The effect of pH on the formation of aroma compounds produced by heating a model system containing l-ascorbic acid with l-threonine/l-serine

The identification of aroma compounds, formed from the reactions of l-ascorbic acid with l-threonine/l-serine at five different pH values (5.00, 6.00, 7.00, 8.00, or 9.55) and 143 ± 2 °C for 2 h, was performed using a SPME–GC–MS technique, and further use of LRI. The results showed 35 aroma compounds. The reaction between l-ascorbic acid and l-threonine/l-serine led mainly to the formation of pyrazines. Many of these were alkylpyrazines, such as 2-methylpyrazine, 2,5-dimethylpyrazine, 2-ethylpyrazine, 2-ethyl-6-methylpyrazine, 2-ethyl-5-methylpyrazine, 3-ethyl-2,5-dimethylpyrazine, 2,3-diethyl-5-methylpyrazine, and 3,5-diethyl-2-methylpyrazine; other compounds identified were furans and aldehydes. More volatiles were generated in l-ascorbic acid with l-threonine systems than in l-ascorbic acid with l-serine systems. The studies showed that furans, such as furfural, 2-furanmethanol, benzofuran, 2,5-furandicarboxaldehyde and 2-furfurylfuran were formed mainly at acidic pH. In contrast, higher pH values could promote the production of pyrazines.     

Structural characterization of barley β-glucan extracted using a novel fractionation technique

A barley β-glucan concentrate prepared according to a novel technology was further purified and subjected to detailed structural characterization by NMR spectroscopy. β-Glucan was hydrolysed with β-glucan-4-glucanohydrolase (lichenase). Fractions of hydrolysate were collected using an HPLC-fraction collector. Intact β-glucan and the major fractions collected were subjected to MALDI-TOF–MS and NMR analyses. The two major oligosaccharides produced by lichenase hydrolysis of purified barley β-glucan were identified as β-d-Glc p-(1 → 4)- β-d-Glc p-(1 → 3)-β-d-Glc p and β-d-Glc p-(1 → 4)-β-d-Glc p-(1 → 4)-β-d-Glc p-(1 → 3)-β-d-Glc p based on ¹³C and ¹H NMR data. Spectrums were similar to those documented for barley β-glucan in the literature.     

Tumorigenicity study of l-theanine administrated orally to mice

l-Theanine was orally administered at a concentration of 0–5% (maximum tolerated dose) of the diet to B6C3F₁ mice for 13 weeks in a subacute test and 78 consecutive weeks, in a chronic toxicity test. There were no significant differences in intake of diet, weight gains or survival rates between treated and control groups. Unignorable differences in tumor incidence were observed at the end of 78 weeks; however, total number of tumors decreased significantly in the l-theanine administered groups. Thus, the long-term oral administration of l-theanine to mice did not show any chronic toxicological evidence or tumorigenic aberration.     

Inhibition of microbiological activity during sole (Solea solea L.) chilled storage by applying ellagic and ascorbic acids

Fresh fish products are commonly sold at storage temperature. However, many undesirable changes can occur in the products during storage, due to microbial growth and fish spoilage, affording evident economic loss. The effects of ellagic acid (EA) and l-ascorbic acid (l-AA)/sodium ascorbate (SA), either alone or in combination, on the microbiological quality of fresh finfish Solea solea during storage at 0 °C, were investigated.The results showed that addition of EA (0.03%) alone or in combination with l-AA (1.71%)/SA (1.98%) significantly delayed the proliferation of aerobic plate counts, psychrotrophic counts and Pseudomonas bacteria extending the product shelf life up to 10 days, versus 8 days for control. During the storage, the pH increase is slowed down when the fish samples are subjected to the treatment with preservative agents. The effect of lowering the pH increase is particularly pronounced when a combination of EA and l-AA/SA is used. Therefore, EA alone or in combination with l-AA and SA can be successfully utilized to reduce the microbial growth, extending the shelf life of fish during storage at 0 °C.     

Key enzymes involved in methionine catabolism by cheese lactic acid bacteria

Cheese microbiota and their enzymatic conversion of l-methionine to volatile sulphur compounds (VSCs) play an important role in aroma formation during cheese ripening. Here, lactic acid bacteria (LAB) strains isolated from raw goats' milk cheeses were screened for the major enzymes critical to the formation of VSCs from l-methionine. A large natural biodiversity in enzyme capabilities and high inter- and intra-species variability was found among the LAB isolates investigated. From those isolates tested, lactococci displayed higher C–S lyase specificities towards the sulphur-containing compounds examined than did Lactobacillus and Leuconostoc, in some cases generating higher levels of VSCs than B. linens, known to be an efficient producer of methanethiol (MTL) and related VSCs. Moreover, these differences in C–S lyase activities (determined spectrophotometrically by measuring the formation of free thiol groups) were shown to correspond with the enzymatic potential of the isolates as determined by visualization of enzymatic activities. This technique could therefore prove valuable for the detection and preliminary characterization of C–S lyase activities among LAB isolates. Lactococci were also found to possess higher aminotransferase activities than lactobacilli and leuconostocs, while glutamate dehydrogenase activities were observed to be highest among Leuconostoc and Lactobacillus spp. Meanwhile, α-keto acid decarboxylase activities were highly variable and were measurable in only a limited number of isolates, mainly lactobacilli. From these data, combining indigenous isolates showing high VSCs-producing capabilities with those that facilitate the completion of the metabolic pathway responsible for degrading l-methionine into volatile compounds may provide an efficient approach to enhance cheese aroma development.     

Accumulation of non-proteolytic aminoacyl derivatives in Parmigiano-Reggiano cheese during ripening

For the first time, the identification of N-γ-glutamyl-, N-pyroglutamyl- and N-lactoyl-amino acids in Parmigiano-Reggiano cheese is reported. Their structures were assigned by electrospray mass spectrometric analysis and demonstrated for several compounds by the synthesis of authentic specimens and sample spiking. The comparison with the synthesised compounds also allowed the determination of their absolute stereochemistry (l,l), which indicated a common enzymatic origin. All these molecules were shown to accumulate in Parmigiano-Reggiano during ripening, probably because of a resistance to proteolysis due to their unusual structures. Thus, these compounds can be used as markers of ripening time and may be involved in the development of the pleasant flavour of aged cheeses. Their presence in other cheeses was also demonstrated.     

Fractionation and identification of ACE-inhibitory peptides from α-lactalbumin and β-casein produced by thermolysin-catalysed hydrolysis

The thermolysin catalysed hydrolysates of α-lactalbumin and β-casein were fractionated by size-exclusion chromatography (SEC) and reversed-phase high performance liquid chromatography (RP-HPLC) in order to identify the peptides responsible for the high ACE-inhibitory activity of these hydrolysates. The SEC fractionation separated many co-eluting peptides into different fractions allowing individual peptides to be isolated in one or two subsequent semi-preparative RP-HPLC fractionation steps. Five potent ACE-inhibitory peptides from α-lactalbumin were isolated. They all contained the C-terminal sequence -PEW, corresponding to amino acid residues 24–26 in α-lactalbumin, and had IC₅₀ values of 1–5 μm. From one SEC fraction of the β-casein hydrolysate two potent ACE-inhibitory peptides were isolated and identified as f58-76 and f59-76 of β-casein A2. They both contained IPP as the C-terminal sequence and had IC₅₀ values of 4 and 5 μm. From another SEC fraction a new but less ACE-inhibitory peptide from β-casein was identified (f192–196; LYQQP).     

Influence of various factors on formation of 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one (DDMP) in a solid-state model system of Maillard reaction

The influences of various factors on the formation of 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one (DDMP) were investigated in a solid-state Maillard reaction system. Thermogravimetric analysis (TG) and differential scanning calorimetry analysis (DSC) showed that the solid-state Maillard reaction mainly occurred at around 150 °C. The DDMP formation from reducing sugars (glucose or fructose) and several selected amino acids (l-proline, l-alanine, l-asparagine, l-threonine, l-tyrosine and l-lysine) was compared. Proline was found to have special activity in DDMP formation when reacted with glucose because of its special catalytic action. The influences of reactant ratios, oxygen, reaction temperature and reaction time on DDMP formation from the reaction of proline with glucose were studied. Reactant molar ratios of glucose to proline played important impact on DDMP formation, and the formation of DDMP was improved when the ratio was higher than 1:1 and achieved the highest level at 2:1. Oxygen had no obvious effects on DDMP formation. The effects of reaction time and temperature were investigated together and revealed the information on the highest yield and minimum temperature for DDMP formation. Furthermore, the kinetics of DDMP formation was studied, and DDMP formation in the initial stage was shown to follow an apparent first-order reaction with an activation energy of 68.8 kJ/mol.     

Effect of addition of selected solid cosolutes on viscoelastic properties of model processed cheese containing pectin

The aim of this study was to investigate the effect of various selected solid low-molecular cosolutes (d-glucose, d-fructose, d-galactose, lactose, saccharose; target concentrations in samples 1% w/w) on viscoelastic properties of model processed cheese (40% w/w dry matter and 50% w/w fat in dry matter) without and with pectin addition (target concentrations in final products: 0.2% w/w and 0.4% w/w, respectively). Dynamic oscillation rheometry with parallel plate geometry was used to evaluate the changes in viscoelastic properties of samples. All applied low-molecular cosolutes (1% w/w) decreased the firmness of processed cheese. The nature of cosolute was not significant, i.e. the firmness of final products was declined by various cosolutes to the similar level regardless of their (i) reducing or non-reducing ability; (ii) structure (mono- or disaccharide, aldose or ketose); or (iii) steric arrangement (comparison of two epimers – d-glucose and d-galactose). The reduction of sample rigidity due to cosolute addition was observed in both cases, i.e. in processed cheese without and also with pectin.     

Decomposition and discoloration of L-ascorbic acid freeze-dried with saccharides

L-Ascorbic acid (vitamin C) was freeze-dried along with the following six types of wall materials: soluble soybean polysaccharide (SSPS), gum arabic (GA), maltodextrin (MD), α-cyclodextrin (α-CD), trehalose (Tre) and lactose (Lac). α-CD, Tre and Lac were used in mixtures with MD (weight ratio of 1:1). The decomposition kinetics of L-ascorbic acid in these powders stored at 80 °C and relative humidities of 11, 51 and 79% were expressed by the Weibull equation. The discoloration of the powders was also monitored by measuring the absorbance at 500 nm for solutions which were obtained by dissolving the systems in water. The L-ascorbic acid powders prepared with GA and SSPS were significantly discolored and the processes could be expressed by a modified Weibull equation. The discoloration of the L-ascorbic acid powders prepared with other saccharides was very slow. The discoloration rate constants were significantly greater at temperatures higher than the glass transition temperatures of the saccharides under the stated storage conditions, while the decomposition rate constant was only slightly affected by the system glass transition temperature.

Industrial relevance

Decomposition and discoloration are examples of food deterioration. Food usually represents a multi-component system in which components interact with each other. Both their state and interaction would affect the deterioration. L-Ascorbic acid is one of the food components which are subject to decomposition and discoloration during storage. The decomposition and discoloration of L-ascorbic acid powders freeze-dried with saccharides were measured under various conditions and kinetically analyzed to understand an aspect of food deterioration.     

Novel casein-derived peptides with antihypertensive activity

In this study, we report novel casein-derived peptide sequences with angiotensin converting enzyme (ACE)-inhibitory activity and antihypertensive activity demonstrated in spontaneously hypertensive rats (SHR). The peptides were obtained by enzymatic hydrolysis of total isoelectric casein with pepsin. To identify ACE-inhibitory peptides, the casein hydrolysate was fractionated by semi-preparative high performance liquid chromatography, and 44 (CN) peptides contained in the active fractions were sequenced by using an ion trap mass spectrometer. Among the identified peptides, three sequences, that corresponded to α_s1-CN f(90–94) (RYLGY), α_s1-CN f(143–149) (AYFYPEL), and α_S2-CN f(89–95) (YQKFPQY), showed IC₅₀ values as low as 0.71 μm, 6.58 μm, and 20.08 μm, respectively. These three peptides also exerted antihypertensive activity when they were orally administered to SHR at a dose of 5 mg kg⁻¹ of body weight. The activity of peptides RYLGY and AYFYPEL in SHR was similar to that found for tripeptide VPP when orally administered at the same dose.     

Determination of <Emphasis Type="SmallCaps">d</Emphasis>,<Emphasis Type="SmallCaps">l</Emphasis>-Amino Acids in Collagen from Pig and Cod Skins by UPLC Using Pre-column Fluorescent Derivatization

A simple and sensitive analytical method for the quantification of d,l-amino acids by ultra-performance liquid chromatography (UPLC) with fluorescence (FL) detection is described. The reaction of the R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R(-)-DBD-PyNCS] with d,l-hydroxyproline (Hyp), glycine (Gly), d,l-aspartic acid (Asp), and d,l-proline (Pro) effectively proceeds at 55 °C for 20 min in the presence of 3% TEA to produce the corresponding fluorescent diastereomers (excitation at 460 nm, emission at 550 nm). The mixture was simultaneously separated within 20 min on an ACQUITY UPLCTM BEH C18 (1.7 μm, 100 mm?×?2.1 mm i.d.) by gradient elutions using water–acetonitrile containing 0.2% formic acid as the mobile phase. Peak resolution was in the range of 1.62 (d,l-Asp), 2.99 (d,l-Pro), and 6.74 (d,l-Hyp). A good linearity was achieved from the calibration curves (r²?>?0.9984) in the range of 1.0~100 pmol; the limit of detection (S/N?=?3) was 42.0–250 fmol, the inter-day and intra-day assay precisions were all less than 6.23%, and the mean recoveries (%) of the d,l-amino acids spiked in the collagen from pig and dried cod skins were 87.58–107.41%. The derivatives of the free d,l-amino acids in the collagen were successfully identified by the proposed procedure. To the best of our knowledge, for the first time, these three kinds of d-amino acids, which were d-Asp, d-Pro, and d-Hyp, were detected in the collagen samples.     

Microwave treatment of dietary gelatin does not generate cis-4-hydroxy-l-proline, an inhibitor of collagen biosynthesis

Aqueous solutions (5?g/100?ml) of commercial preparations of (a) an enzymatic partial hydrolysate of gelatin and (b) type A gelatin were subjected to threefold heating to boiling in a domestic microwave oven at 750?W and to conventional heating. Then samples were totally hydrolyzed (6?M hydrochloric acid, 110??°C, 24?h) and investigated for the presence of eight possible stereoisomers of 3- and 4-hydroxyproline (Hyp) using capillary gas chromatography. Amino acids were analyzed as N(O)-trifluoroacetyl 2-propyl esters on Chirasil-l-Val and detected by selected ion monitoring mass spectrometry. Blanks of (a) and (b) were analyzed in parallel. Relative amounts of 5.0±0.2% cis-4-d-Hyp were generated from native trans-4-l-Hyp as a result of total hydrolysis in all samples and independent of previous treatment. Notably, neither cis-3-l-Hyp nor cis-4-l-Hyp could be detected in either of the gelatin samples. Thus a report on the generation of antifibrotic and therefore potentially hazardous cis-3-l-Hyp and cis-4-l-Hyp from protein-bonded native trans-3-l-Hyp and trans-4-l-Hyp on microwave heating of infant formulae could not be confirmed.     

Structural features of a xylogalactan isolated from Hymenaea courbaril gum

The gum from Hymenaea courbaril (Caesalpiniaceae) produced gum at seed level. The gum is soluble in water, dextrorotatory and less viscous than the gum from Cyamopsis tetragonolobus (guar gum). The polysaccharide, isolated from this gum, contains galactose, glucose, xylose and arabinose. The preparation of degraded products by acid hydrolysis and Smith-degradation process led to obtain degraded gums A and B, and the polysaccharides I and II. Chemical methods in combination with 1D NMR (¹H, ¹³C, DEPT-135) and 2D NMR (COSY, HMQC and HMBC) were applied. The backbone is a xylogalactan; β-d-galactose and β-d-xylose residues are 4-O- and 2-O-linked, respectively. The branches are constituted by xylose, arabinose and galactose. It was also observed 4,6-di-O-substituted galactose residues. This work shows interesting structural features of the polysaccharide isolated from H. courbaril gum.     

Reduction of Acrylamide Formation in Sweet Bread with l-Asparaginase Treatment

Acrylamide, 2-propenamide, has the chemical formula $ \mathrm{CH}2=\mathrm{CH}-\mathrm{CO}-\mathrm{NH}2 $ . It is produced at elevated levels in high temperature fried and baked foods. It has adverse effects on human health and is proven to be neurotoxic, genotoxic, carcinogenic, and toxic to reproductive system. The aim of this paper was to reduce acrylamide formation in bakery products such as sweet bread by enzyme treatment. l-Asparaginase produced from Cladosporium sp. was treated to wheat-based dough at different concentrations (50–300 U). There was no change in the rheological properties of wheat flour and physico-sensory characteristics of bread with l-asparaginase treatment. Moisture, sugars, l-asparagine, acrylamide, and some indicators of Millard reaction (hydroxymethylfurfural (HMF), color, browning) were estimated. With increase in l-asparaginase level the acrylamide formation was reduced. At 300 U, there was 97 % and 73 % reduction of acrylamide formation in the crust and crumb regions of bread, respectively. HMF, a common intermediate product in the Maillard reaction and a genotoxic compound via 5-sulfoxymethylfurfural, also decreased in l-asparaginase-treated bread samples. These results indicated the potential of l-asparaginase enzyme for industrial and domestic applications in reducing harmful Maillard reaction compounds.     

Tyrosinase inhibitory effects and antioxidative activities of novel cinnamoyl amides with amino acid ester moiety

Nine cinnamoyl amides with amino acid ester (CAAE) moiety were synthesized by the conjugation of the corresponding cinnamic acids (cinnamic acid, 4-hydroxy cinnamic acid, ferulic acid and caffeic acid) with amino acid esters, and their inhibitory effects on the activities of mushroom tyrosinase were investigated, using l-3,4-dihydroxyl-phenylalanine (l-DOPA) as the substrate. Among these CAAE amides, ethyl N-[3-(4-hydroxy-3-methoxyphenyl)-1-oxo-2-propen-1-yl]-l-phenylalaninate (b₄) showed the strongest inhibitory activity; the IC₅₀ was 0.18 μM. The IC₅₀ values, inhibition types, inhibition mechanisms and kinetics of all these CAAE amides were evaluated. A structure–activity relationship (SAR) study found that the inhibitory effects were potentiated with the increasing length of hydrocarbon chains at the amino acid esters and also influenced by the substituents at the styrene groups. Furthermore, the hydroxyl radical scavenging and anti-lipid peroxidation activities of four CAAE derivatives were also investigated. Among these compounds, b₃ (ethyl N-[3-(3,4-dihydroxyphenyl)-1-oxo-2-propen-1-yl]-l-phenylalaninate) and b₄ exhibited potential antioxidant activities.     

Structural and mechanical characterization of κ/ι-hybrid carrageenan gels in potassium salt using Fourier Transform rheology

The mechanical and structural properties of κ/ι-hybrid carrageenan gels obtained at various concentrations in the presence of 0.1 m KCl were studied with Fourier Transform rheology (FTR) and cryoSEM imaging. FTR data show that gels formed at concentration below 1.25 wt% exhibit a strain hardening behavior. The strain hardening is characterized by a quadratic increase of the scaled third harmonic with the strain and a third harmonic phase angle of zero degree. Both features are weakly depending on the concentration and conform to predictions from a strain hardening model devised for fractal colloidal gels. However, the phase angle of the third harmonic reveals that κ/ι-hybrid carrageenan gels obtained at higher concentrations show shear thinning behavior. Colloidal gel models used to extract structural information from the concentration scaling of gel equilibrium shear modulus G₀ and the strain dependence of FTR parameters suggest that κ/ι-hybrid carrageenan gels are built from aggregating rod-like strands (with fractal dimension x = 1.13) which essentially stretch under increasing strain. The mechanically relevant structural parameters fairly match the gel fractal dimension (d = 1.66) obtained from the cryoSEM analysis.     

Isolation and characterization of a multienzyme complex (cellulosome) of the Paenibacillus curdlanolyticus B-6 grown on Avicel under aerobic conditions

A multienzyme complex, cellulosome, of the facultatively anaerobic bacterium, Paenibacillus curdlanolyticus B-6 was produced on microcrystalline cellulose (Avicel) under aerobic conditions. During growth on Avicel, the bacterial cells were found to be capable of adhesion to Avicel by scanning electron microscopic (SEM) analysis. The multienzyme complex of P. curdlanolyticus B-6 was isolated from the crude enzyme preparation by gel filtration chromatography on Sephacryl S-300 and affinity purification on cellulose. The isolated multienzyme complex was able to bind to both Avicel and insoluble xylan and consists of cellulolytic and xylanolytic enzymes such as avicelase, carboxymethyl cellulase (CMCase), cellobiohydrolase, β-glucosidase, xylanase, β-xylosidase and α-l-arabinofuranosidase. The molecular mass of the complex was estimated to be 1600 kDa. It composed of at least 12 proteins on SDS-PAGE and 10 CMCases and 11 xylanases on zymograms. The isolated multienzyme complex could degrade the raw lignocellulosic substances effectively.     

Study of the aroma compounds of rose apple (<Emphasis Type="Italic">Syzygium jambos</Emphasis> Alston) fruit from Brazil

Volatiles of the headspace and total aqueous extract from the rose apple fruit were investigated by gas chromatography (GC)–flame ionization detection, GC–mass spectrometry and GC–olfactometry/aroma extract dilution analysis O/AEDA). The headspace fraction was rich in low boiling compounds, while medium and high volatility components predominated in the total aqueous extract. GC–O/AEDA revealed that hexanal, 3-penten-2-one, hexanol, (Z)-3-hexen-1-ol, linalool, isovaleric acid, benzyl alcohol, 2-phenylethylalcohol and (E)-cinnamaldehyde were potent odorants contributing to the headspace aroma. Thus, they might be related to the first odour impression of the fruit. Other potent volatiles, such as caproic and phenylacetic acids, methyl-(E)-cinnamate and (E)-cinnamyl alcohol were identified in the total aqueous extract, only. These results indicated that some volatiles, with medium and high boiling points, might also contribute to the overall rose apple flavour. Besides them, volatiles with lower odour potency, such as 7-octen-4-ol, acetic acid, 2-octen-1-ol and cinnamic acid, could have some additional effect for the overall fruit aroma.