Volatiles of the Balkan endemic Daucus guttatus ssp. zahariadii and cultivated and wild-growing D. carota – A comparison study

The hydrodistilled essential oil obtained from the aerial parts of Daucus guttatus Sibth. & Sm. ssp. zahariadii Heywood, an endemic plant species of the Balkan Peninsula, as well as solvent extracts and essential oils from different parts of Daucus carota L. have been analysed by GC and GC–MS and screened for antimicrobial activity against 12 bacterial and two fungal strains. The volatiles of the two plant taxa differed significantly in both their chemical identity and antimicrobial effect. The dominant constituent of D. guttatus oil was apiol (43.3%), which was absent from all samples of D. carota. The diethyl ether extract of D. carota inhibited the growth of the yeast Candida albicans while the oil of D. guttatus at 25 mg/ml had no effect on the growth of the fungal organisms tested. Additionally, the oil of D. guttatus showed prominent antibacterial activity against a pathogenic Corynebacterium pyogenes.     

Assessment of GH1, CAPN1 and CAST polymorphisms as markers of carcass and meat traits in Bos indicus and Bos taurus–Bos indicus cross beef cattle

The objective of this study was to investigate the effects of bovine GH1, CAPN1 and CAST gene polymorphisms on carcass and meat traits in Nellore and Nellore x Bos taurus beef cattle. Three hundred animals were genotyped for GH1/MspI (TC/G in intron 3), CAPN316 (AF_252504.2:g.5709C > G) and CAST/RsaI (AY_008267.1:g282C > G) and phenotyped for rib eye area, backfat thickness, intramuscular fat, shear force (SF), and myofibrillar fragmentation index (MFI). No significant associations were observed between the GH1/MspI and CAST/RsaI polymorphisms and phenotypes, although the relation between the CAST/RsaI genotypes and meat tenderness evaluated by MFI approached significant. The fact that the CAPN316 polymorphism did not show adequate segregation in Nellore cattle confirms the difficulty of using this marker in breeding programs of different Bos indicus breeds. However, the positive results of the association analysis obtained for Nellore x B. taurus crosses contributed to the validation of previous findings.     

Acidification,crushing and thermal treatments can influence the profile and stability of folate poly-γ-glutamates in broccoli (Brassica oleracea L. var. italica)

The influence of different treatments, i.e., crushing, high temperature short time (90 °C/4 min) (HTST) and low temperature long time (60 °C/40 min) (LTLT) blanching, acidification (pH 4.3), and sequences of these treatments on the folate poly-γ-glutamate profile and stability were investigated. In this study, broccoli was used as a case study. Regarding the folate poly-γ-glutamate profile, endogenous folate poly-γ-glutamates in broccoli florets were found predominantly as hepta- and hexa-γ-glutamates. Crushing raw broccoli, acidification and LTLT blanching enhanced folate deconjugation resulting in monoglutamate, di- and tri-γ-glutamates. Compared to other treatments, HTST blanching preformed prior to crushing resulted in the highest concentration of long chain poly-γ-glutamates. Regarding folate poly-γ-glutamates stability, acidification combined with LTLT blanching decreased folate stability whereas HTST blanching combined with different sequences of blanching and crushing did not affect folate poly-γ-glutamates stability. It was concluded that crushing (prior to heating), acidification and blanching could be strategically applied to increase the folate monoglutamate content of broccoli.     

Occurrence of extended-spectrum β-lactamases among isolates of Salmonella enterica subsp. enterica from food-producing animals and food products,in Portugal

A total of 1120 Salmonella spp. isolates, recovered from poultry, swine and food products of animal origin (bovine, swine and poultry) over the period of 2009–2011, were investigated in order to determine their serotype, susceptibility to a panel of eleven antimicrobials (A, ampicillin; Ct, cefotaxime; Cp, ciprofloxacin; Tm, trimethoprim; Su, sulfamethoxazole; C, chloramphenicol; S, streptomycin; G, gentamicin; T, tetracycline; NA, nalidixic acid; Fl, florfenicol), and the presence of resistance determinants of extended-spectrum cephalosporins. Overall, Salmonella Enteritidis was the most common serotype in all three animal species. In 618 isolates of poultry, 32.8% comprised S. Enteritidis, 18.3% Salmonella Havana and 16.5% Salmonella Mbandaka; in 101 isolates of pigs, 21.8% comprised Salmonella Rissen and Salmonella Typhimurium, 10.9% Salmonella Derby and Salmonella London. Salmonella I 4,[5],12:i:- was the most common serotype recovered from pork and beef food products comprising 32.6% and 30% of isolates respectively, followed by S. Rissen (26% and 24%) and S. Typhimurium (18.2% and 19%), respectively. In poultry products, S. Enteritidis was the most frequent serotype (62.7%), followed by S. Mbandaka (10.2%) and S. Derby (8.5%). Susceptibility profiles differed according to the origin of the isolates. Five multidrug resistant isolates (0.45%) were further characterized as extended-spectrum β-lactamase (ESBL) producers. Polymerase chain reaction and sequencing of the amplicons confirmed the presence of bla_CTX-M-1 (n = 2), bla_CTX-M-14 (n = 1), bla_CTX-M-15 (n = 1) and bla_CTX-M-32 (n = 1); bla_SHV-12 and bla_TEM-1 genes were also detected in two isolates of S. I 4,[5],12:i:-. Four isolates, two S. Havana and two S. I 4,[5],12:i:-, carried class 1 integrons and in three, two S. I 4,[5],12:i:- and one S. Havana, ISEcp1 was identified associated to bla_CTX-M-1, bla_CTX-M-32 and bla_CTX-M-14 genes. Additionally, in one S. I 4,[5],12:i:- isolate, orf477 was identified linked to bla_CTX-M-32. No plasmid mediated quinolone resistance-encoding genes were detected. Here, we report for the first time the presence of bla_CTX-M genes in Salmonella enterica subsp enterica isolates recovered from poultry and food products of swine origin, in Portugal.     

In vitro and in vivo assessment of cytochrome P450-mediated herb–drug interaction of Ssang-hwa-tang

We have evaluated the herb–drug interaction potential of Ssang-hwa-tang (SHT) mediated by cytochrome P450 (CYP) inhibition/induction. Further, the effects of fermentation on the CYP-mediated herb–drug interaction potential were determined. SHT showed inhibitory activity toward CYP1A2, but not 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4 in human liver microsomes. The results of the enzyme kinetic study suggested that the SHT-induced CYP1A2 inhibition is mixed reversible inhibition. The hepatic CYP expression and activity in rats treated with SHT were examined. The expression/activity of CYP2E1 increased as a result of SHT extract treatment (P < 0.005 or P < 0.001, respectively), which raises the possibility that SHT may increase the toxicity of environmental toxicants through the elevation of CYP2E1-mediated metabolic activation. SHT fermentation using Lactobacillus fermentum or Lactobacillus gasseri resulted in attenuation of the SHT-induced CYP1A2 inhibition, but not CYP2E1 induction, suggesting that changes in the chemical composition of SHT through fermentation can affect the inhibition of CYP1A2 activity.     

Survival of three Bifidobacterium animalis subsp. lactis strains is related to trans-vaccenic and α-linolenic acids contents in organic fermented milks

This study aims at relating the survival at 4 °C for 28 days in organic and conventional fermented milks of three strains of Bifidobacterium animalis subsp. lactis (BB12, B94 and BL04), in co-culture with Streptococcus thermophilus TA040 and Lactobacillus bulgaricus LB340, to milk fatty acids profile. Cultivability after 28 days of cold storage was improved in organic fermented milks as compared to conventional products, with slight differences among strains. In addition, the poly-unsaturated fatty acids fraction was higher in organic products, as well as the relative trans-vaccenic (TVA) and α-linolenic (ALA) fatty acids contents that were respectively 1.7 and 2.4 times higher in organic than in conventional fermented milks. From these results, it was concluded that elevated levels of TVA and ALA, together with a lower ratio between linoleic acid and α-linolenic acid, as found in organic products, improved the survival of the bifidobacteria during chilled storage.     

Structure–activity relationships of acetylated flavone glycosides from Galeopsis ladanum L. (Lamiaceae)

Antioxidant activity, neuroprotective effect and acetylcholinesterase inhibitor activity were studied in acetylated flavones from Galeopsis ladanum L. (Lamiaceae), previously isolated and identified by UV-spectra, UPLC–MS/MS and ¹H-NMR spectroscopy. Structure–activity relationships of flavonoids have been determined in many antioxidant assays, generally focused on hydroxyl groups. In this study we have detected new interesting structure–activity relationships for the isolated flavonoids due to the acetylation of sugar moiety of these flavonoids. Methylation at 4′-OH and monoacetylation is beneficial to inhibiting AChE and shows a neouroprotective effect.     

GC–MS quantification and sensory thresholds of guaiacol in orange juice and its correlation with Alicyclobacillus spp.

Guaiacol is a profoundly negative taint/off-flavour produced by the increasingly common microbial contamination of fruit juices with Alicyclobacillus spp. The objectives of this study included: determining sensory thresholds for guaiacol in orange juice (OJ), developing an analytical method whose detection limits were equivalent to sensory thresholds and determining levels of Alicyclobacillus spp. that would produce detectable levels of guaiacol. A 12 member trained panel was used to establish guaiacol detection and recognition thresholds. Guaiacol ortho and retro nasal detection thresholds in OJ were 0.70 and 0.53 μg/l respectively. Odour recognition threshold was 2.0 μg/l. A SPME GC–MS Selected Ion Monitoring (SIM) procedure was optimised to achieve analytical detection limits of 0.5 μg/l. Optimum guaiacol detection limit was achieved using only responses from m/z 109 and 124. Ion ratios (m/z 109/124) and linear retention index value matching were used to confirm the identification of guaiacol. Quantification was achieved using peak areas from standard guaiacol additions in orange juice between 0.5 and 100 μg/l. Alicyclobacillus growth of 2.2 log CFU/ml in OJ produced just detectable levels (0.7 μg/l) of guaiacol.     

Bioassay-guided screening and isolation of α-glucosidase and tyrosinase inhibitors from leaves of Morus alba

In this study, bioassay-guided fractionation of extracts from the leaves of Morus alba L. led to the isolation of 15 bioactive constituents with α-glucosidase and tyrosinase inhibitory activities, among which prenylated stilbenes were proved to be a new group of α-glucosidase inhibitors apart from iminosugars derived from Morus alba. Their structures were identified on the basis of extensive spectroscopic analysis and chemical evidence, as well as comparing with data from the literature. Among them, compounds (2R)/(2S)-Euchrenone a₇ (6a/6b), Chalcomoracin (7), Moracin C (8), Moracin D (9) and Moracin N (10) exhibited a significant degree of α-glucosidase inhibitory activity with IC₅₀ of 6.28, 2.59, 4.04, 2.54 and 2.76 μM, respectively, while (2R)/(2S)-Euchrenone a₇ (6a/6b), Moracin N (10), Quercetin (13), Norartocarpetin (14), the interconvertible epimeric mixture of (2R)/(2S)-7-methoxyl-8-ethyl-2′,4′-dihydroxylflavane-2″-O-β-d-glucopyranoside (1a/1b) and the interconvertible enantiomers of (2R)/(2S)-7-methoxyl-8-hydroxyethyl-2′,4′-dihydroxylflavane (5a/5b) displayed a potent tyrosinase inhibitory effect with IC₅₀ of 0.260, 0.924, 0.523, 0.0824, 0.616 and 0.528 μM, respectively. Especially, (2R)-7-methoxyl-8-ethyl-2′,4′-dihydroxylflavane-2″-O-β-d-glucopyranoside (1a), (2S)-7-methoxyl-8-ethyl-2′,4′-dihydroxylflavane-2″-O-β-d-glucopyranoside (1b), (2S)-8-hydroxyethyl-7,4′-dimethoxylflavane-2′-O-β-d-glucopyranoside (2), (2R)-7-methoxyl-8-hydroxyethyl-2′,4′-dihydroxylflavane (5a) and (2S)-7-methoxyl-8-hydroxyethyl-2′,4′-dihydroxylflavane (5b) were identified as new compounds.     

Impact of the co-culture of Saccharomyces cerevisiae–Oenococcus oeni on malolactic fermentation and partial characterization of a yeast-derived inhibitory peptidic fraction

The present study was aimed to evaluate the impact of the co-culture on the output of malolactic fermentation and to further investigate the reasons of the antagonism exerted by yeasts towards bacteria during sequential cultures. The Saccharomyces cerevisiae D strain/Oenococcus oeni X strain combination was tested by applying both sequential culture and co-culture strategies. This pair was chosen amongst others because the malolactic fermentation was particularly difficult to realize during the sequential culture. During this traditional procedure, malolactic fermentation started when alcoholic fermentation was achieved. For the co-culture, both fermentations were conducted together by inoculating yeasts and bacteria into a membrane bioreactor at the same time. Results obtained during the sequential culture and compared to a bacterial control medium, showed that the inhibition exerted by S. cerevisiae D strain in term of decrease of the malic acid consumption rate was mainly due to ethanol (75%) and to a peptidic fraction (25%) having an MW between 5 and 10 kDa. 0.4 g l⁻¹ of l-malic acid was consumed in this case while 3.7 g l⁻¹ was consumed when the co-culture was applied. In addition, there was no risk of increased volatile acidity during the co-culture. Therefore, the co-culture strategy was considered effective for malolactic fermentation with the yeast/bacteria pair studied.     

Effect of production system on physical–chemical,antioxidant and fatty acids composition of Longissimus dorsi and Serratus ventralis muscles from Iberian pig

The effect of three production systems of Iberian pigs namely Montanera (free-range system and feeding based on acorns and grass), Recebo (free-range system and nutrition based in combination of acorns, grass and mixed feeds) and Intensive (confinement with mixed feeds) on some quality traits of Longissimus dorsi (LD) and Serratus ventralis (SV) muscles were studied. Muscles from pigs raised in the Montanera system showed significantly higher CIE L^∗, a^∗ and b^∗ values and higher haem pigment content than those from Intensive system. Similarly, muscles from pigs raised in the Montanera system had significantly higher contents of α and γ-tocopherol and phenolic compounds contents and higher lipophilic and hydrophilic activity antioxidant than those from pigs raised in the Intensive system. Fatty acids profiles from Montanera pigs had significantly higher monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids and lower saturated fatty acids (SFA) than those from pigs raised in the Intensive system. In relation to muscle effect, LD showed lower intramuscular fat (IMF), α-tocopherol, phenolic compounds, lipid oxidation and PUFA, but higher MUFA than SV.     

Toxicity and growth inhibitory activities of methanol extract and the β-carboline alkaloids of Peganum harmala L. against two coleopteran stored-grain pests

Methanol extract and the β-carboline alkaloids were extracted from the seeds of Peganum harmala (Zygophyllaceae). Their toxicity, growth inhibitory and effects on the progeny production of Tribolium castaneum and Rhyzopertha dominica was studied. To assess any additive effects among the extracted β-carbolines, they were tested as binary mixtures (1:1) or as a crude alkaloid fraction. All extracts exhibited a considerable adulticidal effect with increasing activities in response to increased exposure period. Using the contact toxicity bioassay, the crude β-carboline fraction was the most effective (LC_50’s were 20.1 and 36.7) μg/cm², 48 h post-treatment against R. dominica and T. castaneum, respectively. LC_50’s of (harmaline + harmine), (harmaline + harmane), and methanol extract were (31.2, 39.4), (33.7, 47.2), and (39.8, 65.2) μg/cm², 24 h post-treatment against R. dominica and T. castaneum, respectively. At 48 h post-treatment, LC₅₀ of (harmaline + harmine) reached 22.4 μg/cm² against R. dominica. When mixed with the insect’s diets, toxicity of all extracts were increased with the crude alkaloidal fraction the most toxic (LC_50’s were 7.8 and 14.7) mg/kg grains, 48 h post exposure against R. dominica and T. castaneum, respectively. When the 2nd instar larvae were fed sub-lethal doses-treated grains, development and F1 progeny of both insects were significantly affected (P ≤ 0.001). At 3.5 mg/kg grains of the crude alkaloidal extract, percentages of malformed larvae and pupae of T. castaneum were 19.7 and 33.4%, respectively. In this case, a total life span of 81.3 days was recorded for the treated individuals compared to 44.2 for the control. A reduction in the adult progeny of 56.9, 44.0 and 43.6% was obtained with 3.5 mg/kg of the crude alkaloids, (harmaline + harmine) and methanol extract, respectively. Meanwhile, the reduction in adult progeny of R. dominica reached 79.2% with the same concentration of the crude alkaloid extract.     

Substrate specificities and mechanism of action of multiple α-galactosidases from Streptomyces griseoloalbus

α-Galactosidase or melibiase is a versatile enzyme with many potential biotechnological and industrial applications. The substrate specificities of three α-galactosidases – α-Gal I, α-Gal II, and α-Gal III – from Streptomyces griseoloalbus were studied using different galactose-containing natural substrates like melibiose, raffinose and stachyose. The kinetic parameters K_m and V_max were determined from the Lineweaver–Burk plot. α-Gal I showed highest affinity towards melibiose where as α-Gal II and α-Gal III showed highest affinity towards stachyose. The ¹H NMR studies showed that all the three α-galactosidases had a retaining mechanism of hydrolysis.     

Antioxidant properties of 1,2,3,4,6-penta-O-galloyl-β-d-glucose from Elaeocarpus sylvestris var. ellipticus

The antioxidant potential of 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG), isolated from Elaeocarpus sylvestris var. ellipticus, was investigated by various established systems based on cell-free and cell system experiments, such as radical detection, antioxidant enzyme assay, lipid peroxidation detection, and cell viability assay. PGG was found to quench the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and intracellular reactive oxygen species. PGG recovered the cellular antioxidant enzyme activities of superoxide dismutase, catalase, and glutathione peroxidase, which were reduced by H₂O₂ treatment, thereby resulting in the inhibition of lipid peroxidation. Cytoprotective effects of PGG were based on the results of DNA fragmentation, mitochondrial membrane potential (Δψ), apoptotic body formation, and caspase-3 activity. The results suggest that PGG protects cells against H₂O₂-induced cell damage via antioxidant properties.     

Fractionation of lipids and purification of γ-linolenic acid (GLA) from Spirulinaplatensis

The microalgae, Spirulinaplatensis, is an excellent source of γ-linolenic acid (GLA), an essential polyunsaturated fatty acid and a potent nutraceutical. The fatty acid composition of S.platensis ARM 740 was determined after transmethylation by gas chromatography (GC). Lipid fractionation was achieved on silica gel column chromatography and preparative TLC. Neutral lipids, glycolipids and phospholipids accounted for 77.0%, 15.6% and 7.4%, respectively, of the total lipid fraction. S.platensis ARM 740 was found to contain 94% of the total GLA in the glycolipid fraction. Attempts were made to purify GLA methyl ester by using urea to form inclusion complexes with the saturated and the less unsaturated FAMEs (fatty acid methyl esters), which enhanced the purity of GLA methyl ester to 84%. A further approach to isolate GLA methyl ester with higher purity involved the use of argentated silica gel chromatography. An initial PUFA concentration step frequently adopted by most researchers to increase GLA purity was not necessary in the isolation of GLA from S.platensis. A GLA methyl ester with a purity of >96% and a recovery of 66% was obtained. Purity of the isolated GLA methyl ester was confirmed by GC and IR analysis with respect to authentic standard.     

Enzymatic properties and transglycosylation of α-galactosidase from Penicillium oxalicum SO

Penicillium oxalicum SO α-galactosidase demonstrated weak hydrolysing activity but a high rate of transglycosylation in the reaction with melibiose, where the major product was 6-α-galactosyl melibiose. The transfer ratio was 83.6% and was maintained over a long reaction time of 80 h. The molecular weight was estimated to be 124,000 by SDS–PAGE. The optimal pH was ∼3 and a stable pH, with a range of 2.4–9.5, was found. The optimal temperature was ∼60 °C and the activity was stable below 60 °C. With respect to acceptor specificity, mono-alcohols, sugar alcohols and sugars were poor acceptors, but the di-alcohol ethylene glycol and the tri-alcohol glycerin were good acceptors. The percentage of transglycosylation to glycerin increased up to 41.7%, as that to melibiose decreased, with the initial glycerin concentration of 40%. The production of α-d-galactosylglycerol was 293 mg for each gram of melibiose used by the enzymatic reaction.     

Identification of an acidic α-amylase from Alicyclobacillus sp. A4 and assessment of its application in the starch industry

An acidic α-amylase was purified from thermoacidophilic Alicyclobacillus sp. A4 by ion exchange chromatography with 22% recovery, and showed a molecular mass of 64 kDa by SDS-PAGE. Its amino acid sequence was determined by sequencing three internal peptides and the complete genome of strain A4, and shared highest identity (64%) with Alicyclobacillusacidocaldarius α-amylase. Compared with other reported α-amylases, the purified enzyme had some distinct characteristics. The optimal activity was found to occur at 75 °C and pH 4.2, similar to the glucamylase widely used in the starch industry. The enzyme was Ca²⁺ independent, and had strong ability to digest raw starch (96.71%) with commercial glucamylase in one step. These properties of the purified enzyme make up the deficiency of the commercial α-amylases currently used and avoid repeated adjustment of pH and temperature in double-enzymatic sugar-making process. The purified enzyme will be commercially valuable in the starch industry.     

Gas chromatographic–mass spectrometric characterisation of triterpene alcohols and monomethylsterols in developing Olea europaea L. fruits

Five triterpene alcohols and four 4-monomethylsterols were identified by GC–MS during the ripening of Picholine olive. The quantitative characterisation of these compounds was performed using GC–FID. The results showed that the maximum level of total triterpene alcohols (263.68 mg/100 g oil) was reached at 26th week after the flowering date (WAF) of olive; whilst the highest level of total 4-monomethylsterols (234 mg/100 g oil) was attained at 24th WAF of fruit. The percentage of these two classes represented 20–33% of total phytosterols during olive maturity. 24-Methylene cycloartenol (12–207 mg/100 g oil) and cycloartenol (27–198 mg/100 g oil) were the predominant triterpene alcohols during the ripening of Picholine olive; whereas citrostadienol (30–161 mg/100 g oil) and cycloeucalenol (11–74 mg/100 g oil) were the main 4-monomethylsterol compounds followed by obtusifoliol and gramisterol. β-Amyrin, δ-amyrin and traroxerol were less present in Picholine olive and they accounted for 14% of total triterpene alcohols at complete maturity of fruit. The level of these methylsterols was overwhelmed by the amount of 4-desmethylsterols at each stage of Picholine olive maturity.     

Comparing the in vitro effects of MGO™ Manuka honey and tea tree oil on ocular Demodex viability

Purpose

To compare the in vitro antiparasitic effects of MGO^? Manuka honey and tea tree oil against ocular Demodex.