Digestibility and allergenicity assessment of enzymatically crosslinked β‐casein

Crosslinking enzymes are frequently used in bioprocessing of dairy products. The aim of this study was to examine the effects of enzymatic crosslinking on IgE binding, allergenicity and digestion stability of β‐casein (CN). β‐CN was crosslinked by transglutaminase, tyrosinase, mushroom tyrosinase/caffeic acid and laccase/caffeic acid. The IgE binding to β‐CN was compared in vitro by CAP inhibition assay, ELISA inhibition as well as ex vivo by basophil activation assay. Crosslinked CNs were digested by simulated gastric fluid for 15 and 60 min and obtained digests analyzed for their ability to inhibit IgE binding by CAP inhibition assay and SDS‐PAGE. The ability of crosslinked CNs to activate basophils was significantly reduced in seven patients in the case of CN crosslinked by laccase and moderately reduced in the case of tyrosinase/caffeic acid crosslinked CN (in two cow's milk allergy patients tested with different allergen concentrations). The response to various crosslinked CNs differed individually among patients' sera tested by ELISA inhibition assay. The presence of caffeic acid hampered digestion by pepsin, and this effect was most pronounced for the tyrosinase/caffeic acid crosslinked CN. The laccase/caffeic acid and mushroom tyrosinase/caffeic acid had the highest potential in mitigating IgE binding and allergenicity of the β‐CN out of all investigated enzymes. The presence of a small phenolic compound also increased digestion stability of β‐CN.     

Blood pressure-lowering properties of chokeberry (Aronia mitchurinii,var. Viking)

The blood pressure-lowering properties of lyophilized chokeberry juice and polyphenols were monitored using in vitro angiotensin-converting enzyme (ACE) inhibition measurement and a 10 day in vivo study with spontaneously hypertensive rats (SHR). Juice and polyphenols indicated weak ACE-inhibitory activity. The IC₅₀ values for polyphenols and juice were 1.5–2.5 and 4.5 mg dry matter/ml, respectively. In the SHR study the blood pressure-lowering effects of juice and polyphenol extract seemed to be short-term and were generally highest after 3 h from administration (50 mg/kg/day) when mean reductions in systolic blood pressure were 20 ± 8 and 15 ± 7 mm Hg, respectively. Corresponding mean decreases in diastolic blood pressure were 23 ± 6 and 13 ± 2 mm Hg in juice and polyphenol groups, respectively. It was concluded that both chokeberry juice and polyphenols had blood pressure-lowering effects. We hypothesize that chokeberry polyphenols enhance endothelial nitric oxide production with an ACE-independent mechanism, e.g. by activation of endothelial nitric oxidase enzyme; this is yet to be verified.     

Carcass and meat quality characteristics of immunocastrated male,surgically castrated male,entire male and female pigs

The aim of the study was to evaluate the effect of immunocastration on meat and carcass quality compared with meat from females, entire and surgically castrated males. One hundred and eighteen (Landrace × Duroc) × Pietrain crossbred pigs were assigned to four experimental groups: entire males (EM), females (FE), surgically castrated males (CM) and vaccinated males (IM). Pigs were reared in two pens per sex and slaughtered at an average of 180 days of age. Carcass and meat quality characteristics such as testis size and length, fat depth, lean content, proportion of the carcass represented by each joint, pH, colour and intramuscular fat were evaluated. There was a significant reduction in the size of these sexual organs in IM compared with EM. CM and IM were fatter than FE and EM in the loin area but, in the ham area, CM was the fattest and EM the leanest, while IM and FE were in between. Intramuscular fat of IM (2.1%) was no different from the other sexes evaluated, although it was higher in CM (2.5%) with respect to FE (1.7%) and EM (1.8%). There was no difference between the IM and other treatment groups in meat quality. Regarding ours results we can conclude that from the point of view of meat and carcass quality the immunocastration could be a good alternative to the surgical castration.     

Determination of l- and d-carnitine in dietary food supplements using capillary electrophoresis–tandem mass spectrometry

This paper reports the application of a capillary electrophoresis–electrospray ionisation-tandem mass spectrometry methodology for the unequivocal identification and the quantitative determination of the two enantiomers of the non-protein amino acid carnitine (l- and d-Carn) in 22 dietary food supplements, including drinks, biscuits, capsules and tablets. MS/MS experiments were optimised to achieve the high sensitivity and selectivity required for food analysis. A comparison of the slopes obtained by the external standard and the standard additions calibration methods indicated the absence of matrix interferences in these food samples. Good precision (RSDs ranged from 2.3% to 4.3% for migration times, and from 2.1% to 10.5% for corrected peak areas) and acceptable accuracy established by means of recovery studies (from 85% to 102%) were obtained. The limit of detection was about 10 ng/mL (S/N = 3) enabling the determination of enantiomeric impurities (d-Carn) up to 0.025% of Carn in foods. This chiral method illustrated is suitable for routine qualitative and quantitative analyses of Carn in foods. The results showed contents for l-Carn comprised from 47% to 115% with respect to the labelled content of l-Carn. Percentages obtained for the d-enantiomer ranged from 0.4% to 5.9%, except for one of the samples analysed, that contained the racemate (49.3% d-Carn). The use of the racemate is not allowed by legislation, which corroborated the high potential of the developed method in the control of the quality and safety of foods containing Carn.     

Efficient Platinum(II) Catalyzed Hydroformylation Reaction in Water: Unusual Product Distribution in Micellar Media

The hydroformylation of a variety of terminal and internal alkenes is efficiently performed by cationic platinum triflate complexes of the type [P₂Pt(H₂O)₂](OTf)₂ under mild conditions in an aqueous micellar medium. The use of surfactants is essential to ensure dissolution of the catalyst and substrate in water with catalysts being positioned on the anionic surface of the micelles. Aldehydes are obtained with linear to branched ratios up to >99:1. With styrene derivatives also the corresponding benzaldehydes are formed. The catalyst can be separated by extraction of the organic products with hexane and recycled for at least four times with only a modest loss of activity and no effect on selectivity.     

Use of the SAW Sensor Electronic Nose for Detecting the Adulteration of Virgin Coconut Oil with RBD Palm Kernel Olein

An electronic nose (zNose™) was applied to the detection of adulteration of virgin coconut oil. The system, which is based on a surface acoustic wave sensor was used to generate a pattern of volatile compounds present in the samples. Virgin coconut oil was mixed with refined, bleached and deodorized palm kernel olein at a level of adulteration from 1 to 20% (wt/wt). Adulterant peaks were identified from the chromatogram profile and fitted to a curve using linear regression. The best relationship (R ² = 0.91) was obtained between the peak tentatively identified as methyl dodecanoate and the percentage of palm kernel olein added. Pearson’s correlation coefficients (r) of 0.92 and 0.89 were obtained between adulterant peak methyl dodecanoate and of the iodine and peroxide values, respectively. Principal component analysis (PCA) was used to differentiate between pure and adulterated samples. The PCA provided good differentiation of samples with 74% of the variation accounted for by PC 1 and 17% accounted for by PC 2. Pure samples formed a separate cluster from all of the adulterated samples.     

Thermal cleavage of the fmoc protection group

The Fmoc protection group is among the most commonly used protection groups for the amino function. A fast method for the thermal deavage of this protection group under base-free conditions without the need for dibenzofulvene scavengers is presented. The advantages of this method include straightforward testability by means of a simple high-temperature NMR experiment, usually high yields, and good selectivity towards the BOC protection group and t-butyl ethers.     

Liquid chromatographic analysis and mass spectrometric identification of farnesylated peptides

Farnesylation involves the post-translational attachment of a 15 carbon unit to the C-terminus of proteins, thus allowing them to incorporate into membranes. The farnesylation reaction requires farnesyldiphosphate as the farnesyl group donor and is catalyzed by the farnesyltransferase. Some of the most familiar farnesylated proteins belong to the Ras protein superfamily, well-known oncoproteins. As Ras proteins require the membrane localization for the transduction of extracellular signals, farnesyltransferase inhibitors are discussed as chemotherapeutic agents. Despite the importance of this post-translational modification, farnesylated peptides have been investigated rarely by means of high-pressure liquid chromatography in combination with mass spectrometry. In this study, we examined the liquid chromatographic separation of farnesylated peptides with the help of the multidimensional protein identification technology. The peptides were further ionized by electrospray ionization and subsequently analyzed by tandem mass spectrometry. We demonstrated that farnesylated peptides are more strongly retained by reversed phase than nonfarnesylated peptides. This allowed for the identification of farnesylated peptides, if spiked into complex peptide samples. In some cases the farnesyl group was apparently split off from the peptide during the ionization process, and tandem mass spectra often revealed a neutral loss of the farnesyl moiety.     

Systematic Evaluation of Extraction Methods for Multiplatform-Based Metabotyping: Application to the Fasciola hepatica Metabolome

Combining data from multiple analytical platforms is essential for comprehensive study of the molecular phenotype (metabotype) of a given biological sample. The metabolite profiles generated are intrinsically dependent on the analytical platforms, each requiring optimization of instrumental parameters, separation conditions, and sample extraction to deliver maximal biological information. An in-depth evaluation of extraction protocols for characterizing the metabolome of the hepatobiliary fluke Fasciola hepatica , using ultra performance liquid chromatography and capillary electrophoresis coupled with mass spectroscopy is presented. The spectrometric methods were characterized by performance, and metrics of merit were established, including precision, mass accuracy, selectivity, sensitivity, and platform stability. Although a core group of molecules was common to all methods, each platform contributed a unique set, whereby 142 metabolites out of 14,724 features were identified. A mixture design revealed that the chloroform:methanol:water proportion of 15:59:26 was globally the best composition for metabolite extraction across UPLC-MS and CE-MS platforms accommodating different columns and ionization modes. Despite the general assumption of the necessity of platform-adapted protocols for achieving effective metabotype characterization, we show that an appropriately designed single extraction procedure is able to fit the requirements of all technologies. This may constitute a paradigm shift in developing efficient protocols for high-throughput metabolite profiling with more-general analytical applicability.     

Aminopeptidase from jumbo squid (Dosidicus gigas) hepatopancreas: purification,characterisation, and casein hydrolysis

An aminopeptidase (AP) was partially purified from jumbo squid (Dosidicus gigas) hepatopancreas with 154.24‐fold and yield of 6.15%. The purification procedure consisted of ammonium sulphate fractionation and DEAE‐Sephacel chromatography. The enzyme was approximately 48–53 kDa as estimated by SDS‐PAGE. With l ‐leu‐p‐NA, it had optimum activity at pH 8.0 and 30 °C. The K_m and V_max/K_m values of the enzymes for l ‐leu‐p‐NA were 0.326 mm and 2787 at 37 °C, respectively. Activation energy (E_a) of the enzyme was 53.50 kJ M^?1.The AP showed activity against seven synthetic substrates: l ‐proline>l ‐methionine>Ac. l ‐γ‐glutamic>l ‐glycine>l ‐leucine>l ‐alanine>l ‐lysine‐p‐NA. The enzyme was strongly inhibited by Bestatin, partially inhibited by a metal‐chelating agent and by PCMB, a cystein protease inhibitor. Zn²⁺ and (or) Ca²⁺ seemed to be its metal cofactor(s). Incubation of casein with the partially purified AP resulted in a degree of hydrolysis of 6%.