Stereoisomeric flavour compounds LXXVI: direct enantioseparation, structure elucidation and structure-function relationship of 4-tert-butyl-α-methyldihydrocinnamaldehyde

 Using enantioselective gas chromatography and heptakis(2,3-di-O-acetyl-6-O-tert-butyldimethylsilyl)-β-cyclodextrin (DIAC-TBDMS-β-CD) as the chiral stationary phase, the direct enantioseparation of 4-tert-butyl-α-methyldihydrocinnamaldehyde was achieved. The threshold values and odour characteristics of the enantiomers were investigated by enantioselective gas chromatography/olfactometry. In order to elucidate stereochemical features, the carbonyl-function was oxidized to the corresponding acid and diastereomeric amides were generated with (S)-2-amino-2-phenyl-ethanol [L(+)-α-phenylglycinol] as the enantiopure reagent. After separation and isolation by high-performance liquid chromatography, absolute configurations were deduced from X-ray structure elucidation of a pure stereoisomer. Amide cleavage, reduction and selective oxidation yielded the enantiomers of 4-tert-butyl-α-methyldihydrocinnamaldehyde. Received: 7 October 1996 / Revised version: 18 November 1996     

Changes in cell-wall-degrading enzyme activities in stored olives in relation to respiration and ethylene production Influence of exogenous ethylene

 The activity of various enzymes (α-D-galactosidase; β-D-galactosidase; α-L-arabinofuranosidase; α-D-mannosidase, β-D-N-acetylglucosaminidase, cellulase and polygalacturonase) associated with the cell wall during olive storage was assayed in order to establish the behaviour of the enzymes as a function of the ripening stage and in relation to the production of ethylene. The effect of exogenous ethylene (100 mg/l for 24 h) was also evaluated. In addition, gaseous emissions of CO₂ and ethylene during olive storage were monitored. The results obtained indicate that the high initial CO₂ level in the green olive coincides almost exactly in time with the climacteric maxima when the fruit is on the tree. After the rapid decrease in the respiration rate of green olives during storage, the CO₂ production rate increases as the stage of maturity advances. The results also indicate that ethylene is not capable of stimulating the activity or synthesis of enzymes in green olives, but can produce such a stimulation in black olives. Furthermore, during the first day of storage there were very marked decreases in enzyme activities. Small variations in the conditions of aerobiosis in post-harvest ripening were shown to have notable effects on the normal metabolism of the fruits. Received: 29 January 1996 / Revised version: 1 July 1996     

Covalent Crosslinking in Heated Protein Systems

Changes in the solubility (in water or in 1% sodium dodecyl sulfate plus 1%β-mercaptoethanol), isoelectric point, and degree of browning were followed for a range of food proteins when they were heated to 105 to 145°C at 3 relative humidities (RH). In general, there is a decrease in solubility with increasing RH and temperature of heating, but most proteins also showed an increase in solubility on extensive heating. The order for the proteins, based on the temperatures at which an increase in solubility occurred was: gelatin < gluten < soya isolate, sodium caseinate < egg albumin, bovine serum albumin < whey isolate, milk powder, β-lactoglobulin. The bonds that could be formed and broken during this thermal treatment are considered.     

Purification and application of α-galactosidase from germinating coffee beans (<Emphasis Type="Italic">Coffea arabica</Emphasis>)

The objective of this study was to prepare and purify α-galactosidase (α-Gal) from germinating coffee beans. The molecular weight of purified α-Gal from germinating coffee beans was determined to be 38.8 kDa by SDS-PAGE. Then the purified α-Gal was applied to synthesize galactosyl β-cyclodextrin using melibiose as donor and β-cyclodextrin as acceptor. Galactosyl β-cyclodextrin was isolated by preparative high-performance liquid chromatography (HPLC) and its structure was analyzed and elucidated by HPLC, fourier transform infrared spectrometer, electro spraying ionization-mass spectrometry, and ¹³C nuclear magnetic resonance. Our results strongly demonstrate that the synthesized product is a mono-6-O-α-d-galactopyranosyl-β-cyclodextrin and the purified α-galactosidase could transfer one galactosyl residue directly to the β-cyclodextrin ring and synthesize the mono-6-O-α-d-galactosyl β-cyclodextrin.     

Use of different thermal indices to assess the quality of pasteurized milks

 Lactoperoxidase activity and lactulose, furosine and undenatured whey protein contents were determined in Spanish commercial milks labelled as pasteurized (group A) and high-temperature pasteurized (group B), in order to assess their quality. Three samples of group A and all of the samples of group B were lactoperoxidase negative. Most samples of group A had measurable amounts of lactulose, even though their concentrations of undenatured β-lactoglobulin were higher than 2600 mg/l. In general, samples of group B showed higher lactulose and furosine and lower undenatured whey protein contents. High levels of furosine and lactulose accompanied by high levels of undenatured β-lactoglobulin could indicate the addition of milk heated at high temperatures, whereas high levels of furosine and relatively low levels of lactulose may have been due to the presence of reconstituted milk powder. Received: 29 June 1998     

Use of different thermal indices to assess the quality of pasteurized milks

 Lactoperoxidase activity and lactulose, furosine and undenatured whey protein contents were determined in Spanish commercial milks labelled as pasteurized (group A) and high-temperature pasteurized (group B), in order to assess their quality. Three samples of group A and all of the samples of group B were lactoperoxidase negative. Most samples of group A had measurable amounts of lactulose, even though their concentrations of undenatured β-lactoglobulin were higher than 2600 mg/l. In general, samples of group B showed higher lactulose and furosine and lower undenatured whey protein contents. High levels of furosine and lactulose accompanied by high levels of undenatured β-lactoglobulin could indicate the addition of milk heated at high temperatures, whereas high levels of furosine and relatively low levels of lactulose may have been due to the presence of reconstituted milk powder.     

The antigenic response of<Emphasis Type="Italic"> β</Emphasis>-lactoglobulin is modulated by thermally induced aggregation

The antigenic response of thermally denatured and aggregated -lactoglobulin was determined by an indirect competitive enzyme-linked immunosorbent assay using polyclonal antibodies from the egg yolk of chicken immunized with heat-denatured -lactoglobulin as a measure for the potential antigenicity/allergenicity. The heat denaturation and the aggregation of heated whey protein isolate solutions were followed by reversed-phase high-performance liquid chromatography and photon correlation spectroscopy. Thermally modified whey proteins showed a remarkable increase of antigenicity when heated to 90 °C, possibly as a consequence of the exposure or formerly hidden epitopes. Above 90 °C, the antigenic response decreased owing to the loss of conformational epitopes and masking of sequential epitopes in the course of aggregation to particles. When large and compact particles were formed, the antigenicity was reduced remarkably. Depending the heating conditions applied, the structure and the size of whey protein particles and thus the potential allergenicity may be modulated in a wide range.     

Detection of Maillard products of lactose in heated or processed milk by HPLC/DAD

 Two HPLC methods with diode array detection were used to separate Maillard products from milk components. The Maillard products 4-(β-d-galactopyranosyloxy)-2-hydroxy-2-methyl-2H-pyran-3(6H)-one (2), 1-[3-(β-d-galactopyranosyloxy)-2-furanyl]-1-ethanone (4), 4-(β-d-galactopyranosyloxy)-5-(hydroxymethyl)-2-methyl-3(2H)-furanone (5), isomaltol (7), maltol (9), 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (6), and 5-hydroxymethyl-2-furfuraldehyde (8) were determined using a water-methanol gradient. Furthermore, 4,5-dihydroxy-2-(β-d-galactopyranosyloxy)-5-methyl-2-cyclopenten-1-one (3) was separated from the milk components by applying the interaction reagent octylamine. Several heated or processed milk samples were analyzed, and formation of the Maillard products was determined and quantified. Thus it was found that 2 and 3 are early products of the Maillard reaction in milk, whereas after prolonged heating 4, 5, and particularly 9 and 6 become more important. Compounds 7 and 8 were not detected, even if the samples were heated under stringent conditions. Received: 25 June 1998     

Detection of Maillard products of lactose in heated or processed milk by HPLC/DAD

 Two HPLC methods with diode array detection were used to separate Maillard products from milk components. The Maillard products 4-(β-d-galactopyranosyloxy)-2-hydroxy-2-methyl-2H-pyran-3(6H)-one (2), 1-[3-(β-d-galactopyranosyloxy)-2-furanyl]-1-ethanone (4), 4-(β-d-galactopyranosyloxy)-5-(hydroxymethyl)-2-methyl-3(2H)-furanone (5), isomaltol (7), maltol (9), 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (6), and 5-hydroxymethyl-2-furfuraldehyde (8) were determined using a water-methanol gradient. Furthermore, 4,5-dihydroxy-2-(β-d-galactopyranosyloxy)-5-methyl-2-cyclopenten-1-one (3) was separated from the milk components by applying the interaction reagent octylamine. Several heated or processed milk samples were analyzed, and formation of the Maillard products was determined and quantified. Thus it was found that 2 and 3 are early products of the Maillard reaction in milk, whereas after prolonged heating 4, 5, and particularly 9 and 6 become more important. Compounds 7 and 8 were not detected, even if the samples were heated under stringent conditions.     

Effect of high-pressure treatment on denaturation of bovine β-lactoglobulin and α-lactalbumin

The effect of high-pressure treatment on denaturation of β-lactoglobulin and α-lactalbumin in skimmed milk, whey, and phosphate buffer was studied over a pressure range of 450–700 MPa at 20 °C. The degree of protein denaturation was measured by the loss of reactivity with their specific antibodies using radial immunodiffusion. The denaturation of β-lactoglobulin increased with the increase of pressure and holding time. Denaturation rate constants of β-lactoglobulin were higher when the protein was treated in skimmed milk than in whey, and in both media higher than in buffer, indicating that the stability of the protein depends on the treatment media. α-Lactalbumin is much more baroresistant than β-lactoglobulin as a low level of denaturation was obtained at all treatments assayed. Denaturation of β-lactoglobulin in the three media was found to follow a reaction order of n = 1.5. A linear relationship was obtained between the logarithm of the rate constants and pressure over the pressure range studied. Activation volumes obtained for the protein treated in milk, whey, and buffer were −17.7 ± 0.5, −24.8 ± 0.4, and −18.9 ± 0.8 mL/mol, respectively, which indicate that under pressure, reactions of volume decrease of β-lactoglobulin are favoured. Kinetic parameters obtained in this work allow calculating the pressure-induced denaturation of β-lactoglobulin on the basis of pressure and holding times applied.     

Gas chromatographic evaluation of amino acid epimerisation in the course of gelatin manufacturing and processing

 Using capillary gas chromatography on chiral stationary phases total hydrolysates of gelatins were investigated for their amounts of D-amino acids (D-AAs). Gelatins of different quality and origin were analysed including gelatin-containing final products like candies and meat products. In gelatins manufactured by stepwise extraction (first to fifth extract) with water of increasing temperature (ca. 55–95°C) amounts of 2.7–4.8% of D-aspartic acid (D-Asp) were determined in extracts obtained at ≤ 85°C. The amounts D-Asp were as high as 28.1% in fifth extracts obtained at 95°C (data corrected for 4.3% of D-Asp determined in native collagen as a result of acid-induced epimerisation of L-Asp during total hydrolysis). Epimerisation of aspartyl or asparaginyl residues of proteins in general on heating in water is assumed to proceed via formation of aspartyl succinimide, tautomerisation and release of D-Asp on total hydrolysis. The other AA residues of gelatin showed only a slight increase of epimerisation with increasing thermal treatment. Furthermore, amounts of 3–4% of cis-4-D-hydroxyproline, formed from native trans-4-L-hydroxyproline in collagen on hydrolysis, were detected in total hydrolysates of all gelatins. Received: 8 August 1997 / Revised version: 24 November 1997     

Phenolic composition of rhubarb

Extracts of different parts (leaves, petioles and rhizomes) of domestic garden rhubarb (hybrids of Rheum rhabarbarum L. and Rheum rhaponticum L.) were investigated for their content of phenolic ingredients. Two stilbenes (trans-rhapontigenin, trans-desoxyrhapontigenin), five stilbene glycosides (trans-rhaponticin, cis- and trans-desoxyrhaponticin, trans-resveratrol-4′-O-β-d-glucopyranoside, trans-piceatannol-3′-O-β-d-glucopyranoside) and seven flavonoids [rutin, quercetin-3-O-glucuronide, isovitexin, 6,8-di-C-β-d-glucosylapigenin, 6-C-β-d-glucosyl-8-C-β-d-arabinosylapigenin (schaftoside), 6-C-β-d-arabinosyl-8-C-β-d-glycosylapigenin (isoschaftoside), (+)-catechin] were unequivocally established. Separation was done in two steps. Multilayer countercurrent chromatography was applied to separate different extracts of plant material. Preparative HPLC was then used to obtain pure substances. The purity and identity of isolated compounds was confirmed by different NMR experiments, HR-MS and HPLC-DAD analysis.     

Proteins recovered from milks heated at alkaline pH values

Approximately 95% of available nitrogen can be precipitated from milk on adjustment to pH 4.6 after heating at 90°C × 15 minutes at its natural pH and pH 7.5, while 89% can be precipitated after heating at pH 10.0 at 60°C × 3 minutes. Non-recovered protein includes some serum albumin, β-lactoglobulin, α-lactalbumin and proteose peptones. Protein isolates precipitated from milk heated at pH >7.0 are more soluble in the pH range 6.0–7.0 than those precipitated from milk heated at its natural pH. Whey proteins complex onto the casein micelles after heating milk at its natural pH, while on heating at pH >7.0 whey proteins appear to interact with k-casein in the serum phase. When N-ethylmaleimide is present in milk during heating the percentage protein recovered on pH 4.6 precipitation is decreased, confirming that disulphide linkage is involved in complex formation. However, addition of β-mercaptoethanol to recovered isolates did not result in dissociation of the casein/whey protein complex, suggesting that forces other than disulphide bonding are also involved in maintaining the complex.     

Synthesis of oligosaccharides with lactose and N-acetylglucosamine as substrates by using β-d-galactosidase from Bacillus circulans

In the present study, β-d-galactosidase from Bacillus circulans was proved to be a suitable biocatalyst for the production of N-acetyl-oligosaccharides with lactose and N-acetylglucosamine (GlcNAc) as biocatalyst. During the hydrolysis of lactose, apart from no ultraviolet absorption oligosaccharides such as β-d-Galp-(1 → 6)-d-Glcp (6′-allolactose) and β-d-Galp-(1 → 4)-β-d-Galp-(1 → 4)-d-Glcp (4′-galactosyl-lactose), the formation of four N-acetyl-oligosaccharides was followed by high-performance liquid chromatography with a diode-array detector. The four N-acetyl-oligosaccharides were isolated from the reaction mixture and identified to be as β-d-Galp-(1 → 4)-d-GlcpNAc (LacNAc, I), β-d-Galp-(1 → 6)-d-GlcpNAc (allo-LacNAc, II), β-d-Galp-(1 → 4)-β-d-Galp-(1 → 4)-d-GlcpNAc (III), β-d-Galp-(1 → 4)-β-d-Galp-(1 → 4)-β-d-Galp-(1 → 4)-d-GlcpNAc (IV) by authentic standards and the spike technique or high-resolution mass spectrometry with an electrospray ionization source and nuclear magnetic resonance spectroscopy. Furthermore, the effects of synthetic conditions including reaction temperature, concentration of substrate, molar ratio of donor/acceptor and enzyme concentration on the formation of N-acetyl-oligosaccharides were examined. We found that the optimal synthetic conditions were different for production of oligosaccharides with β-(1 → 4) linkages and β-(1 → 6) linkage. The optimal reaction conditions for I, III and IV were 40 °C, 0.50 M lactose and 0.50 M GlcNAc and 1.0 U/mL of enzyme. Under such conditions, the N-acetyl-oligosaccharides formed were composed of 28.75% of I, 2.29% of II, 9.47% of III and 5.67% of IV. On the other hand, suitable reaction conditions found for II were 40 °C, 0.50 M lactose and 0.50 M GlcNAc and 2.0 U/mL of enzyme.     

Modulation of the thermal stability of β-lactoglobulin by transglutaminase treatment

The influence of microbial transglutaminase (MTGase) treatment in the presence and absence of dithiothreitol (DTT) on the thermal stability of β-lactoglobulin (β-LG) was investigated using size-exclusion chromatography (SEC) combined with on-line multiangle laser light scattering (MALLS) and UV detection. SEC–MALLS–UV analyses showed that moderate MTGase treatment (up to 6 or 9 h) led to the decline in thermal stability of β-LG, while excess treatment (e.g. 23 h) resulted in remarkable increase, irrespective of whether the reducing agent DTT was present or not. The modulation in thermal stability may be attributed to the partial unfolding of protein molecules and the subsequent re-arrangement of conformation.     

Apparent chemical composition of nine commercial or semi-commercial whey protein concentrates, isolates and fractions

Summary Analytical results are given for whey powders prepared on a commercial or semi-commercial scale by three companies. Altogether, five preparations enriched in β-lactoglobulin, four whey protein isolates and a fraction enriched in α-lactalbumin were analyzed for protein composition, including %β-lactoglobulin, α-lactalbumin, bovine serum albumin, casein (glyco) macropeptide and the main triglycerides. Protein composition was determined by high pressure gel permeation and reversed phase liquid chromatography and by capillary zone electrophoresis. The extent of modification of the native β-lactoglobulin structure was also measured through the degree of lactosylation and the fraction of accessible free sulphydryl groups. One significant finding was that the calculated recovery of protein following quantitation of the chromatogram or electropherogram was seldom above 90% and occasionally below 60% of that loaded onto the column or capillary, raising doubts as to the reliability of the analytical results. Extrapolation by linear regression to 100% recovery allowed estimates to be made of the true β-lactoglobulin composition of the samples. The nine samples could be placed into three distinct groups with estimated true β-lactoglobulin weight % of 70.9 ± 1.1, 62.0 ± 3.4 and 39.5 ± 4.9. Physico-chemical properties of the group of samples are reported elsewhere (Holt et al ., 1999).     

Microwave irradiation under different pH conditions induced a decrease in β-lactoglobulin antigenicity

Whey proteins adjusted at pH values 2, 4.6, 9 and at the natural milk pH (pH 6.8) were subjected to microwave irradiation at 300 W for 20 min or 700 W for 10 min. The protein composition of treated and native whey proteins were evaluated by Lowry’s method and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The ability of treated whey to bind IgG polyclonal antibody was determined by an enzyme-linked immunosorbent assay (ELISA) using sera obtained from rabbit immunized to β-Lactoglobulin (β-Lg). Significantly higher losses in soluble protein concentrations were observed for microwave irradiated whey proteins at pH value 4.6 (35.6% at 300 W and 44.33% at 700 W) (P < 0.0001) compared with those irradiated at the natural milk pH (26% at 300 or 700 W). The electrophoretic patterns of these proteins revealed a considerable decrease in intensity of the band corresponding to α-lactalbumin but only a slight modification was observed for the electrophoretic profiles of β-lactoglobulin. The data obtained with a rabbit anti-β-lactoglobulin immunoglobulin indicated a low antigenic response for microwave-irradiated whey proteins at the natural milk pH (up to 29.32% as well as 300–700 W) (P < 0.001). The lowest antigenicity was observed for samples adjusted to pH 4.6 followed by microwave irradiation at 300 or 700 W (46.99% at 300 W and 41.16% at 700 W) (P < 0.0001).     

Proteins recovered from acid whey/skim milk mixtures heated at alkaline pH values

Skim milk and mixtures prepared by combining acid whey with skim milk at volume ratios of 2:1, 1:1, 1:2, 1:3 and 1:4 were adjusted to pH 7.5 and heated at 90°C × 15 min. Protein was isolated from these heated samples by precipitation at pH 4.6 and it was found that 65% of the whey protein was recovered in each case. Non-recovered proteins included the proteose peptones and small quantities of β-lactoglobulin, α-lactalbumin and bovine serum albumin. The solubility of these isolates, which contained from 10–25% whey protein, decreased to > 95% when the whey protein exceeded ˜16%. Further characterization of the isolate, prepared from the 1:1 volume ratio of acid whey and skim milk, showed that ˜50% of the whey protein was insoluble, bound to casein and non-functional while the other ˜50% was complexed with casein and was soluble. The addition of a reducing agent suggests that sulphydryl bonding alone is not responsible for complex formation.     

Protein-backbone-modifications: Formation of imidazolines

The formation of imidazolines within the backbone during heating of peptides and proteins was studied. In model experiments, the generation of 2-methyl-4-carboxy-imidazoline after incubation of N ^α -acetyl-β-aminoalanine was demonstrated by cation-exchange chromatography and NMR-studies. At 60 °C the highest concentrations of this imidazoline were measured at pH 9. With increasing temperature the pH-optimum of the imidazoline formation shifted to lower pH levels. The reaction of N ^α -acetyl-dehydroalanine-methylester with ammonia generated β-aminoalaninoalanine, which was identified in comparison with synthesized references by HMQC-and¹H-NMR-spectroscopy. β-Aminoalaninoalanine, a crosslink-product homologous to lysinoalanine, which reacts easily with the corresponding imidazoline. Thus, the formation of imidazolines in peptide backbones containing β-aminoalanine was shown unequivocally. Further studies were conducted with peptides, synthesized according to the 10–28 sequence of bovine β-caseine with β-aminoalanine respectively β-alanine substituted for a phosphoserine residue. After incubation, the loss of one molecule of water from the β-aminoalanine containing peptide, but not from the native peptide or the peptide containing β-alanine, was detected by HPLC and MS. As a consequence, the loss of water was explained by cyclization and the formation of an imidazoline. These investigations indicate that the formation of imidazolines—besides the formation of thiazolines—takes place during heating of peptides and proteins. Heterocyclic backbone-modifications are a form of posttranslational modifications, which may play a role during the processing of proteins and subsequent formation of unexpected compounds.     

Stability of naturally occurring 2,5-dimethyl-4-hydroxy-3 [2H]-furanone derivatives

The stability, in aqueous buffer solutions at different pH values (pH 2.0–8.0, interval: 1.5 pH units), of 2,5-dimethyl-4-hydroxy-3[2H]-furanone (Furaneol, DMHF, 1), its methoxy derivative 2,5-dimethyl-4methoxy-3[2H]-furanone (methoxyfuraneol, mesifurane, DMMF, 2 and the glycosidically bound forms DMHF β-D-glucopyranoside 3 and DMHF 6′-O-malonyl-β-Dglucopyranoside 4 was investigated over a period of 32 days at 23 °C. Only slight decomposition of 2 and 3 was observed, whereas 1 and 4 were found to be unstable at all pH values. In addition, 3 and 4 were subjected to enzymatic hydrolysis. In contrast to the rapid hydrolysis of 3, the malonylated glycoside, 4, remained unaffected by enzymatic treatment with β-glucosidase (Emulsin). Using a pectinolytic enzyme preparation (Rohapect D5L; R?hm, Darmstadt, Germany) with esterase activities, hydrolysis of 4 was achieved. Received: 25 September 1996