EFFECT OF SUBSTRATE SIZE ON THE ACTIVITY OF TOMATO POLYGALACTURONASE

SUMMARY— Three polygalacturonic acid preparations with widely differing molecular weight distributions were obtained by controlled enzymatic hydrolysis of pectic acid. A study of the action of tomato polygalacturonase on the polygalacturonic acids and pectic acid revealed that the activity h dependent on the molecular size of the substrate. Pectic acid was hydrolyzed optimally at pH 5, with little activity below pH 4. Decreasing the molecular weight of the substrate resulted in a progressive shift of the pH optimum to the acid side. For the smallest substrate, the activity extended to below pH 2. Monovalent cations enhanced the activity at low pH, and this effect was also dependent on the molecular weight of the substrate. Below pH 4, pectic acid inhibited 70% of the hydrolysis of low molecular weight substrates by tomato polygalacturonase. The incomplete inhibition is attributed to the presence of a polygalacturonase isoenzyme which is not inhibited by high molecular weight polygalacturonic acids.     

Ethephon‐induced fractional changes of pectic polysaccharides in developing cape gooseberry (Physalis peruviana L) fruits

Pectic polysaccharides extracted from fruit tissue of cape gooseberry (Physalis peruviana L) at different maturity levels were fractionated and analyzed for galacturonic acid backbone molecules. Preharvest spray of the fruits at 7 days old with ethephon resulted in a higher accumulation of water‐soluble and oxalate‐soluble pectic fractions than non‐treated fruits, while this treatment effectively enhanced the polygalacturonase enzyme activity necessary for maintenance of pectin degradation. Treatment with ethephon not only enhanced the solubilization rate but also resulted in a net increase in total pectic polymer content in developing cape gooseberry fruits. Copyright © 2005 Society of Chemical Industry     

Enzymatic degradation studies of pectin and cellulose from red beets

The influence of structural features of the cell wall polysaccharides pectin and cellulose on the enzymatic degradation of red beet was evaluated. Alcohol‐insoluble substances and acetone‐insoluble residues were prepared from red beets and characterized with respect to the content of dietary fibre, pectin fractions, neutral saccharide composition and water absorption. The high‐methylated and high‐acetylated pectin component was partly soluble in water and in EDTA. Pectin was hardly extractable from alcohol‐insoluble substances as well as from red beets. Isolated pectin could not be completely degraded by pectolytic enzymes. After de‐acetylation, the pectic acid from red beets was degradable in a similar rate like citrus pectic acid. From alcohol‐insoluble substances, several cellulose and lignin fractions were prepared and analysed. A cellulose preparation from red beets was intensely degraded by cellulases with high activities as shown by the release of reducing end‐groups, viscosity and scanning electron microscopy. Cell wall preparations from red beets were able to bind high amounts of water. A decrease in water absorption during enzymatic action or changes in viscosity and flow behaviour are sensitive markers for decomposition or depolymerization processes. Furthermore, an inhibitor of microbial enzymes was isolated from red beets and acetone‐insoluble residues. The main reason for the poor enzymatic liquefaction or maceration of red beets by pectolytic and cellulolytic enzymes is the high degree of acetylation of the pectin component.     

Pectolytic enzymes and degradation of pectin associated with breakdown of sulphited strawberries

Both pectate-based culture filtrates and ripe strawberries, artificially inoculated with Rhizopus stolonifer, Rhizopus sexualis, Mucor piriformis or Aureobasidium pullulans, contained endo-polygalacturonase and pectin esterase. Uninfected strawberries contained traces of exo-polygalacturonase together with pectin esterase. No other pectin-degrading enzymes were detected in either sound or infected fruit. Naturally infected strawberries from commercial sources contained pectin esterase and variable amounts of polygalacturonase, the greatest amounts being associated with fruit carrying the heaviest microbial contamination. Disintegration of strawberries in sulphite liquor was associated with pectin degradation as judged by loss of liquor viscosity, reduction in pectic substance degree of polymerisation and the appearance in liquors of galacturonic acid. Polygalacturonase was detected in liquors during the early stages of disintegration but activity subsequently disappeared due to instability of the enzyme at low pH. Addition of culture filtrates to sound sulphited fruit to give polygalacturonase levels similar to those detected in some commercial liquors reproduced the fruit breakdown symptoms. Disintegration could be substantially reduced if polygalacturonase was inhibited either by lowering the pH, or by addition of anionic surfactant. It is concluded that polygalacturonase secreted by various decay fungi and present in fruit at the time of sulphiting is primarily responsible for breakdown. The possible involvement of non-enzymic synergists is discussed.     

Changes in activities of cell wall degrading enzymes during fermentation of cassava (Manihot esculenta Crantz) with citrobacter freundii

The cell wall degrading enzymes polygalacturonase (EC 3.2.1.15),pectinase (EC 3.1.1.11), cellulose (EC 3.2.1.4) and xylanase (EC 3.2.1.8), as well as α-amylase (EC 3.2.1.1) and phosphorylase (EC 2.4.1.1), were monitored during fermentation of cassava (Manihot esculenta Crantz) with Citrobacter freundii. All the enzymes were detected in cassava at the start. During fermentation, initial decreases in polygalacturonase, cellulase and xylanase were followed by increases which peaked as the tissue softened. There were significant (P<0.05) increases in pectinase, xylanase, cellulase, polygalacturonase and phosphorylase in inoculated cassava and the fermentation medium relative to controls (uninoculated cassava and medium) during the softening period. The control cassava did not ferment, indicating that the textural changes in inoculated cassava were due to enzymes secreted by C freundii. Studies on the effect of enzyme inhibition on fermentation showed that the pectic enzymes and cellulase were of primary importance and that inhibition of α-amylase and phosphorylase had no effect on the process.     

Physical and chemical changes in developing strawberry fruits

Strawberry fruits of thevariety Red Gauntlet were harvested at 7 day intervals after petal fall. Changes in fruit weight, percentages survival on the plant, chlorophyll: carotenoid and anthocyanin, titratable acid, pH of extracts and sugar content were measured. Also changes in soluble and total pectic polysaccharides in alcoholinsoluble residues of harvested fruits were followed during development. Fruit growth was not exponential and in later stages of growth the falling survival rate was correlated to fruit softening. Net synthesis of chlorophyll and carotenoid pigments took place up to 28 days and anthocyanin synthesis commenced 28 to 35 days after petal fall. The sugar content of berries increased with time and titratable acid concentrations increased slightly during development, falling in ripening fruits. The specific viscosity of soluble pectic polysaccharides fell from 28 days after petal fall. There was net synthesis of polyuronide but not neutral polysaccharide during the development of fruits and the amount of insoluble pectic polysaccharide became small and relatively constant compared with the amount of soluble polysaccharide by 21 days after petal fall. Fruits undergoing senescence lost almost all their insoluble pectic polysaccharides. The developmental processes taking place in growing fruits, especially with respect to changes in cell wall structure, and the relevance of results to fruit storage are discussed.     

Degradation of carrot (Daucus carota) fibres with cell-wall polysaccharide-degrading enzymes

Carrot Daucus carota L fibres were degraded with two enzyme preparations, SP249 from Aspergillus aculeatus and Celluclast from Trichoderma reesei. The enzymic activities of these complexes indicate that SP249 was particularly active on pectic polymers, and Celluclast could degrade amorphous and crystalline cellulose. A combination of both preparations degraded carrot fibres with a synergistic effect and led to the solubilisation of 95% of the cell-wall polysaccharides. The kinetics of solubilisation of sugars and gel-permeation chromatography of the soluble products show that pectic polymers were rapidly solubilised and then, in a second stage, degraded mainly to monomers, whereas cellulose was more slowly hydrolysed to cellobiose and glucose. Part (67%) of the polysaccharides were saccharified, the residual soluble material being rhamnogalacturonans containing arabinose residues. Residual insoluble fibres (10% of initial weight) were not liquefied and were composed mainly of lignin, proteins and polymers of glucose, xylose and galacturonic acid.     

多聚半乳糖醛酸酶在黑曲霉中的超量表达及其酶学性质研究

运用基因工程手段,构建高效表达多聚半乳糖醛酸酶的黑曲霉重组菌株,并对重组酶活性及酶学性质进行研究,以期更好的实现多聚半乳糖醛酸酶在食品加工、饲料生产及废水处理中的重要作用。从果胶酶生产菌黑曲霉GJ-1中扩增得到多聚半乳糖醛酸酶基因pgaB的成熟肽编码序列(1232 bp),集成glaA多拷贝强启动子和信号肽,构建其黑曲霉表达载体pSZHG6RS-pgaB,通过农杆菌介导法转化黑曲霉,获得多聚半乳糖醛酸酶黑曲霉重组菌株。聚丙烯酰胺凝胶电泳(SDS-PAGE)检测结果表明,目的蛋白大小约为38 kDa;在发酵第10 d时酶活最高达到4617.8 U·mL-1。酶学性质研究表明,最适温度为50℃,高温时热稳定性较差;最适pH为5.0,碱性耐受能力较强;Ca2+、Fe3+、Fe2+均对重组酶有一定激活作用;重组酶对不同底物的催化能力不同,甲酯化程度越高酶对其降解能力越弱,其中对无甲酯化的多聚半乳糖醛酸催化能力最强,DE值为33.04%。重组菌株产酶活性较高,具有明确的产业化前景。     

Enzymatic degradation studies of pectin and cellulose from red beets.

The influence of structural features of the cell wall polysaccharides pectin and cellulose on the enzymatic degradation of red beet was evaluated. Alcohol-insoluble substances and acetone-insoluble residues were prepared from red beets and characterized with respect to the content of dietary fibre, pectin fractions, neutral saccharide composition and water absorption. The high-methylated and high-acetylated pectin component was partly soluble in water and in EDTA. Pectin was hardly extractable from alcohol-insoluble substances as well as from red beets. Isolated pectin could not be completely degraded by pectolytic enzymes. After de-acetylation, the pectic acid from red beets was degradable in a similar rate like citrus pectic acid. From alcohol-insoluble substances, several cellulose and lignin fractions were prepared and analysed. A cellulose preparation from red beets was intensely degraded by cellulases with high activities as shown by the release of reducing end-groups, viscosity and scanning electron microscopy. Cell wall preparations from red beets were able to bind high amounts of water. A decrease in water absorption during enzymatic action or changes in viscosity and flow behaviour are sensitive markers for decomposition or depolymerization processes. Furthermore, an inhibitor of microbial enzymes was isolated from red beets and acetone-insoluble residues. The main reason for the poor enzymatic liquefaction or maceration of red beets by pectolytic and cellulolytic enzymes is the high degree of acetylation of the pectin component.     

Pectolytic Enzyme Studies for Peeling of Grapefruit Segment Membrane

In vitro studies of commercial pectolytic enzymes with grapefruit membranes and citrus pectin were conducted to determine optimum conditions for peeling membranes from grapefruit segments. Alcohol-insoluble solids residue (AIS) extracted from washed membrane consisted of ca. 46% pectic substances and 25% neutral sugars. The main neutral sugar of AIS was glucose, followed by arabinose and xylose. Optimum conditions for degradation of segment membranes by pectinase “C-80” were: pH between 4.0 and 5.0 at 55°C. Optimum temperature for degradation of commercial pectin was 50°C; this indicates that optimum conditions for enzymatic activation depend also on the substrate degraded. The enzyme was fully inactivated after 1 min at 80°C. This study provides additional basic information for industrial application of enzymatic peeling of citrus segments.     

Combined enzymatic and colorimetric method for determining the uronic acid and methylester content of pectin: Application to tomato products

A simple procedure for determining the galacturonic acid and methanol contents of soluble and insoluble pectins, relying on enzymatic pectin hydrolysis and colorimetric quantification, is described. Pectin samples are incubated with a commercial pectinase preparation, Viscozyme, then the galacturonic acid content of the hydrolyzed pectin is quantified colorimetrically using a modification of the Cu reduction procedure originally described by Avigad and Milner. This modification, substituting the commonly used Folin–Ciocalteau reagent for the arsenic containing Nelson reagent, gives a response that is linear, sensitive, and selective for uronic acids over neutral sugars. This method also avoids the use of concentrated acids needed for the commonly used m-phenylphenol method. Methanol, released by the action of the pectin methylesterase found in the Viscozyme, is quantified using alcohol oxidase and Purpald. This combined enzymatic and colorimetric procedure correctly determined the galacturonic acid and methanol content of purified, soluble citrus pectin. Application of the procedure to water insoluble pectins was evaluated with water insoluble material from apples and oranges. In both cases good agreement was obtained between this method and commonly used methods based on chemical pectin hydrolysis. Good agreement between these procedures was also found in the analysis of both soluble and insoluble pectins from several tomato products.     

超高效液相色谱-质谱联用分析茶叶中酚酸类化合物的组成及分布

采用超高效液相色谱-串联质谱技术建立同时测定茶叶样品中游离型、游离酯型、结合型、可溶性糖苷型及不溶性糖苷型等不同形态酚酸的分析方法。以70%甲醇溶液直接提取样品中的游离型酚酸,该提取液分别以2 mol/L NaOH溶液（含1 g/100 mL抗坏血酸和10 mmol/L乙二胺四乙酸）和1 mol/L HCl溶液水解以释放游离酯型与可溶性糖苷型酚酸;提取后的样品残渣分别以碱水解、酸水解方式释放结合型与不溶性糖苷型酚酸。23 种酚酸类化合物在50～1 000 μg/L范围内呈良好线性关系,相关系数（r）均高于0.996,相对标准偏差小于8.84%;茶叶样品中游离型酚酸的回收率为71.14%～105.43%,游离酯型酚酸和可溶性糖苷型酚酸的回收率分别为82.81%～108.93%、39.09%～102.25%。在茶叶中鉴定出14 种酚酸类化合物,总含量范围为7.84～12.90 mg/g。     

热处理对采后琯溪蜜柚果实汁胞粒化的影响及其与细胞壁代谢的关系

汁胞粒化是柚类果实采后主要的品质劣变现象。本实验以琯溪蜜柚果实为材料,研究热处理对果实贮藏期间品质的影响,探讨其抑制汁胞粒化的作用和适宜处理条件,并探究该抑制作用与细胞壁中木质素和细胞壁多糖代谢的关系。分别以35、42、49、56 ℃热水对琯溪蜜柚果实进行喷淋处理,根据果实冷藏期内汁胞粒化指数、水分质量分数、可溶性固形物和可滴定酸质量分数变化筛选较优处理温度,并评价该温度处理对果实汁胞木质素质量分数、木质素合成酶活力、细胞壁多糖含量及其降解酶活力的影响。结果表明：与其他处理相比,42 ℃热水处理15 min能够较好地延缓琯溪蜜柚果实贮藏期间汁胞粒化指数升高和水分质量分数降低,维持汁胞可溶性固形物和可滴定酸质量分数;与对照组相比,42 ℃热水处理抑制了汁胞中苯丙氨酸解氨酶、肉桂醇脱氢酶和过氧化物酶活性,减缓了木质素质量分数增加;并且抑制了纤维素酶、果胶甲酯酶、多聚半乳糖醛酸酶和β-半乳糖苷酶的活性,延缓了纤维素和半纤维素含量的升高、共价结合态果胶含量降低、离子结合态果胶和水溶性果胶含量的增加。因此,42 ℃热水喷淋处理可有效延缓采后琯溪蜜柚果实汁胞粒化等品质劣变现象,其减缓汁胞粒化的作用可能与木质素合成相关酶和细胞壁多糖降解酶活性受抑制,延缓汁胞木质化、纤维化和果胶降解,使细胞壁组分和结构得到较好的维持有关。     

Degradation characteristics of isolated and in situ cell wall lucerne pectic polysaccharides by mixed ruminal microbes

Two pectic polysaccharide fractions were isolated form lucerne (Medicago sativa L) leaves and used in fermentation experiments with mixed ruminal microbes. Both fractions were similar in chemical composition, containing galacturonic acid (52-58 mol%) and the neutral sugars arabinose (14-18 mol%), galactose (6-8 mol%) and rhamnose (8-12 mol%). Fermentation of both fractions was rapid and complete with a half-life of approximately 4 h. Production of total volatile fatty acids matched the degradation profile reaching a maximum level shortly after the rate of degradation began to decrease. The fermentation characteristics of citrus pectin and polygalacturonic acid were similar to those of the lucerne pectic fractions but galacturonic acid was much slower in its rate of degradation while soluble arabinogalactan from larchwood was virtually undegraded. Leaves of early bud stage lucerne and lower nodes and internodes of stems from full bloom lucerne were also fermented by mixed ruminal microbes. Pectic polysaccharides were rapidly and extensively degraded from both tissues. Initial rates were faster for leaves than for stems and the extent of pectic degradation was greater in leaves (8% residual) than in stems (17% residual). Selection of forage lines with increased pectic polysaccharides would provide greater amounts of rapidly available energy that could result in more efficient utilisation of the rapidly degraded protein in lucerne.     

挤压蒸煮加工米糠可溶和不溶膳食纤维对米淀粉性质的影响及其相互作用分析

研究未挤压、挤压蒸煮加工米糠可溶和不溶膳食纤维对米淀粉糊化性质、热性质、回生性质、结晶性质、微观结构的影响,并采用质构分析、核磁共振、傅里叶变换红外光谱等方法探究挤压蒸煮米糠膳食纤维与米淀粉之间的相互作用。结果表明：与未挤压蒸煮加工米糠膳食纤维相比,挤压蒸煮加工米糠可溶和不溶膳食纤维分别使米淀粉的崩解值显著增加了74.09%和128.36%,并均显著降低米淀粉的峰值黏度、谷值黏度、终值黏度、峰值时间、糊化温度。米糠经过挤压蒸煮加工后,米糠可溶膳食纤维使淀粉凝胶的自由水向强结合水转化,米糠不溶膳食纤维使淀粉凝胶的自由水向弱结合水转化。与未挤压蒸煮加工相比,挤压蒸煮加工米糠可溶和不溶膳食纤维分别使米淀粉的回生值降低了62.59%和44.81%,也均降低了米淀粉凝胶的回生率、相对结晶度、硬度、内聚性、回复性、胶黏性、咀嚼性、1 047 cm－1与1 022 cm－1处吸收峰的峰高比,添加挤压蒸煮米糠可溶、不溶膳食纤维的淀粉凝胶表面较光滑,凝胶结构出现较大的裂缝,说明挤压蒸煮加工米糠提高了米糠膳食纤维对米淀粉回生的抑制效果,且挤压蒸煮可溶膳食纤维比挤压蒸煮不溶性膳食纤维效果好。     

核桃内种皮不同形态酚酸类化合物的溶剂提取率比较

该文研究溶剂对核桃内种皮中不同形态酚酸类化合物的提取率,建立了适用于核桃内种皮中酚酸类化合物的提取方法.以新疆核桃为原料,比较不同比例的甲醇、乙醇溶液对核桃内种皮中可溶性游离态、可溶性酯化态和不溶性结合态酚酸的提取率.结果表明,50％(体积分数,下同)甲醇适合提取可溶性游离态绿原酸、没食子酸;50％(体积分数,下同)乙...     

VISCOSITY AS AFFECTED BY VARIOUS CONSTITUENTS OF TOMATO JUICE

SUMMARY— Degradation of proteins of tomato juice with pronase caused a relatively small loss of viscosity. Commercial cellulase freed of pectolytic activity with 8 M urea greatly decreased the viscosity of washed tomato solids. Further degradation with pectinase was ineffective in reducing viscosity. Cellulase extracted from the tomato lowered viscosity of tomato juice. Enzymatic degradation as indicated by chemical analysis did not always result in a loss of viscosity. By extracting the solids with water, viscosity of tomato juice was lowered drastically. The extent of the reduction was not affected by previous treatment with cellulase or pectinase. Viscosity was not affected by extraction with versene. The greater reduction in viscosity of tomato juice by extraction of the solids with water than with ethanol indicates that the high molecular polymers associated with the insoluble solids contribute greatly to viscosity. The data did not indicate a significant contribution of the uronides to the viscosity of tomato juice.     

Fractionation of water insoluble solids and extraction of pectins from residues of juice-extracted satsuma mandarin fruit by wet grinding

The waste biomass residue from satsuma mandarin juice production was extracted by wet-fine grinding and separated by centrifugation into insoluble and soluble fractions. The insoluble material was wet-screened into four fractions and analysed for crude fibre and total hydrolysable sugars. Crude fibre was almost exclusively in fractions containing particles over 50 μm, comprising about 65% of the insoluble material; this could provide a raw material high in crude fibre. The remaining insoluble fraction was almost devoid of crude fibre.
The soluble extract contained up to 63% of the total pectic substances with very similar methoxyl contents and degree of esterification, but double the viscometric molecular weight of a standard citrus pectin. The method provides an alternative to that of hot acid extraction.     

Pomelo Citrus grandis (L.) osbeck peel as an economical alternative substrate for fungal pectinase production

Polygalacturonase production by Aspergillus niger LFP-1 was studied in solid state fermentation (SSF) using pomelo (Citrus grandis) peel as a substrate. This local agricultural waste product is rich in lignocellulolytic material, including pectin, which can act as an inducer of pectinase production. Using the parameters of 5 g of 0.75 mm (particle size) pomelo peel as a substrate, moisture content ratio of 1:1 (w/v), inoculum size of 1×10⁷ spores/mL, cultivation temperature of 30°C, and no mixing, static fermentation conditions with addition of 1.2% ammonium nitrate produced the highest polygalacturonase production rate of 8.90 U/g of substrate and a fungal growth rate of 2.07mg of glucosamine/g of substrate on day 5 of cultivation. A large increase (1,434.5%) in enzyme production occurred after improvement of the growth parameters. Under optimum bioprocess conditions, pomelo peel can be used as solid substrate for production of pectinase.     

Texture and distribution of pectic substances of mango as affected by infusion of pectinmethylesterase and calcium

Fresh cut mangos were infused with pectinesterase (PME) and calcium chloride, and the effect on textural properties, distribution of pectic substance and degree of esterification was determined. Temperature gradient infusion with PME and/or calcium chloride increased gumminess and chewiness, but had no impact on hardness and adhesiveness. The distribution of pectic substances, as protopectin or alkaline soluble pectin, was approximately twice that of water‐ or chelator‐soluble pectin. The degree of esterification of water‐ and chelator‐soluble pectic substances was near 50–60%, and less than 10%, respectively. Heat‐sensitive PME inhibitor in mango was detected. The initial hardness of Kent mango was variable, and differences in distribution of pectic substances were observed. Texture of Kent mango is most likely moderated by changes in the solubility of insoluble pectin or by non‐pectin components in the cell wall. Copyright © 2004 Society of Chemical Industry