Preparation of eicosapentaenoic acid concentrates from sardine oil by Bacillus circulans lipase

An extracellular lipase derived from Bacillus circulans, isolated from marine macroalga, Turbinaraia conoides, was used to prepare n-3 polyunsaturated fatty acid (PUFA) concentrates from sardine oil triglycerides. The enzyme was purified 132-fold with specific activity of 386 LU/mg. The purified lipase was able to enrich sardine oil with 37.7 ± 1.98% 20:5n-3 and 5.11 ± 0.14% 18:3n-3 in the triglyceride fraction after 3 h of hydrolysis. Lower hydrophobic constants of n-3 fatty acids (18:3n-3_logP = 5.65; 20:5n-3_logP = 5.85, respectively) than n-6 (20:4n-6_logP = 6.16) resulted in higher hydrolytic resistance of the former toward lipase, leading to their enrichment in the triglyceride fraction. Lipase-catalysed hydrolysis of sardine oil for 3 h, followed by urea complexation, provided free fatty acids containing 51.3 ± 4.65% 20:5n-3. The purified methyl ester of 20:5n-3 (68.29 ± 2.15%) from the urea concentrate was attained by chromatography on argentated neutral alumina.     

Phenolic acid contents of kale (Brassica oleraceae L. var. acephala DC.) extracts and their antioxidant and antibacterial activities

Nine phenolic acids were identified and quantified by HPLC–MS in leaves and 10 in seeds of kale (black cabbage). The free, ester (methanol-soluble), glycoside and ester-bound (methanol-insoluble) phenolic acid contents of the leaves were 487, 532, 4989 and 6402 ng/g fresh weight, respectively. Ferulic and caffeic acids (total contents; 4269 and 4887 ng/g, respectively) were the most abundant. The seed contents of these fractions were 1993, 1477, 1231 and 4909 ng/g dry weight (DW), respectively, and sinapic acid was the most abundant (5037 ng/g DW). The fractions’ total phenolic contents, determined colorimetrically, were highly correlated with their DPPH scavenging capacity, and in antimicrobial activity assays, with nine test organisms representing a wide array of taxa, all of the fractions were effective against Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis and (most strongly) Moraxella catarrhalis. Antimicrobial and antioxidant activities of kale phenolics in free and conjugated forms are discussed.     

Kinetics of browning onset in white wines: influence of principal redox-active polyphenols and impact on the reducing capacity

The browning capacity of white wines was studied, employing an accelerated test and samples produced and stored under identical conditions. Browning was approached from a kinetic point of view and efforts were focussed on the investigation of plausible correlations with major redox-active polyphenols, including substances with an o-diphenol feature, such as gallic acid, caftaric acid, 2-S-glutathionylcaftaric acid (GRP), caffeic acid, catechin, and epicatechin. Over a period of ten days, browning development was shown to obey zero-order kinetics from the third day of the treatment, and browning rate constants (k) varied from 15.3 to 74.5 × 10⁻³ day⁻¹. Regression analysis between k values and concentration of individual phenolics provided strong evidence that epicatechin is the principal browning agent (r² = 0.8033, P < 0.01). Furthermore, the monitoring of the reducing power (P_R), throughout treatments, indicated that increases in browning are accompanied by a commensurate decline in the reducing ability, raising concerns about the impact of browning reactions on the in vitro antioxidant properties of white wines.     

Development and validation of an HPLC-method for determination of free and bound phenolic acids in cereals after solid-phase extraction

Whole cereal grains are a good source of phenolic acids associated with reduced risk of chronic diseases. This paper reports the development and validation of a high-performance liquid chromatography–diode array detection (HPLC–DAD) method for the determination of phenolic acids in cereals in either free or bound form. Extraction of free phenolic acids and clean-up was performed by an optimised solid-phase extraction (SPE) protocol on Oasis HLB cartridges using aqueous methanol as eluant. The mean recovery of analytes ranged between 84% and 106%. Bound phenolic acids were extracted using alkaline hydrolysis with mean recoveries of 80–95%, except for gallic acid, caffeic acid and protocatechuic acid. Both free and bound phenolic extracts were separated on a Nucleosil 100 C₁₈ column, 5 μm (250 mm × 4.6 mm) thermostated at 30 °C, using a linear gradient elution system consisting of 1% (v/v) acetic acid in methanol. Method validation was performed by means of linearity, accuracy, intra-day and inter-day precision and sensitivity. Detection limits ranged between 0.13 and 0.18 μg/g. The method was applied to the analysis of free and bound phenolic acids contents in durum wheat, bread wheat, barley, oat, rice, rye, corn and triticale.     

Multivariate classification of rice wines according to ageing time and brand based on amino acid profiles

Discrimination of Chinese rice wines according to ageing time and brand using amino acid profiles was presented in this study. Free amino acids (16) in 98 rice wines were simultaneously determined using high-performance liquid chromatograph-diode array detection (HPLC-DAD). Then the experimental data was subjected to multivariate analysis. Principal component analysis (PCA) was employed to differentiate samples from various ageing times (3, 9, 11 and 15 months) and brands (“pagoda”, “kuaijishan”, and “guyuelongshan”). Partial least square discriminant analysis (PLS-DA) and full (leave-one-out) cross-validation were used to develop classification models. The overall correct classification rate for different ageing times and brands was 99.7% and 94.9%, respectively. The proposed method shows an effective strategy for the detection of mislabelling of rice wines.     

A simple method for the analysis of trans fatty acid with GC–MS and AT-Silar-90 capillary column

The study was aimed to evaluate a simplified gas chromatography method based on the AOAC method 996.06 to analyze the trans fat content in food samples. The gas chromatograph was equipped with mass spectrometer and Alltech AT^™-Silar-90 capillary column. Ten kinds of the trans fatty acid standard were separated completely from the cis standard and the chemical composition of the peaks was verified by using the mass spectrum. Under the optimized conditions, the recovery rate for triheptadecanoin was 99.0%, the correlation coefficients of trans fatty acid calibration curve was 0.9998 or higher. It demonstrated that the methylation and hexane extraction procedures used in this method was effective and the result was consistent. The major fatty acids found in the shortening sample were 16:0, 18:0, trans-18:1, cis-18:1, cis-18:2, and cis-18:3. The total trans fat content in the sample was 283.6 ± 18.2 mg/g. The current method was more convenient. It is adequate for the routine analysis of trans fat content in food products with low free fatty acid content.     

Analysis and formation of trans fatty acids in hydrogenated soybean oil during heating

Hydrogenated oil has been widely used for production of shortenings or margarine, however, the presence of trans fatty acids may be detrimental to human health. The objectives of this study were to develop an improved method for analysis of trans fatty acids and evaluate their formation in both unhydrogenated and hydrogenated soybean oil during heating at 160, 180 and 200 °C for varied length of time. Results showed that among the four columns tested, an Agilent HP-88 column (100 × 0.25 mm I.D., 0.2-μm film thickness) could resolve eight trans fatty acids and nine cis fatty acids simultaneously within 31 min with injector temperature 240 °C, detector temperature 250 °C, and column temperature 170 °C in the beginning, maintained for 24 min, increased to 220 °C at 7.5 °C/min, 230 °C at 10 °C/min, and maintained for 5 min. The contents of both cis and trans fatty acids showed a decreased trend for the increase of heating time or temperature. No trans fatty acid formation was observed even after extensive heating of unhydrogenated and hydrogenated soybean oil for 24 h. This phenomenon demonstrated that trans fatty acids can only be formed under severe conditions.     

Cytoprotective and antioxidant activity of free,conjugated and insoluble-bound phenolic acids from swallow root (Decalepis hamiltonii)

Swallow root (Decalepis hamiltonii) was extracted for free (SRFP), conjugated (SRCP) and insoluble-bound phenolic acids (SRIBP), and evaluated for cytoprotectivity, 1,1,diphenyl-2-picrylhydrazyl (DPPH) scavenging ability, reducing power and protection to DNA damage. In addition, the constituent phenolic acids in the extracts were also analysed. Results indicated a total phenol content of 20.72, 7.97 and 11.52 mg gallic acid equivalents (GAE)/g for SRFP, SRCP and SRIBP extracts, respectively. At 0.12 μg/mL concentration SRCP showed 87% cytoprotection (on NIH 3T3 cells) compared to SRFP (47%) and SRIBP (65%). DPPH radical scavenging activity indicated an IC₅₀ of 0.046, 0.06 and 0.128 μg/mL for SRCP, SRIBP and SRFP, respectively. Also, SRCP showed higher reducing power and DNA protectivity (80%). HPLC analysis of phenolic acid extracts showed the presence of hydroxybenzoate and cinnamate derivatives. Among the phenolics identified gallic, gentisic, protocatechuic and p-coumaric acids were the major contributors to antioxidant activity.     

Evolution of 5-hydroxymethylfurfural (HMF) and furfural (F) in fortified wines submitted to overheating conditions

As furfural (F) and 5-hydroxymethylfurfural (HMF) are essentially formed from sugar dehydration, especially in food submitted to heat, they can be found in beverages, as well as fortified sweet wines. In order to assess the impact of temperature on Madeira winemaking, three traditional varieties of Madeira wines (Malvasia, Sercial and Tinta Negra Mole) were studied to evaluate the F and HMF contents. The wines were produced by two vinification processes, following traditional and modern methodologies, heated at standard conditions (30 °C and 45 °C, for 4 months) and compared with the same wines submitted to overheating conditions (55 °C, for 4 months). The RP-HPLC-DAD methodology used for the control of F and HMF during the process showed no significant changes in the wines maintained at 30 °C (canteiro) and a noticeable but controlled increase in the wines heated at 45 °C (estufagem) where values up to about 150 mg/L of HMF could be found in sweet wines. The strong relation of this compound with the sugar content and baking temperature stood out in the wines submitted to overheating conditions where values higher than 1 g/L could be found for sweeter wines, with HMF level being in general higher than F.The results clearly suggest that the amount of HMF in these fortified wines can be easily controlled when submitted to adequate conditions of heating during estufagem and storage. Furthermore, different temperatures for the baking of sweet and dry wines may be considered.     

Effect of ammonium nitrogen supplementation of grape juice on wine volatiles and non-volatiles composition of the aromatic grape variety Albariño

Vitis vinifera cv. Albariño is an aromatic variety characterised by terpenes present largely as glycosidic flavour precursor compounds, which depends on fermentation to reveal its aromatic varietal potential. Clarified Albariño must containing 250 mg N/L (control) was supplemented with diammonium phosphate to 350 and 450 mg N/L before fermentation with a high-nitrogen-demand wine strain of Saccharomyces cerevisiae (M05). Ammonium supplementation had a significant effect on the chemical composition (titratable acidity and ethanol) and volatile profile of Albariño wines. Varietal compounds in the free fraction (limonene, linalool, α-terpineol, α-ionone and β-damascenone) and bound (limonene, linalool and α-terpineol), as well as most yeast-derived fermentation products (including esters, higher alcohols and volatile acids) were associated with moderate nitrogen concentrations. Free β-ionone, bound geraniol, bound α-ionone, bound β-damascenone and 1-hexanol were associated with high nitrogen concentrations. OAV wines made with moderate nitrogen exhibited a higher total odorant activity, driven by ethyl esters, terpenes and C₁₃-norisoprenoids, resulting in fruity and floral aroma attributes.     

Influence of major polyphenols on antioxidant activity in Mencía and Brancellao red wines

The purpose of this work was to examine the relationship of the total polyphenol index (TPI) as measured spectrophotometrically and individual phenols as determined by HPLC to antioxidant activity in red wines made from two Vitis vinifera grape varieties grown in NW Spain (viz. Mencía and Brancellao) during bottled storage in the dark for 12 months. Antioxidant activity was determined by using various methods based on inhibition of the coupled oxidation of the β-carotene/linoleic acid mixture. Also, free radical scavenging activity was determined with 2,2-diphenyl-1-picrylhydrazyl (DPPH). Antioxidant activity as measured by DPPH after storage for 3 months was higher in Mencía wines than in Brancellao wines (4.5 ± 0.60 vs. 3.7 ± 0.30 mmol/L trolox equivalents). Beyond 3 months of storage, radical scavenging activity increased in both types of wine (from 4.5 ± 0.60 to 6.0 ± 0.80 mmol/L in Mencía wines and from 3.7 ± 0.30 to 4.7 ± 0.71 mmol/L trolox equivalents in Brancellao wines). The significant correlation found between antioxidant activity and TPI in all wines (r > 0.88) is indicative of the significance of condensation and polymerisation products to the total antioxidant activity of the wines. The relatively high correlation of total flavonols (r = 0.89) and acylated anthocyanins (r = 0.70) as measured by HPLC with to the overall antioxidant capacity suggests that these two polyphenol classes can substantially influence the antioxidant properties of these wines. The linoleic acid/β-carotene assay exposed a higher antioxidant capacity in Brancellao wines than in Mencía wines after 3 months of storage (6.0 ± 0.80 vs. 4.7 ± 0.70). Most of the studied wines lost an average 45% antioxidant activity during bottled storage. Antioxidant activity as measured with the linoleic acid/β-carotene assay was closely correlated with both hydroxycinnamic acids (r = 0.90) and flavanols (r = 0.71 and 0.61 for monomeric and polymeric forms, respectively).     

Effects of feeding flaxseed or sunflower-seed in high-forage diets on beef production,quality and fatty acid composition

Yearling steers were fed 70:30 forage:concentrate diets for 205 d, with either grass hay (GH) or red clover silage (RC) as the forage source, and concentrates containing either sunflower-seed (SS) or flaxseed (FS), each providing 5.4% oil to diets. Feeding diets containing SS versus FS significantly improved growth and carcass attributes (P < 0.05), significantly reduced meat off-flavor intensity (P < 0.05), and significantly increased intramuscular proportions of vaccenic (t11-18:1), rumenic (c9,t11-CLA) and n − 6 fatty acids (FA, P < 0.05). Feeding diets containing FS versus SS produced significantly darker and redder meat with greater proportions of atypical dienes (P < 0.05). A significant forage × oilseed type interaction (P < 0.05) was found for n − 3 FA, α-linolenic acid, and conjugated linolenic acid, with their greatest intramuscular proportions found when feeding the RC-FS diet. Feeding GH versus RC also significantly improved growth and carcass attributes, sensory tenderness (P < 0.05) and significantly influenced intramuscular FA composition (P < 0.05), but overall, forage effects on FA profiles were limited compared to effects of oilseed.     

Glycosylation,amino acid analysis and kinetic properties of a major Kunitz-type trypsin inhibitor from Acacia victoriae Bentham seeds

An Acacia victoriae trypsin inhibitor (AvTI) was purified from the seeds of prickly wattle (A. victoriae Bentham) by salt precipitation, ion-exchange and gel filtration chromatography, and its degree of glycosylation, amino acid composition, and kinetic properties were determined. Gel electrophoresis revealed at least four glycoprotein bands in the crude extract, salt-precipitated and ion-exchange protein fractions, while the purified AvTI showed only one band and a degree of glycosylation of 2.06%. Glutamate (13.3%), aspartate (10.3%), leucine (7.62%) and lysine (7.01%) were the major amino acids in AvTI while the contents of sulphur-containing amino acids, cysteine (1.38%) and methionine (0.75%), as well as of tryptophan (1.17%) were low. Its dissociation constant (K_i) for the inhibition of bovine trypsin was found to be 1.06 × 10⁻⁸ M, indicating a high affinity between AvTI and this enzyme, and its role as a competitive inhibitor was confirmed by a double reciprocal plot. These results complement our earlier studies which indicated the presence of three isoforms of this Kunitz-type trypsin inhibitor in prickly wattle seed.     

Antimicrobial properties and analytical profile of traditional Eruca sativa seed oil: Comparison with various aerial and root plant extracts

The present study investigates the antimicrobial activity of various solvent extracts of Eruca sativa (aerial and root) and seed oil against-antibiotic resistant Gram-negative (Escherichia coli, Pseudomoms aeruginosa and Shigella flexneri) and Gram-positive (Staphylococcus aureus and Bacillus subtilis) bacteria. Among the various preparations, seed oil was the most active, exhibiting a maximum zone inhibition of 97% for Gram-positive bacteria and of 74–97% for Gram-negative bacteria. The MIC of the seed oil was found to be 65–75 and 60–70 μg/ml for Gram-negative and Gram-positive bacteria, respectively. Analytical investigation on main volatile and non-volatile components was performed on seed oil. Among the formers allyl isothiocyanate (40 μg/g), 3-butenyl isothiocyanate (260 μg/g), 4-methylsulfinybutyl isothiocyanate (sulforaphane 743 μg/g), 2-phenylethyl isothiocyanate (159 μg/g) and bis(isothiocyanatobutyl)disulphide (∼5000 μg/g) were determined by head space/SPME/GC–MS analysis. Free fatty acids were 1.6% w/w of the oil and overall 25 fatty acids were identified. Erucic and oleic acids were the main fatty acids both in the free (7.8 and 2.1 mg/ml) and esterified forms (50.6% w/w and 14.9% w/w of total fatty acids). Unsaponifiable fraction was 1.8% w/w.     

Meat quality,proximate composition and muscle fatty acid profile of young llamas (Lama glama) supplemented with hay or concentrate during the dry season

Thirty llamas were used to study the effect of a 90 day feed supplementation on meat quality, chemical composition and muscle fatty acid profile. Treatments were: GR = llama on native pasture until slaughter; GR + SH = like GR, but with overnight free access to barley/alfalfa hay; and GR + SC = like GR, but with overnight free access to a wheat bran/sorghum grain concentrate. The supplementation had no effect on postmortem pH and temperature decline in the Longissimus lumborum muscle (LLM), cooking losses nor Warner–Bratzler shear force values (P > 0.05). Meat from GR + SC llama had higher fat content in LLM (P < 0.05) compared to GR and GR + SH llama. Intramuscular fat from GR + SH llama showed higher (P < 0.01) proportions of polyunsaturated fatty acids, higher (P < 0.05) polyunsaturated fatty acids/saturated fatty acids and desirable fatty acids ratio, lower (P < 0.05) omega-6/omega-3 (n − 6/n − 3) ratio, and higher (P < 0.01) conjugated linoleic acid.     

Chemical composition,antioxidative activity and cell viability effects of a Siberian pine (Pinus sibirica Du Tour) extract

Siberian pine (Pinus sibirica Du Tour) seeds, commonly known as cedar nuts, are ascribed a number of medicinal properties. In this study, we report the qualitative–quantitative composition, antioxidant activity and cell viability-related properties of a defatted aqueous-acetone-soluble P. sibirica seed extract. The total phenolic and total tannin contents were estimated at 266 ± 3.9 mg gallic acid/g and 115 ± 7.8 mg tannic acid/g, respectively. Reverse-phase chromatographic analysis of the crude extract indicated the presence of a chromatographic hump indicative of the presence of proanthocyanidins. After acid hydrolysis, the presence of hydroxylated benzoic and cinnamic acids, flavanones and flavan-3-ols was confirmed. After thiolysis, (+)-catechin was identified as more abundant than (−)-epicatechin, suggesting that this molecule was the main terminal unit of the proanthocyanidins within this extract. The extract demonstrated iron(III)-reductive (AscAE = 650 ± 5.10 μmol ascorbic acid/g) and iron(II) chelating (EC₅₀ = 20.1 ± 2.1) activities and the ability to scavenge 1,1-diphenyl-2-picrylhydrazyl (IC₅₀ = 257 ± 2.36 μg/ml) and hydroxyl (IC₅₀ = 338 ± 6.49 μg/ml) free radicals. When the effects of P. sibirica extract were assessed in a tumourigenic SH-SY5Y neuroblastoma cell line, it was found that the cell viability was diminished in the presence of P. sibirica extract (0.2–1.0 mg/ml), as indicated by decreased membrane integrity (LDH assay) and mitochondrial metabolic activity (MTT assay), but the level of p53 protein was not changed (Western blot).     

Nutrient contents of kale (Brassica oleraceae L. var. acephala DC.)

Fructose, glucose and sucrose, as the major soluble sugars and citric and malic acids, as the major organic acids, were identified and determined in kale (Brassica oleraceae L. var. acephala DC., black cabbage) leaves. Fructose was the predominant sugar (2011 mg 100 g⁻¹ dry wt) identified, followed by glucose (1056 mg 100 g⁻¹ dry wt) and sucrose (894 mg 100 g⁻¹ dry wt). The contents of citric and malic acids were at 2213 and 151 mg 100 g⁻¹ dry wt in the leaves. The 16:0, 18:2n − 6 and 18:3n − 3 fatty acids were the most abundant fatty acids in the leaves. Considering the level of these fatty acids, 18:3n − 3 was found to be the highest (85.3 μg g⁻¹ dry wt), contributing 54.0% of the total fatty acid content. Linoleic acid (18:2n − 6), being the second most abundant fatty acid was present at 18.6 μg g⁻¹ dry wt, contributing 11.8% of the total fatty acid content. In the seed oil of kale, 22:1n − 9 was the most abundant fatty acid (4198 μg g⁻¹ dry wt, 45.7%), with 18:2n − 6 (1199 μg g⁻¹ dry wt, 12.3%) and 18:1n − 9 (1408 μg g⁻¹ dry wt, 14.8%) being the second next most abundant fatty acids. The most abundant amino acid was glutamic acid (Glu) which was present at 33.2 mg g⁻¹ dry wt. Aspartic acid, which was the second most abundant amino acid, was present at 27.6 mg g⁻¹ dry wt and accounted for 10.2% of the total amino acid content of kale leaf. The amino acid content was assessed by comparing the percentages of the essential amino acids in kale leaf versus those of a World Health Organization (WHO) standard protein. The protein of kale leaf compares well with that of the WHO standard. Only one amino acid, lysine, had a score that fell below 100%; the lysine score of kale leaf was 95%. This study attempts to contribute to knowledge of the nutritional properties of the plant. These results may be useful for the evaluation of dietary information.     

Polyphenolic content of Vranec wines produced by different vinification conditions

Macedonian Vranec wines were analysed by HPLC coupled with DAD and MS detections and by spectrophotometric methods. ESI-IT MS and MS–MS methods with alternating ionisation polarity were used for identification of the phenolic compounds. Both, nonflavonoids (stilbens, hydroxybenzoic and hydroxycinnamic acids and derivatives) and flavonoids (flavonols, flavan-3-ols and anthocyanins) were detected in the samples. Vranec wines were produced under different fermentation conditions: maceration time of 3, 6 and 10 days, two doses of SO₂ (30 and 70 mg l⁻¹) and two yeasts for fermentation, in order to examine their effects on the extraction of phenolic compounds from grapes into the wine.     

Determination of free phenolic acids and antioxidant activity of methanolic extracts obtained from fruits and leaves of Chenopodium album

In this study, determination of phenolic acids as well as investigation of antioxidant activity of methanolic extracts from the fruits and leaves of Chenopodium album is described. Extracts were subjected to acidic hydrolysis in order to obtain total free phenolic acids. However, some of phenolic acids were identified and quantified by HPLC-DAD. The results were confirmed by LC-MS equipped with MS-ESI. In addition, Folin–Ciocalteu method was applied to determine the total phenolic contents. The antioxidant activity of C. album extracts was examined by using DPPH and hydroxyl radical-scavenging activity assays. Results revealed that the leaves extract exhibits better performance in antioxidant assays and in the higher total phenolic contents (3066 mg of GAE/100 g) when compared to fruits extract (1385 mg of GAE/100 g). From these results it has been revealed that the methanolic extracts of C. album from fruits and leaves have great potential as a source for natural health products.     

Evaluation of sequential inoculation of Saccharomyces cerevisiae and Oenococcus oeni strains on the chemical and aromatic profiles of cherry wines

The current study was carried out to elucidate the effect of sequential inoculation of Saccharomyces cerevisiae (RC212, D254) and Oenococcus oeni (SG26, Lalvin 31 and Uvaferm Alpha) on the production of cherry wines, especially on the chemical and aromatic characteristics. SI-D culture required the shortest period (23 d) to complete the fermentation, while other inoculations needed longer time. Analysis from chemical composition showed that titratable acidity and content of l-malic acid exhibited evident differences among the samples after MLF. For volatile compounds, 49 major components were identified, mostly comprising of alcohols, acids and esters. Cherry wines obtained from SI-B and SI-C showed higher contents of total volatile alcohols, and SI-D wines produced the greatest amount of volatile acids. According to the odour active value (OAV), 9 out of 49 studied volatile components had OAV >1 in all the analyzed wines, while six volatile components showed OAV >1 only for some of them. Furthermore, a sensory analysis was performed to compare the sensory profile of these cherry wines, and results evidenced that wines resulting from different inoculations presented diverse sensory profiles. These findings suggest that sequential inoculations posed a great potential in affecting and modulating the aromatic profile of cherry wines.