Direct Derivatization vs Aqueous Extraction Methods of Fecal Free Fatty Acids for GC-MS Analysis.

A comprehensive and accurate determination of free fatty acids (FFA) is required for fecal metabolomic investigations. The present study compares three aqueous extraction methods (1) ULTRA-TURRAX(®), (2) whirl mixing and (3) basic ULTRA-TURRAX extraction of fecal FFA with a direct derivatization approach using ethyl chloroformate as the derivatization reagent before determination by gas chromatography-mass spectrometry. The direct derivatization method resulted in significantly higher estimations (P < 0.01) of short- and long-chain fatty acids than was the case when applying the aqueous extraction methods using ULTRA-TURRAX, whirl mixing, or basic ULTRA-TURRAX extraction before the derivatization step. Thus, avoiding an aqueous extraction before derivatization reduces the loss of volatile short-chain FFA and the less water-soluble long-chain FFA.

Time-saving design of experiment protocol for optimization of LC-MS data processing in metabolomic approaches.

We describe a time-saving protocol for the processing of LC-MS-based metabolomics data by optimizing parameter settings in XCMS and threshold settings for removing noisy and low-intensity peaks using design of experiment (DoE) approaches including Plackett-Burman design (PBD) for screening and central composite design (CCD) for optimization. A reliability index, which is based on evaluation of the linear response to a dilution series, was used as a parameter for the assessment of data quality. After identifying the significant parameters in the XCMS software by PBD, CCD was applied to determine their values by maximizing the reliability and group indexes. Optimal settings by DoE resulted in improvements of 19.4% and 54.7% in the reliability index for a standard mixture and human urine, respectively, as compared with the default setting, and a total of 38 h was required to complete the optimization. Moreover, threshold settings were optimized by using CCD for further improvement. The approach combining optimal parameter setting and the threshold method improved the reliability index about 9.5 times for a standards mixture and 14.5 times for human urine data, which required a total of 41 h. Validation results also showed improvements in the reliability index of about 5-7 times even for urine samples from different subjects. It is concluded that the proposed methodology can be used as a time-saving approach for improving the processing of LC-MS-based metabolomics data.

Chemical composition and sensory quality of bovine milk as affected by type of forage and proportion of concentrate in the feed ration.

BACKGROUND: The objective of this study was to investigate how some small changes in the forage content of maize and lucerne silage and in the ration between forage and concentrate in the diet of dairy cows affect milk quality. Milk quality was assessed by quantitative descriptive sensory analysis and by analysis of tocopherols and carotenoids as well as fatty acid composition. RESULTS: Changing the ratio between maize silage and lucerne silage from 5:1 to 2:1 increased milk fat content of carotenoids (23-27%) and C18:3 n3 (15%), and reduced stale aroma and creamy flavour. Increasing the proportion of concentrates in the feed ration from 0.2 to 0.4 increased energy corrected milk yield (26%), reduced fat content (-10%), increased C18 fatty acids (8-62%) and reduced C16 (-20%) content in milk fat. In addition, this milk type was described by the sensory panel as less oily, less saturated and less yellow. The changes in milk composition were related to differences in feed composition. CONCLUSION: The study revealed the potential to produce milk with a distinct composition and sensory quality based on even small changes in the feed composition that are straightforward to implement by farmers.

Site-specific detection of radicals on α-lactalbumin after a riboflavin-sensitized reaction, detected by immuno-spin trapping, ESR, and MS.

Free radicals and other oxidation products were characterized on α-lactalbumin with electron spin resonance (ESR), immuno-spin trapping, and mass spectrometry (MS) after riboflavin-mediated oxidation. Radicals were detected using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in immuno-spin trapping with both enzyme-linked immunosorbent assay (ELISA) and Western blotting and further characterized with mass spectrometry. A DMPO-trapped radical was identified at His68 and another at one of the tyrosine residues, Tyr50 or Tyr36, respectively, generated by a type II or I mechanism. Not all tyrosyl radicals were trapped, as the secondary oxidation product, 3,4-dihydroxyphenylalanine (DOPA), was detected by mass spectrometry at Tyr18 and Tyr50. A further oxidation of DOPA resulted in the DOPA o-semiquinone radical, which was characterized by ESR. Both surface exposure and the neighboring residues in the local environment of the tertiary structure of α-lactalbumin seem to play a role in the generation of DMPO trapped radicals and secondary oxidation products.

Effect of antioxidants on oxidation during the production of whey fat concentrate.

Whey fat has a relatively high level of unsaturated fatty acids, and as such, whey products with a high fat content are vulnerable to oxidation. The purposes of the present study were to assess the oxidative development in whey fat concentrate (WFC) during production and investigate the effect of the addition of antioxidants. Green tea extract (GTE) or a mixture of ascorbyl palmitate and tocopherol (AP/TOC) were used, each in two concentrations. Samples were taken before and after pasteurization of WFC and after drying. The level of volatile oxidation products decreased during processing, while dityrosine concentrations increased during drying. GTE reduced oxidation in both unpasteurized and pasteurized WFC, while the effect of AP/TOC was nonsignificant. In the WFC powder, there was no significant effect of the antioxidants. In conclusion, results indicated that GTE was able to inhibit oxidation in WFC during production and that AP/TOC addition had no effect.

Fluorescence spectroscopy: a rapid tool for analyzing dairy products.

This paper gives a critical evaluation of the use of fluorescence spectroscopy for measuring chemical and physical changes in dairy products caused by processing and storage. Fluorescence spectroscopy is able to determine various properties of foods without use of chemicals and time-consuming sample preparation. This is shown by examples where the measurement of a given chemical parameter has been appropriately described and validated, as well as situations showing potential applications, but where further research and validation is required. The interpretation of fluorescence spectroscopic data is complex due to absorbance by other molecular groups, changes caused by variation in the sample matrix, etc. It is illustrated how advanced data analytical techniques are required to obtain optimal interpretation of the data. Even though the review focuses on examples from the dairy industry, the principles are broader and can be applied to other fields of food and agricultural research.

Effect of specific wavelengths on light-induced quality changes in Havarti cheese.

The effects of exposure of slices of Havarti cheeses to monochromatic light of wavelengths 366 nm, 405 nm, and 436 nm, respectively, were studied by tristimulus colorimetry, solid-phase microextraction gas chromatographic analysis of volatiles, and open-end fluorescence spectroscopy. Having determined the photon fluxes of the three wavelengths by ferrioxalate actinometry, it was possible to quantify the effects of light exposure in an absolute manner. For all analyses, the most severe effects were caused by visible light, leading to colour bleaching, change in hue, riboflavin degradation, and formation of the secondary oxidation products hexanal, 1-pentanol, and 1-hexanol. Apparent quantum yields for formation of hexanal and 1-pentanol were found to be insignificantly different for 405 nm and 436 nm exposures, having values of (3-5) x 10(-5) mol x einstein(-1) and (9-13) x 10(-5) mol x einstein(-1), respectively. These compounds were not formed when exposed to 366 nm light. In contrast, 1-hexanol was formed when exposing cheese to all three wavelengths, resulting in apparent quantum yields of (2-6) x 10(-5) mol x einstein(-1). The results obtained are discussed in relation to the interplay between inherent product colorants, light sources, and transmission characteristics of the packaging materials.

Comparison of peroxide value methods used for semihard cheeses.

The objective was to evaluate alternatives to the peroxide value method of choice in the dairy industry, the method issued by the International Dairy Federation. Furthermore, the study evaluated the feasibility of alternative solvents for extracting lipids and subsequent peroxide value determinations. Packaged cheeses were stored under illuminated display at 4 degrees C to obtain samples with various peroxide contents but with uniform gross composition. The hydroperoxide contents were measured during 3 weeks of storage by applying two lipid extraction methods, Folch and Bureau of Dairy Industry (BDI) extractions, and three different hydroperoxide extraction solutions [chloroform/methanol (7:3, v/v), hexane/2-propanol/methanol (5:7:2, v/v/v), and methanol/decanol/hexane (3:2:1, v/v/v)], prior to standard colorimetric measurements. Extraction yields of fat from Havarti cheeses using the Folch and BDI extraction methods were approximately 109 and 61%, respectively, of the yields obtained by the International Dairy Federation gravimetric reference method. Although differences in fat extraction yields were compensated for, significantly higher peroxide values resulted from the Folch extraction method than from the BDI extraction method. The peroxide values obtained by the three methods were all in the same range, and pronounced linear correlations between peroxide contents determined using the three solutions were noted (r (2) in the range of 0.951-0.983). Peroxide value levels were not significantly different in samples stored in the dark or exposed to light.

Light-induced oxidation in semihard cheeses. Evaluation of methods used to determine levels of oxidation.

Light-induced oxidation in Havarti cheese (38% fat) stored in the dark and exposed to fluorescent light was evaluated by an array of chemical, physical, and spectroscopic methods. Light-induced changes were noticeable already after short exposure times (<12 h). A clear differentiation between samples stored in the dark and samples exposed to 1000 lx fluorescent light was obtained by means of the following methods: color measurements (a values), peroxide value determinations, and evaluations of volatile oxidation products by solid-phase microextraction gas chromatography (SPME-GC). The expected changes in peroxide values in relation to storage time were not evident. Measuring free radicals by electron spin resonance spectrometry could not be done to distinguish between samples, possibly due to the conversion of radicals during sample preparation. However, significant light-exposure effects on secondary oxidation products, detected by SPME-GC, were noted for 1-pentanol, 1-hexanol, nonanal, and benzaldehyde.