Levels of histamine and other biogenic amines in high-quality red wines

Biogenic amines in wine may impair sensory wine quality and cause adverse health effects in susceptible individuals. In this study, histamine and other biogenic amines were determined by HPLC after amine derivatisation to dansyl chloride conjugates in 100 selected high-quality red wines made from seven different cultivars. Amine levels varied considerably between different wines. The most abundant amines were putrescine (median?=?19.4?mg?l^?1, range?=?2.9–122), histamine (7.2?mg?l^?1, 0.5–26.9), and tyramine (3.5?mg?l^?1, 1.1–10.7), whereas lower levels were found for isoamylamine (median?=?0.25?mg?l^?1), phenylethylamine (0.16?mg?l^?1), cadaverine (0.58?mg?l^?1), spermidine (1.8?mg?l^?1) and tryptamine (0.06?mg?l^?1). Positive correlations were observed between isoamylamine and phenylethylamine, and between histamine, putrescine and tyramine levels. Amine concentrations were similar in all wine cultivars except Pinot noir and St. Laurent wines, which showed significantly higher tryptamine and cadaverine levels. The results indicate that levels of histamine and other biogenic amines may vary considerably between red wines independent of grape variety and that high amounts can also be found in high-rated wines. Adopting a legal histamine threshold level of 10?mg?l^?1 in the European Union, as formerly introduced in other countries, would have excluded 34% of the investigated wines from the market.     

Classification of the geographical origin of Italian donkey's milk based on differences in inorganic anions

The content of chlorides, nitrites, nitrates, phosphates and sulphates was used to classify 45 donkey's milk samples collected from different Italian regions. A method employing ion exchange chromatography with conductivity detector and chemical suppression was used. The quantitative results indicated phosphates (569.4–1304.4?mg?kg^?1) and chlorides (545.9–1757.9?mg?kg^?1) as being the most abundant anions, followed by sulphates (109.5–200.7?mg?kg^?1). The concentrations of nitrites and nitrates were found to be lower at 5.6 and 5.5?mg?kg^?1 respectively. The data set was subdivided into three groups according to the region of origin of milk, and was statistically evaluated by analysis of variance (ANOVA). Concentrations of chlorides and nitrites showed a significant difference among farms (p?<?0.001). In a first discriminant analysis procedure, functions based on linear combinations of the log _e -transformed element concentrations of anions were generated to classify donkey's milk samples from different regions. In an alternative approach, a three-step discriminant analysis procedure to classify a milk sample was tested. The results obtained led to a correct classification of donkey's milk samples based on their anions content with 91–98% of the samples being correctly classified. The procedure proved to be very simple, so it could be used as an evaluation method for the traceability of donkey's milk, thus defending this unique product against fraud or commercial disputes.     

Determination of the caffeine contents of various food items within the Austrian market and validation of a caffeine assessment tool (CAT)

The caffeine content of 124 products, including coffee, coffee-based beverages, energy drinks, tea, colas, yoghurt and chocolate, were determined using RP-HPLC with UV detection after solid-phase extraction. Highest concentrations of caffeine were found for coffee prepared from pads (755?mg?l^?1) and regular filtered coffee (659?mg?l^?1). The total caffeine content of coffee and chocolate-based beverages was between 15?mg?l^?1 in chocolate milk and 448?mg?l^?1 in canned ice coffee. For energy drinks the caffeine content varied in a range from 266 to 340?mg?l^?1. Caffeine concentrations in tea and ice teas were between 13 and 183?mg?l^?1. Coffee-flavoured yoghurts ranged from 33 to 48?mg?kg^?1. The caffeine concentration in chocolate and chocolate bars was between 17?mg?kg^?1 in whole milk chocolate and 551?mg?kg^?1 in a chocolate with coffee filling. A caffeine assessment tool was developed and validated by a 3-day dietary record (r ^2?=?0.817, p?<?0.01) using these analytical data and caffeine saliva concentrations (r ^2?=?0.427, p?<?0.01).     

Sensitive determination of cadmium in brown rice and spinach by flame atomic absorption spectrometry with solid-phase extraction

A sensitive flame atomic absorption spectrometry (FAAS) method was developed for the determination of cadmium (Cd) in brown rice and spinach. The method involves extraction with 1?M hydrochloric acid (HCl), followed by a selective pre-concentration by solid-phase extraction (SPE). The pH of the loading sample solution was adjusted to 4.0 for the brown rice and to 5.0 for the spinach. The masking agents, tartrate and citrate, were required for the spinach before pH adjustment. The SPE step achieved a 20-fold enrichment of the sample solution. The limits of quantification (LOQs) were 0.0054?mg?kg^?1 for the brown rice and 0.0022?mg?kg^?1 for the spinach, being more sensitive than those of AOAC Official method 999.10. A single-laboratory validation was performed by testing spiked samples at 0.04 and 0.08?mg?kg^?1 for the brown rice, and 0.02 and 0.04?mg?kg^?1 for the spinach. The average recoveries were 93.3–96.9% with relative standard deviations (RSDs) of 4.1–8.2% for brown rice, and 90.5–91.9% with RSDs of 5.8–10.0% for spinach.     

Aflatoxins and cyclopiazonic acid in feed and milk from dairy farms in São Paulo,Brazil

The occurrence of aflatoxins (AF) B₁, B₂, G₁, G₂ and cyclopiazonic acid (CPA) in feeds, and AFM₁ and CPA in milk was determined in dairy farms located in the northeastern region of São Paulo state, Brazil, between October 2005 and February 2006. AF and CPA determinations were performed by HPLC. AFB₁ was found in 42% of feed at levels of 1.0–26.4 µg kg^?1 (mean: 7.1 ± 7.2 µg kg^?1). The concentrations of AFM₁ in raw milk varied between 0.010 and 0.645 µg l^?1 (mean: 0.104 ± 0.138 µg l^?1). Only one sample was above the tolerance limit adopted in Brazil (0.50 µg l^?1) for AFM₁ in milk. Regarding CPA in feed, six (12%) samples showed concentrations of 12.5–153.3 µg kg^?1 (mean: 57.6 ± 48.7 µg kg^?1). CPA was detected in only three milk samples (6%) at levels of 6.4, 8.8 and 9.1 µg l^?1. Concentrations of aflatoxins and CPA in feed and milk were relatively low, although the high frequency of both mycotoxins indicates the necessity to continuously monitor dairy farms to prevent contamination of feed ingredients.     

Toward a criterion for suspect thiouracil administration in animal husbandry

Thyreostats are growth-promoters banned in Europe since 1981. The identification of thiouracil (TU) in animal biological matrices can, however, no longer be systematically interpreted as a consequence of illegal administration. Indeed, some experimental results have indicated a causal link between cruciferous-based diet and the presence of TU in urine of bovines. The present study aims at investigating, on a large scale (n?>?1300), the natural occurrence of thiouracil in urine samples collected from different animal species. TU was identified in main breeding animal species: bovine, porcine and ovine. The natural distribution of TU allowed proposing threshold values to differentiate compliant from suspect urine samples. Suggested values are 5.7 and 9.1?µg?l^?1 in male adult bovines (6–24?months), 3.1 and 8.1?µg?l^?1 in female adult bovines (6–24 months), 7.3 and 17.7?µg?l^?1 in calves (<6 months), 3.9 and 8.8?µg?l^?1 in female bovines (>24 months), and 2.9 and 4.1?µg?l^?1 in porcines at a 95 and 99% confidence level, respectively.     

Herbicide contamination of live armclad rockfish,clam and pen shell by moss-control agents used in aquariums of seafood restaurants in Korea

Determination of simazine and diuron by high-performance liquid chromatography-ultraviolet detection (HPLC-UV) in moss-control agents, seawater and fish in aquariums was investigated and validated. The detection limits are 0.2 (simazine) and 0.4?µg l^–1 (diuron) in blank seawater, and 0.20 (simazine) and 0.30 µg kg^?1 (diuron) in blank fish homogenate, while the recoveries ranged from 93.9% to 102.4% with a relative standard deviation?≤?9.8% for simazine and diuron. The method was successfully used in the study of simazine and diuron contamination in live fish stored in seafood restaurant aquaria in Korea. It was found that 0.4–2.3% of simazine and <0.10–3.8% of diuron were included the in moss-control agents tested. Of the 66 sampled aquarium seawaters, simazine was found to be present in four samples (3.8–42?µg?l^?1) while diuron was detected in two samples (1.3–1.6?µg?l^?1). For fish homogenates used in a bioconcentration study, simazine content ranges from 0.17 to 1.8?mg?kg^?1.     

Survey of aflatoxin M1 in cow's milk for human consumption in Kerman Province of Iran

The aim of this study was to assess levels of aflatoxin M₁ (AFM1) in milk samples from Kerman, Iran. AFM1 was detected in 72 samples, ranging in concentration from <0.01 to 0.41?µg?l^?1. The samples were analyzed using immunoaffinity column for clean-up and HPLC for determining AFM1. Milk samples were collected from six dairy farms. AFM1 was found in ～50% of the milk samples. The average level of AFM1 was below the tolerance limit (0.05?µg?l^?1), but 50% of the samples had greater levels than the maximum tolerance limit accepted by EU and the Iranian national standard. The method detection limit and limit of quantification were 0.01 and 0.03?µg?l^?1, respectively, and recovery of the method was 87%. The results showed that AFM1 contamination is a serious problem for public health. To achieve a low level of AFM1 in milk, cattle feed must be monitored regularly for aflatoxin contamination and protected from fungal contamination as much as possible.     

Impact of non-selective fungicides on the growth and production of ochratoxin A by Aspergillus ochraceus and A. carbonarius in barley-based medium

The aim of this study was to assess the influence of the non-selective fungicides mancozeb, copper oxychloride, and sulfur on the growth and capability for producing ochratoxin A (OTA) of ochratoxigenic isolates of Aspergillus carbonarius and A. ochraceus in barley-based medium. Lag phases and growth rates were determined for each fungicide at different doses, at 15°C and 25°C and at 0.97?a_w . Mancozeb at 40?mg?l^?1 inhibited fungal growth and provided lag phases >24 days at 10–20?mg?l^?1 and 15°C. OTA was observed only at 25°C and doses <10?mg?l^?1. At 15°C, copper oxychloride proved inhibitory at 800?mg?l^?1, while at 25°C growth was not delayed and only high doses decreased OTA levels. Sulfur was inhibitory or provided large lag phases at 5–8?g?l^?1 (at 15°C) while at 25°C growth took place even at 8?g?l^?1, although OTA levels were low or undetectable. The antifungal activity decreased in the order mancozeb?>?copper oxychloride?>?sulfur, and was lower at 25°C than at 15°C. OTA accumulation was affected by the type of fungicide, dose, temperature and time. The efficacy of these fungicides on the growth of A. carbonarius and A. ochraceus and OTA production in barley-based medium is assessed for the first time.     

Rapid determination of nitrites in food using a diffuse UV-visible reflectance method

A simple, low-cost, sensitive and selective method for the determination of trace quantities of nitrite in foods such as cheese and cured meat using diffuse ultraviolet-visible reflectance was developed. It is based on the reaction of nitrite with sulphadiazine and α-naphthol, which produces a coloured product in basic medium. The reaction is carried out directly in the measuring cell. For cheese the limit of detection (LOD), expressed as NaNO₂, was estimated to be about 2.0?×?10^?2?mg?l^?1 (2.9?×?10^?7?mol?l^?1) in the final measuring solution and 0.17?mg?kg^?1 in cheese (2.5?×?10^?6?mol?kg^?1). The relative standard deviation (RSD) varied from 5% to 8% depending on the sample. For meat the LOD was estimated to be about 2.0?×?10^?2?mg?l^?1 (2.9?×?10^?7?mol?l^?1) in the final measuring solution and 0.13?mg?kg^?1 in meat (1.9?×?10^?6?mol?kg^?1). The RSD varied from 3% to 6% depending on the sample. The results of the proposed method were also compared with those obtained with the official method using the statistical Student's t-test and F-test procedures.     

Enhanced screening efficiency for endocrine-disrupting chemicals in milk and powdered milk using UPLC/QTOF-MS by the introduction of dansyl chloride derivatisation

This study developed and validated a sensitive analytical method for simultaneous screening of four classes of endocrine-disrupting chemicals (i.e. progestogens, androgens, oestrogens and phenols) in milk and powdered milk using ultra-performance liquid chromatography (UPLC) coupled with electrospray ionisation quadrupole time-of-flight mass spectrometry (QTOF-MS). Dansylation of oestrogens and phenols enhanced the ionisation efficiency and shifted the ionisation mode from negative to positive, which allowed for the simultaneous analysis of four EDCs in one chromatographic run. An efficient sample pre-treatment minimised the matrix effects. The mass errors for the precursor and product ions for 26 target compounds varied between ?2.8 and 2.3?mDa; and the limits of detection (signal-to-noise ratio?=?3) for milk and powdered milk were less than 0.04?µg?l^?1 and 0.10?µg?kg^?1, respectively. The proposed method was successfully used to analyse multiple types of real samples, including normal temperature whole milk, infant formula and whole powdered milk. In 11 samples, two target compounds, progesterone and androstenedione, were detected. The progesterone concentrations ranged from 8.1 to 12.7?µg?l^?1 in milk, and from 1.2 to 32.0?µg?kg^?1 in infant formulas and whole powdered milks. The androstenedione concentrations varied from 0.39 to 0.79?µg?l^?1 in milks, and from 0.29 to 1.2?µg?kg^?1 in infant formulas and whole powdered milks. Two post-target compounds, one isomer of oestriol and 5α-dihydroprogesterone, were tentatively identified by post-target analysis in two of 11 real samples.     

Simultaneous determination of antimony and boron in beverage and dairy products by flame atomic absorption spectrometry after separation and pre-concentration by cloud-point extraction

A new cloud-point extraction (CPE) method was developed for the pre-concentration and simultaneous determination of Sb(III) and B(III) by flame atomic absorption spectrometry (FAAS). The method was based on complexation of Sb(III) and B(III) with azomethine-H in the presence of cetylpyridinium chloride (CPC) as a signal-enhancing agent, and then extraction into the micellar phase of Triton X-114. Under optimised conditions, linear calibration was obtained for Sb(III) and B(III) in the concentration ranges of 0.5–180 and 2.5–600 μg l^?1 with LODs of 0.15 and 0.75 μg l^?1, respectively. Relative standard deviations (RSDs) (25 and 100 μg l^?1 of Sb(III) and B(III), n = 6) were in a range of 2.1–3.8% and 1.9–2.3%, respectively. Recoveries of spiked samples of Sb(III) and B(III) were in the range of 98–103% and 99–102%, respectively. Measured values for Sb and B in three standard reference materials were within the 95% confidence limit of the certified values. Also, the method was used for the speciation of inorganic antimony. Sb(III), Sb(V) and total Sb were measured in the presence of excess boron before and after pre-reduction with an acidic mixture of KI-ascorbic acid. The method was successfully applied to the simultaneous determination of total Sb and B in selected beverage and dairy products.     

Optimal conditions for determination of zinc bacitracin,polymyxin B,oxytetracycline and sulfacetamide in animal feed by micellar electrokinetic capillary chromatography

A separation technique for zinc bacitracin, polymyxin B, oxytetracycline and sulfacetamide in animal feedstuffs by micellar electrokinetic capillary chromatography (MEKC) was developed. The running buffer was 20 mmol l^?1 borate, 20 mmol l^?1 phosphate, pH 8.4, containing 20 mmol l^?1 sodium dodecylsulphate and 10% (v/v) methanol. MEKC was performed at 25°C; the applied voltage was 25 kV with a running pressure of 10 mbar. Simultaneous UV detection for all analytes was at 215 nm. The method was validated for specificity, accuracy, linearity, precision and robustness. It was shown to be specific, accurate (recoveries were 99.7?±?0.3, 99.9?±?0.9, 99.8?±?1.0 and 99.5?±?0.4, respectively, for oxytetracycline-, sulfacetamide-, polymyxin B- and zinc bacitracin-spiked samples of feed for cow, pigs, chicken and cattle), linear over the tested range (correlation coefficients ≥0.9987) and precise (RSDs below 1.8% for each analyte). The method was applied to determine zinc bacitracin, polymyxin B, oxytetracycline and sulfacetamide as additives in animal feed.     

Validation of a lateral flow test (MRLAFMQ) for the detection of aflatoxin M1 at 50 ng l−1 in raw commingled milk

Aflatoxin M₁ contamination in dairy products is a risk when feedstuff contaminated with aflatoxin B₁ produced by moulds is consumed by milk-producing animals. Milk can be screened for aflatoxin M₁ at the European Union maximum limit of 50 ng l^?1 by a lateral flow test, the MRLAFMQ (Aflatoxin M1) Test. The method takes 15 min with no milk dilution or a sample preparation step. The lateral flow assay was validated at the Technology and Food Science Unit of the Institute for Agricultural and Fisheries Research (ILVO-T&V) according to European Union guidelines using fortified raw milk samples. A detection capability of 50 ng l^?1 was demonstrated with a false negative rate lower than 2% at 50 ng l^?1 and a false positive rate of less than 0.3%. Quantitative readings had a mean bias of +2 to 6 ng l^?1 at 50 ng l^?1 with a standard deviation of 5–8 ng l^?1. Based on the validation results, the test could be considered appropriate for milk screening prior to milk unload at dairies.     

Colour and label evaluation of commercial pasteurised red juices and related drinks

Despite growing demand by consumers for healthy beverages, artificial colours are still widely used. Levels of anthocyanins and artificial colours were determined by HPLC with UV-Vis detection in red orange juices and other red beverages (nectar, juice-based, health, carbonated and sports drinks). The contribution of pigments to the visible colour of the beverage was calculated. Red orange juice samples contained about 34?mg?l^?1 of anthocyanins, which were responsible for about 92% of the visible colour. Red juice-based drinks, containing from 0% to 30% of red orange, berry, grape or pomegranate juices, had low levels of anthocyanins (about 7?mg?l^?1) and high levels of E129 (about 32?mg?l^?1), which were responsible for about 90.7% of the colour of these beverages. Red health drinks, enriched with vitamins and polyphenols, contained from 3% to 50% of red fruit juices. Also in this case the E129 levels were higher (about 22?mg?l^?1) than anthocyanins (about 9?mg?l^?1), and were responsible for the colour of the beverages (76.1%). High levels of artificial colours were found in red orange carbonated drinks, but in comparable amounts with those found in the other beverage samples, while anthocyanins were only present in trace amounts. Although all of the beverages claimed to contain red fruits on the labels, no correlation was found between the level of anthocyanins and the declared percentage of red fruits. These labels generally conformed with the requirements of the law, but food product labels can often be misleading to consumers about the real characteristics of the product.     

Influence of post-harvest application rates of cyprodinil,treatment time and temperature on residue levels and efficacy in controlling green mould on ‘Valencia’ oranges

The effectiveness of heat treatments with water and cyprodinil in controlling post-harvest green mould caused by Penicillium digitatum was investigated on artificially inoculated ‘Valencia’ oranges. Residue levels of cyprodinil were determined in the oranges as a function of active ingredient concentration, temperature and treatment time. Cyprodinil residues were significantly dependent on treatment time when applied at 600 mg l^?1 and 20°C, but not when fruit were treated at 150–300 mg l^?1. The application of cyprodinil at 50 or 100 mg l^?1 at 55°C for 30 s produced similar residue levels, while residues increased when the application rate was 150 mg l^?1. Cyprodinil at 100 mg l^?1 and 60°C produced a significant increase in residues compared to treatment at 50 mg l^?1; no significant increase in residues was found when the application rate was raised from 100 to 150 mg l^?1. In comparison to treatments performed at 20°C, the application of a heated cyprodinil mixture resulted in significantly higher residues in fruit. All treatments with cyprodinil at 20°C similarly reduced green mould after 7 days of storage at 20°C. After 18 days, treatment with cyprodinil at 600 mg l^?1 for 30 s was more effective than at 150–300 mg l^?1. When dip time was extended to 90 or 180 s, treatment efficacy was positively related to fungicide concentration. Treatments with water at 55°C for 30 s were as effective as cyprodinil at 50–100 mg l^?1, but less effective than cyprodinil at 150 mg l^?1. After 7 days, treatment with water or cyprodinil at 50–150 mg l^?1 and 60°C were equally effective in controlling green mould; while, after 18 days, treatment with cyprodinil at 150 mg l^?1 was consistently more effective than at 50–100 mg l^?1 or hot water alone.     

Analysis of isothiazolinone biocides in paper for food packaging by ultra-high-performance liquid chromatography–tandem mass spectrometry

A novel and simple method to detect isothiazolinone-type biocides (2-methyl-3-isothiazolinone (MI), 5-chloro-2-methyl-3-isothiazolinone (CMI), 1,2-benzisothiazolinone (BIT) and 2-octyl-3-isothiazolinone (OIT)) in paper used for food packaging by ultrasonic extraction coupled with UPLC-MS/MS was developed. Parameters affecting process efficiency such as extraction solvents, UPLC mobile phase, gradient elution procedure and MS/MS conditions were studied to optimise the operating conditions. Using the optimised gradient elution procedure, the retention time was less than 6?min. The limits of detection (LODs) were found to be between 0.001 and 0.010?mg?kg^?1, which was validated using actual concentrations. After diluting the standard solution with a blank matrix, the linear calibration curve ranges were 0.002–1.000?mg?kg^?1 for BIT and OIT, 0.005–1.000?mg?kg^?1 for MI, and 0.020–1.000?mg?kg^?1 for CMI, with correlation coefficients higher than 0.9985 (n?=?6). A good level of precision with a mean recovery greater than 81.3% and a relative standard deviation (RSD) less than 6.2% were also obtained. A methodology has been proposed for the analysis of isothiazolinones in paper.     

Elemental composition of commercial sea cucumbers (holothurians)

Toxic and essential elements in 11 different sea cucumber species were determined and compared with daily intake recommendations and maximum allowed levels. The contents of macro-elements contents in dried sea cucumber samples were found to be 25,000–152,000?mg?kg^–1 for Na, 4000–8600?mg?kg^?1 for Mg, 1100–5200?mg?kg^?1 for K, 15,000–68,000?mg?kg^?1 and 36,300–251,000?mg?kg^?1 for Cl. Trace element concentrations in dried sea cucumber samples were found to be 11–100?mg?kg^?1 for Zn, 41–660?mg?kg^?1 for Fe, 3–74?mg?kg^?1 for Cu, 1.1–16?mg?kg^?1 for Mn, 1.4–3.7?mg?kg^?1 for Se, 1.1–9.6?mg?kg^?1 for Cr, and 0.3–5.1?mg?kg^?1 for Ni. All sea cucumber species were rich sources of Na, Cl, Mg, Ca, Fe, Cu, Se and Cr for human consumption. Regarding contaminants, As, Cd and Pb concentrations in dried sea cucumbers were in the ranges of 1.1–6.1, 0.03–0.06 and 0.11–0.69?mg?kg^?1, respectively. Moreover, Hg values of 11 sea cucumbers were below the detection limit (0.01?mg?kg^?1).     

Undeclared allergenic ingredients in foods from animal origin: survey of an Italian region's food market, 2007–2009

Several EC Directives have been promulgated to protect allergic individuals but no rule has been established with regard to allergen cross-contamination caused by shared transport vehicles or common processing equipment. The aim of this research was to quantify, by enzyme-linked immunosorbent assay (ELISA) or real-time polymerase chain reaction, the presence in meat- or fish-based foods of four allergens (milk, egg, crustaceans and molluscs) that was not indicated either in the list of ingredients or in the label alert. In the time frame of 2007–2009, a total of 723 samples were subjected to 1983 analyses. The percentage of samples scoring positive ranged between 1.8% and 6.8% over the 3 years, and the concentrations of undeclared allergens found were 0.3–13.3?mg?kg^?1 for milk (β-lactoglobulin) and 0.21–12?mg?kg^?1 for egg white proteins. On this basis, the possibility of cross-contamination serious enough to raise public health concern cannot be dismissed.     

Content of conjugated linoleic acid in neutral and polar lipid fractions of milk of different ruminant species

The aim of this research was to investigate the distribution of conjugated linoleic acid (CLA) in the neutral and polar milk lipid fractions of samples of bovine, ovine and caprine milk. Lipids were fractionated by thin-layer chromatography to obtain triglyceride, diglyceride and monoglyceride fractions. Phospholipids were separated by solid-phase extraction. The CLA content was quantitatively determined after transmethylation and addition of internal standard. As expected, 95–97% of the CLA was found in the triglyceride fraction. The main differences between the species were observed in CLA content of the diglyceride and phospholipid fraction, while the amount of CLA in the monoglycerides fraction was negligible. Cows' milk showed comparable contents of CLA in both diglyceride and phospholipid fractions (4.2 and 5.5 mg 100 g^?1 of lipids, respectively). The level of CLA in phospholipids of ewes' (38 mg) and goats' (20 mg) milk lipids was four times higher than the level of CLA in diglycerides (7 and 5 mg, respectively). In terms of the phospholipid content, CLA accounted for 2–4% (calculated as a percentage of total fatty acids esterified in phospholipids) in caprine and ovine and less than 1% in bovine milk.