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.     

An explorative study on the cell wall polysaccharides in the pulp and peel of dragon fruits (Hylocereus spp.)

The pectic and hemicellulosic cell wall polysaccharides from the pulp and the peel of white-flesh and red-flesh dragon fruits (Hylocereus spp.) were isolated and compared in terms of degree of methoxylation (DM), solubility properties (relative content of uronic acids and neutral sugars in different fractions), neutral sugar composition, molar mass distribution, and affinity toward some specific anti-pectin antibodies. Hereto, the alcohol-insoluble residues were extracted and sequentially fractionated using hot water, a chelating agent, sodium carbonate, and potassium hydroxide solutions to obtain different pectin fractions and a hemicellulose fraction. Chemical analyses were used to characterize these polysaccharide fractions. The results show that cell wall polysaccharides of the pulp and especially of the peel from white-flesh and red-flesh dragon fruits contain significant amounts of pectic substances that are lowly methyl-esterified. The cell wall polysaccharides of the peel as well as those of the pulp contain high amounts (38–47 %) of loosely bound (water-soluble) pectic substances. In the water-soluble fraction (WSF) of the peel samples, uronic acids are the predominant monomers. On the contrary, rhamnogalacturonan-I type neutral sugars, and especially arabinose and galactose, contribute equally, as compared to uronic acid, to the WSF of the pulp samples. Despite the low average DM value of pulp and peel pectin, pectic substances in both samples showed affinity for antibodies with different specificities indicating that a wide range of epitopes, including long blocks of unesterified galacturonic acids (GalA) residues as well as (short) blocks of esterified GalA residues, is present. No large differences between the pectic substances among the different dragon fruit varieties were observed.     

Untersuchungen an Kartoffelpülpe als Ballaststoffquelle. Die Zusammensetzung der Kartoffelpülpe nach Einwirkung von Pektinasen und Cellulasen sowie enzymatischem Stärkeabbau

Investigations on Potato Pulp as a Dietary Fiber Source. Composition of Potato Pulp After Influence of Pectinases and Cellulases and Enzymatic Degradation of Starch. The composition of alcohol insoluble substances (AIS) of potato pulp have been investigated after succesive degradation with different pectolytic and cellulolytic enzymes followed by starch degrading enzymes. The pretreatment with pectinase/cellulase combinations has a significant influence on the amount and the properties of the residues after starch degradation. Furthermore the most important changes in the composition were found in the receiving residues after action of amylolytic enzymes. A correlation exists between the content of residual starch in the residues and the action of the cell wall degrading enzymes. The amount of residual starch correlates negatively with the intensity of cell wall degradation. The enzymatic susceptibility of starch depends on the damage of cell wall of potato pulp, especially on the degree of degradation of the cellulose. The water binding capacity decreases intensively after action of pectinase/cellulase combinations. The treatment with amylolytic enzymes causes only a negligible variation of the water binding capacity.     

Cloning, sequence analysis and overexpression of a Saccharomyces cerevisiae endopolygalacturonase-encoding gene (PGL1)

Only a few yeast strains produce pectin-degrading enzymes such as pectin esterases and depolymerases (hydrolases and lyases). Strain SCPP is the only known Saccharomyces strain to produce these pectinases. One of these pectolytic enzymes. PGL1-encoded endopolygalacturonase (EC 3.2.1.15), hydrolyses the alpha-1,4-glycosidic bonds within the rhamnogalacturonan chains in pectic substances. This paper presents the cloning and sequencing of the first S. cerevisiae gene involved in pectin degradation. Few differences were found between the two deduced amino acid sequences encoded by PGL1-1 from a pectolytic (PG+) strain (SCPP) and PGL1-2 from a non-pectolytic (PG-) strain (X2180-1B). Similarities were found with other polygalacturonases from plants and other microorganisms. Of the two S. cerevisiae genes, only the one isolated from strain SCPP was able, by overexpression, to confer endopolygalacturonase activity to a laboratory strain of S. cerevisiae. Overexpression of PGL1-1 gene in a non-pectolytic strain resulted in halo formation on polygalacturonic acid-containing agar plates stained with ruthenium red.     

Gamma-Radiation Affects Cell Wall Composition of Strawberries

Tissue softening limits the use of gamma irradiation for controlling postharvest microbial development on some produce. The carbohydrate composition of the cell wall of the strawberry (Fragaria ananassa Duch. cv. Chandler) fruit and of cell wall fractions was compared in untreated controls and in fruit irradiated at 4 kGy, a dose causing tissue softening. Cell wall polysaccharides were partially degraded, particularly cellulose and pectic substances. However, neutral sugars from the pectic and hemicellulose fractions were not affected by irradiation, in contrast with cell wall degradation during ripening.     

Molecular Characteristics of Pectins from Enzyme-Treated Apple Juices

Red Delicious apple mashes treated by two commercial pectic enzymes were investigated by high performance size exclusion chromatography (HPSEC). Pectins isolated from the juice samples were fractionated into water-soluble, chelator-soluble, and alkali-soluble pectin fractions. Distinct pectin breakdowns were observed with HPSEC. Number average molecular weights of pectin fractions were determined by intrinsic viscosity measurements.     

Isolation and characterization of colloidal soluble polysaccharides in raspberry juice

Depending on the preparation method, the colloid content of raspberry juices treated with pectolytic enzymes, can be up to 2.5 g/L. Analysis of the polysaccharides shows that there are in particular, arabinans, arabinogalactans type II and rhamnogalacturonans. While the content of arabinans could be reduced by the action of arabinases, the arabinogalactans and rhamnogalacturonans were not degraded by conventional pectic enzymes. Besides these cell-wall polysaccharides, it was possible to isolate a high molecular-mass beta-glucan, which is thought to originate from Botrytis cinerea. Technical problems in juice clarification or filtration may be caused by this beta-glucan or the high content of residual pectic substances.     

Degradation of apple cell wall material by commercial enzyme preparations

The action of commercial enzyme preparations on the release of cell wall constituents from alcohol-insoluble substance prepared from apples without skins and cores as well as their influence on the water binding of remaining residues is described as a model for the enzymatic cell wall destruction during production of liquid fruit products. Besides 'normal' enzyme concentrations adapted from the usual industrial dosage, 'tenfold' enzyme concentrations were applied. Dependent on enzyme spectrum and activities, concentrations of dietary fibre, e.g., pectin, increased in the soluble fractions using conditions of enzymatic 'mash treatment'. A further release of these cell wall constituents occurred when cellulase containing enzyme preparations were used under conditions of 'pomace treatment', especially with the 'tenfold' enzyme dosage. The partial enzymatic degradation of the cell wall material is connected with a decrease in water binding of the remaining residues during both simulated mash treatment of pomace treatment. Alcohol-insoluble substance from apples is a suitable model for the determination of complex enzymatic actions of enzyme preparations containing pectolytic, hemicellulolytic, and/or cellulolytic activities under standardised conditions.     

Cross-flow filtration of pectic substances during the enzymatic treatment of mandarin segment membranes

 This study is concerned with the cross-flow filtration of pectic substances, produced by the enzymatic treatment of membranes of mandarin segments, using several filtration membranes with pore sizes of between 0.45 μm and 30 kDa. Pectic substances solubilised in the digestion medium were polymers with an average molecular weight of 90–100 kDa. Pectin was retained by the membrane with a 100-kDa pore size. As a result of the addition of enzymes, pectin was degraded to a mixture of galacturonic acid and galacturonic acid oligomers, which appeared to be distributed between the retentate and the permeate. The final filtration rate of galacturonic acid oligomers was close to 100% when enzymes were present at the filtration membranes. In the absence of enzymes, the filtration rate ranged from 5% to 15%, and the permeation flux tended to decrease. The enzymatic treatment had an important effect on the resistances of the filtration membranes, and also caused a decrease in viscosity. These two factors were responsible for the increase in the permeate flux after the addition of enzymes, where this flux was almost as high as when filtration commenced. Received: 27 October 1997     

Cross-flow filtration of pectic substances during the enzymatic treatment of mandarin segment membranes

 This study is concerned with the cross-flow filtration of pectic substances, produced by the enzymatic treatment of membranes of mandarin segments, using several filtration membranes with pore sizes of between 0.45 μm and 30 kDa. Pectic substances solubilised in the digestion medium were polymers with an average molecular weight of 90–100 kDa. Pectin was retained by the membrane with a 100-kDa pore size. As a result of the addition of enzymes, pectin was degraded to a mixture of galacturonic acid and galacturonic acid oligomers, which appeared to be distributed between the retentate and the permeate. The final filtration rate of galacturonic acid oligomers was close to 100% when enzymes were present at the filtration membranes. In the absence of enzymes, the filtration rate ranged from 5% to 15%, and the permeation flux tended to decrease. The enzymatic treatment had an important effect on the resistances of the filtration membranes, and also caused a decrease in viscosity. These two factors were responsible for the increase in the permeate flux after the addition of enzymes, where this flux was almost as high as when filtration commenced. Received: 27 October 1997     

Pectic substances in onion and garlic skins

Pectic substances from onion (white and red varieties) and garlic skins were isolated by extraction with ammonium oxalate. White onion and garlic skins were found to contain 11 to 12% pectin which can be recovered as a by-product in the dehydration industries. Characterisation of these pectic substances in terms of jelly grade, molecular weight, degree of esterification, methoxyl and uronide content was attempted. Pectins from white onions were superior to red onions in terms of jelly grade. Both types of onion pectins appeared to be of the rapid set type while the garlic skin pectin was of the medium set variety. Equivalent weight, methoxyl content and degree of esterification by themselves did not give any clear indication of pectin grade. Intrinsic viscosity values gave good correlation between jelly grade and molecular weight. The pectic substances from garlic skin differed from onion skin in certain respects and most remarkably in its viscosity behaviour.     

Evaluation of pectolytic activities of enological interest in industrial enzyme preparations

 The purpose of this research was to determine the activity of several pectolytic enzymes, such as pectin methyl esterase, endopolymethylgalacturonase, endopolygalacturonase, exopolymethylgalacturonase, endopolygalacturonase, pectin transeliminase and pectic acid transeliminase, from within preparations that are available on the Portuguese market. The selected preparations were as follows: 15 pectolytic enzyme preparations, one hemicellulolytic preparation and one glucanase preparation. The measurements of enzyme activity were carried out using model solutions with 50 mg·l^–1 SO₂, pH 3.2, 25°C, with and without 12% ethanol (v/v), and a 24-h incubation period. Quantitatively, the results proved to be heterogeneous, significantly varying from preparation to preparation: all pectolytic enzyme activities in the 17 preparations analysed in this study were significantly different. The presence of ethanol reduced the enzymatic activity, apart from that of endopolymethylgalacturonase, and, in general, an increase of enzyme concentration raised the pectolytic enzyme activity. Received: 17 March 1997 / Revised version: 11 June 1997     

Evaluation of pectolytic activities of enological interest in industrial enzyme preparations

 The purpose of this research was to determine the activity of several pectolytic enzymes, such as pectin methyl esterase, endopolymethylgalacturonase, endopolygalacturonase, exopolymethylgalacturonase, endopolygalacturonase, pectin transeliminase and pectic acid transeliminase, from within preparations that are available on the Portuguese market. The selected preparations were as follows: 15 pectolytic enzyme preparations, one hemicellulolytic preparation and one glucanase preparation. The measurements of enzyme activity were carried out using model solutions with 50 mg·l^–1 SO₂, pH 3.2, 25°C, with and without 12% ethanol (v/v), and a 24-h incubation period. Quantitatively, the results proved to be heterogeneous, significantly varying from preparation to preparation: all pectolytic enzyme activities in the 17 preparations analysed in this study were significantly different. The presence of ethanol reduced the enzymatic activity, apart from that of endopolymethylgalacturonase, and, in general, an increase of enzyme concentration raised the pectolytic enzyme activity. Received: 17 March 1997 / Revised version: 11 June 1997     

Effects of High Pressure and Heat Treatment on Pectic Substances and Related Characteristics in Guava Juice

Effects of high pressure treatment on guava juice pectic substances and related juice characteristics were investigated and compared with heat treatment. The viscosity and turbidity of guava juice pressurized at 600 MPa and 25°C for 10 min increased slightly; whereas the viscosity of juice heated at 95°C for 5 min decreased while turbidity increased. There were no apparent changes in water soluble, oxalate soluble and alkali soluble pectin in pressurized juice. However, heat treatment of juice decreased water and alkali soluble pectins and slightly increased oxalate soluble pectin. High pressure treatment resulted in no marked changes in pectic and cloud substances of guava juice, and maintained the natural viscous of the juice.     

Cell wall break down of pitanga fruit (Eugenia uniflora L.) is associated with pectic solubilisation and softening

Pitanga fruit stands out for its exotic flavour and antioxidant properties. The objective of this work was to evaluate changes in the physical, chemical and biochemical variables of red pitanga variety, in four stages of development (green, yellow, orange and red) and associate them to the cell wall break down. Analysis of pH, titratable acidity, soluble solids, firmness, soluble pectin, pectinamethylesterase (PME), polygalacturonase (PG), cell wall swelling and microstructure were performed. pH and levels of soluble solids and soluble pectin increased, in addition to the cell wall swelling and reduction of firmness, throughout the development of pitanga. From the SEM, it was possible to observe the degradation of the fruit cell wall during ripening, suggesting mainly the degradation of pectic polysaccharides, verified by the solubilisation of these substances. PG and PME enzymes were not active, which suggests that other enzymes may be associated with the cell wall break down.     

Effective diffusivity of total solids and pectic substances from apple tissue

The effective diffusivities of total solids and pectic substances from apple tissue were determined at 40 and 60°C in water and buffer; the addition of pectolytic enzyme in buffer at 40°C was also investigated. the Fickian model for unsteady-state diffusion was applicable in all cases. Effective diffusivity values both for total solids and soluble pectic substances in deionized water were temperature dependent. the effective diffusivity values for total solids loss were reduced by the presence of buffer at 40°C but not at 60°C. the effective diffusivity values for soluble pectic substances were not affected by buffer at 40°C but were noticeably lower at 60°C compared to deionized water at the same temperature. the addition of a pectolytic enzyme preparation did not improve the mass transfer of either total solids or soluble pectic substances.     

Hindrance of Hemicellulose and Cellulose Hydrolysis by Pectic Substances

Treating the tissue of the grapefruit segment membrane with pectinase and cellulase decreased the content of xylose and glucose significantly more than the sum of the separate effects. The synergistic effect obtained by the combination of pectinase and cellulase showed that the pectic substance sterically masked the hemicellulose and cellulose. Breaking down the barrier of the pectic substance with pectinase allowed a significant increase in the hydrolysis of the hemicellulose and cellulose. Preparation of alchol-insoluble solids from fresh tissue modified the structure of the pectin in the cell wall and prevented its steric hindrance to breakdown of hemicellulose and cellulase. Extraction of pectic substances from the tissue by NaOH greatly increased cellulose hydrolysis by cellulase.     

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     

果胶及果胶酶研究进展

果胶分子是由HGA、RG-Ⅰ和RG-Ⅱ3个结构区域构成，后二者为结构复杂的杂多糖成分，因此参与果胶分解的酶类也是复杂多样的，逐渐延伸了果胶酶概念的内涵．其应用已不再停留在最初的食品加工上，而是在纺织、造纸、化妆品等行业都有所发展，在天然产物提取以及果胶低聚糖的生理活性研究等方面也有了阶段性的进展，果胶酶的固定化研究也在逐步深入．