Protopectinase production by Paenibacillus polymyxa Z6 and its application in pectin extraction from apple pomace

Paenibacillus polymyxa Z6 was screened as protopectinase (PPase) producing strain and its PPase activity was 44.4 U/mL. The factors influencing PPase production were identified by a two‐level Plackett–Burman design with seven variables. The results indicated that Ca²⁺ concentration, fermentation time, and temperature were the most influential factors on the PPase production, which were applied in the Box–Behnken design. The predicted maximum PPase activity was 219 U/mL and the experimental maximum PPase activity was 221 U/mL, under the predicted optimum conditions, 170 mg/L Ca²⁺, 27 °C, and 29 hr of fermentation. The present PPase was composed of both type‐A PPase, polygalacturonase; and type‐B PPase, arabinanase, and rhamnogalacturonase. Finally, the PPase was applied for the pectin extraction from apple pomace and achieved an average yield of 11.9% with properties like 8.5% moisture content, 1.6% ash content, 3.8 mPa?S viscosity, and pH 6.1 of 1% solution.

Practical applications

Protopectinase (PPase) is a “green” way for pectin extraction with the advantages of low emission, low energy consumption, and environment‐friendly. PPase includes different types, which can work on different region of protopectin in cell wall of plants and then release highly polymerized soluble pectin applicable in food industry. Present PPase from the strain Paenibacillus polymyxa Z6, contains both A‐type PPase reacting with the smooth regions of protopectin composed of partially methoxylated galacturonic acid, polygalacturonase; and B‐type PPase reacting with the hairy regions consisting of rhamnogalacturonan and neutral side‐chains of protopectin, arabinanase, and rhamnogalacturonase. This PPase was used in pectin extraction from apple pomace and harvested pectin with lower moisture, lower ash, and higher viscosity compared with the chemically produced one, which indicates that this PPase has potential for application in food industry.     

Purification and Characterization of a Novel Protopectin-Solubilizing Enzyme from Aspergillus niger Z-25

A mutant, Aspergillus niger Z-25 derived from A. niger XZ-131 with N⁺ supplementation, produced a protopectin-solubilizing enzyme (protopectinase). The enzyme was purified to homogeneity by a procedure involving ammonium sulfate fractionation and gel chromatographies on DEAE-Sephadex A-50 and Sephadex G-100 columns. The molecular weight of the protopectinase was estimated at 68.4 KD by SDS-polyacrylamide gel electrophoresis. The enzyme was stable from pH 3.0 to 7.0 and up to 60°C. The optimum pH and temperature for enzyme activity was 4.0 and 50°C, respectively. The purified enzyme had 268-U/mL PPase (protopectinase) activity on citrus protopectin, and also showed 100-U/mL PGase (polygalacturonase) activity, which catalyzed the hydrolysis of polygalacturonic acid. The Km value for protopectin was 0.215 mg/mL, while the Km value for polygalacturonic acid was 39.09 mg/ml. The PPase/PGase q-value was 2.68, more than 0.5. Therefore the enzyme was considered a novel pectinase and classified as protopectinase.     

Pectin lyase and polygalacturonase of Aspergillus niger pathogenic for yam tubers (Diascorea rotundata)

Polygalacturonase and pectin lyase of Aspergillus niger partially purified by ethanol, ammonium sulphate precipitation, DEAE-cellulose and Sephadex G-150 column chromatography were characterized. Polygalacturonase gave optimum activity at pH 4–5, and at 35°C. It was stable at pH 3–7 and at 20–50°C. The molecular weight was 38020. For pectin lyase optimum activity occurred at pH 5 and 45°C. The enzyme was stable at pH 3–4 and at 40–50°C. The molecular weight was 30 900. Yam tissue was optimally macerated at pH 4–5 by the enzymes. At pH 4.5, potassium sorbate (0.6 mg/ml), benzoic acid (0.8 mg/ml) and sodium benzoate (1.0 mg/ml) caused complete inhibition of polygalacturonase activity. With pectin lyase, this effect was achieved with potassium sorbate and benzoic acid each at 0.9 mg/ml, but not with sodium benzoate.     

Degradation of cell wall materials from sweetpotato, cassava, and potato by a bacterial protopectinase and terminal sugar analysis of the resulting solubilized products

Cell wall materials (CWMs) from sweetpotato, cassava, and potato starch residues were degraded using a crude enzyme solution from the culture filtrate of a Bacillus sp. isolated from soil, Bacillus sp. M4. This organism has been found to secrete polygalacturonic acid lyase (PGL) and glycan depolymerase activities, especially arabinanase, but cellulase activity was nearly absent. Sugar analysis of the solubilized product after enzyme treatment at pH 7.0 revealed that it is mainly composed of galacturonic acid, galactose, and arabinose, the sugars found commonly in the pectin fraction. This suggested the presence of a protopectinase (PPase) activity in the culture filtrate. The presence of EDTA completely inhibited PGL but PPase activity was almost retained, suggesting that the PGL is not the primary activity responsible for pectin solubilization. The mode of action of the crude enzyme was determined by terminal sugar analysis using HPAEC-PAD after hydrolysis of the reduced products. Results revealed that galactose is the main neutral sugar at the reducing terminal of the products, although rhamnose was also present in the higher molecular weight component. This suggested that at neutral pH, the primary activity in the culture filtrate of Bacillus sp. M4 is a B-type PPase, which attacked the galactan as well as rhamnogalacturonan moieties of the protopectin, resulting in the release of a soluble pectin fraction.     

A study of factors affecting in-situ De-esterification of mango (Mangifera indica) pectin

De-esterification of mango peel pectin in situ by the action of the enzyme pectinesterase (PE) has been investigated. The rate of enzymic deesterification was highest between pH 8.5 and 9.5 with chemical deesterification also being important. In-situ PE activity declined with fruit ripening but remained relatively constant when assayed on citrus pectin. The most likely explanation for this difference is a change in suitability of the pectin substrate within the fruit during ripening. A reduction in the degree of esterification (DE) of the pectin to 36-42% was obtained following incubation of the mango peels at pH 8.5 for 90 min. A crude preparation of exogenous PE from mango peel and lime pulp was incubated at pH 8.5 with heat-treated (PE inactive) peel as a means of increasing the rate and extent of de-esterification. The PE activity of both these exogenous enzyme preparations was lower on the peel than that of the in-situ mango enzyme, but considerably higher on a hot-water-soluble fraction extracted from the peel. It is suggested that enzyme solubility and pectin accessibility are the major factors affecting in-situ de-esterification of pectin in mango peel.     

Optimising growth conditions for the pectinolytic activity of Kluyveromyces wickerhamii by using response surface methodology

This present study was undertaken to find optimum conditions of pH, temperature and, period of incubation for the pectinolytic activity of Kluyveromyces wickerhamii isolated from rotting fruits and to assess the effect of these factors by use of response surface methodology (RSM). A central composite rotatable design was used as an experimental design for the analysis of the allocation of treatment combinations. A second order polynomial regression model was fitted and was found adequate, with an R(2) of 0.94469 (P<0.001). The effects of temperature and pH were the most significant factors in influencing enzyme production. Estimated optimum conditions were as follows: pH 5.0, temperature, 32 degrees C and an incubation period of 91 h. Pectinesterase (PE), pectin lyase (PL), and cellulase activities were not detected. Pectinase production was partially constitutive. Pectin was degraded by the isolated strain of K. wickerhamii in the current study, and the pectinolytic activity is referred to as polygalacturonase (PG) activity. Crude enzyme extract was thermostable at various temperatures and, stimulated by the presence of Ca(2+) ions but inhibited by other ions like Mg(2+), Zn(2+), Co(2+), Mn(2+) and Na(+).     

Characterisation of pectins extracted from banana peels (Musa AAA) under different conditions using an experimental design

An experimental design was used to study the influence of pH (1.5 and 2.0), temperature (80 and 90 °C) and time (1 and 4 h) on extraction of pectin from banana peels (Musa AAA). Yield of extracted pectins, their composition (neutral sugars, galacturonic acid, and degree of esterification) and some macromolecular characteristics (average molecular weight, intrinsic viscosity) were determined. It was found that extraction pH was the most important parameter influencing yield and pectin chemical composition. Lower pH values negatively affected the galacturonic acid content of pectin, but increased the pectin yield. The values of degree of methylation decreased significantly with increasing temperature and time of extraction. The average molecular weight ranged widely from 87 to 248 kDa and was mainly influenced by pH and extraction time.     

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.     

Purification and characterization of pyrophosphatase from bighead carp (Aristichthys nobilis)

Pyrophosphatase (PPase, EC 3.6.1.1) responsible to the hydrolysis of pyrophosphate (PPi) in muscle was purified from bighead carp (Aristichthys nobilis), and characterized in detail herein for the first time. PPase was extracted with 20 mmol/L Tris-HCl buffer (pH 7.0) containing 0.05 mol/L KCl, followed by heat treatment and ammonium sulfate precipitation. Then it was purified by deithylaminoethyl-cellulose (DE52) and Sephacryl S-300 chromatography, subsequently resulted in a 114.5-fold purification. The molecular mass of the PPase was defined to be 50 kDa with two subunits. The optimum pH of the purified PPase was around 8.0, and its optimum temperature was confirmed to be 50 °C. Magnesium ion was necessary to the activity of PPase. An excess of PPi over Mg²⁺ resulted in inhibition of PPase. When the Mg²⁺/PPi molar ratio was 1:1, the maximal enzyme activity was obtained. In addition, PPase converted PPi to orthophosphate (Pi) stoichiometrically with a K_m of 1.98 mmol/L under the condition of 5 mmol/L Mg²⁺. Ethylene diamine tetraacetate (EDTA) activated the activity of PPase at low concentration, however, it consumingly did inhibit the enzyme activity at high concentration.     

黑曲霉果胶裂解酶和聚半乳糖醛酸酶合成的发酵调控

通过对黑曲霉(Aspergillus niger)wz 003液态发酵过程的调控,制备了高活性的果胶裂解酶,并讨论了发酵过程中聚半乳糖醛酸酶活力的变化情况。实验表明,发酵产酶培养基组成为(g/L):麸皮60,玉米粉40,酵母膏20,胡萝卜12,CaCO_3 10。培养条件为:初始pH 6.5,30℃,接种量8%,培养2 d。此时果胶裂解酶活力为375.3 U/mL,为基础条件下的6.225倍,而聚半乳糖醛酸酶的合成得到了有效控制,酶活仅为29.4 U/mL。改变发酵条件,有利于聚半乳糖醛酸酶的发酵生产,此时酶活力为基础条件下的5.05倍。     

白鲢鱼背侧肌焦磷酸酶的纯化及酶学特性

通过粗分离、50%～80%饱和度硫酸铵分级沉降、DE-52阴离子交换柱层析等步骤,从白鲢鱼背侧肌中分离纯化出焦磷酸酶。通过变性凝胶电泳分析,该酶在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳图谱中仅有一个分子质量为40 kD的条带。酶学特性研究表明,白鲢鱼背侧肌焦磷酸酶可专一性地水解焦磷酸盐,反应初速率时间范围为0～15 min,最适反应温度为45 ℃,最适反应pH值为7.5。Mg2＋对该酶有明显的激活作用,在Mg2＋浓度为5 mmol/L时酶活力最高。5 mmol/L的Ca2＋、Zn2＋、乙二胺四乙酸（ethylenediaminetetraacetic acid,EDTA）-Na2、EDTA-Na4、KIO3和1 mmol/L的NaF均对该酶有强烈的抑制作用。该酶水解焦磷酸四钠的最大反应速率为0.051 U/mg,米氏常数为0.54 mmol/L。     

猪背最长肌焦磷酸酶的分离纯化与酶学特性

通过离心、50%～70%饱和硫酸铵沉淀、DEAE-52离子交换柱层析,从猪背最长肌中分离纯化出焦磷酸酶(PPase)。变性聚丙烯酰胺凝胶电泳图谱显示,PPase分子质量约72kD。焦磷酸酶酶学特性研究表明,最适反应温度和pH值分别为50℃和7.5。Mg2+是PPase的激活剂,在浓度4.75mmol/L时,酶活力最强。但Na+和K+都能抑制酶的活力,且Na+的抑制效果强于K+。PPase水解焦磷酸钠(TSPP)的动力学参数Vmax为0.086μmol/(L.min)),Km为0.36mmol/L。     

Rheological and high gelling properties of mango (Mangifera indica) and ambarella (Spondias cytherea) peel pectins

Ambarella and mango peels are good sources of pectins (15–20%), with high degree of methylation (60–78%) and high molar masses. Ambarella and mango ( Améliorée and Mango varieties) peel pectins were extracted using HCl or oxalic acid/ammonium oxalate (OAAO). Purified pectins were analysed for their flow behaviour and phase diagrams were established at pH 3 as sucrose vs. pectin concentration. The gelation kinetics and mechanical spectra of these pectin gels were studied and compared to those of commercial citrus (lime) pectins. At a concentration of 1% (w/v), all pectic solutions had a shear thinning behaviour but at 0.6% (w/v), only OAAO-extracted pectins exhibited such behaviour. Phase diagrams showed that at pH 3, gelation of OAAO mango extracted pectins was possible at low polymer concentration (0.2%; w/w) for a sucrose concentration of 60% (w/w). OAAO-extracted pectins exhibited a higher gelling ability than HCl-extracted ones. Sucrose (45–50%) and pectin (0.2–0.6%) concentration had a deep impact on the gel strength. Our results enable to conclude that the OAAO extraction from mango and ambarella peels allowed the recovery of pectins that exhibit high gelling properties.     

CP/MAS 13C NMR技术分析酸提取对烟草果胶产率和结构的影响

为研究烟草果胶的精细结构和掌握果胶提取的关键因素,采用交叉极化/魔角旋转固态核磁光谱技术(CP/MAS13C NMR)考察了酸提取条件(pH、温度和提取时间)对烟草果胶产率、纯度(质量分数)及结构的影响。结果表明:①在pH 1.5和85℃条件下提取1.5 h时,烟草果胶得率最高,为10.87%,但在pH 2.0和95℃条件下提取1.5 h时,聚半乳糖醛酸(PGA)的纯度最高,为78.4%。②提取条件对烟草果胶的结构也有一定的影响,pH=1.5~2.5时,果胶的甲酯度(DM)和乙酰度(DA)均随pH升高而增大,随温度升高而降低,随提取时间延长而显著下降。      

Effect of extraction conditions on the quality characteristics of pectin from passion fruit peel (Passiflora edulis f. flavicarpa L.)

Passion fruit (Passiflora edulis f. flavicarpa L.) yellow variety is composed of 50-55 g peel per 100 g of fresh fruit which is discarded as waste during processing. Utilization of passion fruit peel for pectin extraction was studied. Passion fruit peel obtained after juice extraction was blanched in boiling water for 5 min, dehydrated in a cross flow hot air drier at 60 ± 1 °C to a moisture content of 4 g/100 g of dried peel. The dehydrated passion fruit peel was used for extraction experiments of pectin. The effect of pH, peel to extractant ratio, and number of extractions, extraction time and temperature on the yield and quality characteristics of pectin were investigated. The optimized conditions for extraction of pectin from passion fruit peel yielded 14.8 g/100 g of dried peel. Pectin extracted from the dried peels had a methoxyl content of 9.6 g/100 g, galacturonic acid content of 88.2 g/100 g and jelly grade of 200. Extraction of pectin from dried peels of passion fruit may be considered for effective utilization of passion fruit processing waste.     

ISOLATION and CHARACTERIZATION of MANGO PEEL PECTINS

An efficient method for manufacture of pectin from Totapuri mango peels was standardized by studying various factors that govern the recovery and quality of pectin. Among the different organic and inorganic acids, 0.05 N HCl was found to be the best for recovery of pectin from mango peels. Optimum yield of pectin was obtained by taking two extractions each for one-hour duration employing a peel: extractant ratio of 1:2 and by alcohol precipitation method. Dried mango peels could be stored for six months at ambient conditions (14.5–33.9C) without any significant effect on the recovery of pectin. Pectin extract, an intermediate product in the manufacture of pectin, could be stored for one month either at low temperature (6C±2) or at ambient conditions (24.5–33.0C) by the addition of 700 ppm SO₂ with minimum loss in the recovery of pectin. Using the optimum extracting conditions about 20.8% (DWB) of purified pectin was obtained from mango peels. the powdered pectin could be stored for over 6 months without any deterioration in quality when packed in airtight containers at ambient conditions.     

连续逆流萃取法从桔皮中提取果胶

桔皮是一种理想的高酯果胶原料。从桔皮中提取果胶常采用批量萃取法,本实验中,采用一定pH的盐酸溶液提取果胶,对连续逆流萃取法和批量萃取法进行了比较。在液/固比为20:1时,pH1.0-3.2范围内,以及pH2.0时,液/固比在16:1-25:1范围内,连续逆流萃取法的果胶提取率均高于批量萃取法。在较宽的pH和液/固比范围内,连续逆流萃取法都获得了较高的果胶提取率。由此,与批量法相比,连续逆流萃取法在使用较少提取剂和较小设备的情况下也可获得足够高的提取率,从而起到减少废水量和节约运行成本的目的。     

Improved lycopene extraction from tomato peels using cell-wall degrading enzymes

Four commercial enzyme preparations with pectinolytic, cellulolytic and hemicellulolytic activities were tested for their ability to enhance lycopene extraction from tomato peels. Screening experiments were performed at 25 °C by subjecting the peels to a 4-h enzyme incubation followed by 1-h hexane extraction. Peclyve EP and LI were the most efficient, with an almost 20-fold increase in extraction yield. Peclyve LI was used to evaluate the influence of solvent type and enzyme incubation time on lycopene recovery. Hexane, ethyl acetate and the mixture hexane/acetone/ethanol 50:25:25 (v/v) were used as solvents. Under the best extraction conditions (1-h enzyme incubation followed by a 3-h solvent extraction at 40 °C) up to 440 mg of lycopene per 100 g of dry tomato peels were obtained. The percentage recoveries were in the range of 3–30%, for the untreated peels, and 77–98% for the enzymatically treated material.     

Structural Characteristics of Pumpkin Pectin Extracted by Microwave Heating

Abstract: To improve extraction yield of pumpkin pectin, microwave heating was adopted in this study. Using hot acid extraction, pumpkin pectin yield decreased from 5.7% to 1.0% as pH increased from pH 1.0 to 2.0. At pH 2.5, no pectin was recovered from pumpkin flesh powder. After a pretreatment at pH 1.0 and 25 °C for 1 h, pumpkin powder was microwave‐extracted at 120 °C for 3 min resulting in 10.5% of pectin yield. However, premicrowave treatment at 60 °C for 20 min did not improve extraction yield. When microwave heating at 80 °C for 10 min was applied after premicrowave treatment, final pectin yield increased to 11.3%. When pH was adjusted to 2.0, the yield dropped to 7.7% under the same extraction conditions. Molecular shape and properties as well as chemical composition of pumpkin pectin were significantly affected depending on extraction methods. Galacturonic acid content (51% to 58%) of pumpkin pectin was lower than that detected in commercial acid‐extracted citrus pectin, while higher content of neutral sugars and acetyl esters existed in pumpkin pectin structure. Molecular weight (M_w) and intrinsic viscosity (η_w) determined for microwave‐extracted pumpkin pectins were substantially lower than acid‐extracted pectin, whereas polydispersity was greater. However, microwave‐extracted pectin at pH 2.0 had more than 5 times greater M_w than did the pectin extracted at pH 1.0. The η_w of microwave‐extracted pectin produced at pH 2.0 was almost twice that of other microwave‐extracted pectins, which were comparable to that of acid‐extracted pectin. These results indicate that extraction yield of pumpkin pectin would be improved by microwave extraction and different pectin structure and properties can be obtained compared to acid extraction. Practical Application: Pumpkin is a promising alternative source for pectin material. Pumpkin pectin has a unique chemical structure and physical properties, presumably providing different functional properties compared to conventional commercial pectin sources. Depending on the conditions to produce pumpkin pectin, diverse molecular structures can be obtained and utilized in various food applications.     

响应面法优化提取甜橙皮渣中果胶的研究

通过单因素试验研究料液比、提取温度、提取时间和pH 值4 因素对甜橙皮渣中果胶得率的影响。在单因素试验的基础上,采用三因素三水平的Box-Behnken Design(BBD)中心试验设计研究响应值以及最佳变量的组合。结果表明：随着pH 值的降低和温度的增加果胶得率增加;处理时间和料液比对果胶得率也有积极的影响。通过响应面法综合评价提取甜橙皮渣中果胶的最佳提取条件为温度90℃、时间1.3h、pH1.1,在此条件下的果胶得率预测值为0.998%。