30：1（mL/g）. The maximum yield of KA is 0.708 mg/kg, with mean purity of 6.09%, indicating that ultrasound-assisted extraction is a feasible and useful method for extracting KA from R. excisa.

Application of Statistically Based Experimental Designs to Optimize Cellulase Production and Identification of Gene

A natural bacterial strain identified as Bacillus amyloliquefaciens MBAA3 using 16S rDNA partial genome sequencing has been studied for optimization of cellulase production.Statistical screening of media components for production of cellulase by B.amyloliquefaciens MBAA3 was carried out by Plackett–Burman design.Plackett–Burman design showed CMC,MgSO4 and pH as significant components influencing the cellulase production from the media components screened by Plackett-Burman fractional factorial design.The optimum concentrations of these significant parameters were determined employing the response surface central composite design,involving three factors and five levels was adopted to acquire the best medium for the production of cellulase enzyme revealed concentration of CMC(1.84 g),MgSO4(0.275 g),and pH(8.5)in media for highest enzyme production.Response surface counter plots revealed that middle level of MgSO4 and middle level of CMC,higher level of CMC and lower level of pH and higher level of MgSO4 with lower level of pH increase the production of cellulase.After optimization cellulase activity increased by 6.81 fold.Presence of cellulase gene in MBAA3 was conformed by the amplification of genomic DNA of MBAA3.A PCR product of cellulase gene of 1500 bp was successfully amplified.The amplified gene was conformed by sequencing the amplified product and sequence was deposited in the gene bank under the accession number KF929416.

Optimization of Culture Conditions for Production of Cellulase by a Thermophilic Bacillus Strain

The production of cellulase in Bacillus amyloliquefaciens UNPDV-22 was optimized using response surface methodology （RSM）. Central composite design （CCD） was used to study the interactive effect of culture conditions （temperature, pH, and inoculum） on cellulase activity. Results suggested that temperature and pH all have significant impact on cellulase production. The use of RSM resulted in a 96% increase in the cellulase activity over the control of non-optimized basal medium. Optimum cellulase production of 13 U/mL was obtained at a temperature of 42.24 ℃, pH of 5.25, and inoculum size of 4.95% （v/v） in a fermentation medium containing wheat bran, soybean meal and malt dextrin as major nutritional factors.

Application of Box-Behnken Design for Optimization of Different Pretreatment Conditions for Cellulase Production

Effects of dilute acid and acid steam pretreatments were inspected for cellulose production of Eucalyptus leaves through Box-Behenken design, a three variable factors for response surface methodology by Bacillus subtilus K-18. Maximum cellulose production performed in 250 mL erlenmeyer flask with submerged fermentation attained at 50＂C, pH 5, 140 r· min-1 for 24 h. Results showed the efficient cellulose production from acid steam pretreatrnent （being autoclaved at 15 Psi for 15 rain） than acid pretreatment. The optimum condition for maximum carboxymethyl cellulas （CMCase） was 1.811 IU·mL-1·min-1 （0.8% acid cone., 10 g biomass loading, 6 h reaction time） and filter paper activity （FPase） was 2.255 IU·mL·-1·min-1 （1% acid conc., 10 g biomass loading, 8 h reaction time）. Whereas, the acid steam maximum CMCase activity recorded was 2.585 IU·mL-1·min-1 （0.8% acid cone., 15 g substrate loading and 8 h reaction time） and the highest FPase activity was 2.055 IU·mL-1·min-1 （0.8% cone., 10 g biomass, 6 h reaction time then autoclaved）. Results revealed that acid pretreated Eucalyptus leaves were better lignocellulosic biomass for cellulose production by submerged fermentation.

双水相协同超声法提取枇杷叶中绿原酸的工艺研究

试验采用响应面法优化双水相协同超声法提取枇杷叶中绿原酸的工艺,探讨提取液中乙醇体积分数、超声温度、超声时间、磷酸氢二钾用量和超声功率对绿原酸提取率的影响。结果显示,双水相协同超声法提取枇杷叶中绿原酸最佳提取工艺条件为乙醇体积分数61%、超声温度61℃、超声时间25 min、磷酸氢二钾用量4.6 g,此条件下绿原酸提取率1.53%,与预测值相接近。研究表明,双水相协同超声法提取枇杷叶中绿原酸的提取工艺稳定可行,可为枇杷叶的资源化开发与利用提供参考。

粉煤灰基充填膏体制备过程中氨气释放的超声强化特性

将粉煤灰作为主要胶凝材料的膏体充填开采不仅能有效控制地面沉降、解决“三下”压煤问题、提高煤炭采出率,还可解决燃煤电厂粉煤灰堆积难题。而粉煤灰基充填膏体的氨气释放会对充填作业面的气体环境造成污染,亟需治理。提出一种利用超声波促进膏体氨气释放的新思路。通过对粉煤灰浆液进行超声处理,采用以超声时长(A)、超声功率(B)、固液比(C)作为考察因素,进行单因素实验,结果表明:随着超声时长、超声功率以及固液比的的增加氨气释放速率呈现出先增大后减小的趋势,并在超声时长、超声功率以及固液比分别为30 min、80 W和1∶10时达到最大值。在单因素实验结果的基础上进行响应面实验建立回归模型。通过响应面优化确定最佳实验参数。结果表明:影响因素主次顺序为:A>B>C,AB>AC>BC;去除氨气最佳实验参数为:超声时长为32 min,超声功率为80 W,固液比为1∶7.7。验证结果表明:实测值与预测值仅存在-1.6%的相对误差。利用优化后的实验参数处理粉煤灰,并制备成充填膏体测定氨气释放量,发现膏体氨气释放量平均减少了95.94%,氨气释放速率提高84.02%,膏体3 d和28 d的单轴抗压强度分别提高185.00%和86.50%,坍塌度、泌水率和凝结时间均降低,同时减少了膏体内部多害孔数量,使孔隙率由33.58%降低到28.13%,为粉煤灰基充填膏体的氨气释放治理提供新的方法与思路。

响应面法优化超声提取多花黄精多糖工艺及其生物活性研究

通过响应面法和超声波辅助法优化多花黄精多糖的提取工艺,并研究多糖的抗氧化活性。采用单因素试验及Box-Behnken Design方法,以提取物中多糖的含量作为评估指标,以确定最佳提取工艺。结果显示,超声功率对多花黄精多糖含量影响有显著影响,液料比、超声波时间、超声波温度对实验结果影响不大。多花黄精多糖的最佳提取工艺条件为:液料比41 mL/g、超声时间40 min,超声功率151 W,超声温度59℃,最佳提取工艺条件下多花黄精多糖含量可达(16.67±0.53)%,且具有较强的抗氧化活性,30 mg/mL的多花黄精多糖对DPPH·的清除率为(61.35±0.96)%,对ABTS^(+)·的清除率为(97.58±0.70)%。因此,基于对多花黄精多糖的超声辅助进行响应面优化的提取工艺可靠、提取率高,可为进一步开发利用多花黄精提供依据。

酸化甘油-超声辅助提取杨梅花青素工艺优化及其抗氧化活性研究

目的 通过响应面实验确定酸化甘油-超声辅助提取杨梅花青素最佳提取参数,并探究其体外抗氧化活性。方法 研究了酸化甘油浓度、提取时间、超声功率和提取温度4个单因素对杨梅花青素得率的影响,并优化花青素提取工艺参数。此外,考察了最佳提取工艺下杨梅花青素对3种氧化自由基的清除率和还原力等指标,评估了其抗氧化活性。结果 优化后的酸化甘油-超声辅助提取杨梅花青素的工艺条件为:酸化甘油浓度16%、提取时间42 min、超声功率204 W、提取温度46℃,此条件下杨梅花青素的得率为(6.915±0.027) mg CGE/g DW,接近预测值(6.961 mg CGE/g DW)。提取的杨梅花青素质量浓度为100μg/mL时,对超氧阴离子自由基、羟基自由基、1,1-二苯基-2-三硝基苯肼自由基清除率分别为59.35%、46.77%和61.65%。该浓度下,杨梅花青素的还原力为维生素C的39.84%。结论 本研究成功地应用响应面法优化了酸化甘油-超声辅助提取杨梅花青素的提取参数,并且提取物展现出良好的抗氧化活性。

纤维素酶辅助超声提取丁香叶黄酮工艺优化及抗氧化性分析

为研究丁香叶中总黄酮的高效提取方法,本文首次采用纤维素酶辅助超声法对4种丁香叶中的总黄酮进行提取。以总黄酮提取得率为指标,单因素实验分析纤维素酶用量、pH、超声时间、超声功率、液料比、温度、乙醇体积分数对小叶丁香叶总黄酮提取得率的影响,应用响应面法优化,得到最优提取工艺,并通过体外抗氧化检测丁香叶总黄酮对DPPH自由基和OH自由基的清除能力。结果表明,最优提取工艺为加酶量2%,pH5.5,超声时间40 min,超声功率400 W,乙醇体积分数63%,温度56℃,液料比30:1(mL/g),在该条件下,小叶丁香叶总黄酮提取得率为32.21±0.16 mg/g,与回归模型的预测值32.57 mg/g接近。抗氧化活性结果表明,4种丁香叶总黄酮对DPPH自由基的清除能力为小叶丁香>暴马丁香>白丁香>紫丁香;对OH自由基的清除能力为小叶丁香>暴马丁香>紫丁香>白丁香。纤维素酶辅助超声法能高效提取丁香叶中的总黄酮,且丁香叶总黄酮具有较好的抗氧化性,具有一定的开发潜力。

基于遗传算法-反向传播神经网络优化高压-超声-酶解法提取羊皮胶原蛋白工艺

采用高压-超声-酶解法提取羊皮胶原蛋白,对比遗传算法-反向传播(genetic algorithm-back propagation,GA-BP)神经网络模型和响应面模型的优化效果,确定最佳工艺参数。结果表明:GA-BP神经网络在模型拟合和预测方面表现优于响应面模型;最佳提取参数为高压时间23 min、超声时间22 min、酶添加量3.2%、酶解时间222 min,羊皮胶原蛋白提取率达到(80.5±1.6)%,较传统的木瓜蛋白酶法提高40%;紫外-可见吸收光谱和傅里叶变换红外光谱结果显示,此条件下提取的羊皮胶原蛋白结构完整,高压-超声-酶解法对胶原蛋白的破坏较小。

塔拉多糖超声提取及其自由基清除能力研究

为研究塔拉多糖的提取工艺及抗氧化活性,选用超声辅助提取法对塔拉多糖进行提取,围绕液料比、超声功率、提取温度和提取时间展开单因素试验,并通过响应面法对影响参数进行优化。经过统计分析和数学回归模型拟合得知,决定系数(R~2)为0.956,即精确系数为95.60%,表明该模型吻合度较好,适用于塔拉多糖超声提取工艺的优化,其最佳参数为10∶1 mL/g, 20 min, 150 W,40℃,实际平均得率为24.58%,接近理论期望得率25.07%。此外,又对塔拉多糖自由基的清除能力进行一阶动力学方面的研究,以维生素C为对照,根据试验数据得知,塔拉多糖的DPPH、ABTS~+对应IC 50分别为524.07和3 496.88μg/mL,说明其在自由基清除能力方面表现较为优异,因此在抗氧化性方面具有较好的应用价值。研究成果最终可为植物多糖的提取、纯化与应用提供可靠的理论指导与数据支撑。

无花果原花青素超声辅助提取工艺优化及抗氧化性研究

以无花果为原料,采用单因素实验结合响应面法对超声辅助提取无花果原花青素(Ficus carica L.Proanthocyanidin,FCPC)工艺进行优化,同时测定DPPH自由基清除率、羟基自由基清除率和总抗氧化能力来综合评价其抗氧化活性。结果表明,FCPC最优提取条件为乙醇体积分数68%,液料比41∶1(mL∶g),提取时间29 min,超声温度47℃,FCPC平均提取量为(15.75±0.28)mg/g,接近模型预测值(15.60 mg/g)。FCPC具有良好的抗氧化能力,对DPPH和OH自由基均具有较强的清除能力,当FCPC质量浓度为0.013 2 mg/mL时,对DPPH和OH自由基的清除率分别可达为94.64%和70.73%。优化的FCPC提取工艺条件稳定可靠,能在实际生产中应用;提取的FCPC具有较强的抗氧化活性,其抗氧化能力大于同质量浓度的维生素C。

超声波辅助酶法提取猪皮胶原蛋白的工艺优化及胶原纤维的湿法纺丝制备

胶原纤维材料是一种重要的生物医用纺织品,广泛用于各类外科手术。为了提高猪皮源胶原蛋白的提取率,设定酶浓度、超声强度、超声时间为影响因素,胶原提取率为响应值,采用超声波辅助酶法,通过单因素实验和Box-Behnken响应面法探究最佳提取条件,并采用湿法纺丝技术制备猪皮源胶原纤维。结果表明:影响胶原提取率的显著性顺序为:酶浓度>超声时间>超声强度,最佳提取工艺的酶浓度为93 U/g、超声强度为162 W、超声时间为58 min,所制备的胶原提取率56.21%,与响应面回归方程预测值相近,说明该回归模型能够较好地优化提取工艺条件。同时,紫外光谱、红外光谱和X射线衍射分析表明猪皮源胶原的类型为Ⅰ型,具有完整的三螺旋结构;纤维形貌分析表明猪皮源胶原具有良好的成纤性;力学性能分析表明纺丝液为4.0%浓度的胶原纤维具有最佳的力学性能。

金花茶多酚制备、成分分析及其体内外抗氧化活性分析

为了探究金花茶多酚最佳提取工艺条件及纯化后总多酚成分及体内外抗氧化活性分析。本研究以金花茶为原料,在单因素实验的基础上,设计响应面试验,筛选超声功率、提取温度、乙醇浓度和料液比最佳提取参数。采用Lx-8型大孔树脂对提取物进行纯化,并以高效液相色谱法对5种多酚单体化合物进行定性定量分析,随后对提取物进行体内外抗氧化实验。结果表明,最优工艺条件为超声功率290 W,提取温度35℃,乙醇浓度55%,料液比1:30 g/mL,在此条件下,金花茶多酚得率为3.53%,茶多酚纯度为63.73%。5种多酚单体化合物没食子酸、绿原酸、表儿茶素、芦丁、鞣花酸含量分别为0.47、11.18、59.03、18.34、16.41 mg/g。体外抗氧化实验结果表明,金花茶花多酚能够有效清除DPPH自由基、ABTS+自由基和超氧阴离子自由基,其IC50值分别为0.38、0.16、0.39 mg/mL。此外,金花茶多酚还能够通过显著提高线虫体内SOD酶活力及降低MDA含量,有效增强秀丽隐杆线虫对氧化损伤的抵抗力。

超声辅助酶法提取滇刺枣多酚工艺优化及抗氧化活性研究

目的优化滇刺枣多酚(Zizyphus mauritiana Lam.polyphenols,ZMP)超声辅助酶法提取工艺条件并测定ZMP抗氧化性。方法以液料比、乙醇体积分数、超声温度、超声时间、超声功率、酶用量作为考察因素,在单因素实验考察基础上设计响应面法实验优化ZMP提取条件,并对ZMP清除DPPH自由基(DPPH·)、羟基自由基(·OH)的能力进行研究。结果ZMP超声辅助酶法提取最佳工艺条件为:液料比为40∶1(mL∶g)、超声温度为41℃、超声功率为304W、酶用量为4.1%(m/m)、乙醇体积分数为60%、超声时间为50min,此时,ZMP提取量平均值为(18.41±0.16)mg·g^(-1),接近预测值(18.33mg·g^(-1))。抗氧化性测定结果显示,当ZMP质量浓度为3.0μg·mL^(-1)时,对DPPH·、·OH的清除率分别为75%、72%,二者清除率均大于对照药品维生素C。结论响应面法实验优化的ZMP超声辅助酶法提取工艺稳定、可行,具有较强的抗氧化性,ZMP可作为一种抗氧化剂开发利用。

超声辅助低共熔溶剂提取韩信草总黄酮的工艺优化

为提高韩信草中总黄酮的提取率,实验采用超声辅助低共熔溶剂——氯化胆碱-乙二醇提取韩信草中的总黄酮。首先采用冷冻干燥法合成氯化胆碱-乙二醇低共熔溶剂,以单因素试验确定含水率、料液比、超声功率、超声时间对韩信草中总黄酮提取量的影响,通过Plackett-Burman法结合Box-Behnken响应面法进一步对超声提取工艺进行优化。试验结果表明,该提取工艺的最佳条件为含水率59.30%,液料比25.90∶1,超声功率121 W,总黄酮含量为7.924 mg/g。相同条件下采用氯化胆碱-乙二醇为提取溶剂明显高于其他传统溶剂,可实现总黄酮高效提取的优点。因此,通过优化后的超声辅助低共熔溶剂氯化胆碱-乙二醇法能明显提高总黄酮的提取率。

响应面法优化兰雪醌超声辅助提取工艺的研究

目的优化白花丹中兰雪醌超声辅助提取的最佳工艺。方法采用紫外分光光度法分析兰雪醌含量。采用单因素试验考察超声时间、提取温度和料液比对兰雪醌得率的影响,并在单因素试验的基础上使用Box-Behnken响应面法优化得到最佳工艺。结果该模型的调整决定系数(R_(Adj)^(2))为0.8394,回归模型的概率值(P<0.005)显示出较高的显著性。通过Box-Behnke响应面法得到超声提取兰雪醌的最佳工艺参数为超声时间41 min,提取温度42℃,料液比1∶23(g/mL),预测兰雪醌得率为1.823%。在最佳条件下进行了5次验证试验,得到兰雪醌平均得率为1.8941%,与预测值基本吻合。结论利用超声辅助提取方法提取白花丹中兰雪醌的工艺稳定可靠。

超声辅助低共熔溶剂提取当归药渣粗多糖的工艺优化

为研究当归(Angelica sinensis)药渣提取粗多糖的工艺,本研究以当归药渣粗多糖(Angelica sinensis Residue Crude Polysaccharide, ARP)的提取率为指标,考察低共熔溶剂(Deep Eutectic Solvent, DES)的种类、组成成分的摩尔比、DES含水量、提取温度、提取时间和液料比对ARP提取率的影响,并结合响应面分析法优化提取工艺参数。研究结果表明,氯化胆碱和乙二醇组成的低共熔溶剂更适合提取ARP,最优提取工艺为提取温度82℃、提取时间40 min、摩尔比6∶1。该工艺条件下的ARP提取率为(14.43±0.10)%,ARP总糖含量为(47.54±1.23)%;而传统水提醇沉法的ARP提取率为(9.85±0.12)%,ARP总糖含量为(25.45±1.37)%。因此,氯化胆碱和乙二醇组成的低共熔溶剂是一种较好的提取溶剂,可替代传统溶剂进行多糖的提取,具有实际应用价值。