The active sites of feruloyl esterases from mesophilic and thermophilic sources were probed using... more The active sites of feruloyl esterases from mesophilic and thermophilic sources were probed using methyl esters of phenylalkanoic acids. Only 13 out of 26 substrates tested were significant substrates for all the enzymes. Lengthening or shortening the aliphatic side chain while maintaining the same aromatic substitutions completely abolished activity for both enzymes, which demonstrates the importance of the correct distance between the aromatic group and the ester bond. Maintaining the phenylpropanoate structure but altering the substitutions of the aromatic ring demonstrated that the type-A esterase from the mesophilic fungus Fusarium oxysporum (FoFaeA) showed a preference for methoxylated substrates, in contrast to the type-B esterase from the same source (FoFaeB) and the thermophilic type-B (StFaeB) and type-C (StFaeC) from Sporotrichum thermophile, which preferred hydroxylated substrates. All four esterases hydrolyzed short chain aliphatic acid (C2-C4) esters of p-nitrophenol, but not the C12 ester of laurate. All the feruloyl esterases were able to release ferulic acid from the plant cell wall material in conjunction with a xylanase, but only the type-A esterase FoFaeA was effective in releasing the 5,5' form of diferulic acid. The thermophilic type-B esterase had a lower catalytic efficiency than its mesophilic counterpart, but released more ferulic acid from plant cell walls.
The active site of the recombinant Talaromyces stipitatus type-C feruloyl esterase (TsFaeC) was p... more The active site of the recombinant Talaromyces stipitatus type-C feruloyl esterase (TsFaeC) was probed using a series of C 1 -C 4 alkyl ferulates and methyl esters of phenylalkanoic and cinnamic acids. The enzyme was active on 23 of the 34 substrates tested. Lengthening or shortening the aliphatic side chain while maintaining the same aromatic substitutions completely abolished the enzyme activity. Maintaining the phenylpropenoate structure but altering the substitutions of the aromatic ring demonstrated the importance of hydroxyl groups on meta and/or para position of the benzoic ring. The highest catalytic efficiency of TsFaeC for methyl cinnamates was shown on methyl 3,4-dihydroxy cinnamate and on its hydro form (3,4-dihydroxy-phenyl-propionate). Maintaining the ferulate structure but altering the esterified alkyl group, the comparison of k cat and k cat /K m values showed that the enzyme hydrolysed faster and more efficiently than ethyl ferulate. Alkyl ferulates were applied also for substrate selectivity mapping of feruloyl esterase to catalyze feruloyl group transfer to l-arabinose, using as a reaction system a ternary water-organic mixture consisting of n-hexane, t-butanol and water. The reaction parameters affecting the feruloylation rate and the conversion of the enzymatic synthesis, such as the composition of the reaction media, temperature, substrate and enzyme concentration have been investigated.
Aims: To evaluate the antimicrobial properties of flavonoid-rich fractions derived from bergamot ... more Aims: To evaluate the antimicrobial properties of flavonoid-rich fractions derived from bergamot peel, a byproduct from the Citrus fruit processing industry and the influence of enzymatic deglycosylation on their activity against different bacteria and yeast. Methods and Results: Bergamot ethanolic fractions were tested against Gramnegative bacteria (Escherichia coli, Pseudomonas putida, Salmonella enterica), Gram-positive bacteria (Listeria innocua, Bacillus subtilis, Staphylococcus aureus, Lactococcus lactis) and the yeast Saccharomyces cerevisiae. Bergamot fractions were found to be active against all the Gram-negative bacteria tested, and their antimicrobial potency increased after enzymatic deglycosylation. The minimum inhibitory concentrations of the fractions and the pure flavonoids, neohesperidin, hesperetin (aglycone), neoeriocitrin, eriodictyol (aglycone), naringin and naringenin (aglycone), were found to be in the range 200 to 800 lg ml )1 . The interactions between three bergamot flavonoids were also evaluated. Conclusion: The enzyme preparation Pectinase 62L efficiently converted common glycosides into their aglycones from bergamot extracts, and this deglycosylation increased the antimicrobial potency of Citrus flavonoids. Pairwise combinations of eriodictyol, naringenin and hesperetin showed both synergistic and indifferent interactions that were dependent on the test indicator organism. Significance and Impact of the Study: Bergamot peel is a potential source of natural antimicrobials that are active against Gram-negative bacteria.
Wheat bran, the major side-stream generated in the milling of wheat grains in the production of w... more Wheat bran, the major side-stream generated in the milling of wheat grains in the production of white flour, contains significant quantities of carbohydrate and proteins. While not interfering with flour utilization, the bran could be considered as an important feedstock within a biorefinery concept. Wheat bran also contains some amounts of lipids that can be used as a source of valuable phytochemicals. Gas chromatography and mass spectrometry analysis of the lipid composition of destarched wheat bran demonstrated that the predominant lipids found in wheat bran were free fatty acids (ca. 40% of total lipids), followed by acylglycerols (40%). Additionally, important amounts of alkylresorcinols (13% of total lipids) and steroid compounds (hydrocarbons, ketones, free sterols, sterol glycosides, sterol esters, and sterol ferulates) (7% of total lipids) were also present among the lipids of wheat bran. The use of wheat bran as a valuable source of phytochemicals of interest in the context of a wheat bran biorefinery is discussed.
Bergamot (Citrus bergamia Risso) is a less commercialized Citrus fruit, mainly used for its essen... more Bergamot (Citrus bergamia Risso) is a less commercialized Citrus fruit, mainly used for its essential oil extracted from the peel. Bergamot peel (BP) represents about 60% of the processed fruits and is regarded as primary waste. However, it contains good amounts of useful compounds, such as pectins and flavonoids. Many of the bioactivities of Citrus flavonoids appear to impact vascular endothelial cells. Herein, we report the protective effect of two flavonoid-rich extracts from BP (endowed with radical-scavenging properties and lacking genotoxic activity) against alterations in cell modifications induced by the pleiotropic inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) on human umbilical vein endothelial cells (HUVECs), as demonstrated by monitoring intracellular levels of malondialdehyde/4-hydroxynonenal, reduced and oxidized glutathione and superoxide dismutase activity, and the activation status of nuclear factor-kappaB (NF-kappaB). Thus, BP appears to be a potential source of natural antioxidant/anti-inflammatory phytocomplexes to be employed as ingredients of nutraceutical products or functional foods.
Opuntia ficus-indica cladodes represent the green stem of the plant and are generally used as ani... more Opuntia ficus-indica cladodes represent the green stem of the plant and are generally used as animal feed or disposed of in landfills. The present work investigated the anatomical and chemical composition of Opuntia cladodes, which form the basis of their pharmacological effects. Glucose and galacturonic acid were the main sugars of Opuntia cladodes, whereas high-performance liquid chromatography (HPLC) analysis showed the presence of mainly kaempherol and isorhamnetin glycosides (glucoside and rhamnoside). The presence of high amounts of calcium oxalate crystals was demonstrated by light microscopy on fresh and lyophilized cladodes. No antimicrobial activity was observed even after enzymatic treatment. O. ficus-indica cladodes may retain material tightly associated with cell-wall components, and this property will have the potential to greatly reduce the bioavailability of bioactive compounds.
Brewers' spent grain (BSG), a high-volume coproduct from the brewing industry, primarily contains... more Brewers' spent grain (BSG), a high-volume coproduct from the brewing industry, primarily contains proteins, barley cell wall carbohydrates, and lignin. To create new possibilities for the exploitation of this large biomass stream, the solubilization of BSG by the combined action of carbohydrases (Depol 740 and Econase) and peptidase (Alcalase and Promod 439) was explored. Hydrolysis protocols were optimized with respect to temperature (influencing both microbial contamination and rate of enzymatic hydrolysis), pH, enzyme dose, order of enzyme addition, and processing time. On the basis of this approach, one-and two-step protocols are proposed taking 4-8 h and yielding combined or separate fractions of hydrolyzed oligosaccharides and liberated hydrolyzed protein. Optimized procedures resulted in the solubilization of >80% of the proteinaceous material, up to 39% of the total carbohydrates, and up to 42% of total dry matter in BSG. Of the original xylan present in BSG, 36% could be solubilized. Sequential and simultaneous treatments with the two enzyme types gave similar results. In sequential processes, the order of the carbohydrase and peptidase treatments had only minor effects on the outcome. Depol 740 released more pentoses than Econase and gave slightly higher overall dry matter solubilization yields.
Pectinolytic and cellulolytic enzymes (Pectinase 62L, Pectinase 690L, and Cellulase CO13P) were u... more Pectinolytic and cellulolytic enzymes (Pectinase 62L, Pectinase 690L, and Cellulase CO13P) were used to evaluate the solubilization of carbohydrates and low molecular weight flavonoids from bergamot peel, a major byproduct of the essential oil industry. The enzymes were characterized for main-chain and side-chain polysaccharide hydrolyzing activities and also against pure samples of various flavonoids previously identified in bergamot peel to determine various glycosidase activities. The addition of Pectinase 62L or 690L alone, or the combination of Pectinase 62L and Cellulase CO13P, was capable of solubilizing between 70 and 80% of the bergamot peel, and up to 90% of the flavonoid glycosides present were cleaved to their aglycones. Cellulase CO13P alone solubilized 62% of the peel but had no deglycosylating effect on the flavonoid glycosides. Over a 24-h time course, a rapid release of cell wall carbohydrates was observed after treatment with Pectinase 62L, with a concurrent gradual hydrolysis of the flavonoid glycosides. Size-exclusion chromatography of the solubilized extract showed that after 24-h incubation, the majority of the solubilized carbohydrates were present as monosaccharides with a smaller proportion of oligosaccharides.
... James A. Robertson, and Gary Williamson. ... Preparation of AIR and Bran Cell Wall Samples. T... more ... James A. Robertson, and Gary Williamson. ... Preparation of AIR and Bran Cell Wall Samples. The preparation of coarse and fine wheat bran cell wall samples (Ryden and Robertson, 1995a) and AIR of sugarbeet samples (Ryden and Robertson, 1995b) have been described. ...
Hydroxycinnamic acid content and ferulic acid dehydrodimer content were determined in 11 barley v... more Hydroxycinnamic acid content and ferulic acid dehydrodimer content were determined in 11 barley varieties after alkaline hydrolysis. Ferulic acid (FA) was the most abundant hydroxycinnamate with concentrations ranging from 359 to 624 microg/g dry weight. p-Coumaric acid (PCA) levels ranged from 79 to 260 microg/g dry weight, and caffeic acid was present at concentrations of <19 microg/g dry weight. Among the ferulic acid dehydrodimers that were identified, 8-O-4'-diFA was the most abundant (73-118 microg/g dry weight), followed by 5,5'-diFA (26-47 microg/g dry weight), the 8,5'-diFA benzofuran form (22-45 microg/g dry weight), and the 8,5'-diFA open form (10-23 microg/g dry weight). Significant variations (p < 0.05) among the different barley varieties were observed for all the compounds that were quantified. Barley grains were mechanically fractionated into three fractions: F1, fraction consisting mainly of the husk and outer layers; F2, intermediate fraction; and F3, fraction consisting mainly of the endosperm. Fraction F1 contained the highest concentration for ferulic acid (from 77.7 to 82.3% of the total amount in barley grain), p-coumaric acid (from 78.0 to 86.3%), and ferulic acid dehydrodimers (from 79.2 to 86.8%). Lower contents were found in fraction F2, whereas fraction F3 exhibited the lowest percentages (from 1.2 to 1.9% for ferulic acid, from 0.9 to 1.7% for p-coumaric acid, and <0.02% for ferulic acid dehydrodimers). The solid barley residue from the brewing process (brewer's spent grain) was approximately 5-fold richer in ferulic acid, p-coumaric acid, and ferulic acid dehydrodimers than barley grains.
The enzymatic hydrolysis of brewers' spent grain (BSG) has been investigated through treatment wi... more The enzymatic hydrolysis of brewers' spent grain (BSG) has been investigated through treatment with commercial carbohydrases and proteases. Resultant residues were then chemically fractionated and delignified. Enzymatic treatments released 25À30% of the BSG mass and yielded precursors suitable for subsequent conversion to potentially value-added products. Controlled chemical fractionation selectively solubilized arabinoxylan but with no differences apparent due to prior enzyme treatment. The loss of non-polysaccharide components during alkali treatment suggests the presence of a high proportion of alkalisoluble lignin. Further delignification of the alkali-insoluble residues and further chemical fractionation released the remaining hemicellulose, to yield a residue which was >90% cellulose. Further knowledge of the properties and interaction between BSG polymers will facilitate an improved enzyme-assisted total deconstruction of BSG and hence the exploitation of its biomass.
Bergamot peel is an underutilized byproduct of the essential oil and juice-processing industry. A... more Bergamot peel is an underutilized byproduct of the essential oil and juice-processing industry. As with other Citrus peels, it still contains exploitable components, such as pectins and flavonoids. Commercial glycoside hydrolases, specifically a combination of pectolytic and cellulolytic enzymes, solubilized a high percentage of the material (81.94%). The flavonoid profile of the peel consisted of characteristic Citrus species flavanone rutinosides and neohesperosides derived from naringenin, eriodictyol, and hesperetin. In addition, a number of minor flavanone and flavone glycosides, not found in orange and lemon peels, were identified. The majority of flavonoids were extracted in the two 70% v/v EtOH extractions. Processing this material clearly has economic potential leading to low environmental impact.
Analysis of wheat bran and spent grain shows that concentrations of ferulate and diferulates offe... more Analysis of wheat bran and spent grain shows that concentrations of ferulate and diferulates offer considerable scope to modify the cross-linking of feruloylated polysaccharides and hence the mechanical properties of these residues. In solution ferulic acid (FA) can be readily polymerized by horseradish peroxidase, but when esterified to a polysaccharide, the profile of diferulates becomes restricted. This situation also exists in muro and suggests structural constraints may limit the availability of FA for cross-linking. At relatively low polysaccharide concentration, (∼3%), steric restrictions were apparent in gels prepared using isolated polysaccharides. Mechanical properties such as swelling also appear to be fixed at these relatively low polysaccharide concentrations. This limits the potential to modify mechanical properties in muro through oxidoreductase activity. To modify mechanical properties such treatments will need to focus on increasing the "flexibility" of the cell wall matrix and the accessibility of enzymes to the cell wall matrix.
Ferulic acid esterase (EC 3.1.1.73) cleaves the feruloyl groups substituted at the 5′-OH group of... more Ferulic acid esterase (EC 3.1.1.73) cleaves the feruloyl groups substituted at the 5′-OH group of arabinosyl residues of arabinoxylans and is known to modulate their functional properties. In this study, ferulic acid esterase from 96 h finger millet malt was purified to apparent homogeneity by three-step purification with a recovery of 3% and a fold purification of 22. The substrate pnitrophenylferulate (PNPF) was synthesized and used to assay this enzyme spectrophotometrically. The products liberated from ragi and wheat water-soluble polysaccharides by the action of purified ragi ferulic acid esterase were identified by ESI-MS. The pH and temperature optima of the enzyme were found to be 6.0 and 45°C, respectively. The pH and temperature stabilities of the enzyme were found to be in the range of 5.5-9.0 and 30°C, respectively. The activation energy of the enzymatic reaction was found to be 4.08 kJ mol -1 . The apparent K m and V max of the purified ferulic acid esterase for PNPF were 0.053 µM and 0.085 unit mL -1 , respectively. The enzyme is a monomer with a molecular mass of 16.5 kDa. Metal ions such as Ni 2+ , Zn 2+ , Co 2+ , and Cu 2+ and oxalic and citric acids enhanced the enzyme activity. The enzyme was completely inhibited by Fe 3+ . Group specific reagents such as p-chloromercuric benzoate and iodoacetamide inhibited the enzyme, indicating the possible presence of cysteine residues in the active site pocket. All chemicals were purchased from Sisco Research Laboratories (Mumbai, India) or Sigma Chemical Co. (St. Louis, MO). Protein molecular mass markers for SDS (sodium dodecyl sulfate) were obtained from Genei (Bangalore, India). DEAE-cellulose, Biogel P-30 was obtained from Pharmacia fine chemicals (Uppsala, Sweden). Precoated silica gel plates (0.25 mm layer thick) with an UV254 indicator were from Merck (Darmstadt, Germany).
Dietary supplementation with prebiotics may result in the stimulation of the growth of beneficial... more Dietary supplementation with prebiotics may result in the stimulation of the growth of beneficial bacteria such as lactobacilli and bifidobacteria in the human gastrointestinal tract. The effect of water-unextractable arabinoxylans (WU-AX) derived from wheat on the modulation of gut bacterial composition was investigated using a mixed culture fermentation system. A prebiotic index (PI) score of 2.03 was obtained after addition of 1% (w/v) WU-AX to a pH-controlled stirred anaerobic fermentation vessel. Pretreatment of the WU-AX with endo-β-1,4-xylanase resulted in significantly higher PI value (3.48) indicating that pretreatment provided oligomers that were better utilised by the gut bacteria. The extracellular hydrolytic enzymes xylanase and ferulic acid esterase are both required for bacterial metabolism of WU-AX and both activities were present in supernatants derived from the mixed batch cultures. Addition of the WU-AX substrates to the batch cultures produced several fold increases of bacterial synthesis of both enzymes, and these increases were greater when the WU-AX substrate was pretreated with xylanase.
With the yearly accumulation of agro-industrial waste-material generated by the milling, brewing ... more With the yearly accumulation of agro-industrial waste-material generated by the milling, brewing and sugar industries in Europe, the importance of extracting high value residues must be considered to offset the cost of treating and disposing of the residues. This work ...
The cDNA encoding Aspergillus niger cinnamoyl esterase (FAEA) with its native signal sequence was... more The cDNA encoding Aspergillus niger cinnamoyl esterase (FAEA) with its native signal sequence was isolated by reverse transcriptasepolymerase chain reaction, sequenced, and expressed in Pichia pastoris. Secretion yields up to 300 mg l 31 were obtained in buffered medium. The recombinant FAEA was purified to homogeneity using a one-step purification protocol and found to be identical to the native enzyme with respect to size, pI, immunoreactivity and N-terminal sequence. Specific activity, pH and temperature optimum, and kinetic parameters were also found similar to the native esterase. FAEA is thus the first fungal esterase efficiently produced using a heterologous system. ß Abbreviations : AOX1, alcohol oxidase; BMGY, bu¡ered minimal glycerol-complex medium; BMMY, bu¡ered minimal methanol-complex medium ; ESI, electrospray ionisation ; faeA, cDNA encoding A. niger cinnamoyl esterase; FAEA, A. niger cinnamoyl esterase; reFAEA, recombinant FAEA; HPLC, high performance liquid chromatography ; MFA, methyl ferulate (3-methoxy-4-hydroxycinnamic acid); MOPS, morpholino-propanesulfonic acid; MS, mass spectrometry ; MSA, methyl sinapate (3,5dimethoxy-4-hydroxycinnamic acid); RT-PCR, reverse transcriptase-polymerase chain reaction ; SDS, sodium dodecyl sulfate; YNB, yeast nitrogen base
Lactase phlorizin hydrolase (LPH; EC 3.2.1.62) is a membrane-bound, family 1 L L-glycosidase foun... more Lactase phlorizin hydrolase (LPH; EC 3.2.1.62) is a membrane-bound, family 1 L L-glycosidase found on the brush border of the mammalian small intestine. LPH, purified from sheep small intestine, was capable of hydrolysing a range of flavonol and isoflavone glycosides. The catalytic efficiency (k cat / K m ) for the hydrolysis of quercetin-4P P-glucoside, quercetin-3glucoside, genistein-7-glucoside and daidzein-7-glucoside was 170, 137, 77 and 14 (mM 31 s 31 ) respectively. The majority of the activity occurred at the lactase and not phlorizin hydrolase site. The ability of LPH to deglycosylate dietary (iso)flavonoid glycosides suggests a possible role for this enzyme in the metabolism of these biologically active compounds.
The active sites of feruloyl esterases from mesophilic and thermophilic sources were probed using... more The active sites of feruloyl esterases from mesophilic and thermophilic sources were probed using methyl esters of phenylalkanoic acids. Only 13 out of 26 substrates tested were significant substrates for all the enzymes. Lengthening or shortening the aliphatic side chain while maintaining the same aromatic substitutions completely abolished activity for both enzymes, which demonstrates the importance of the correct distance between the aromatic group and the ester bond. Maintaining the phenylpropanoate structure but altering the substitutions of the aromatic ring demonstrated that the type-A esterase from the mesophilic fungus Fusarium oxysporum (FoFaeA) showed a preference for methoxylated substrates, in contrast to the type-B esterase from the same source (FoFaeB) and the thermophilic type-B (StFaeB) and type-C (StFaeC) from Sporotrichum thermophile, which preferred hydroxylated substrates. All four esterases hydrolyzed short chain aliphatic acid (C2-C4) esters of p-nitrophenol, but not the C12 ester of laurate. All the feruloyl esterases were able to release ferulic acid from the plant cell wall material in conjunction with a xylanase, but only the type-A esterase FoFaeA was effective in releasing the 5,5' form of diferulic acid. The thermophilic type-B esterase had a lower catalytic efficiency than its mesophilic counterpart, but released more ferulic acid from plant cell walls.
The active site of the recombinant Talaromyces stipitatus type-C feruloyl esterase (TsFaeC) was p... more The active site of the recombinant Talaromyces stipitatus type-C feruloyl esterase (TsFaeC) was probed using a series of C 1 -C 4 alkyl ferulates and methyl esters of phenylalkanoic and cinnamic acids. The enzyme was active on 23 of the 34 substrates tested. Lengthening or shortening the aliphatic side chain while maintaining the same aromatic substitutions completely abolished the enzyme activity. Maintaining the phenylpropenoate structure but altering the substitutions of the aromatic ring demonstrated the importance of hydroxyl groups on meta and/or para position of the benzoic ring. The highest catalytic efficiency of TsFaeC for methyl cinnamates was shown on methyl 3,4-dihydroxy cinnamate and on its hydro form (3,4-dihydroxy-phenyl-propionate). Maintaining the ferulate structure but altering the esterified alkyl group, the comparison of k cat and k cat /K m values showed that the enzyme hydrolysed faster and more efficiently than ethyl ferulate. Alkyl ferulates were applied also for substrate selectivity mapping of feruloyl esterase to catalyze feruloyl group transfer to l-arabinose, using as a reaction system a ternary water-organic mixture consisting of n-hexane, t-butanol and water. The reaction parameters affecting the feruloylation rate and the conversion of the enzymatic synthesis, such as the composition of the reaction media, temperature, substrate and enzyme concentration have been investigated.
Aims: To evaluate the antimicrobial properties of flavonoid-rich fractions derived from bergamot ... more Aims: To evaluate the antimicrobial properties of flavonoid-rich fractions derived from bergamot peel, a byproduct from the Citrus fruit processing industry and the influence of enzymatic deglycosylation on their activity against different bacteria and yeast. Methods and Results: Bergamot ethanolic fractions were tested against Gramnegative bacteria (Escherichia coli, Pseudomonas putida, Salmonella enterica), Gram-positive bacteria (Listeria innocua, Bacillus subtilis, Staphylococcus aureus, Lactococcus lactis) and the yeast Saccharomyces cerevisiae. Bergamot fractions were found to be active against all the Gram-negative bacteria tested, and their antimicrobial potency increased after enzymatic deglycosylation. The minimum inhibitory concentrations of the fractions and the pure flavonoids, neohesperidin, hesperetin (aglycone), neoeriocitrin, eriodictyol (aglycone), naringin and naringenin (aglycone), were found to be in the range 200 to 800 lg ml )1 . The interactions between three bergamot flavonoids were also evaluated. Conclusion: The enzyme preparation Pectinase 62L efficiently converted common glycosides into their aglycones from bergamot extracts, and this deglycosylation increased the antimicrobial potency of Citrus flavonoids. Pairwise combinations of eriodictyol, naringenin and hesperetin showed both synergistic and indifferent interactions that were dependent on the test indicator organism. Significance and Impact of the Study: Bergamot peel is a potential source of natural antimicrobials that are active against Gram-negative bacteria.
Wheat bran, the major side-stream generated in the milling of wheat grains in the production of w... more Wheat bran, the major side-stream generated in the milling of wheat grains in the production of white flour, contains significant quantities of carbohydrate and proteins. While not interfering with flour utilization, the bran could be considered as an important feedstock within a biorefinery concept. Wheat bran also contains some amounts of lipids that can be used as a source of valuable phytochemicals. Gas chromatography and mass spectrometry analysis of the lipid composition of destarched wheat bran demonstrated that the predominant lipids found in wheat bran were free fatty acids (ca. 40% of total lipids), followed by acylglycerols (40%). Additionally, important amounts of alkylresorcinols (13% of total lipids) and steroid compounds (hydrocarbons, ketones, free sterols, sterol glycosides, sterol esters, and sterol ferulates) (7% of total lipids) were also present among the lipids of wheat bran. The use of wheat bran as a valuable source of phytochemicals of interest in the context of a wheat bran biorefinery is discussed.
Bergamot (Citrus bergamia Risso) is a less commercialized Citrus fruit, mainly used for its essen... more Bergamot (Citrus bergamia Risso) is a less commercialized Citrus fruit, mainly used for its essential oil extracted from the peel. Bergamot peel (BP) represents about 60% of the processed fruits and is regarded as primary waste. However, it contains good amounts of useful compounds, such as pectins and flavonoids. Many of the bioactivities of Citrus flavonoids appear to impact vascular endothelial cells. Herein, we report the protective effect of two flavonoid-rich extracts from BP (endowed with radical-scavenging properties and lacking genotoxic activity) against alterations in cell modifications induced by the pleiotropic inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) on human umbilical vein endothelial cells (HUVECs), as demonstrated by monitoring intracellular levels of malondialdehyde/4-hydroxynonenal, reduced and oxidized glutathione and superoxide dismutase activity, and the activation status of nuclear factor-kappaB (NF-kappaB). Thus, BP appears to be a potential source of natural antioxidant/anti-inflammatory phytocomplexes to be employed as ingredients of nutraceutical products or functional foods.
Opuntia ficus-indica cladodes represent the green stem of the plant and are generally used as ani... more Opuntia ficus-indica cladodes represent the green stem of the plant and are generally used as animal feed or disposed of in landfills. The present work investigated the anatomical and chemical composition of Opuntia cladodes, which form the basis of their pharmacological effects. Glucose and galacturonic acid were the main sugars of Opuntia cladodes, whereas high-performance liquid chromatography (HPLC) analysis showed the presence of mainly kaempherol and isorhamnetin glycosides (glucoside and rhamnoside). The presence of high amounts of calcium oxalate crystals was demonstrated by light microscopy on fresh and lyophilized cladodes. No antimicrobial activity was observed even after enzymatic treatment. O. ficus-indica cladodes may retain material tightly associated with cell-wall components, and this property will have the potential to greatly reduce the bioavailability of bioactive compounds.
Brewers' spent grain (BSG), a high-volume coproduct from the brewing industry, primarily contains... more Brewers' spent grain (BSG), a high-volume coproduct from the brewing industry, primarily contains proteins, barley cell wall carbohydrates, and lignin. To create new possibilities for the exploitation of this large biomass stream, the solubilization of BSG by the combined action of carbohydrases (Depol 740 and Econase) and peptidase (Alcalase and Promod 439) was explored. Hydrolysis protocols were optimized with respect to temperature (influencing both microbial contamination and rate of enzymatic hydrolysis), pH, enzyme dose, order of enzyme addition, and processing time. On the basis of this approach, one-and two-step protocols are proposed taking 4-8 h and yielding combined or separate fractions of hydrolyzed oligosaccharides and liberated hydrolyzed protein. Optimized procedures resulted in the solubilization of >80% of the proteinaceous material, up to 39% of the total carbohydrates, and up to 42% of total dry matter in BSG. Of the original xylan present in BSG, 36% could be solubilized. Sequential and simultaneous treatments with the two enzyme types gave similar results. In sequential processes, the order of the carbohydrase and peptidase treatments had only minor effects on the outcome. Depol 740 released more pentoses than Econase and gave slightly higher overall dry matter solubilization yields.
Pectinolytic and cellulolytic enzymes (Pectinase 62L, Pectinase 690L, and Cellulase CO13P) were u... more Pectinolytic and cellulolytic enzymes (Pectinase 62L, Pectinase 690L, and Cellulase CO13P) were used to evaluate the solubilization of carbohydrates and low molecular weight flavonoids from bergamot peel, a major byproduct of the essential oil industry. The enzymes were characterized for main-chain and side-chain polysaccharide hydrolyzing activities and also against pure samples of various flavonoids previously identified in bergamot peel to determine various glycosidase activities. The addition of Pectinase 62L or 690L alone, or the combination of Pectinase 62L and Cellulase CO13P, was capable of solubilizing between 70 and 80% of the bergamot peel, and up to 90% of the flavonoid glycosides present were cleaved to their aglycones. Cellulase CO13P alone solubilized 62% of the peel but had no deglycosylating effect on the flavonoid glycosides. Over a 24-h time course, a rapid release of cell wall carbohydrates was observed after treatment with Pectinase 62L, with a concurrent gradual hydrolysis of the flavonoid glycosides. Size-exclusion chromatography of the solubilized extract showed that after 24-h incubation, the majority of the solubilized carbohydrates were present as monosaccharides with a smaller proportion of oligosaccharides.
... James A. Robertson, and Gary Williamson. ... Preparation of AIR and Bran Cell Wall Samples. T... more ... James A. Robertson, and Gary Williamson. ... Preparation of AIR and Bran Cell Wall Samples. The preparation of coarse and fine wheat bran cell wall samples (Ryden and Robertson, 1995a) and AIR of sugarbeet samples (Ryden and Robertson, 1995b) have been described. ...
Hydroxycinnamic acid content and ferulic acid dehydrodimer content were determined in 11 barley v... more Hydroxycinnamic acid content and ferulic acid dehydrodimer content were determined in 11 barley varieties after alkaline hydrolysis. Ferulic acid (FA) was the most abundant hydroxycinnamate with concentrations ranging from 359 to 624 microg/g dry weight. p-Coumaric acid (PCA) levels ranged from 79 to 260 microg/g dry weight, and caffeic acid was present at concentrations of <19 microg/g dry weight. Among the ferulic acid dehydrodimers that were identified, 8-O-4'-diFA was the most abundant (73-118 microg/g dry weight), followed by 5,5'-diFA (26-47 microg/g dry weight), the 8,5'-diFA benzofuran form (22-45 microg/g dry weight), and the 8,5'-diFA open form (10-23 microg/g dry weight). Significant variations (p < 0.05) among the different barley varieties were observed for all the compounds that were quantified. Barley grains were mechanically fractionated into three fractions: F1, fraction consisting mainly of the husk and outer layers; F2, intermediate fraction; and F3, fraction consisting mainly of the endosperm. Fraction F1 contained the highest concentration for ferulic acid (from 77.7 to 82.3% of the total amount in barley grain), p-coumaric acid (from 78.0 to 86.3%), and ferulic acid dehydrodimers (from 79.2 to 86.8%). Lower contents were found in fraction F2, whereas fraction F3 exhibited the lowest percentages (from 1.2 to 1.9% for ferulic acid, from 0.9 to 1.7% for p-coumaric acid, and <0.02% for ferulic acid dehydrodimers). The solid barley residue from the brewing process (brewer's spent grain) was approximately 5-fold richer in ferulic acid, p-coumaric acid, and ferulic acid dehydrodimers than barley grains.
The enzymatic hydrolysis of brewers' spent grain (BSG) has been investigated through treatment wi... more The enzymatic hydrolysis of brewers' spent grain (BSG) has been investigated through treatment with commercial carbohydrases and proteases. Resultant residues were then chemically fractionated and delignified. Enzymatic treatments released 25À30% of the BSG mass and yielded precursors suitable for subsequent conversion to potentially value-added products. Controlled chemical fractionation selectively solubilized arabinoxylan but with no differences apparent due to prior enzyme treatment. The loss of non-polysaccharide components during alkali treatment suggests the presence of a high proportion of alkalisoluble lignin. Further delignification of the alkali-insoluble residues and further chemical fractionation released the remaining hemicellulose, to yield a residue which was >90% cellulose. Further knowledge of the properties and interaction between BSG polymers will facilitate an improved enzyme-assisted total deconstruction of BSG and hence the exploitation of its biomass.
Bergamot peel is an underutilized byproduct of the essential oil and juice-processing industry. A... more Bergamot peel is an underutilized byproduct of the essential oil and juice-processing industry. As with other Citrus peels, it still contains exploitable components, such as pectins and flavonoids. Commercial glycoside hydrolases, specifically a combination of pectolytic and cellulolytic enzymes, solubilized a high percentage of the material (81.94%). The flavonoid profile of the peel consisted of characteristic Citrus species flavanone rutinosides and neohesperosides derived from naringenin, eriodictyol, and hesperetin. In addition, a number of minor flavanone and flavone glycosides, not found in orange and lemon peels, were identified. The majority of flavonoids were extracted in the two 70% v/v EtOH extractions. Processing this material clearly has economic potential leading to low environmental impact.
Analysis of wheat bran and spent grain shows that concentrations of ferulate and diferulates offe... more Analysis of wheat bran and spent grain shows that concentrations of ferulate and diferulates offer considerable scope to modify the cross-linking of feruloylated polysaccharides and hence the mechanical properties of these residues. In solution ferulic acid (FA) can be readily polymerized by horseradish peroxidase, but when esterified to a polysaccharide, the profile of diferulates becomes restricted. This situation also exists in muro and suggests structural constraints may limit the availability of FA for cross-linking. At relatively low polysaccharide concentration, (∼3%), steric restrictions were apparent in gels prepared using isolated polysaccharides. Mechanical properties such as swelling also appear to be fixed at these relatively low polysaccharide concentrations. This limits the potential to modify mechanical properties in muro through oxidoreductase activity. To modify mechanical properties such treatments will need to focus on increasing the "flexibility" of the cell wall matrix and the accessibility of enzymes to the cell wall matrix.
Ferulic acid esterase (EC 3.1.1.73) cleaves the feruloyl groups substituted at the 5′-OH group of... more Ferulic acid esterase (EC 3.1.1.73) cleaves the feruloyl groups substituted at the 5′-OH group of arabinosyl residues of arabinoxylans and is known to modulate their functional properties. In this study, ferulic acid esterase from 96 h finger millet malt was purified to apparent homogeneity by three-step purification with a recovery of 3% and a fold purification of 22. The substrate pnitrophenylferulate (PNPF) was synthesized and used to assay this enzyme spectrophotometrically. The products liberated from ragi and wheat water-soluble polysaccharides by the action of purified ragi ferulic acid esterase were identified by ESI-MS. The pH and temperature optima of the enzyme were found to be 6.0 and 45°C, respectively. The pH and temperature stabilities of the enzyme were found to be in the range of 5.5-9.0 and 30°C, respectively. The activation energy of the enzymatic reaction was found to be 4.08 kJ mol -1 . The apparent K m and V max of the purified ferulic acid esterase for PNPF were 0.053 µM and 0.085 unit mL -1 , respectively. The enzyme is a monomer with a molecular mass of 16.5 kDa. Metal ions such as Ni 2+ , Zn 2+ , Co 2+ , and Cu 2+ and oxalic and citric acids enhanced the enzyme activity. The enzyme was completely inhibited by Fe 3+ . Group specific reagents such as p-chloromercuric benzoate and iodoacetamide inhibited the enzyme, indicating the possible presence of cysteine residues in the active site pocket. All chemicals were purchased from Sisco Research Laboratories (Mumbai, India) or Sigma Chemical Co. (St. Louis, MO). Protein molecular mass markers for SDS (sodium dodecyl sulfate) were obtained from Genei (Bangalore, India). DEAE-cellulose, Biogel P-30 was obtained from Pharmacia fine chemicals (Uppsala, Sweden). Precoated silica gel plates (0.25 mm layer thick) with an UV254 indicator were from Merck (Darmstadt, Germany).
Dietary supplementation with prebiotics may result in the stimulation of the growth of beneficial... more Dietary supplementation with prebiotics may result in the stimulation of the growth of beneficial bacteria such as lactobacilli and bifidobacteria in the human gastrointestinal tract. The effect of water-unextractable arabinoxylans (WU-AX) derived from wheat on the modulation of gut bacterial composition was investigated using a mixed culture fermentation system. A prebiotic index (PI) score of 2.03 was obtained after addition of 1% (w/v) WU-AX to a pH-controlled stirred anaerobic fermentation vessel. Pretreatment of the WU-AX with endo-β-1,4-xylanase resulted in significantly higher PI value (3.48) indicating that pretreatment provided oligomers that were better utilised by the gut bacteria. The extracellular hydrolytic enzymes xylanase and ferulic acid esterase are both required for bacterial metabolism of WU-AX and both activities were present in supernatants derived from the mixed batch cultures. Addition of the WU-AX substrates to the batch cultures produced several fold increases of bacterial synthesis of both enzymes, and these increases were greater when the WU-AX substrate was pretreated with xylanase.
With the yearly accumulation of agro-industrial waste-material generated by the milling, brewing ... more With the yearly accumulation of agro-industrial waste-material generated by the milling, brewing and sugar industries in Europe, the importance of extracting high value residues must be considered to offset the cost of treating and disposing of the residues. This work ...
The cDNA encoding Aspergillus niger cinnamoyl esterase (FAEA) with its native signal sequence was... more The cDNA encoding Aspergillus niger cinnamoyl esterase (FAEA) with its native signal sequence was isolated by reverse transcriptasepolymerase chain reaction, sequenced, and expressed in Pichia pastoris. Secretion yields up to 300 mg l 31 were obtained in buffered medium. The recombinant FAEA was purified to homogeneity using a one-step purification protocol and found to be identical to the native enzyme with respect to size, pI, immunoreactivity and N-terminal sequence. Specific activity, pH and temperature optimum, and kinetic parameters were also found similar to the native esterase. FAEA is thus the first fungal esterase efficiently produced using a heterologous system. ß Abbreviations : AOX1, alcohol oxidase; BMGY, bu¡ered minimal glycerol-complex medium; BMMY, bu¡ered minimal methanol-complex medium ; ESI, electrospray ionisation ; faeA, cDNA encoding A. niger cinnamoyl esterase; FAEA, A. niger cinnamoyl esterase; reFAEA, recombinant FAEA; HPLC, high performance liquid chromatography ; MFA, methyl ferulate (3-methoxy-4-hydroxycinnamic acid); MOPS, morpholino-propanesulfonic acid; MS, mass spectrometry ; MSA, methyl sinapate (3,5dimethoxy-4-hydroxycinnamic acid); RT-PCR, reverse transcriptase-polymerase chain reaction ; SDS, sodium dodecyl sulfate; YNB, yeast nitrogen base
Lactase phlorizin hydrolase (LPH; EC 3.2.1.62) is a membrane-bound, family 1 L L-glycosidase foun... more Lactase phlorizin hydrolase (LPH; EC 3.2.1.62) is a membrane-bound, family 1 L L-glycosidase found on the brush border of the mammalian small intestine. LPH, purified from sheep small intestine, was capable of hydrolysing a range of flavonol and isoflavone glycosides. The catalytic efficiency (k cat / K m ) for the hydrolysis of quercetin-4P P-glucoside, quercetin-3glucoside, genistein-7-glucoside and daidzein-7-glucoside was 170, 137, 77 and 14 (mM 31 s 31 ) respectively. The majority of the activity occurred at the lactase and not phlorizin hydrolase site. The ability of LPH to deglycosylate dietary (iso)flavonoid glycosides suggests a possible role for this enzyme in the metabolism of these biologically active compounds.
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