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Fruit softening exacerbates mechanical damage incurred during shipping and handling and the increase in pathogen susceptibility. Here, oligogalacturonides (OGs) produced by fungal polygalacturonase (PG) delayed fruit softening in tomato and maintained fruit firmness at 8.37 ± 0.45 N at 13 d of storage, which was consistent with the fruit firmness level of 5 d in the control groups. From RNA sequencing data in line production of phytohormones, we confirmed ethylene and jasmonic acid signals, the MAPK signaling cascade, and calmodulin involved in the OG-mediated firmness response of whole fruit.

Thermostable enzymes have many advantages for industrial applications. Therefore, in this study, computer-aided design technology was used to improve the thermostability of a highly active endo-polygalacturonase from

Thermostability is a key property of industrial enzymes. Endo-polygalacturonases of the glycoside hydrolase family 28 have many practical applications, but only few of their structures have been determined, and the reasons for their stability remain unclear. We identified and characterized the Talaromyces leycettanus JCM12802 endo-polygalacturonase TlPGA, which differs from other GH28 family members because of its high catalytic activity, with an optimum temperature of 70 °C. Distinctive features were revealed by comparison of thermophilic TlPGA and all known structures of fungal endo-polygalacturonases, including a relatively large exposed polar accessible surface area in thermophilic TlPGA. By mutating potentially important residues in thermophilic TlPGA, we identified Thr284 as a critical residue. Mutant T284A was comparable to thermophilic TlPGA in melting temperature but exhibited a significantly lower half-life and half-inactivation temperature, implicating residue Thr284 in the kinetic stability of thermophilic TlPGA. Structure analysis of thermophilic TlPGA and mutant T284A revealed that a carbon-oxygen hydrogen bond between the hydroxyl group of Thr284 and the Cα atom of Gln255, and the stable conformation adopted by Gln255, contribute to its kinetic stability. Our results clarify the mechanism underlying the kinetic stability of GH28 endo-polygalacturonases and may guide the engineering of thermostable enzymes for industrial applications.

Sweet osmanthus (

Microbial fermentation of the viscous pulp surrounding cocoa beans is a crucial step in chocolate production. During this process, the pulp is degraded, after which the beans are dried and shipped to factories for further processing. Despite its central role in chocolate production, pulp degradation, which is assumed to be a result of pectin breakdown, has not been thoroughly investigated. Therefore, this study provides a comprehensive physicochemical analysis of cocoa pulp, focusing on pectic polysaccharides, and the factors influencing its degradation. Detailed analysis reveals that pectin in cocoa pulp largely consists of weakly bound substances, and that both temperature and enzyme activity play a role in its degradation. Furthermore, this study shows that pulp degradation by an indigenous yeast fully relies on the presence of a single gene (PGU1), encoding for an endopolygalacturonase. Apart from their basic scientific value, these new insights could propel the selection of microbial starter cultures for more efficient pulp degradation.

An endo-polygalacturonase gene (pga-zj5a) was cloned by reverse transcription from cDNAs synthesized from Aspergillus niger ZJ5 total RNA. The open reading frame of pga-zj5a was 1089 base pairs encoding 362 amino acids. Pga-zj5a lacking a signal peptide sequence was successfully amplified using A. niger ZJ5 cDNA as the template and was ligated into the pPIC9 vector. The resulting plasmid was transformed into competent cells of Pichia pastoris GS115 for heterologous expression. The polygalacturonase showed a maximum activity level of 10436 U/mL in the culture supernatant from a 3 L fermenter. Assays of enzymatic properties showed that the optimal pH and temperature of the recombinant PGA-ZJ5A were 4.5 and 40 °C, respectively. PGA-ZJ5A was effective in pear juice clarification, increased the volume of pear juice by 41.8%, and improved its light transmittance 3-fold.

S-Allylcysteine (SAC), produced in large amounts during the aging process of garlic via enzymatic hydrolysis, is known as a key compound responsible for the multiple pharmacological activities of aged black garlic. This study investigated the effects of enzyme- and high hydrostatic pressure (HHP)-assisted extraction on the content of the bioactive compounds, including SAC, in black garlic juice (BGJ) and evaluated the antidiabetic effects of SAC-enriched BGJ in streptozotocin (STZ)-treated mice. The aging process increased the contents of SAC, total polyphenols, and total flavonoids in garlic juice. More importantly, pretreatment of pectinase cocktail with HHP resulted in a greater increase in those compounds during aging. Enzyme-treated BGJ reduced hyperglycemia and improved islet architecture and β-cell function in STZ-treated mice. Moreover, these effects were more potent than those of BGJ prepared by the conventional aging process. These findings provide useful information for the production of black garlic with improved bioactivities.

Histone deacetylase (HDAC) performs important functions in plant growth and development, including fruit ripening. As a complex biological process, fruit ripening involves the histone acetylation modification of ripening-associated genes. Histone deacetylase genes (

The acquisition of susceptibility to necrotrophy over the course of ripening is one of the critical factors limiting shelf life. In this study, phytopathology and molecular biology were employed to explore the roles of pectinase in fruit susceptibility and ripening. Solanum lycopersicum fruit softened dramatically from entirely green to 50% red, which was accompanied by a continuously high expressed SlPG2 gene. The necrotrophic fungus Botrytis cinerea further activated the expression of SlPGs and SlPMEs to accelerate cell wall disassembly, while most of the polygalacturonase inhibitor proteins encoding genes expression were postponed in ripe fruit following the pathogen attack. Pectin induced the antagonistic yeast to secrete pectinolytic enzymes to increase fruit resistance against gray mold. The activities of pathogenic pectinase of B. cinerea were correspondingly depressed in the pectin-inducible yeast enzyme elicited ripe fruit. These data suggest that pectinase is a molecular target for regulation of disease resistance during fruit ripening.

Since Saccharomyces cerevisiae strains display no to weak pectinase activity, the utilization of external pectinase is a common practice in winemaking to enhance the extraction of compounds located in the grape berry skins during maceration. In this study, the activity of the native endopolygalacturonase of a Kluyveromyces marxianus strain, isolated from grape juice, was characterized in Shiraz grape must during alcoholic fermentation with or without prefermentative cold maceration. The wines made with K. marxianus had a higher methanol concentration, more free-run wine, an altered volatile compound profile, and displayed pectinase activity in cell-free wine samples. Moreover, the results strongly suggest that K. marxianus' pectinase released polygalacturonic acid soluble fragments, unlike fungal pectinases, which mostly release monomers. Overall, this study shows that K. marxianus is an effective pectinase producer in wine with potential benefits for wine properties.

Pectinase treatment was used to facilitate protein recovery from defatted rapeseed (Brassica rapa) cold-pressing residue in water-lean conditions and without pH adjustment. Effect of extraction pH on protein yield and physiochemical properties of the protein concentrates was assessed. Enzymatic hydrolysis of carbohydrates was feasible at high (40%) solid content and improved protein recovery at pH 6. Comparable protein yields (40-41% of total protein) from enzyme-aided water extraction (pH 6) and nonenzymatic alkaline extraction (pH10) at 10% solid content suggested that after enzymatic treatment, rapeseed protein could be extracted without exposure to alkali. However, water extraction required dilute conditions, whereas alkaline extraction was feasible also at 20% solid content. The water extracts possessed better protein solubility, higher ?- potential, and smaller particle size than isoelectric precipitates from alkaline extraction, indicating higher dispersion stability. This is suggested to be mediated by electrostatic interactions between proteins and pectic carbohydrates in the water extracts.

Pomelo peel is an abundant pectin-rich biomass waste in China and has the potential to serve as a source of fuels and chemicals. This study reports a promising way to deal with pomelo peel waste and to utilize it as raw material for ethanol production via simultaneous saccharification and fermentation (SSF). An integrated strategy, incorporating hydrothermal treatment, multienzyme formulation, and fed-batch operation, was further developed to enhance the ethanol production. The results show that hydrothermal treatment (120 °C, 15 min) could significantly reduce the use of cellulase (from 7 to 3.8 FPU g(-1)) and pectinase (from 20 to 10 U g(-1)). A multienzyme complex, which consists of cellulase, pectinase, β-glucosidase, and xylanase, was also proven to be effective to improve the hydrolysis of pretreated pomelo peel, leading to higher concentrations of fermentative sugars (36 vs 14 g L(-1)) and galacturonic acid (23 vs 9 g L(-1)) than those with the use of a single enzyme. Furthermore, to increase the final ethanol concentration, fed-batch operation by adding fresh substrate was employed in the SSF process. A final solid loading of 25% (w/v), which is achieved by adding 15% fresh substrate to the SSF system at an initial solid loading of 10%, produced 36 g L(-1) ethanol product in good yield (73.5%). The ethanol concentration is about 1.73-fold that at the maximum solid loading of 14% for batch operation, whereas both of them have a closed ethanol yield. The results indicate that the use of the fed-batch mode could alleviate the decrease in ethanol yield at high solid loading, which is caused by significant mass transfer limitation and increased inhibition of toxic compounds in the SSF process. The integrated strategy demonstrated in this work could open a new avenue for dealing with pectin-rich biomass wastes and utilization of the wastes to produce ethanol.

International audience; Commercial pectinase preparations are applied in winemaking to improve wine processing and final quality. These preparations contain pectolytic enzyme activities such as polygalacturonases, pectin esterases, pectin lyases, and rhamnoglacturonases. These enzymes modify the polysaccharide and oligosaccharide composition of wines. The influence of various commercial enzyme preparations on wine oligosaccharide composition was studied, on Merlot wines from the Bordeaux area. Wine oligosaccharides were isolated by high-resolution size-exclusion chromatography on a Superdex-30 HR column. The glycosyl residue and glycosyl linkage compositions of the oligosaccharide fractions obtained were determined. The MS spectra of the Merlot oligosaccharide fractions from control and enzyme-treated wines were recorded on an AccuTOF mass spectrometer equipped with an electrospray ionization (ESI) source and a time-of-flight (TOF) mass analyzer. Oligosaccharides in the control wines were partly methylated homogalacturonans, corresponding to smooth regions of pectins, whereas those of the enzyme-treated wines were mostly rhamnogalacturonan-like structures linked with neutral lateral chains, arising from the hairy regions. The enzyme preparations used thus cleaved the rhamnogalacturonan backbone of the hairy zones and demethylated and hydrolyzed the smooth regions. Besides, different structures were detected, depending on the enzyme preparation used, indicating that they contained rhamnogalacturonase activities with different specificities. The oligosaccharide profiles can serve as a marker of enzymatic treatments.

Pectinase was covalently immobilized onto the macroporous polyacrylamide (PAM) microspheres synthesized via an inverse suspension polymerization approach, resulting in 81.7% immobilization yield. The stability of the macroporous PAM support, which has a large surface area, is not impeded by the adsorbed proteins despite the fact that up to 296.3 mg of enzyme is immobilized per gram of the carrier particles. The immobilized enzyme retained more than 75% of its initial activity over 30 days, and the optimum temperature/pH also increased to the range of 50-60 °C/3.0-5.0. The immobilized enzyme also exhibited great operational stability, and more than 75% residual activity was observed after 10 batch reactions. The kinetics of a model reaction catalyzed by the immobilized pectinase was finally investigated. Moreover, the immobilized pectinase could be recovered by centrifuging and showed durable activity at the process of recycle.

A new allele, the EPG1-2 gene, which codes for an endopolygalacturonase in Kluyveromyces marxianus CECT1043, has been cloned. The gene has 1086 bp and the protein 362 amino acids, one more than the previously described Epg1p. Epg1-2p shows a high degree of similarity with the polygalacturonases of fungi and yeasts. The sequences common to all of the polygalacturonases of prokaryotes, fungi, and higher plants are also conserved in Epg1-2p. The EPG1-2 gene has been expressed in Pichia pastoris , and, when fused with the signal peptide of the alpha-factor of Saccharomyces cerevisiae , the protein is properly secreted into the media. The recombinant enzyme does not appear to be fully glycosylated by P. pastoris or not glycosylated in the same manner as in K. marxianus, but it maintains the same optimum temperature (55 degrees C) and pH (4.5) and the same stability at different temperatures and pH values as the native enzyme, also showing the same hydrolytic behavior. The recombinant strain produces 200-fold more enzyme than the wild-type strain of K. marxianus, making it a yeast of potential industrial interest for the production of endopolygalacturonase for the food industry.

Precursor extracts obtained from different grape varieties were submitted to harsh acid hydrolysis (pH 2.5, 100 degrees C, 1 h) and enzymatic hydrolysis (AR2000, pH 5, 40 degrees C, 16 h) and were also added to a synthetic must (200 g L(-1) glucose), which was fermented (yeast strain Stellevin NT 116), to compare the "natural hydrolysis" carried by yeast with alternative "fast" hydrolytic strategies. In all cases, released volatile compounds were extracted by SPE and determined by GC-MS. Leaving aside Muscat, differences between varieties were not relevant, although Grenache and Chardonnay presented some key peculiarities. In general, alcoholic fermentation showed the lowest potential to release volatile compounds from aromatic precursors, whereas enzymatic hydrolysis was the most efficient but also the most different. Practically, this implies that the predictive ability of this hydrolytic strategy is rather poor. In contrast, harsh acid hydrolysis can be considered to much more adequately measure the aroma potential of grapes for winemaking, which suggests that transformations taking place during fermentation include relevant chemical rearrangements in acid media that are better predicted by acid hydrolysis.

Attractive color is one of the most important sensory characteristics of fruit and berry products, and elderberry juice is widely used as natural colorant. When pectinase preparations were used in the production of elderberry juice for clarification, a concomitant decrease of anthocyanins and thus a color loss were observed. This paper demonstrates that this is due to side glycosidase activities contained in commercial pectinase preparations from Aspergillus sp. Using LC-MS, sequential deglycosylation of cyanidin-3-sambubioside, cy-3-glucoside, cy-3-sambubioside-5-glucoside, and cy-3,5-diglucoside was found to be catalyzed by specific glycosidases contained in the pectinase preparations. There was no big difference in the deglycosylation rate between monoglucosidic or diglucosidic anthocyanins. However, the degradation rate was decreased when rutinose was attached to cyanidin, whereas the structure of the aglycone itself had almost no influence. Pure beta-glucosidases from Agrobacterium species and Aspergillus niger and the beta-glucosidase N188 from A. niger did not show any conversion of anthocyanins, indicating the presence of specific glycosidases. Thus, an activity gel based assay was developed to detect anthocyanin-specific glycosidase activity in enzyme preparations, and according to LC-MS peptide mass mapping of digested bands, homologies to a beta-glucosidase from Aspergillus kawachii were found.

The isolation and utilization of pectin lyase (PL) from commercial pectic enzyme for methanol reduction in wine production was investigated. PL can be separated from pectinesterase (PE) and polygalacturonase (PG) on HM-CL-AIS affinity chromatography at pH 4; however, it is difficult to further distinguish PE from PG. Some desirable physicochemical properties such as transmittance, lightness, redness, and lower total pectin content are found in the external enzyme adding groups (PL, PE and PG, and pectic enzyme groups) in comparison to the control group. Methanol contents in pectic enzyme and the PE and PG groups increase from 628 +/- 13 (control group) to 3103 +/- 16 and 1736 +/- 67 mg/L ethanol in the final products, respectively. Nevertheless, the adding of PL does not cause any increase in methanol content. The results present in this study suggest that the HM-CL-AIS column is a simple, inexpensive, convenient, and effective method for PL purification. Moreover, the partial purified PL is a potential replacement of commercial pectic enzyme for pectin depolymerizing, methanol content reducing, and wine quality improving in wine production.

The unraveling of crushed grapes by maceration enzymes during winemaking is difficult to study because of the complex and rather undefined nature of both the substrate and the enzyme preparations. In this study we simplified both the substrate, by using isolated grape skin cell walls, and the enzyme preparations, by using purified enzymes in buffered conditions, to carefully follow the impact of the individual and combined enzymes on the grape skin cell walls. By using cell wall profiling techniques we could monitor the compositional changes in the grape cell wall polymers due to enzyme activity. Extensive enzymatic hydrolysis, achieved with a preparation of pectinases or pectinases combined with cellulase or hemicellulase enzymes, completely removed or drastically reduced levels of pectin polymers, whereas less extensive hydrolysis only opened up the cell wall structure and allowed extraction of polymers from within the cell wall layers. Synergistic enzyme activity was detectable as well as indications of specific cell wall polymer associations.

A pectolytic enzyme preparation from Aspergillus niger (pectinase AN) decreased most rutin content and antioxidant activity of asparagus juice. To investigate the mechanism of such loss, we analyzed several possible related enzyme activities in pectinase AN. We found that the activity of pectinase AN to oxidize guaiacol had no significant difference with or without the presence of H2O2; thus it was laccase activity, not peroxidase (PO) activity, that pectinase AN contained. We did not find any polyphenol oxidase (PPO) activity in pectinase AN. Laccase in pectinase AN could be the major cause of loss of rutin and antioxidant activity of asparagus juice. When most laccase activity of pectinase AN was inactivated after heating at 70 degrees C for 1.5 min and incubated with asparagus juice, the loss rate of rutin was only 9% of that treated with unheated pectinase AN, and the antioxidant activity was even increased. Rhamnosidase activity was detected in pectinase AN and can change rutin in asparagus juice to quercetin-3-glucoside, which has higher antioxidant activity than rutin. This may explain the increase of antioxidant activity of asparagus juice treated with heated pectinase AN that still contained some rhamnosidase activity. The discovery of our research is helpful to produce juice with high antioxidant activity and high health benefits in the juice industry.

The effect of high-pressure processing (HPP) on cell wall polysaccharides in berries was investigated. HPP decreased the degree of methyl esterification (DM), probably by activation of pectin methyl esterase (PME), and improved the extractability of pectins. When commercial enzyme mixtures were added to mashed berries, a synergistic effect was observed between treatment with commercial enzymes and HPP. Compared to treatment at atmospheric pressure, pectic polysaccharides were degraded to a larger extent when HPP was used. In contrast, hemicelluloses were hardly affected by the added enzymes when HPP was included, although they were degraded during similar treatment at atmospheric pressure. Additionally, the activity of rhamnose-releasing enzymes present in minor quantities might be enhanced after HPP, resulting in a decrease of rhamnose in the polymeric cell wall material. These results exploring the effect of HPP at representative conditions clearly point out the potential of HPP for polysaccharide modification.

Commercial pectolytic enzymes were investigated for their influence on phenolics and antioxidant activities of asparagus juice. The antioxidant activity of asparagus juice was analyzed according to 2,2‘-diphenyl-l-picrylhydrazyl and 2,2‘-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) methods. The enzymes, with the exception of pectinase from Rhizopus sp., contained rutinase, which hydrolyzed rutin to quercetin. Asparagus juice treated with Viscozyme had the highest quercetin content without exhibiting a significant increase in the antioxidant activity. For a pectinase from Aspergillus niger, the antioxidant activity of asparagus juice was markedly reduced. Caution should be paid in the selection of pectolytic enzyme preparations for production of antioxidant activity-rich juice. Keywords: Asparagus juice; pectolytic enzyme preparation; rutinase; antioxidant activity; rutin; quercetin

The objective of this study was to digest okara in high yield by food-processing enzymes. Autoclaving of okara was effective in increasing cellulase digestion for the primary cell wall, and the digestion was accelerated by the formation of single cells by stirring. Most of the residual okara after autoclaving and cellulase digestion was found to be the secondary cell walls compared with the cellulase-treated soybean single cells. The secondary cell wall was found to be composed of galacturonic acid, neutral sugars, and protein and was considered to be a complex of these compositions. Many cellulolytic and proteolytic enzymes could not digest the secondary cell wall; however, it was found that two pectinases could digest the secondary cell wall. A series of digestions resulted in yields of 83-85% from the raw okara, and the final residues were identified as oil body complexes in the soybean cells and fiber-like organ between the cells.

Tomato polygalacturonase (PG) was extracted from ripe tomatoes and purified by cation exchange and gel filtration chromatography. Cation exchange chromatography yielded two peaks with PG activity: the first peak was identified as PG2 (the heat labile form) and the second one as PG1 (the heat stable form). Both PG2 and PG1 presented a molar mass of 42 kDa when analyzed by SDS-PAGE and an isoelectric point >9.3. Thermal inactivation of purified tomato PG2, at pH 4.4, in the temperature range from 53 to 63 degrees C, followed first-order kinetics. Combined pressure-temperature inactivation of tomato PG2 was studied at 5-55 degrees C/100-600MPa. Under all pressure-temperature conditions, PG2 inactivation followed first-order kinetics. Purified tomato PG1, although more thermostable than PG2, showed a pressure stability very similar to that of PG2. These results indicate that high-pressure processing is an efficient alternative to inactivate tomato PG without the need for applying high temperatures.

A study on the determination and standardization of endopolygalacturonase (EPG) activity is reported, with emphasis on the influence of the degree of substrate esterification using pure yeast EPG. Differences in the results, depending on how the EPG activity unit was defined, are described and discussed. From a theoretical analysis of the expressions established, a general equation for expressing EPG activity in standard international units was obtained, together with the proportional coefficient for each of the substrates studied. It was observed that for a wide range of enzyme concentrations good linear correlations were obtained. Analysis of the comparison ratio (CR) values calculated revealed that these do not differ significantly, except for low-methoxyl apple pectin, confirming the validity of the general expression obtained for pectins with different degrees of esterification. The anomalous CR value found for low-methoxyl (LM) apple pectin is discussed.

Processing tomato lines suppressed in the accumulation of ripening-related polygalacturonase or expansin were generated by introduction of transgenes to silence expression of the LePG and LeExp1 genes, respectively. The rheological properties of juice and juice reconstituted from paste produced from lines suppressed in one of these genes, or in both, were compared with azygous controls. When assayed by measuring Bostwick consistency, paste produced from either suppressed LePG or suppressed LeExp1 lines and diluted to 5 degrees Brix was approximately 18% more viscous than that produced from controls. Simultaneous suppression of LePG and LeExp1 produced a small additional increase in viscosity of 4%. Rheometric flow analysis at 5 or 10 degrees Brix also showed substantial increases in the consistency index due to suppression of either LePG or LeExp1 alone, and a small additional increase when both genes were suppressed in the same transgenic line. Measurements by laser diffraction and [1H]NMR showed that suppression of LePG or LeExp1 accumulation altered the size distribution of insoluble particles and modified their surface properties. The data are consistent with suppression of LePG increasing serum viscosity, and suppression of either LePG or LeExp1 altering the properties of the insoluble particles and improving some aspect of particle-particle or particle-serum interaction, or both. However, relative to that caused by suppression of either gene alone, the additional increase in viscosity caused by simultaneous suppression of LePG and LeExp1 together was slight.

Kinetic parameters for the thermal inactivation of several enzymes in carrot and potato homogenates have been determined. In carrots the most heat-resistant activity was polygalacturonase, followed by peroxidase and pectinmethylesterase. In potatoes peroxidase was the most resistant, followed by pectin methylesterase, polyphenol oxidase, and lipoxygenase. There were several notable similarities between the inactivation kinetics in the two vegetables. In both cases peroxidase activity gave simple first-order inactivation kinetics but yielded a curved Arrhenius plot for the temperature dependence. Pectin methylesterase in both commodities consisted of a labile and a resistant form. The relative amounts of the two forms and the temperature dependences for their inactivation were also similar.

Oil palm is one of the most productive oil-producing crops and can store up to 90% oil in its fruit mesocarp. Oil palm fruit is a sessile drupe consisting of a fleshy mesocarp from which palm oil is extracted. Biochemical changes in the mesocarp cell walls, polyamines, and hormones at different ripening stages of oil palm fruits were studied, and the relationship between the structural and the biochemical metabolism of oil palm fruits during ripening is discussed. Time-course analysis of the changes in expression of polyamines, hormones, and cell-wall-related genes and metabolites provided insights into the complex processes and interactions involved in fruit development. Overall, a strong reduction in auxin-responsive gene expression was observed from 18 to 22 weeks after pollination. High polyamine concentrations coincided with fruit enlargement during lipid accumulation and latter stages of maturation. The trend of abscisic acid (ABA) concentration was concordant with GA₄ but opposite to the GA₃ profile such that as ABA levels increase the resulting elevated ABA/GA₃ ratio clearly coincides with maturation. Polygalacturonase, expansin, and actin gene expressions were also observed to increase during fruit maturation. The identification of the master regulators of these coordinated processes may allow screening for oil palm variants with altered ripening profiles.

High-pressure processing (HPP) can inactivate pathogenic microorganisms and degradative enzymes without the use of heat, thereby minimizing the destruction of flavors, nutrients, and other quality attributes. Lipoxygenase plays a role in the off-flavor production of tomatoes, whereas pectinesterase and polygalacturonase impact tomato texture. The purpose of this study was to determine HPP's ability to inactivate lipoxygenase, pectinesterase, and polygalacturonase in diced tomatoes. Processing conditions used were 400, 600, and 800 MPa for 1, 3, and 5 min at 25 and 45 degrees C. The magnitude of applied pressure had a significant effect on inactivating lipoxygenase and polygalacturonase (p < 0.05), with complete loss of activity occurring at 800 MPa. Pectinesterase was very resistant to pressure treatment. Percent soluble solids, pH, titratable acidity, and color a/b values did not differ significantly among the high-pressure-processed samples as compared to the control, but color L values increased. This change in L values was not considered of practical importance. Apparent protein content decreased in the pressure-processed samples, due possibly to protein denaturation, loss of solubility, and/or a decrease in dye binding sites to assay protein content.

The effect of high-pressure treatment (200-600 MPa for 20 min) on the texture of cherry tomatoes and on the key softening enzymes (pectinmethylesterase and polygalacturonase) was investigated. When subjected to high-pressure treatment whole cherry tomatoes showed increasing textural damage with increasing pressures up to 400 MPa. However, treatment at pressures above 400 MPa (500-600 MPa) led to less apparent damage than treatment at 300 and 400 MPa; the tomatoes appearing more like the untreated samples. These visual changes were reflected in the texture (firmness) and amount of cell rupture in the tomatoes, with the least firmness and the most cell rupture being seen after treatment at 400 MPa. Light and scanning electron microscopy supported these observations. Although a sample of purified commercial pectinmethylesterase was partially inactivated at pressures above 200 MPa, irrespective of pH (4-9), in the whole cherry tomatoes no significant inactivation was seen even after treatment at 600 MPa, presumably because other components in the tomato offered protection or the isoenzymes were different. Polygalacturonase was more susceptible to pressure, being almost totally inactivated after treatment at 500 MPa. It is concluded that the textural changes in tomato induced by pressure involve at least two related phenomena. Initially, damage is caused by the greater compressibilty of the gaseous phase (air) compared to liquid-solid components, giving rise to a compact structure which, on pressure release, is damaged as the air rapidly expands, leading to increases in membrane permeability. This permits egress of water, and the damage also enables enzymatic action to increase, causing further cell damage and softening. The major enzyme involved in the further softening is polygalacturonase, which is inactivated at 500 MPa and above, and not pectinmethylesterase, which in the whole fruit, is barotolerant.

Orange juice processed from Huanglongbing (HLB) affected fruit is often associated with bitter taste and/or off-flavor. HLB disease in Florida is associated with Candidatus Liberibacter asiaticus (CLas), a phloem-limited bacterium. The current standard to confirm CLas for citrus trees is to take samples from midribs of leaves, which are rich in phloem tissues, and use a quantitative real-time polymerase chain reaction (qPCR) test to detect the 16S rDNA gene of CLas. It is extremely difficult to detect CLas in orange juice because of the low CLas population, high sugar and pectin concentration, low pH, and possible existence of an inhibitor to DNA amplification. The objective of this research was to improve extraction of DNA from orange juice and detection of CLas by qPCR. Homogenization using a sonicator increased DNA yield by 86% in comparison to mortar and pestle extraction. It is difficult to separate DNA from pectin; however, DNA was successfully extracted by treating the juice with pectinase. Application of an elution column successfully removed the unidentified inhibitor to DNA amplification. This work provided a protocol to extract DNA from whole orange juice and detect CLas in HLB-affected fruit.

A novel 5-hydroxymethyl-2-furfural (HMF; 1) derivative, which is named asfural (compound 2), was isolated from enzyme-treated asparagus extract (ETAS) along with HMF (1) as a heat shock protein 70 (HSP70) inducible compound. The structure of compound 2 was elucidated on the basis of its spectroscopic data from HREIMS and NMR, whereas the absolute configuration was determined using chiral HPLC analysis, compared to two synthesized compounds, (S)- and (R)-asfural. As a result, compound 2 derived from ETAS was assigned as (S)-(2-formylfuran-5-yl)methyl 5-oxopyrrolidine-2-carboxylate. When compound 2, synthesized (S)- and (R)-asfural, and HMF (1) were evaluated in terms of HSP70 mRNA expression-enhancing activity in HL-60 cells, compound 2 and (S)-asfural significantly increased the expression level in a concentration-dependent manner. HMF (1) also showed significant activity at 0.25 mg/mL.

Muscadine grapes (Vitis rotundifolia) are widely grown in the southern United States, as the more common Vitis vinifera cannot be cultivated due to Pierce's disease. There is interest to determine if certain cultivars can be used for good-quality wine production. This study compared the effect of pectolytic enzyme pretreatment with conventional skin-contact fermentation on Muscadine (Noble, Vitis rotundifolia) wine major volatiles, aroma active volatiles, and volatile sulfur compounds (VSCs). Volatile composition, aroma activity, and VSCs in the initial juice and wine samples after 3 years were determined by gas chromatography in combination with mass spectrometry (GC-MS), olfactory detection (GC-O), and pulsed flame photometric detection (GC-PFPD). Forty-three nonethanol MS volatiles were common to all samples. Total ion chromatogram (TIC) MS peak area increased 91% in the skin-contact wines from the initial juice but only 24% in the enzyme-treated wine. Thirty-one VSCs were detected. Twenty-four sulfur volatiles were identified by matching their retention characteristics on polar and nonpolar columns with those of standards or MS spectrum matches. Six of these (sulfur dioxide, 1-propanethiol, 3-mercapto-2-pentanone, 3-mercapto-2-butanone, 2,8-epithio-cis-p-menthane, and 1-p-menthene-8-thiol) were reported for the first time in muscadine wine. Five additional VSCs were tentatively identified by matching standardized retention values with literature values, and two remain unidentified. Total sulfur peak areas increased 400% in the skin-contact wine and 560% in the enzyme-treated wine compared to the initial juice. There were 42 aroma-active volatiles in the initial juice, 48 in the skin-contact wine, and 66 in the enzyme-treated wine. Eleven aroma-active volatiles in the skin-contact wine and 16 aroma volatiles in the enzyme-treated wine appear to be due to sulfur volatiles. Pectolytic enzyme-treated wines contained less total volatiles but more sulfur and aroma-active volatiles than the traditional skin-contact wine. University of Florida, IFAS, CREC, Lake Alfred, FL

The interactions between xylan/lignin and cellulase enzymes play a key role in the effective hydrolysis of lignocellulosic biomass. Organosolv pretreated loblolly pine (OPLP) and sweetgum (OPSG) were used to quantitatively elucidate the distinct roles of residual xylan and lignin on enzymatic hydrolysis, based on the initial hydrolysis rates and the final hydrolysis yields. The initial hydrolysis rates of OPLP and OPSG were 1.45 (glucose) and 1.19 g/L/h (glucose), respectively, under the enzyme loading of 20 FPU/g glucan. The final glucan hydrolysis yields of OPLP and OPSG at 72 h were 76.4 and 98.9%, respectively. By correlating the amount of residual lignin and xylan to the initial hydrolysis rate and the final hydrolysis yield in OPLP and OPSG, a more accurate fundamental understanding of the roles of xylan and lignin in limiting the enzymatic hydrolysis has been developed. The higher amount of residual xylan (9.7%) in OPSG resulted in lower initial hydrolysis rate (1.19 g/L/h). The higher amount of residual lignin in OPLP (18.6%) resulted in lower final hydrolysis yield of glucan (76.4%). In addition, we observed in the simultaneous saccharification and fermentation (SSF) that ethyl xyloside was produced by the enzymatic catalysis of xylose/xylan and ethanol.

The effects of different commercial enzymatic mash treatments on yield, turbidity, color, and polyphenolic and sediment of procyanidins content of cloudy apple juice were studied. Addition of pectolytic enzymes to mash treatment had positive effect on the production of cloud apple juices by improving polyphenolic contents, especially procyanidins and juice yields (68.3% in control samples to 77% after Pectinex Yield Mash). As summary of the effect of enzymatic mash treatment, polyphenol contents in cloudy apple juices significantly increased after Pectinex Yield Mash, Pectinex Smash XXL, and Pectinex XXL maceration were applied but no effect was observed after Pectinex Ultra-SPL I Panzym XXL use, compared to the control samples. The content of polymeric procyanidins represented 50-70% of total polyphenols, but in the present study, polymeric procyanidins were significantly lower in juices than in fruits and also affected by enzymatic treatment (Pectinex AFP L-4 and Panzym Yield Mash) compared to the control samples. The enzymatic treatment decreased procyanidin content in most sediment with the exception of Pectinex Smash XXL and Pectinex AFP L-4. Generally in samples that were treated by pectinase, radical scavenging activity of cloudy apple juices was increased compared to the untreated reference samples. The highest radical scavenging activity was associated with Pectinex Yield Mash, Pectinex Smash XXL, and Pectinex XXL enzyme and the lowest activity with Pectinex Ultra SP-L and Pectinex APFL-4. However, in the case of enzymatic mash treatment cloudy apple juices showed instability of turbidity and low viscosity. These results must be ascribed to the much higher hydrolysis of pectin by enzymatic preparation which is responsible for viscosity. During 6 months of storage at 4 degrees C small changes in analyzed parameters of apple juices were observed.

The potential of sugar beet pulp as raw material for neutral and acidic oligosaccharide production by direct enzymatic treatment was evaluated. The effect of the polygalacturonase to solid ratio (PGaseSR), cellulase activity to polygalacturonase activity ratio (CPGaseR), and reaction time (t) on several dependent variables (selected to quantify the mass of recovered liquors, the conversion of each polysaccharide into monosaccharides, and the conversion of each polysaccharide into oligomers) was studied. Mathematical models suitable for reproducing and predicting the experimental results were also developed. Operational conditions leading to a maximum oligomer production were calculated from models being PGaseSR = 10 U/g, CPGaseR = 0.725 filter paper units/U, and t = 12.82 h. Under these conditions, the models predicted that 906 kg of liquors containing 26.7 kg of oligosaccharides can be obtained from 100 kg of SBP, the distribution being as follows: 5.9 kg of glucooligosaccharides, 2.4 kg of galactooligosaccharides, 9.7 kg of arabinooligosaccharides, and 8.7 kg of oligogalacturonides. Therefore, this study demonstrated that pectic oligomers (for which prebiotic properties have been reported) can be obtained from SBP at high yield by direct enzymatic hydrolysis using mixtures of cellulases and pectinases.

Polymer nanocomposite microspheres (PNCMs) as solid supports can improve the efficiency of immobilized enzymes by reducing diffusional limitation as well as by increasing the surface area per mass unit. In this work, pectinase was immobilized on Fe(3)O(4)/SiO2-g-poly(PSStNa) nanocomposite microspheres by covalent attachment. Biochemical studies showed an improved storage stability of the immobilized pectinase as well as enhanced performance at higher temperatures and over a wider pH range. The immobilized enzyme retained50% of its initial activity over 30 days, and the optimum temperature and pH also increased to the ranges of 50-60 degrees C and 3.0-4.7, respectively. The kinetics of a model reaction catalyzed by the immobilized pectinase was finally investigated by the Michaelis-Menten equation. The PSStNa support presents a very simple, mild, and time-saving process for enzyme immobilization, and this strategy of immobilizing pectinase also makes use of expensive enzymes economically viable, strengthening repeated use of them as catalysts following their rapid and easy separation with a magnet.

Oleosomes, with their unique structural proteins, the oleosins, are known to be useful in cosmetics and other emulsion applications. A procedure to fractionate intact oleosomes to produce soybean oil without the use of organic solvents was investigated. Process parameters, enzyme treatment, filtration, cell lysis, and centrifugation, were studied. Successive extractions of the residue, eliminating the filtration step, pressurization, or ultrasonication of soybean flour prior to enzyme treatment and enzyme treatment on the residue, were the key steps. A mixture of Multifect Pectinase FE, Cellulase A, and Multifect CX 13L in equal proportion gave 36.42-63.23% of the total soybean oil from oleosomes, respectively, for 45 and 180 s of blending time, compared to the conventional method with lower yields (34.24 and 28.65%, respectively, for 45 and 180 s of blending time). Three successive extractions of the residue increased the oil yield to a maximum of 84.65% of the total soybean oil recovered in intact oleosomes. The percentage of lipid in the supernatant fraction decreased to a minimum value of 0.33% with the use of the enzymes at a 3% dosage. The results are considered to be useful for developing large-scale and efficient extraction of oleosomes from soybean.

It is generally assumed that polyphenols, such as anthocyanins in fruit juice, exist in a free soluble state and are readily available for absorption in the gastro-intestinal tract. In the present study, we have investigated the interaction of polyphenols with soluble carbohydrate polymers, such as pectin and lipid nanovesicles, that are generated during homogenization of the fruit tissue during juice extraction. A commercially available grape juice concentrate contained nearly 25% of polyphenol fraction bound to macromolecules that were nondialyzable. Treatment of dialyzed juice with cellulase, pectinase, and beta-galactosidase did not cause the release of bound polyphenols; however, treatment with triton X-100 caused an increased release of bound polyphenols. The dialyzate contained relatively more -3-O glucosides and -3-O-acetoyl glucosides in comparison to the bound fraction which was enriched in -3-O-coumaroyl glucosides, suggesting qualitative differences in the bound and the free anthocyanin composition. Electron microscopic analysis of the juice fractions revealed the presence of electron-dense nanovesicle-fiber complexes ranging from 10 to 200 nm in diameter. Such complexes were absent in the dialyzate fraction. Cellulase treatment did not change the morphology of the complexes; however, treatment with pectinase and beta-galactosidase disrupted the complexes, releasing vesicular structures, suggestive of the pectin nature of the fibrous matrix. The dialyzed and the dialyzate fractions also showed differences in their 1H NMR and fluorescence spectral characteristics. The dialyzed fraction containing polyphenol-pectin complexes showed no superoxide scavenging capacity, reduced hydroxyl radical scavenging activity, and high 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, indicating potential changes in functionality because of the complex formation.

Long-term monoculture of watermelon leads to frequent occurrence of watermelon fusarium wilt caused by Fusarium oxysporum f.sp. niveum (FON). Some allelochemicals contained in watermelon root exudates and decaying residues are possibly responsible for promoting the wilt disease. The purpose of this study was to evaluate the allelopathic effect of artificially applied cinnamic acid on FON. Results demonstrated that hyphal growth of FON was strongly inhibited by cinnamic acid. At the highest concentration of cinnamic acid, the biomass in liquid culture was decreased by 63.3%, while colony diameter, conidial germination on plates, and conidial production in liquid culture were completely inhibited. However, mycotoxin production and activity of phytopathogenic enzymes were greatly stimulated. Mycotoxin yield, pectinase activity, proteinase activity, cellulase activity, and amylase activity were increased by 490, 590, 760, 2006, and 27.0%, respectively. It was concluded that cinnamic acid dramatically stimulated mycotoxin production and activities of hydrolytic enzymes by FON but inhibited growth and germination of FON. The findings presented here indicate that cinnamic acid is involved in promoting watermelon fusarium wilt.

A technically simple and inexpensive discontinuous turbidity assay for qualitative and/or quantitative assessments of polygalacturonic acid depolymerase activity is presented. The enzyme reaction is initiated by the addition of enzyme preparation to a reaction mixture containing 0.02% polygalacturonic acid (PGA) in acetate buffer. The progress of the reaction is monitored by terminating aliquots of the reaction mixture (via heat treatment at appropriate times), subsequently adding poly(diallyldimethylammonium chloride) (PDADMAC) for turbidity development (approximately 30 min), and then measuring the turbidity (typically at 420 nm) of the resulting PGA-PDADMAC complex-containing solution. PGA depolymerase activity causes a decrease in the observed turbidity of PGA-PDADMAC-containing solutions because the stability of the interpolyelectrolyte PGA-PDADMAC complex is a function of the degree of polymerization of the PGA. The rate of turbidity change is herein shown to be proportional to a relatively wide range of enzyme concentrations. The assay is demonstrated using a commercial pectinase preparation and tomato fruit extracts.

Conventional clarification with gelatin and silica sol removes a considerable amount of antioxidant phenolics from berry juices. This study examined the clarification and haze-diminishing effects of alternative clarification strategies on black currant juice including centrifugation and addition of acidic protease and pectinolytic enzyme preparations and gallic acid. Centrifugation of freshly pressed juice (10,000 g for 15 min) resulted in a approximately 95% reduction of immediate turbidity and had a decreasing effect on haze development in the juice during cold storage without significantly compromising the total phenols levels. The extent of clarification and haze diminishment varied after individual treatments with five different acidic proteases, but one of the protease preparations, Enzeco, derived from Aspergillus niger, consistently tended to perform best. The individual and interactive effects on juice turbidity, total phenols, and total anthocyanin contents of clarification treatments involving the use of two selected acid proteases (Enzeco and Novozyme 89L), a pectinase (Pectinex BE 3-L), and gallic acid were evaluated in a full factorial 2(4) experimental design. Haze development during cold storage decreased when gallic acid or any of the enzyme preparations were employed individually, but negative interaction effects resulted when the pectinase was employed in combination with any of the proteases. After 28 storage days at 2 degrees C, the lowest levels of haze formation were achieved when the Enzeco protease preparation, added at 0.025 g/L, was added with 0.050 g/L of gallic acid and allowed to react in the juice for 90 min at 50 degrees C. The corresponding anthocyanin reduction was approximately 12% (compared to approximately 30% with gelatin silica sol treatment). The data support the hypothesis that phenol-protein interactions are involved in juice turbidity development during cold storage of berry juices and demonstrate that precentrifugation and protease-assisted clarification show promise as an alternative, phenolics-retaining clarification strategy in black currant juice processing.

The objective of this work was to investigate the role played by the protein fraction of soy soluble polysaccharide (SSPS) during its adsorption at oil/water interfaces. SSPS was separated in a high (HMF; 310 kDa) and low (LMF; 20 kDa) molecular weight fraction by gel filtration. SSPS/HMF consisted of 91.6% carbohydrate and 2.2% protein and showed better emulsifying properties than those of the whole SSPS, whereas SSPS/LMF seemed to affect negatively the adsorption behavior of SSPS. SDS-PAGE of the protein fraction obtained from SSPS/HMF showed a molecular mass of 50 kDa, was composed predominantly of proline (23.1%) and glutamic acid (15.2%), and still contained 8.8% of neutral sugar and 5.3% of uronic acid. Results indicated that not all of the protein material present in SSPS contributes to SSPS functionality but that only the material associated with HMF aids in the adsorption of SSPS onto oil/water interfaces.

Tomatoes are grown for fresh consumption or for processing of the fruit. Some ripening-associated processes of the fruit can either contribute to or degrade attributes associated with both fresh and processing quality. For example, cell wall disassembly is associated with loss of fresh fruit firmness as well as with loss of processed tomato product viscosity. Several enzymes contribute to cell wall polysaccharide disassembly. Polygalacturonase (PG, poly[1,4-alpha-d-galactouronide] glucanohydrolase, EC 3.2.1.15) is among the most abundant polysaccharide hydrolases in ripening tomato fruit and is the major contributor to pectin depolymerization. Expansin (LeExp1) is also abundant in ripening fruit and is proposed to contribute to cell wall disassembly by nonhydrolytic activity, possibly by increasing substrate accessibility to other enzymes. Suppression of either LePG or LeExp1 expression alone results in altered softening and/or shelf life characteristics. To test whether simultaneous suppression of both LePG and LeExp1 expression influences fruit texture in additive or synergistic ways, transgenic Lycopersicon esculentum var. Ailsa Craig lines with reduced expression of either LePG or LeExp1 were crossed. Fruits from the third generation of progeny, homozygous for both transgenic constructs, were analyzed for firmness and other quality traits during ripening on or off the vine. In field-grown transgenic tomato fruit, suppression of LeExp1 or LePG alone did not significantly increase fruit firmness. However, fruits suppressed for both LePG and LeExp1 expression were significantly firmer throughout ripening and were less susceptible to deterioration during long-term storage. Juice prepared from the transgenic tomato fruit with reduced LePG and LeExp1 expression was more viscous than juice prepared from control fruit.

Thermal inactivation kinetics have been determined for pectin methylesterase (PME), polygalacturonase (PG), and peroxidase (POD) in tomato juice. Two parameters, the inactivation rate constant (k) at a reference temperature and the activation energy for inactivation (E(a)), were determined for each enzyme. For PME and PG, the k and E(a) values reported here do not agree with those in several previously published reports. These differences can be explained either by the differences in pH values used for inactivation determinations or by inadequacies in the heating methods used in some previous studies. POD showed simple first-order inactivation kinetics and was less thermally stable than either PME or PG. When different cultivars of tomatoes were evaluated, there was no difference in the thermal inactivation kinetics of these enzymes.

The aim of the present work was to study the effect of both vintage and the use of four types of pectinolytic enzymatic preparations on the quality of red wine using factor analysis. We also studied the evolution of the factors obtained along two years of storage of the wine. The results obtained from the factor analysis revealed that the addition of pectinolytic enzymes enriched the phenolic composition and elicited an improvement in the visual aspect of the wine, which persisted during the storage period considered.

Marigold flowers are the most important source of carotenoids for application in the food industry. However, the extraction gives almost 50% losses of the carotenoids depending on conditions for silaging, drying, and solvent extraction. In the past decades, macerating enzymes have been successfully applied to improve the extraction yield of valued compounds from natural products. In this work, an alternative extraction process for carotenoids is proposed, consisting of a simultaneous enzymatic treatment and solvent extraction. The proposed process employs milled fresh flowers directly as raw material, eliminating the inefficient silage and drying operations as well as the generation of hard to deal with aqueous effluents present in traditional processes. The process developed was tested at the 80 L scale, where under optimal conditions a carotenoid recovery yield of 97% was obtained.

During plant maturation, degradability of alfalfa (Medicago sativa L.) stems declines due to accumulation of highly lignified xylary tissue. Xylem and nonxylem tissues dissected from lower alfalfa internodes were analyzed for cell wall constituents and degradability. Cell walls comprised 740 mg g(-1) of xylem and 533 mg g(-1) of nonxylem tissues. Xylem tissues contributed about 60% of the cell wall mass in internodes. Xylem walls contained 28% lignin, 4% pectin, 29% hemicellulose, and 39% cellulose as compared to 15% lignin, 25% pectin, 30% hemicellulose, and 30% cellulose in nonxylem walls. Fungal enzymes hydrolyzed 22 and 73% of the structural carbohydrates in xylem and nonxylem walls, respectively. In both cell wall fractions, the release of xylose was 56-90% lower than that of other sugars, indicating that lignin preferentially restricted xylan degradation in secondary walls and xyloglucan degradation in primary walls. Elucidation of lignin-xylose interactions may reveal strategies for improving fiber degradability of alfalfa.

Defatted untoasted soybean cotyledons and hulls were fractionated as water solutes (WSc and WSh) and water unextractable (WUc and WUh). Further fractionation of WUc through deproteinization yielded the isolation of a water unextractable solid (WUS) fraction that was mainly composed (molar percent) of galactose (28.1%), glucose (27.8%), arabinose (13.3%), and uronic acids (17.6%), which accounted for 76% of the water insoluble polysaccharides in soybean cotyledons (WUc). The cell wall (WUS) was sequentially fractionated with chelating agents (chelating agent soluble solids, ChSS) and a gradient of agents (dilute alkali, DASS; 1 M alkali, 1MASS; and 4M alkali, 4MASS), which gave a final cellulosic residue. The ChSS and DASS extracts were characterized as pectin-rich fractions, whereas 1MASS and 4MASS were hemicellulose- and cellulose-rich fractions. Incubation in vitro of the WUc fraction with pectinase, cellulase, and xylanase resulted in the release of low amounts (not more than 5% bound basis) of monosaccharides, mostly uronic acids, xylose, and arabinose. Protein extraction hardly increased this release after enzymatic incubation (7%). However, progressive fractionation of the cell wall matrix markedly increased the release of monosaccharides from pectin- (ChSS and DASS) and hemicellulose-rich fractions (1MASS and 4MASS). Significant degradation of cellulose (up to 20%) was achieved only after complete protein, pectin, and hemicellulose extraction.