Alterations in d-amino acid concentrations and microbial community structures during the fermentation of red and white wines

Alterations in d-amino acid concentrations and microbial community structures during the fermentation of red and white wines were analyzed to clarify the relationship between the occurrence of d-amino acids and the actions of fermentative microorganisms. Relatives of Saccharomyces cerevisiae and Oenococcus oeni were detected in red wine samples, and relatives of S. cerevisiae, O. oeni, and Gluconacetobacter saccharivorans were detected in white wine samples. The S. cerevisiae relatives were detected throughout the fermentation process, whereas the O. oeni relatives were detected at the late stage of fermentation in both the red and white wine samples. The G. saccharivorans relative was detected in the early stage of fermentation. The amino acid analysis showed that d-alanine, d-glutamic acid, and d-lysine were present in both the red and white wine samples. The concentrations of these d-amino acids increased as the fermentation continued, especially from the malolactic fermentation stage to the end of the fermentation processes. These increases seem to be linked to the presence of O. oeni relatives.     

Identification and characterization of a novel l-amino acid ligase from Photorhabdus luminescens subsp. laumondii TT01

l-Amino acid ligase catalyzes dipeptide synthesis from unprotected l-amino acids in an ATP-dependent manner. We recently identified a new member of l-amino acid ligase, the plu1440 protein, from Photorhabdus luminescens subsp. laumondii TT01 by in silico analysis. This protein was found to synthesize dipeptides containing l-asparagine at the N-terminus, which is a novel substrate specificity.     

Screening of d-amino acid oxidase inhibitor by a new multi-assay method

This paper describes an investigation of the inhibition of a d-amino acid oxidase (DAAO) activity in several kinds of food additives and fruit. To screen for an inhibitor of DAAO, the method employed checked for two kinds of indicators, namely, pyruvic acid (indicator-1) and hydrogen peroxide (indicator-2), both of which are formed by digestion by DAAO of d-alanine (the standard d-amino acid used in this study). For measurement of pyruvic acid, a reliable and authenticated method was employed: the presence of pyruvic acid was determined by converting it into a chromophore derivative by tagging with 2,4-dinitro phenylhydrazine (DNP), followed by measurement of absorbance at 445 nm. The pyruvic acid thus determined is referred to as “(colorimetric) indicator-1”. To measure hydrogen peroxide, a highly sensitive fluorogenic substrate, 3-(p-hydroxyphenyl)propionic acid (HPPA), was employed in the present study. Hydrogen peroxide was detected by measuring fluorescence intensity (Ex. = 320 nm, Em. = 405 nm) of 2,2′-dihydroxybiphenyl-5,5′-dipropionic acid (oxidize-HPPA), which is produced by horseradish peroxidase (HRP)-catalyzed oxidation of HPPA with hydrogen peroxide. The hydrogen peroxide thus determined is referred to as “(flourometric) indicator-2” After optimizing the multi-assay procedures, the inhibition of DAAO activity against d-alanine (used as the standard d-amino acid) in 11 kinds of food additives and five kinds of fruit was evaluated, first in terms of indicator-1. Then, two substrates, potassium sorbate and apple juice, that were screened by indicator-1, were further evaluated by indicator-2 to conform the inhibition of DAAO activity. This is the first demonstration of the inhibition of DAAO activity by potassium sorbate, which is widely used as a synthetic preservative in food and apple juice.     

Optical resolution of n-butyl d- and l-lactates using immobilized lipase catalyst

n-Butyl d- and l-lactates (BuDLa and BuLLa) were incubated with immobilized lipase. ¹H-NMR showed that BuDLa reacted to oligomers, while BuLLa did not react. A mixture containing 90.4% of BuLLa and 9.6% of BuDLa was incubated with the enzyme for 72 h, then distilled. The purity of BuLLa increased to 98.6%.     

Mechanism of formation of sulphur aroma compounds from l-ascorbic acid and l-cysteine during the Maillard reaction

The sulphur aroma compounds produced from a phosphate-buffered solution (pH 8) of l-cysteine and l-, l-[1-¹³C] or l-[4-¹³C] ascorbic acid, heated at 140 ± 2 °C for 2 h, were examined by headspace SPME in combination with GC-MS. MS data indicated that C-1 of l-ascorbic acid was not involved in the formation of sulphur aroma compounds. The sulphur aroma compounds formed by reaction of l-ascorbic acid with l-cysteine mainly contained thiophenes, thiazoles and sulphur-containing alicyclic compounds. Among these compounds, 1-butanethiol, diethyl disulphide, 5-ethyl-2-methylthiazole, cis and trans-3,5-dimethyl-1,2,4-trithiolane, thieno[2,3-b]thiophene, thieno[3,2-b]thiophene, cis and trans-3,5-diethyl-1,2,4-trithiolane, 1,2,5,6-tetrathiocane, 2-ethylthieno[2,3-b]thiophene, 2,4,6-trimethyl-1,3,5-trithiane and cyclic octaatomic sulphur (S₈) were formed solely by l-cysteine degradation, and the rest by reaction of l-ascorbic acid degradation products, such as hydroxybutanedione, butanedione, acetaldehyde, acetol, pyruvaldehyde and formaldehyde with l-cysteine or its degradation products, such as H₂S and NH₃. A new reaction pathway from l-ascorbic acid via its degradation products was proposed.     

Substrate specificity of a recombinant d-lyxose isomerase from Providencia stuartii for monosaccharides

The specific activity and catalytic efficiency (k_cat/K_m) of the recombinant putative protein from Providencia stuartii was the highest for d-lyxose among the aldose substrates, indicating that it is a d-lyxose isomerase. Gel filtration analysis suggested that the native enzyme is a dimer with a molecular mass of 44 kDa. The maximal activity for d-lyxose isomerization was observed at pH 7.5 and 45 °C in the presence of 1 mM Mn²⁺. The enzyme exhibited high isomerization activity for aldose substrates with the C2 and C3 hydroxyl groups in the left-hand configuration, such as d-lyxose, d-mannose, l-ribose, d-talose, and l-allose (listed in decreasing order of activity). The enzyme exhibited the highest activity for d-xylulose among all pentoses and hexoses. Thus, d-lyxose was produced at 288 g/l from 500 g/l d-xylulose by d-lyxose isomerase at pH 7.5 and 45 °C for 2 h, with a conversion yield of 58 % and a volumetric productivity of 144 g l^− 1 h^− 1. The observed k_cat/K_m (920 mM^− 1 s^− 1) of P. stuartiid-lyxose isomerase for d-xylulose is higher than any of the k_cat/K_m values previously reported for sugar and sugar phosphate isomerases with monosaccharide substrates. These results suggest that the enzyme will be useful as an industrial producer of d-lyxose.     

Aroma compounds generated from thermal reaction of l-ascorbic acid with l-cysteine

The reaction of l-ascorbic acid with l-cysteine in heated aqueous solution (141 ± 1 °C) at five different pH values (5.00, 6.00, 7.00, 8.00, or 9.00) for 2 h, resulted in the formation of a complex mixture of aroma volatiles. The volatile compounds generated were analysed by SPME–GC–MS. The results gave 43 aroma compounds. The reaction between l-ascorbic acid and l-cysteine led mainly to the formation of alicyclic sulphur compounds, thiophenes, thienothiophenes, thiophenones, thiazoles and pyrazines, most of which contain sulphur. Many of these volatiles had meaty flavour. The origin of many of the compounds was explained. The studies showed that thienothiophenes and thienones were formed mainly at acidic pH. In contrast, higher pH values could promote the production of thiophenes, thiazoles and pyrazines.     

Results of an European inter-laboratory comparison study on the determination of the 15 + 1 EU priority polycyclic aromatic hydrocarbons (PAHs) in liquid smoke condensates

A collaborative study on the determination of the 15 + 1 EU priority PAHs in Primary Smoke Condensate (PSC) investigating the performance profile of EU Member States’ laboratories and supporting the work carried out by the European Food Safety Authority on smoke flavourings was organised. Two spiked liquid smoke condensate materials were employed in this study. The results of 25 laboratories from across the European Union, using either high performance liquid chromatography with fluorescence detection or gas chromatography with mass selective detection, were evaluated by application of robust statistics. The assessment of the data indicated broadened Gaussian distributions for all analytes. For benzo[a]pyrene (BaP), benzo[a]anthracene (BaA), chrysene and 5-methylchrysene more than 80% of the respective reported values gave rise to a satisfactory score of |z| ? 2. For benzo[b]fluoranthene, benzo[k]fluoranthene, and dibenzo[a,l]pyrene 70–80%, for the remaining analytes less than 70%, and for dibenzo[a,i]pyrene 52% of the scores were satisfactory. No systematic differences could be detected between values reported by laboratories using methods based on HPLC and the values related to methods based on GC for most of the analytes, except for benzo[k]fluoranthene and benzo[c]fluorene. In both cases laboratories using GC based methods reported about 50% higher values than laboratories using HPLC based methods. The overview on all z-scores sorted by laboratory revealed broad distributions and/or laboratory biases for several laboratories. An assessment of the reported method performance parameters revealed that for the two regulated compounds, BaP and BaA, only two thirds of the reported data were in compliance with Regulation (EC) 627/2006. Overall the methods used in the participating laboratories were – with ample room for improvement – well on the way to comply with European legislation.     

Effect of different fermentation parameters on l-lactic acid production from liquid distillery stillage

Expansion of lactic acid applications, predominantly for the preparation of biodegradable polymers increased the research interest for new, economically favourable production processes. Liquid stillage from bioethanol production can be an inexpensive, valuable source of nutrients for growth of lactic acid bacteria. Utilisation of residual biomass with spent fermentation media as a functional animal feed can greatly influence the process value and its ecological aspect. In this paper, the kinetics of lactic acid and biomass production on liquid stillage by Lactobacillus rhamnosus ATCC 7469 was studied. In addition, the impact of temperature, inoculum concentration, shaking and pH control by addition of CaCO₃ was evaluated. Maximal lactic acid yield of 73.4%, as well as high biomass production (3 × 10⁸ CFU ml⁻¹) were achieved under selected conditions (41 °C, 5% (v/v) of inoculum, 1% (w/v) of CaCO₃, initial pH of 6.5 and shaking rate of 90 rpm). These results were achieved without supplementation of the stillage with nitrogen or mineral sources.     

Physiochemical properties and kinetics of glucoamylase produced from deoxy-d-glucose resistant mutant of Aspergillus niger for soluble starch hydrolysis

Glucoamylases (GAs) from a wild and a deoxy-d-glucose-resistant mutant of a locally isolated Aspergillus niger were purified to apparent homogeneity. The subunit molecular mass estimated by SDS–PAGE was 93 kDa for both strains, while the molecular masses determined by MALDI-TOF for wild and mutant GAs were 72.876 and 72.063 kDa, respectively. The monomeric nature of the enzymes was confirmed through activity staining. Significant improvement was observed in the kinetic properties of the mutant GA relative to the wild type enzyme. Kinetic constants of starch hydrolysis for A. niger parent and mutant GAs calculated on the basis of molecular masses determined through MALDI-TOF were as follows: k_cat = 343 and 727 s⁻¹, K_m = 0.25 and 0.16 mg mL⁻¹, k_cat/K_m (specificity constant) = 1374 and 4510 mg mL⁻¹ s⁻¹, respectively. Thermodynamic parameters for soluble starch hydrolysis also suggested that mutant GA was more efficient compared to the parent enzyme.     

Seasonal variation in the fatty acid composition of three Mediterranean fish – sardine (Sardina pilchardus), anchovy (Engraulis encrasicholus) and picarel (Spicara smaris)

The total fat contents and the fatty acid compositions of three common Mediterranean fish, namely sardine (Sardina pilchardus), anchovy (Engraulis encrasicholus) and picarel (Spicara smaris) were determined at bimonthly intervals for a one-year period. The purpose of this work was to study the seasonal variation of the fatty acids in the three fish that are some of the best sources of n − 3 fatty acids. The fat and fatty acid content of the investigated fish species show a significant seasonal dependency. Two of the fish (anchovy and picarel) have the highest fat content during the late winter – spring period. On the other hand, sardine shows the highest fat concentrations during the spring-early summer period. The fish that showed the highest variation in fatty acid composition was the anchovy. The sardine was found to be the best source of n − 3 fatty acids during the one-year period (35.35 g/100 g fatty acids). Finally the picarel had the highest oleic acid content (on average, 13.89/100 g fatty acids).     

Determination of selenium in garlic (Allium sativum) and onion (Allium cepa) by electro thermal atomic absorption spectrometry

A separation/enrichment procedure has been developed for the determination of selenium in garlic and onion samples by electrothermal atomic absorption spectrometry (ET-AAS) as a slurry sampling after preconcentration with 3,3-diaminobenzidine (DAB) reagent on the activated carbon. The influences of pH, time, amount of carbon and complexing reagent were outlined. The effect of acids used in the digestion of samples was also studied and compared. Selenium level was found to be 0.024 μg g⁻¹ for onion (n = 5; LOD – 0.5 μg L⁻¹; LOQ – 1.7 μg L⁻¹) and 0.015 μg g⁻¹ for garlic (n = 5; LOD – 1.3 μg L⁻¹; LOQ – 3.3 μg L⁻¹). Three different samples of garlic were analyzed by k₀-instrumental neutron activation analysis (k₀-INAA) at the Jozef Stefan Institute (JSI). The data obtained by k₀-INAA show that the content of selenium overlapped the results obtained by ET-AAS.     

Polyphenol composition and in vitro antioxidant activity of amaranth,quinoa buckwheat and wheat as affected by sprouting and baking

This study examined the polyphenol composition and antioxidant properties of methanolic extracts from amaranth, quinoa, buckwheat and wheat, and evaluated how these properties were affected following two types of processing: sprouting and baking. The total phenol content amongst the seed extracts were significantly higher in buckwheat (323.4 mgGAE/100 g) and decreased in the following order: buckwheat > quinoa > wheat > amaranth. Antioxidant capacity, measured by the radical 2,2-diphenyl-1-picylhydrazyl scavenging capacity and the ferric ion reducing antioxidant power assays was also highest for buckwheat seed extract (p < 0.01). Total phenol content and antioxidant activity was generally found to increase with sprouting, and a decrease in levels was observed following breadmaking. Analysis by liquid chromatography coupled with diode array detector revealed the presence of phenolic acids, catechins, flavanol, flavone and flavonol glycosides. Overall, quinoa and buckwheat seeds and sprouts represent potential rich sources of polyphenol compounds for enhancing the nutritive properties of foods such as gluten-free breads.     

Hydroxymethylfurfuraldehyde and amylase contents in Australian honey

The quality of Australian honey samples (processed and unprocessed) was assessed using HPLC techniques. 5-Hydroxymethylfurfuraldehyde (HMF) was used as the main quality indicator. Sampling included four commercially-processed honeys (Australian rainforest, Beechworth, Homebrand and Leabrook) and three unprocessed (Banksia, Grey box and Mallee). All honey samples, except Leabrook and Beechworth, showed an initial HMF content less than the Codex Alimentarius and International Honey Commission standard (40 mg/kg). HMF contents in Leabrook and Beechworth were 50.8 ± 1.34 and 74.9 ± 2.34 mg/kg, respectively. Heating unprocessed honey at 85 °C for 2 min caused significant (p ? 0.05) increment in HMF contents. The amounts of HMF in Mallee samples increased from 34.0 ± 0.31 to 42.3 ± 0.37 mg/kg after 2 min at 85 °C. All honey samples showed amylase activity above the minimum limit (8 Gothes). The physiochemical properties of honey showed significant variations among samples. The results revealed also that heating was not the only factor influencing HMF formation in honey, but also honey composition, pH value and floral source can contribute to these variations. Consequently, the amount of HMF may be an insufficient sole indicator of honey quality.     

Elucidation of the flavonoid and furocoumarin composition and radical-scavenging activity of green and ripe chinotto (Citrus myrtifolia Raf.) fruit tissues,leaves and seeds

Citrus myrtifolia Raf. (chinotto) flavedo, albedo and carpel membranes from green and ripe fruits, as well as seeds and leaves, were investigated for the first time for their flavonoid and furocoumarin composition. Twenty-three unique compounds distributed in the different fruit/plant parts were identified and quantified. All samples analysed contained flavanone O-glycosides, flavone C- and O-glycosides and furocoumarins; flavedos and albedos also contained significant amounts of polymethoxyflavones. Flavedo and albedo extracts were found to be richest in flavonoids and furocoumarins, containing up to 1.95 g/kg fresh weight. Flavedo, albedo and carpel membranes from ripe fruits were found to be richer than the corresponding tissues from unripe fruits. The remarkable radical-scavenging activity of all the extracts was tested by DPPH^•, ABTS^•+ and FRAP methods, revealing that (i) they were particularly efficient in quenching ABTS^•+ radical cations (up to 9.8 mM Trolox equivalents), and (ii) flavedo and albedo extracts, on average, showed the highest antioxidant capacity.     

Profiling the chlorogenic acids of sweet potato (Ipomoea batatas) from China

The leaves, stem and root of sweet potato cultivated in China have been analysed qualitatively for chlorogenic acids by structure-diagnostic LC–MS³. Chlorogenic acids were not detected in the root. Caffeoylquinic acids were quantitatively the major subgroup of chlorogenic acids detected in the stem and the only subgroup detected in the leaves. This subgroup was dominated by 5-caffeoylquinic acid. The stem also contained three feruloylquinic acids, 3,5- and 4,5-dicaffeoylquinic acid, and small amounts of at least four caffeoyl-feruloylquinic acids. This is the first report of feruloylquinic and caffeoyl-feruloylquinic acids from sweet potato. Two chemically unrelated and coeluting substances with the same molecular mass (M_r = 530) extracted from the Chinese sweet potato interfered with the characterisation of the caffeoyl-feruloylquinic acids. At least five caffeoyl-feruloylquinic acids were detected in the peel of sweet potato cultivated in Tanzania that lacked these interfering substances.     

GC-MS quantification of fatty acid profile including trans FA in the locally manufactured margarines of Pakistan

Ten margarine brands of Pakistan were analyzed for their fatty acid composition with emphasis on trans fatty acids (TFA) using GC-MS. Saturated, cis-monounsaturated and polyunsaturated fatty acids were present at 24.2–58.1, 5.7–35.4 and 3.8–37.4% of total fatty acids, respectively. Among the saturated fatty acids, palmitic acid (16.9–33.8%) was dominant in all analyzed margarine brands and its higher amount indicates that palm oil was a major contributor in the margarine manufacturing. Among samples tested only one contained a low level of TFA (2.2%) while the rest contained very high amounts of TFA (11.5–34.8%) which clearly shows that hydrogenated oils were used in the formulation of margarines. Fatty acid profiles demonstrated that all samples belong to the hard margarine category containing high amounts of trans and saturated fatty acids which is an alarming issue for the health of consumers.     

Mate (Ilex paraguariensis St. Hilaire) saponins induce caspase-3-dependent apoptosis in human colon cancer cells in vitro

Saponins are naturally occurring metabolites associated with several health benefits. The objective was to quantify and purify saponins from mate dry leaves, and to assess their anti inflammatory and apoptotic mechanisms in human colon cancer cells in vitro. Matesaponins were extracted with methanol from dry leaves, partially purified and quantified. Leaves contained 10–15 mg/g dry weight total saponins, predominantly matesaponins 1 and 2. HPLC and LC/ESI-MS-MS identified saponins in six preparative chromatographic fractions (A, B, C, D, E, and F). Major matesaponins were identified as 1 [M–H]⁻ = 911 and 2 [M–H]⁻ = 1057, with trace amounts of 3 [M–H]⁻ = 1073, 4 [M–H]⁻ = 1219, and 5 [M–H]⁻ = 1383. Fractions D, E, and F significantly inhibited iNOS (IC₃₅ = 36.3, 29.5, 43.7 μM), PGE₂ (IC₃₅ = 23.1, 22.3, 11.7 μM) and COX-2 (IC₃₅ = 45.7, 32.4, 17.0 μM). Fraction F reduced nuclear translocation of nuclear factor-κB subunits p50 (49.8%) and p65 (49.0%) and induced apoptosis through suppression of Bcl-2 and increased Bax protein expressions and activated caspase-3 activity. Saponins in leaves of mate prevent inflammation and colon cancer in vitro.     

In vivo investigation on the potential of galangin,kaempferol and myricetin for protection of d-galactose-induced cognitive impairment

The potential of three natural flavonols (galangin, kaempferol and myricetin) to protect against d-galactose-induced cognitive impairment in mice was investigated. After 8 weeks treatment, the mice were assessed by behavioural tests. The levels of oxidative stress, the amount of Na⁺,K⁺-ATPase and extracellular signal-regulated kinases (ERK)-cyclic AMP response element binding protein (CREB) signaling pathway in hippocampus were also analysed. It was found that all the three dietary flavonols could ameliorate the oxidative stress, enhance the activity of Na⁺,K⁺-ATPase and regulate the expression of ERK-CREB pathway in mice. However, only kaempferol and myricetin could significantly improve the learning and memory capability when compared with d-galactose model. Our results suggest that the presence of hydroxyl groups in the B ring of flavonols may have contribution to the neuroprotective activity.     

Colour properties,stability, and free radical scavenging activity of jambolan (Syzygium cumini) fruit anthocyanins in a beverage model system: Natural and copigmented anthocyanins

The colour and stability properties of jambolan anthocyanins, both natural and copigmented forms, were investigated in beverage model as well as their radical scavenging ability. Natural anthocyanins of jambolan revealed low colour intensity due to glycosylation structure of the anthocyanins as diglucoside. The intermolecular copigmentation of anthocyanins with sinapic acid, caffeic acid, ferulic acid, and rosemary polyphenolic extract could enhance the colour intensity, which was observed through spectrometric parameters, such as hyperchromic effect (ΔA_vis-max) and bathochromic shift (Δλ_vis-max). In addition of sinapic acid, caffeic acid, and rosemary polyphenolics also increased the stability of the anthocyanin colour during exposure to white fluorescent light and storage at refrigeration and room temperatures, whereas on high thermal treatments, this phenomenon was not observed. Furthermore, beverage model coloured with natural or copigmented anthocyanins revealed DPPH radical-scavenging activity. The AEAC values indicated that the beverage models with copigmented anthocyanins had higher scavenging activity than the beverage with natural anthocyanins.