黑曲霉YY-22产酸性果胶酶的分离纯化

本文研究了从黑曲霉YY-22发酵产酸性果胶酶粗酶液中分离出较高纯度果胶裂解酶(PL)、聚半乳糖醛酸酶(PG)、果胶酯酶(PE)的方法,同时考察了主要组分PL在各分离阶段的纯化效果。依次采用硫酸铵盐析、疏水相互作用层析及离子交换层析对果胶酶PG、PE、PL进行分离。结果表明:果胶酶粗酶液中硫酸铵饱和浓度为65%时,沉淀中PL回收率最大,部分杂蛋白在盐析过程中被分离出来;沉淀复溶后经Phenyl-Sepharose FF疏水相互作用层析首先分离出PE活性组分,又经Q-Sepharose HP强阴离子交换层析分别得到PG和PL活性组分。果胶酶粗酶液中主要组分PL经三步纯化后的比活力达到79.37 U/mg蛋白,纯化倍数为13.30,活力回收为33.05%。较高纯度PL、PG、PE的获得,为进一步研究酶的基本性质及其在果蔬加工和葡萄酒中的应用奠定基础。     

葡萄酒酿造用商业果胶酶中主要功能酶活性研究

对8种商业果胶酶中聚半乳糖醛酸酶(PG)、果胶裂解酶(PL)、果胶甲酯酶(PME)、纤维素酶、内切-1-4-β-D-木聚糖酶以及β-D-葡萄糖苷酶共6种酶的活性进行测定并进行使用效果估测。结果表明,不同商业果胶酶中的此6种酶活性存在差异,聚半乳糖醛酸酶活性最强,其次是内切-1-4-β-D-木聚糖酶和果胶甲基酯酶酶活,而β-D-葡萄糖苷酶活性较低;与红葡萄酒用果胶酶相比较,白葡萄酒用果胶酶中纤维素酶活性较低。     

亚麻脱胶过程中常用酶类的动态变化

对亚麻快速生物脱胶和温水沤麻过程中的果胶酶、木聚糖酶、纤维素酶、微生物、pH值和还原糖等进行了动态变化研究。结果表明,2种脱胶过程中果胶酶和木聚糖酶的变化趋势基本相似,酶活性脱胶前期均增加缓慢,中后期迅速增加;纤维素酶活性的变化趋势有明显的区别,快速生物脱胶的纤维素酶酶活性增加幅度小,酶活性低(0.05 IU/mL左右),温水沤麻的纤维素酶在发酵72 h后迅速增加,脱胶完成后酶活性达0.337 9 IU/mL;微生物的变化趋势基本相似,在脱胶前期迅速增加、脱胶完成时开始下降;pH值和还原糖量的变化趋势分别呈“U”型和“M”型。     

胡椒果皮高效脱胶菌的筛选、鉴定及初步应用

为实现胡椒生物酶法高效脱皮,从海南胡椒种植园的土样中采集样品,以茵株产果胶酶活力的大小和胡椒脱皮效果为筛选指标,分离、筛选出1株胡椒果皮高效脱胶茵WC 0021;对其进行生理生化及16SrDNA序列分析,初步鉴定为蜡状芽孢杆菌(Bacillus cereus).将菌株WC0021发酵生产的果胶酶用于胡椒鲜果脱皮试验,结果表明,在pH为7.5,温度为35℃的条件下,所产果胶酶2天内可将胡椒果皮脱除干净,所得白胡椒中挥发油和胡椒碱的含量分别是3.37 mL/100 g和4.25 g/100 g,菌落总数为1.0×103 cfu/g,感官评价为优.     

果胶酶产生菌的筛选鉴定和酶学性质研究

以果胶为唯一碳源,从海南采集的土样中筛选出一株能够利用果胶生长并产生果胶酶的菌株,采用3,5-二硝基水杨酸定糖法测定果胶酶酶活,通过生理、生化特性以及16S rRNA基因序列分析比对对菌株进行鉴定,并对其所产果胶酶的酶学性质进行研究。结果表明,分离筛选出一株果胶酶产生菌,编号为YY01,被鉴定为Bacillus niabensis。菌株YY01所产果胶酶的最适pH值为7.0,最适反应温度为45 ℃;在pH 10.0孵育12 h,仍有52.5%残余酶活力,50 ℃孵育6 h,仍有64%的残余酶活力。结果显示该酶有较好的耐碱性及较高的热稳定性,具有应用于果汁加工和胡椒脱皮的应用前景。     

果胶酶在葡萄酒酿造中的作用及其实践应用

果胶酶是现代葡萄酒酿造中的重要辅料,大多数是由黑曲酶（Aspergillus niger）经特殊工艺制成的液体果胶酶或固体果胶酶。葡萄酿酒中常用的果胶酶通常是复合果胶酶,含有果胶裂解酶（Pectinlyase,PL）,果胶酯酶（Pectin esterase,PE）和聚半乳糖醛酸酯酶（Polygalacturonases,PG）等。     

果胶酶在葡萄酒酿造中的作用及其实践应用

果胶酶是现代葡萄酒酿造中的重要辅料,大多数是由黑曲酶(Aspergillus niger)经特殊工艺制成的液体果胶酶或固体果胶酶.葡萄酿酒中常用的果胶酶通常是复合果胶酶,含有果胶裂解酶(Pectinlyase,PL),果胶酯酶(Pectin esterase,PE)和聚半乳糖醛酸酯酶(Polygalacturonases,PG)等.在复合果胶酶的协同作用下,可促进葡萄汁澄清,果香浸提和颜色浸渍,对葡萄酒品质的改善和提升起到显著效果[1].     

微生物果胶酶的研究进展

果胶酶(pectinases)是指能协同分解果胶质的一组酶的总称,主要包括聚半乳糖醛酸酶(PG)、果胶裂解酶(PL)和果胶酯酶(PE)等,广泛应用于食品工业、饲料工业、造纸工业和纺织工业.近年来,随着我国国家政策和产业结构的调整,果汁和果酒行业得到迅猛发展.果胶酶作为果汁加工的重要酶制剂,市场需求量巨大.果胶酶生产普遍采用微生物发酵法,本文主要概述并比较了国内外微生物发酵法生产果胶酶的研究和应用情况.对国内果胶酶生产菌种的选育、微生物产果胶酶的发酵工艺及培养条件等进行了重点阐述,并简要介绍了微生物果胶酶的酶学性质和果胶酶的固定化情况.旨在发现当前我国果胶酶研究和开发中存在的问题,明确今后我国果胶酶研究中的前进方向,为研究和开发出同国际接轨的新型、高效果胶酶提供参考.     

黑曲霉固态发酵凉茶渣产酶工艺研究

为探索利用黑曲霉固态发酵凉茶渣进行资源化利用的可行性,以内切葡聚糖酶、木聚糖酶和果胶酶的活力为指标,对氮源、碳源、含水量、发酵时间、浸泡液pH值和温度共6个单因素进行考察;根据单因素试验结果,添加4%硫酸铵作为氮源,2%葡萄糖为碳源,通过正交试验,以3种酶活力的总和为指标,优化制备工艺,发现含水量为70%,浸泡液pH值为9.00,温度为31℃,发酵168 h,是3种酶的最佳生产工艺。最佳工艺条件下发酵产物的内切葡聚糖酶活力为(5.72±0.23)U/g,木聚糖酶活力为(42.43±2.50)U/g,果胶酶活力为(29.81±0.69)U/g,3种酶活力总和为(77.96±1.08)U/g。     

光叶楮白皮生物脱胶制浆及其机理研究

对影响光叶楮白皮生物脱胶制浆的主要因素及脱胶酶特性进行了研究。结果表明，生物脱胶制浆的最优工艺条件为：初始pH值7，温度30℃，液比1：15～1：25，接种量3％；加入氨源助剂能够显著提高成浆效果，且以用量为1．5％。2．5％的尿素为佳。最优条件下的制浆结果为：得率70．O％，高锰酸钾值14，6，白度43，2％ISO，成浆时间94h，废液pH值4．0，CODc。值为O．337L／t浆。脱胶酶活力分析结果表明：发酵初期，酶活力都随时间延长而升高，发酵至36h，果胶酶活力达到峰值，木聚糖酶活力在发酵至60h达到峰值，峰值过后，2种酶的活力随时间延长逐渐降低，纤维素酶活力在整个周期内缓慢增加，没有峰值出现。     

Pectolytic Enzymes in Sweet Bell Peppers (Capsicum annuum L.)

Low levels of polygalacturonase (PG) and pectinesterase (PE) activities were quantitated in sweet bell peppers at four stages of maturity based on surface ground color (dark green, light green, turning, and red). The PG activity increased during pepper ripening and was maximal at the turning stage while the PE activity declined during ripening and was maximal at the light green stage. Pepper texture measurements were found to decline concomitantly with the increase in PG activity. Pepper PG showed pH optimum between 4.8 to 5.0 and increased temperature stability with advanced ripening. Compared to tomatoes, pepper PG activity was 116- to 164-fold lower and pepper PE activity was 429- to 1900-fold lower than the corresponding tomato enzymes.     

Temperature and water activity effects on growth and temporal deoxynivalenol production by two Argentinean strains of Fusarium graminearum on irradiated wheat grain

The ability of six strains of Pichia anomala, four strains of Pichia kluyveri and two strains of Hanseniaspora uvarum predominant during coffee processing to produce polygalacturonase (PG), pectin esterase (PE) and pectin lyase (PL) in yeast polygalacturonic acid medium (YPA) and in coffee broth (CB) was studied. For comparison, a reference strain of Kluyveromyces marxianus CCT 3172 isolated from cocoa and reported to produce high amount of PG was included.

Initial screening of PG activity using YPA medium showed that K. marxianus CCT 3172, P. anomala S16 and P. kluyveri S13Y4 had the strongest activity. Enzymatic assays showed that the four yeast species secreted PG, but none of the yeasts investigated was found to produce PE or PL. P. anomala S16 and P. kluyveri S13Y4 were found to produce higher amounts of PG when grown in CB than in YPA. When K. marxianus CCT 3172, P. anomala S16 and P. kluyveri S13Y4 were grown in YPA broth adjusted to pH of 3.0–8.0 and incubated at temperatures of 15–40 °C, the three yeast species secreted the highest amount of PG at pH 6.0 and at 30 °C. For PG secreted by K. marxianus CCT 3172 and P. anomala S16, the optimum pH and temperature for the enzymatic activity were 5.5 and 40 °C, respectively. On the other hand, PG produced by P. kluyveri S13Y4 showed the highest activity at pH 5.0 and 50 °C.

Significant differences in the extracellular activity of PG were found between the yeasts species as well as between strains within same species. High amounts of PG were produced by two strains of P. anomala and P. kluyveri. It is therefore likely that strains of those two species may be involved in the degradation of pectin during coffee fermentation.     

Flax retting by degumming composite enzyme produced by Bacillus licheniformis HDYM-04 and effect on fiber properties

Flax enzymatic retting with composite enzyme produced by microbes with inexpensive substrates is widely researched due to less contamination and lower cost. Bacillus licheniformis HDYM-04, isolated from a liquid sample of flax retting pool, efficiently produced degumming enzymes after 48 h of fermentation with inexpensive konjaku flour, consisted of 587.5 U/mL pectinase, 365.2 U/mL mannanase, and 140.1 U/mL xylanase. Almost half the maximum activity of three above-mentioned degumming enzymes was maintained at pH 4.0–6.0 which demonstrated its stability in pH condition of flax retting. After 120 h of retting with this composite enzyme, scanning electronic microscopy showed more significant reduction in gummy components on the fiber surface than those of water retting. The fiber strength was 182.4 ± 9.3 N, 14.3% higher than water-retted samples. The long fiber rate and fiber yield also verified higher fiber productivity. The results permitted this degumming composite enzyme an applicable potential in flax retting.     

Effect of edible coatings on enzymes,cell-membrane integrity,and cell-wall constituents in relation to brittleness and firmness of Huanghua pears (Pyrus pyrifolia Nakai,cv. Huanghua) during storage

To investigate the effects of edible coatings, such as shellac and Semperfresh™ (sucrose-polyester based coating) on the brittleness and firmness of Huanghua pears (Pyrus pyrifolia Nakai, cv. Huanghua), the changes in the cell-membrane permeability and cell-wall constituents, such as total pectin (TP), Na₂CO₃-soluble pectin (NSP), CDTA-soluble pectin (CSP), water-soluble pectin (WSP), hemicellulose and cellulose were periodically measured, for up to 60 days of cold storage (4 °C) after harvesting. The activities of peroxidase (POD), pectinesterase (PE), polygalacturonase (PG), and cellulose were also assayed. The data suggested that high POD activity and low activity of cell-wall-degrading enzymes, such as PE, PG, and cellulase in the coated pears were associated with a high integrity of the cell membrane and few changes in the cell-wall constituents, which contributed to high levels of brittleness and firmness in the pears during storage; further, the shellac coating provided a better effect than Semperfresh coating.     

两种碳源对猴头菇液体发酵中的多糖分泌及胞外酶活性的影响

目的 探讨葡萄糖与红糖在猴头菇液体发酵过程中对菌丝多糖分泌及胞外酶活性的影响, 筛选出猴头菇的最适碳源。方法 分别以葡萄糖和红糖为碳源进行猴头菇菌丝液体培养, 以紫外分光光度法测定不同发酵时间猴头菇的总糖、还原糖与多糖含量及纤维素酶、木聚糖酶与果胶酶活性。结果 两种碳源对猴头菇液体发酵的影响较大, 除菌丝生物量影响不显著外, 其他指标均呈现显著差异(P<0.05)。其中, 以葡萄糖为碳源时, 发酵液始终为酸性, 并且pH随发酵时间延长显著降低, 最低可达4.36; 总糖与还原糖含量随发酵时间延长显著降低; 纤维素酶活力在发酵第11 d时显著升高并达到最高值(392 U/L), 木聚糖酶活性在发酵至第7 d时显著升高至最大值(1809 U/L), 但第11 d时显著降低, 果胶酶在发酵至第3 d时活力最高, 可达7349 U/L, 3种胞外酶的活性最高值交错出现。以红糖为碳源时, pH先升至7.28, 后降低至弱酸性(5.88); 总糖含量随发酵时间延长无显著变化, 还原糖含量显著下降(P<0.05), 而多糖含量升高极显著; 纤维素酶活性随发酵时间延长显著降低, 但木聚糖酶及果胶酶活性无显著变化, 3种酶活性最高值均低于以葡萄糖为碳源的酶活性最高值。结论 以葡萄糖为碳源培养猴头菇, 猴头菇3种胞外酶的分泌具有一定的规律性, 使其可以迅速分解果胶, 然后为木聚糖, 最后大量利用纤维素, 而以红糖为碳源有利于猴头菇胞外多糖的分泌, 为猴头菇液体发酵后续药理作用研究与生产开发利用提供参考。     

复合微生物的筛选及其发酵秸秆的应用效果

为提高秸秆的发酵效果,通过筛选纤维素酶活力和木聚糖酶活力高的一定比例的复合菌株,并进行秸秆复合菌株发酵应用研究。结果表明,最佳复合菌株为：白腐菌+黑曲霉+米曲霉+芽孢杆菌+放线菌（1∶1∶1∶1∶1）,其纤维素酶活最高值为203.64 U/g,木聚糖酶活最高值为10 017.41 U/g。发酵田间大堆小麦秸秆时,试验组发酵38 d的秸秆,中性洗涤纤维、粗纤维比发酵前显著降低（P＜0.05）,粗蛋白显著升高（P＜0.05）。与对照组发酵38 d的秸秆相比,中性洗涤纤维显著降低（P＜0.05）,粗蛋白显著升高（P＜0.05）。发酵过程中温度有高温期、中温期和低温期,发酵结束时秸秆pH值（7.73）为微碱性,符合发酵堆肥标准。发酵结束后试验组的秸秆比对照组的秸秆腐化程度高。因此,混菌发酵有助于提高秸秆的发酵效果。     

β-甘露聚糖酶产生菌HDYM-04的分离鉴定及产酶条件优化

从实验室温水沤麻液中分离并筛选到了一株产β-甘露聚糖酶的细菌HDYM-04,经生理生化试验及16S rDNA序列分析鉴定为地衣芽孢杆菌(Bacillus licheniformis)。研究了HDYM-04菌株72h连续发酵过程中产酶及生长的动态变化,通过单因素及序贯正交试验优化得到其摇瓶发酵产酶的最佳培养基组分及条件：碳源60g/L魔芋粉,氮源30g/L蛋白胨,MgSO4•7H2O 0.2g/L,K2HPO4 5g/L,初始pH 8.0,装液量100ml/250ml三角瓶,接种量2%,37℃振荡培养48h。在研究条件下发酵液酶活力4890U/ml,比优化前提高了3.2倍。     

大麻快速生物脱胶过程中发酵液成分变化

为了探明大麻快速生物脱胶的机理,在实验室条件下,利用脱胶高效菌株Dm111,对大麻韧皮进行快速脱胶试验,定期测定了胶质去除率和发酵液中的相关指标。结果表明,在脱胶前期和中期,胶质去除率、脱胶菌活菌量不断增加,后期趋于平缓;果胶酶和木聚糖酶酶活均是脱胶前期增加缓慢,中期迅速增加,后期下降,而纤维素酶活在脱胶过程中变化不大,且酶活性很低;pH值呈“V”型变化;还原糖出现2个峰值,呈近似“M”型变化。发酵液的COD、蛋白质和残渣量与脱胶时间呈正相关。至脱胶完成时,残渣量占大麻韧皮的27％左右。