Luis Javier R. Barron

Methods for the analysis of triacylglycerols

This article discusses the methods most commonly employed in the analysis of the triacylglycerols (TAGs) in natural fats and considers the main advantages and disadvantages of each and the techniques for optimising analytical conditions. Complete analysis of the composition of a natural fat calls for a method of extracting and purifying the triglyceride fraction, normally by preparatory thin-layer and column chromatography. Determination of the individual components of triglyceride mixtures still entails certain difficulties, namely, the separation and identification of the TAGs in natural fats. High-performance liquid chromatography (HPLC) offers significant advantages over gas and thin-layer chromatography. Many workers have developed non-aqueous, reversed-phase HPLC systems capable of successfully resolving complex mixtures of TAGs, and combining reversed-phase (RP) HPLC and argentation chromatography may improve the results. Identification of the TAGs separated by HPLC becomes an extremely complex task if many different fatty acids are involved and if the sn-stereoscopic positions on the glycerol are to be determined. Enzymatic analysis and chiral-phase chromatography are capable of localising fatty acids on the TAG molecule. In closing, some of the most interesting biomedical applications of TAG analysis are summarised.

Coagulating and lipolytic activities of artisanal lamb rennet pastes

Lamb rennet pastes were prepared by the procedure most commonly used by Idiazabal cheese manufacturers. We studied the effects on their coagulating and lipolytic activities of the state of the stomach at the time of death (full of milk or empty), the amount of NaCl added, the origin of the lambs and paste storage time. Coagulating activities were generally between 155 and 363 units/g tissue. Pastes prepared from stomachs of lambs from slaughterhouse flocks had significantly higher coagulating activities than those of lambs from separate flocks. No significant decrease in coagulating activity was observed after 1 year storage at 4 degrees C. Chymosin represented 75-80% of the total coagulating activity with the remainder being pepsin. Rennet paste extracts with pH < 4.7 did not have increased coagulating activities when their pH was lowered to 2.0, while those with pH > 5.2 had activities 1.5-fold those before treatment. Lipase activity was higher in extracts of rennet pastes prepared using the stomachs of lambs that arrived at the slaughterhouse in the morning just prior to slaughter than in those prepared with the stomachs of lambs that had arrived on the previous evening. However, the reverse was the case for esterase activity. Activating the coagulating activity by pH cycling completely destroyed both lipolytic activities. Storage at 4 degrees C for > 1 year did not affect esterase activity but lipase activity decreased substantially after 4-5 months. Lipase, but not esterase, activity was responsible for the liberation of short-chain free fatty acids from ovine milk fat.

Determination of free fatty acids in cheese: comparison of two analytical methods

Two methods were compared for the determination of free fatty acids (FFA) from acetic to long-chain acids in samples with a large excess of triacylglycerols (TG) (1[ratio ]200, w/w), such as cheese and other dairy products. In method 1, after fat extraction, FFA were separated from TG by aminopropyl-bonded phase chromatography, injecting the fraction containing FFA directly into the gas chromatograph. In method 2, extracted fat was treated with tetramethylammonium hydroxide, the methyl ester derivatives being formed in the injector. Cheese samples and standard mixtures of FFA and TG in different proportions were analysed by both methods. The cheese sample contained 2·4 times more FFA when analysed by method 2 as compared with the result obtained with method 1. The composition of the standard mixtures analysed by method 1 closely reflected that of the original mixture and gave 90–100% recovery of FFA, regardless of their chain length and the ratio of FFA[ratio ]TG (1[ratio ]1 or 1[ratio ]200, w/w). The composition of samples with a FFA[ratio ]TG ratio of 1[ratio ]200 (w/v) was severely distorted (as compared with the original composition of the sample) when analysed by method 2. Varying recoveries of FFA were also obtained, the largest differences being found for the shorter-chain components. We conclude that the FFA fraction should be separated from the TG fraction before derivatization and chromatographic analysis, particularly for samples in which the FFA represent a minor fraction of the TG.

Seasonal changes in the composition of bulk raw ewe's milk used for Idiazabal cheese manufacture

Seasonal changes in the composition of bulk raw ewes milk used for Idiazabal cheese manufacture and its influence on the cheese yield are reviewed. This review is based on biochemical and microbiological data previously published on the composition of a bulk raw ewes milk collected at three different times of the year. In order to characterize, in a more extensive perspective, the seasonal changes in the composition of this bulk raw ewes milk and its influence on actual cheese yield, multivariate statistical analyses were applied to the compositional data of the milk and the actual cheese yield obtained at the factory. The results show pronounced seasonal changes in the composition of bulk raw ewes milk. Changes in cheese yield, lipolytic activity and microbiological quality of the bulk raw ewes milk are the principal aspects seasonally affected during the cheesemaking period. Also, the seasonal changes observed in the bulk raw ewes milk composition strongly affect the quality of Idiazabal cheeses manufactured over the cheesemaking period.

Lamb rennet paste in ovine cheese (Idiazabal) manufacture. Proteolysis and relationship between analytical and sensory parameters

Abstract Cheeses were manufactured with 2 levels of lamb rennet paste or bovine rennet, at two times of the year, to study the effect of artisanally prepared lamb rennet paste (at high and low levels) on the concentrations of proteolysis products (nitrogen fractions, casein degradation and free amino acids). Cheeses made with the lamb rennet paste presented a significantly different evolution of the αs1-I-casein fraction during ripening, as measured by urea polyacrylamide gel electrophoresis. The time of the year affected the concentrations of 39% of the individual free amino acids, as determined by reversed-phase high-pressure liquid chromatography. The relationship between some biochemical parameters (gross composition, degrees of lipolysis (Lamb rennet paste in ovine cheese manufacture. Lipolysis and flavour. International Dairy Journal (2003) in press) and proteolysis) and the sensory characteristics of the cheese (Virto et al., 2003) were studied by a principal component analysis. The effect of the technological parameters “type of rennet” and “time of the year” on these analytical variables and sensory characteristics of the cheese was described by three principal components which explained 80.8% of the total variance. The factor “rennet paste” included concentrations of short chain free fatty acids, sensory descriptors related to strong odours and flavours, an unidentified peptide A1, and αs1-I-caseins. The effect of the ‘time of the year’ was described by two other factors: the “secondary proteolysis” factor, which included the concentrations of free amino acids and could be related to the different endogenous microflora of the raw milk, and the “seasonal milk composition” factor, which included the fat content and the plasmin activity of the milk.

Changes in the free amino acid content during ripening of Idiazabal cheese: influence of starter and rennet type

The effect of starter and rennet type on free amino acid release during ripening of Idiazabal cheese was studied. Four batches of cheeses were manufactured depending on the rennet used, commercial calf rennet or artisanal lamb rennet, and the addition or not of starter culture. Cheese samples contained 24 individual free amino acids Leu, Glu, Val and Phe showing the highest contents during the ripening. The results indicated that the release of the free amino acids during ripening was strongly affected by starter added to the cheeses, and that this effect varied markedly with the rennet used for cheesemaking. Total amounts of free amino acids were higher for the cheeses made with commercial calf rennet than for those made with artisanal lamb rennet, regardless of starter addition. Likewise, the highest total free amino acid levels were found in the cheeses made with starter, regardless of the type of rennet used.

Determination of rat liver triglycerides by gas–liquid chromatography and reversed-phase high-performance liquid chromatography

Rats fed with a fat-free or an olive oil-rich diet were employed to compare the response of two chromatographic techniques in the determination of rat liver triglyceride (TG) molecular species composition. Gas-liquid chromatography (GLC) on polarizable liquid phase and reversed-phase high-performance liquid chromatography (RP-HPLC) have been commonly employed for TG analysis, obtaining a similar number of chromatographic peaks when used for animal tissue TG determination. In the present study similar results were achieved with regard to most relevant chromatographic peaks, however, important differences were found in the content of minor TGs. Indeed, RP-HPLC permitted separation of long chain polyunsaturated fatty acids, which were not detected by GLC, while the latter technique reported a higher number of myristoyl-containing TG species. RP-HPLC analysis reported a greater number of TGs, with more similarity to a random composition, made up from the liver fatty acid composition. Therefore, it was concluded that utilization of both techniques would be helpful for liver TG analysis as the use of only one of them does not provide a complete profile of liver TGs. Nevertheless RP-HPLC seems to be more useful for this purpose since revealed a more extensive profile.

Simultaneous analysis of carotenoids and tocopherols in botanical species using one step solid-liquid extraction followed by high performance liquid chromatography.

Carotenoids and tocopherols from botanical species abundant in Atlantic mountain grasslands were simultaneously extracted using one-step solid-liquid phase. A single n-hexane/2-propanol extract containing both types of compounds was injected twice under two different sets of HPLC conditions to separate the tocopherols by normal-phase chromatography and carotenoids by reverse-phase mode. The method allowed reproducible quantification in plant samples of very low amounts of α-, β-, γ- and δ-tocopherols (LOD from 0.0379 to 0.0720 μg g(-1) DM) and over 15 different xanthophylls and carotene isomers. The simplified one-step extraction without saponification significantly increased the recovery of tocopherols and carotenoids, thereby enabling the determination of α-tocopherol acetate in plant samples. The two different sets of chromatographic analysis provided near baseline separation of individual compounds without interference from other lipid compounds extracted from plants, and a very sensitive and accurate detection of tocopherols and carotenoids. The detection of minor individual components in botanical species from grasslands is nowadays of high interest in searching for biomarkers for foods derived from grazing animals.

Winter/Spring Changes in Fatty Acid Composition of Farmhouse Idiazabal Cheese Due to Different Flock Management Systems

Typically, two different flock managements are employed by basque sheepherders in winter and spring. Thus, seasonal changes in the fatty acid (FA) composition of Idiazabal PDO farmhouse cheeses were studied. Ewes raw milk cheeses elaborated in winter and spring were collected after 120 days of ripening from 10 Idiazabal PDO farmhouses. In winter, concentrate and conserved forages were fed, whereas a part-time grazing system was adopted from spring onward. Spring cheeses had less (P <or= 0.05) saturated FA and higher (P <or= 0.05) content of unsaturated FA, including trans-FA (mainly trans-vaccenic acid) and conjugated linoleic acid (CLA), branched-chain FA (BCFA), and n-3 FA. Principal component analysis (PCA) separated winter and spring cheeses into two groups by the combination of two principal components (84.2% of variance). Fresh pasture in the diet enhanced desirable FA and lowered atherogenicity index in cheeses, supporting the benefits of using a part-time grazing system for the consumer.

Characterization of the fatty acid composition of lamb commercially available in northern Spain: Emphasis on the trans-18:1 and CLA content and profile

A survey of commercially available lamb meat was performed in northern Spain in order to evaluate their fatty acid (FA) composition with emphasis on trans fatty acid (TFA) and conjugated linoleic acid (CLA) isomers. Samples were collected in spring (n=24) and winter (n=24) of 2013, and were obtained in about equal numbers from grocery stores and butcher-shops. Subcutaneous fat, known to be a sensitive indicator of TFA content in ruminants, was analyzed by GC-FID. In general, very few differences were observed between collection periods and type of stores because of the high variability within the groups that was believed to be associated with differences in genetics and feeding strategies. However, the 10t/11t ratio of all samples showed two clearly identifiable groups irrespective of the source: 1) when 10t/11t was >1, 10t-shifted samples; 2) when 10t/11t was ≤1, non-shifted samples where 11t-18:1 was the predominant isomer. These two groups were clearly identified and associated with distinct FAs using principal component analysis.