动力学法优化碱性果胶酶比活的测定方法

本文通过优化蛋白浓度测定方法和酶活力测定方法,并以酶解动力学曲线考察方法优化的合理性,以验证终点法测定碱性果胶酶比活的准确性。结果表明,果胶酶蛋白的最佳测定波长为550 nm,其线性和截距明显优于传统方法,可以忽略果胶酶稀释倍数对蛋白测定的影响。钙离子对该酶具有激活作用;聚半乳糖醛酸比果胶更适合作为酶活力测定底物,其最适pH为9.5,最佳浓度为2 mg/mL,果胶酶活力测定的最佳波长为232 nm,所测酶活力在吸光度值0~2范围内均保持零级反应状态,酶蛋白浓度与酶解速率线性相关且回归曲线截距更接近原点,因此,该测定条件不仅可用于终点法,酶解时间也可灵活掌握,而非拘泥于一个固定的时间段。以酶解30 min为例,其检测限(LOD)为0.15 mU/mL。由于DNS法的灵敏度较低,其半乳糖醛酸标准曲线不过原点,从而导致因酶液稀释倍数不同而产生测量误差,因此,紫外法测定碱性果胶酶活力为最佳选择。     

苎麻果胶的提取及在果胶酶活测定中的应用

研究了苎麻中纤维果胶的提取,优化的提取工艺为:温度90℃,草酸铵质量浓度5g/L,液料比为30:1,反应时间90min。以纤维果胶和苹果果胶为底物,采用DNS法测定了棉精练用碱性果胶酶的酶活力。结果表明,以纤维果胶为底物测定的棉织物精练用碱性果胶酶酶活力为151.6U/mL,远高于苹果果胶的5.3U/mL,更适合用来表征棉织物精练用果胶酶的酶活力。     

基于复合酶解工艺优化的速溶桂圆粉研制

以桂圆肉为主要原料,经过软化、打浆,得到果浆;在单一酶酶解基础上通过正交设计优化复合酶的影响因素,获取最佳果胶复合酶组成:果胶裂解酶(酶活力为1万U/mL)0.15 g/L,纤维素酶(酶活力为1万U/g)0.20 g/L,果胶酶(酶活力为3万U/g)0.15 g/L,可溶性固形物得率可达85.8%;复配、真空喷雾干燥后,速溶桂圆粉得率50.2%,溶解度达到98%。     

酶法澄清石榴果汁的研究

探讨了果胶酶添加量、温度、时间、果汁稀释倍数对石榴汁澄清的影响,在单一因素基础上,果胶酶用量1830￣2550U/mL、酶促反应温度为40￣55℃、澄清时间1.5￣2.5h、稀释倍数0￣5,透光率达59.1%。进行正交实验确定最佳条件,结果表明:石榴果汁中果胶酶用量1830U/mL、澄清时间2h、温度50℃、稀释倍数0,可达到最好的澄清效果。     

双水相分离果胶酶测定方法的研究

研究了果胶酶活力测定中酶活力与稀释倍数的关系，确定了在用PEG／（NH4）2SO4双水相系统进行果胶酶提取分离过程中酶活力测定的最佳稀释倍数，即酶液浓度应控制在消耗0．05mol／LNa2S2O3标准溶液（A-B）之差在0．5mL～1．0mL范围内，且该稀释倍数范围与果胶浓度有关。     

DNS法测定D-氨基葡萄糖含量方法的研究

建立了DNS法测定调味品中色素D-氨基葡萄糖含量的方法。研究了反应的测定波长、DNS试剂用量、反应时间、反应温度、反应液体积以及反应产物在5h内的显色稳定性对测定结果的影响。结果表明:DNS法测定D-氨基葡萄糖含量的最适波长为520nm,D-氨基葡萄糖浓度在100~300μg/mL时,DNS试剂用量为1.0mL,沸水浴反应8min,反应液体积4mL,反应产物在5h内显色性质稳定,该方法精密度良好,RSD为0.08%,回收率为98.86%。     

Bacillus subtilis Z-5产果胶酶发酵条件的优化及其应用

为进一步提高果胶酶的产量,采用响应面法优化了Bacillus subtilis Z-5产果胶酶的发酵培养基和发酵条件,并将优化后得到的果胶酶应用于梨汁澄清实验。通过单因素实验探究9个因子对B. subtilis Z-5产果胶酶活力的影响,然后采用Plackett-Burman试验设计筛选出3个显著影响产酶的因素：酵母粉含量、pH、培养时间;再结合响应面设计法确定了最佳的发酵条件。结果表明最佳的发酵培养基成分是果胶10 g/L,酵母粉7.99 g/L,MgSO₄·7H₂O 0.5 g/L;最佳的发酵条件为初始pH5.66,发酵温度37℃,培养时间72 h,接种量为6%(v/v),装液量50/250 mL,底物浓度为10 g/L;在此基础上,B. subtilis Z-5产果胶酶酶活力由879.00 U/mL提高到1549.62 U/mL,是优化前的1.76倍。在梨汁澄清实验中表明果胶酶的最适添加量为4 mL,最适酶解时间为2.5 h,最适酶解温度为65℃,最适酶解pH为6.0,此时透光率最大为79.77%,与商品酶相比,自制果胶酶是一种具有多种...     

两次压榨与酶解结合提取菠萝果汁工艺技术研究

本文研究了果胶酶和纤维素酶对菠萝出汁率的影响.最适工艺条件为:果胶酶(活力单位为353.13 U/mL)用量为0.05%,纤维素酶(活力单位为10000U/g)用量为0.025%,时间为1 h,温度为35℃.同时对不同提汁工艺对菠萝渣出汁率的影响进行了研究,确定二次压榨与酶处理结合提汁法可以提高出汁率15.94%.     

果胶酶生产及其在苹果汁澄清中的应用

对黑曲霉(Aspergillus niger)LLW-1菌株固态发酵生产酸性果胶酶的发酵培养基组成和发酵条件以及利用果胶酶澄清苹果汁的工艺条件进行了研究。固态发酵产酶条件为:250 mL三角瓶装10 g基料(豆饼∶麸皮=2.0 g∶8.0 g),(NH4)2SO420 g/kg,Tween-80 1.5 g/kg,玉米浆50 mL/kg,KH2PO43.0 g/kg,CaCl21.0 g/kg,MgSO4.7H2O 1.0 g/kg(均相对于基料),料水比1∶1.2,自然pH;31℃静置培养72 h,期间在为24 h翻曲1次,果胶酶活力达2 418 IU/g(干曲)。酸性果胶酶澄清苹果汁的工艺条件为:果汁自然pH值,果胶酶用量500IU/L(果汁),明胶添加量0.05 g/L(果汁),酶解温度和时间分别为45℃和1 h。     

海洋扩展青霉(Penicillium expansum)产耐盐型果胶酶的固态发酵优化

目的:提高海洋扩展青霉(Penicillium expansum)产耐盐型果胶酶的酶活力。方法:测定海洋扩展青霉所产果胶酶的耐盐性;通过单因素和正交实验,对海洋扩展青霉产耐盐型果胶酶进行固态发酵优化;利用SPSS 20.0分析实验结果。结果:该果胶酶在底物所含Na Cl浓度为30g/L时,酶活力最高,在Na Cl浓度为0~60g/L范围内较稳定。最优培养基为:麸皮7g,果胶粉0.42g,氯化铵0.28g,人工海水8.4m L;最优培养条件为:接种量3m L(107cfu/m L),培养温度28℃,250m L三角瓶中培养72h。此优化条件下酶活力可达到6639.79U/g。结论:该果胶酶耐盐性好;固态发酵优化后,酶活力达到6639.79U/g,为原来的酶活1239.80U/g的5.36倍。     

枯草芽孢杆菌产碱性果胶酶发酵条件的优化

通过单因素实验对枯草芽孢杆菌菌株发酵产碱性果胶酶的培养基组分及培养条件进行优化。利用单因素实验确定了产酶的最优培养基:30 g/L豆饼粉、35 g/L马铃薯淀粉、20 g/L果胶、2.775 g/L氯化钙、4.025 g/L硫酸锌、113.6 g/L Na 2HPO 4。同时对温度、接种量、发酵pH进行优化,得到最优发酵条件:温度35℃、接种量3%、发酵过程控制pH=7.4,在此基础上进行补料流加实验,补料配方为350 g/L葡萄糖、10 g/L果胶,补料控制总糖浓度为20 ug/mL,并调整转速和风量控制溶氧30%~40%,最终酶活达到6120 U/mL,较初始酶活1061 U/mL提高了4.77倍。     

糠醛渣复合材料固定果胶酶的制备

本文以糠醛渣为原始材料进行磺化预处理,利用静自电组装技术将壳聚糖包覆表面,得到糠醛渣-壳聚糖和磺化糠醛渣-壳聚糖复合材料。以FT-IR、SEM等技术对以上制备的复合材料的进行分析表征;然后将两种复合材料利用戊二醛交联后进行果胶酶的固定化。采用单因素变量法研究新型固定化酶的最佳催化性能和稳定性。未磺化糠醛渣复合材料固定酶的最佳催化条件:pH 3.5,果胶酶浓度50 mg/mL,果胶浓度15 mg/mL,反应时间120 min,反应温度45℃;磺化糠醛渣复合材料固定酶的最佳催化条件:pH 3.5,果胶酶浓度20 mg/mL,果胶浓度10 mg/mL,反应时间60 min,反应温度50℃。其中糠醛渣复合材料的固定化果胶酶的最大载酶量为197.20 mg/g,在重复循环使用8次后剩余相对酶活可达81.78%,磺化糠醛渣复合材料的固定化果胶酶在4℃下储存32 d后仍剩余88.98%的相对酶活。两种固定酶都表现出较好的操载酶量和储存稳定性,有较好的经济价值和应用前景。     

嗜热碱性果胶酶产生菌的筛选及产酶条件优化

从极端土壤中分离到1株高效产碱性果胶酶菌株RH214,经鉴定为Bacillus smithii。为了进一步提高其产果胶酶活性,对产酶的培养基成分进行了优化。首先采用单因素试验筛选出最佳碳源为可溶性淀粉,最佳氮源为蛋白胨,金属盐为KH2PO4。根据单因素结果确定可溶性淀粉浓度、蛋白胨浓度和KH2PO4浓度为主因素,利用Box-Behnken试验设计和响应面法分析确定各因素的最优水平,得出菌株RH214的最优产酶条件:可溶性淀粉的浓度为85.6 g/L、蛋白胨浓度11.8 g/L、KH2PO4浓度为3.8 g/L、酵母粉1 g/L、FeSO41g/L、KCl 0.5 g/L、起始pH9.0、装液量80 mL/250 mL、温度40℃、摇床转速150 r/min,培养时间为16 h。在此条件下,进行验证实验,获得产酶活性为(88±0.86)U/mL,比未优化前的20.50 U/mL提高了4.3倍。     

Synergistic Effect and Mechanisms of Combining Ultrasound and Pectinase on Pectin Hydrolysis

The current study aimed to investigate the synergistic effect of ultrasound and pectinase on pectin hydrolysis. Effects of ultrasound on the hydrolysis rate of pectin, enzymatic kinetics parameters and pectinase structures (determined by the DNS method, Michaelis–Menten equation, chemical reaction kinetics model, fluorescence spectroscopy and circular dichroism spectroscopy) were also studied in order to illuminate the mechanisms of the synergistic effect. The hydrolysis rate of pectin achieved maximum value with ultrasound treatment at 4.5 W mL^?1 intensity and ultrasound time of 10 min, increasing by 32.59 % over the control. The optimum temperature for the hydrolysis reaction was 50 °C and kept unchanged with ultrasound treatment. Besides, the value of V _max increased whereas K _m decreased in the sonoenzymolysis reaction compared with that in the routine enzymolysis reaction. Results indicated that under ultrasound irradiation, pectin was hydrolyzed at an elevated rate and the pectinase exhibited stronger affinity to the substrate. Fluorescence spectra revealed that ultrasound favorably decreased the amount of tryptophan on the pectinase surface; while the far-UV circular dichroism spectra showed an increased fraction of β-sheet and a reduced fraction of random coil in the secondary conformation. Changes in the pectinase structures contributed to the enhancement of pectinase activity and the consequent promotion of the hydrolysis process. Results of pectin degradation kinetics certified the synergistic effect of ultrasound and pectinase at the temperature range of 20–50 °C, which were evidenced from the positive values of the synergistic coefficients.     

柑橘酒酿造工艺研究

研究了柑橘酒发酵过程中甲醇含量的变化及其影响因素。结果表明,柑橘汁中添加果胶后发酵得到的柑橘酒甲醇含量更高;添加果胶的柑橘果汁中添加一定量果胶酶或蔗糖,甲醇含量也有所增加;添加金属离子,甲醇含量减少。正交试验结果表明果胶酶、CaCl2、蔗糖的含量分别为40mg/L、1.1g/L、180g/L时,柑橘酒中甲醇含量由150.48mg/L降低到15mg/L。     

高温混菌发酵生产果胶酶的研究

为了降低底物抑制效应,提高黑曲霉产果胶酶的能力,在黑曲霉发酵过程中引入酒精酵母,成功构建了黑曲霉GJ-2与酒精酵母J-1混菌发酵生产果胶酶的体系,确定了酒精酵母的最佳接入时间和最佳接入量分别为12h和5mL/50mL,并对其产酶条件进行响应面优化,实验中发现高温对产酶有促进作用,确定最佳产酶条件为:橙子皮29.3g/L,麸皮30.0g/L,硫酸铵11.8g/L,吐温-802.0g/L,发酵温度34.1℃,在此条件下果胶酶酶活为188.12U/mL,比黑曲霉单一体系发酵生产果胶酶提高了63.6%。     

<Emphasis Type="Italic">Saccharomyces boulardii</Emphasis>: Optimization of simultaneous saccharification and fermentation of cell production in organic and conventional apple substrate pulp

A face-centered factorial design was used to study the influence of temperature, cellulase, and pectinase concentration on the production of Saccharomyces boulardii cells during simultaneous saccharification and fermentation of organic and conventional apple substrate pulp. The effects of the variables fermentation temperature (25–35 °C), pectinase concentration (5–25 μL/100 g), and cellulase concentration (4–8 μL/100 g) were analyzed by multiple regression and polynomial models of second order, providing the ideal conditions for yeast cultivation. Cellular production of apple substrates was expressed in log CFU/mL. The optimum condition for temperature was 27.5 °C, and 20 and 5 μL/100 g for pectinase and 8 and 7 μL/100 g for cellulase concentrations for organic and conventional apple pulp, respectively. The observed viability values were in agreement with the predicted values of 8.352 log CFU/mL (organic) and 8.317 log CFU/mL (conventional) apple pulps, thus proving the effectiveness of the models.     

Pectinase and naringinase help to improve juice production and quality from pummelo (Citrus grandis) fruit

Pectinase and naringinase were investigated for improving the production of pummelo juice by increasing the juice yield and eliminating juice bitterness. Compared to a control, the enzymatic treatment significantly (p<0.05) increased the juice yield, soluble pectin and total soluble solid (TSS) contents, and the clarity, while decreasing the concentrations of the bitter chemicals naringin, limonin, and nomilin. A combined processing treatment of peeling and enzymatic hydrolyses using 5U/g of pectinase and 0.4 U/g of naringinase at 50oC for 60 min resulted in a juice yield of 42.3%, a TSS content of 11.4oBx, a titrable acidity (TA) of 0.96%, a vitamin C concentration of 38.4 mg/100 mL, and concentrations of naringin, limonin, and nomilin of 42.4 μg/mL, 33.5 μg/mL, and 13.3 μg/mL, respectively. The enzymatic method was effective and practical for production of high quantity pummelo juice.