产柚苷酶菌株的初步筛选

柚苷酶作为沙田柚和柑桔果实中主要苦味物质的分解酶有望在深加工生产中得到应用。本研究在沙田柚树林下土壤和沙田柚皮腐烂液中分离了多株产柚苷酶菌株,其中A09产酶最高,摇瓶产酶达263U／mL,初步确定为黑曲霉。研究发现菌株A09的产酶过程需要柚苷诱导,该菌株具有较好的选育潜力。     

Naringinase Immobilization in Packaging Films for Reducing Naringin Concentration in Grapefruit Juice

Naringin, a bitter compound in citrus, may be converted to a nonbitter form by enzymic hydrolysis. Our objective was to determine the feasibility of immobilizing naringinase in a food contact approved packaging film. Naringinase from Penicillium sp. was immobilized in cellulose acetate films with up to 23% efficiency at 7°C. Kinetic studies showed that the free enzyme had an optimum pH=3.5 and the immobilized enzyme pH=4.0. Activation energy decreased upon immobilization (from 14.2 to 11.0 Kcal/mol), thus providing an increased catalytic efficiency for immobilized naringinase. The Michaelis constant for immobilized naringinase (K_m=2.1 mM) was lower than for free enzyme (K_mm=3.6 mM). Keeping films under dry storage for 1 mo at room temperature did not cause decreased enzyme activity. A film area/volume ratio (cm²/mL of 10° Brix grapefruit juice) of 7.2 hydrolyzed 60% of the naringin in 15 days at 7°C.     

黑曲霉产柚苷酶的发酵条件优化

利用高效液相色谱法,检测发酵液中α-鼠李糖苷酶和柚苷酶的活力,考察碳源、pH值、搅拌转速、发酵温度对柚苷酶产量的影响,对黑曲霉DB056产柚苷酶的7L罐发酵工艺进行优化.研究结果表明,以20g/L柚皮苷为唯一碳源,发酵黑曲霉DB056产柚苷酶的效果最佳.发酵过程的pH值、搅拌转速和发酵温度的最优控制值分别为6.0、700 r/min和32℃,此时产酶效果最理想,发酵过程α--鼠李糖苷酶和柚苷酶最大酶活力分别达到1 133.00和788.42U/mL.将优化的发酵工艺放大到200 L发酵罐进行试验,α-鼠李糖苷酶和柚苷酶的最大酶活力分别为1 069.30 U/mL和727.44 U/mL.中试放大效果良好.     

超声波及热处理对柚皮苷酶稳定性及其二级结构的影响

研究了超声波及热处理对柚皮苷酶的活性及热稳定性的影响,通过圆二色谱分析超声波及热处理对柚皮苷酶二级结构的影响。结果表明,随着温度的升高,酶失活速率加快,在80℃下保温150min,柚皮苷酶残留酶活仅为原酶的15.69%。超声功率为40～120W时,短时间的超声处理对柚皮苷酶活性有促进作用,而功率大于160W时超声波处理对柚皮苷酶活性有钝化作用。80W处理对柚皮苷酶的热稳定性有促进作用,处理5min后60℃保温150min,酶活性比未经处理的柚皮苷酶活性增加了64.34%;而280W处理对柚皮苷酶的热稳定有钝化作用,处理40min后60℃保温150min,酶活性比未经处理的柚皮苷酶活性降低了42.61%。圆二色谱分析结果表明,未经处理的柚皮苷酶中α-螺旋含量较少,主要结构为β-折叠和无规则卷曲;热处理及超声波处理对α-螺旋结构几乎没有影响,β-折叠、β-转角及无规则卷曲含量的变化分别在0.4%～1.1%、0.1%～0.4%及0～0.9%之间。     

柚苷酶产生菌Penicillium sp.1523分批发酵动力学研究

分析柚苷酶产生菌青霉(Penicillium sp.)1523在5 L自控发酵罐中的分批发酵动力学。探讨了青霉1523在5 L罐分批发酵过程中菌体生长、柚苷酶合成及基质消耗的变化规律,结果表明:菌体生长呈典型S型曲线,酶的合成与菌体生长趋势呈现部分相关性,属于部分偶联型。基于这些过程曲线的变化规律,结合Logistic方程和Luedeking-Piret方程,建立了柚苷酶产生菌青霉1523分批发酵过程的动力学模型,并通过数学推导和非线性拟合获得了模型中各参数的最佳取值,最终确定了能够较好表征青霉1523分批发酵过程中菌体生长、产物柚苷酶合成以及基质消耗的动力学方程。     

柚苷酶在柑桔果醋加工中的应用

在柑桔果醋加工中,利用柚苷酶进行脱苦实验,结果表明：发酵温度在25℃-28℃,酸度控制在0．5－0．8 /100mL,柚苷酶加量在15μ/mL,柚苷酶有较佳的活性;酒精度为6．0％－8．0％（V）时,有利于醋酸发酵,制得的果醋清亮透明,酸甜可口,果香明显,基本无苦味。     

微生物来源柚苷酶的研究进展及应用

柚皮苷是影响柚子汁品味的最主要因素,柚苷酶具有将柚皮苷分解为普鲁宁、鼠李糖、葡萄糖和柚皮素等物质的功能,并且这些物质已广泛用于制药和饮料行业。本文介绍了近年来国内外微生物来源的柚苷酶的研究、酶学特性、酶活测定、结构与功能等,表明细菌来源的柚苷酶最适温度为37～70℃,最始pH值为4.5～8.0;真菌来源的柚苷酶最适温度为30～75℃,最始pH值为4.0～11.0。介绍了柚苷酶及其相关产物在食品、饮料和制药工业中的应用进展,发现柚苷酶不仅具有脱苦的作用,还对人类的身体健康具有重要作用。展望了今后的研究方向,应该从柚苷酶高产菌株的选育、发酵产酶的工艺优化和酶的应用等方面开展工作,大幅度提高柚苷酶的产酶水平。     

磁性硅基壳聚糖微球固定化柚苷酶水解柚皮苷的研究

本文在磁性Fe3O4外包覆一层SiO2,再在其外包裹壳聚糖制备出磁性硅基壳聚糖微球（MSC）,对MSC进行环氧基修饰后用于柚苷酶的固定化研究,并对磁性硅基壳聚糖微球固定化柚苷酶水解柚皮苷的pH、温度、操作和储藏稳定性进行了考察。通过单因素实验,确定了环氧基修饰的磁性硅基壳聚糖微球（MSCE）固定化柚苷酶的最佳工艺条件为：pH 3.0,温度30 ℃,时间4 h、给酶量57.48 U/mL。在此条件下,MSCE固定化柚苷酶的载酶率、酶活回收率和酶比活力分别为31.29%、88.92%和409.33 U/g。与游离柚苷酶相比,MSCE固定化柚苷酶用于水解柚皮苷具有良好的pH稳定性和温度稳定性,重复使用8次后仍具有53.36%的相对酶活力,4 ℃条件储存一个月后仍具有80.97%的相对酶活力。     

Covalent immobilization of naringinase for the transformation of a flavonoid

Naringinase (EC 3.2.1.40) from Penicillium sp was immobilized by covalent binding to woodchips to improve its catalytic activity. The immobilization of naringinase on glutaraldehyde‐coated woodchips (600 mg woodchips, 10 U naringinase, 45 °C, pH 4.0 and 12h) through 1% glutaraldehyde cross‐linking was optimized. The pH–activity curve of the immobilized enzyme shifted toward a lower pH compared with that of the soluble enzyme. The immobilization caused a marked increase in thermal stability of the enzyme. The immobilized naringinase was stable during storage at 4 °C. No loss of activity was observed when the immobilized enzyme was used for seven consecutive cycles of operations. The efficiency of immobilization was 120%, while soluble naringinase afforded 82% efficacy for the hydrolysis of standard naringin under optimal conditions. Its applicability for debittering kinnow mandarin juice afforded 76% debittering efficiency. Copyright © 2005 Society of Chemical Industry