Lucia Monti

Capillary electrophoresis of sialylated oligosaccharides in milk from different species

Oligosaccharides are relevant components of human milk, which have been quite well studied for their pre-biotic effect and their capacity in stimulating the immune system. Since oligosaccharides from milk of non-human mammals received so far less attention, the aim of this work was the application of capillary electrophoresis (CE) for the analysis of sialylated oligosaccharides in cow, goat and equine (mare and donkey) milk to possibly identify potential sources of oligosaccharides to use as health promoting ingredients in functional foods. Human milk was used as reference milk. A recent CE technique was applied to resolve and quantify 3-sialyllactose (3-SL), 6-sialyllactose (6-SL) and disialyl-lacto-N-tetraose (DSLNT). Analysis of non-human milk samples confirmed differences among species and individuals: DSLNT, which was the most abundant compound in human milk (455-805μg/mL) was missing in most of the samples. In most cases, 3-SL showed to be the most concentrated of the quantified analytes, with values ranging from 12 to 77μg/mL.

Development of a near Infrared Method for the Quantification of the Main Classes of Fatty Acids Obtained from Raw Milk by Solvent-Free Extraction

The determination of the fatty acid (FA) profile of milk fat, which is the most complex dietary fat, is more and more for both the definition of nutritional labelling and the study of the genetic variability in bovine milk fat quality. For these reasons, the development of infrared techniques for the rapid evaluation of FA profiles is gaining much wider interest. In this research, a method for the rapid determination of saturated, unsaturated, monounsaturated, polyunsaturated and trans FAs, expressed on 100g of total milk FAs, is reported. The FA profile was estimated from the transmission spectrum (optical pathlength of 6 mm) obtained from the fat extracted by hot centrifugation, without solvents. Authentic triglyceride standards were used in order to perform the spectral band assignments. The whole data set included 135 raw milk samples collected from cows fed either a total mixed ration or grazed on mountain pastures. Ninety five samples were selected for model development (calibration set); the prediction models were then validated by an independent validation set composed by 40 samples. The prediction model for the saturated FA content showed a correlation coefficient of determination (r2) = 0.96, standard error of prediction (SEP) = 0.97 (g 100 g−1 of total FAs), ratio of errors (RER) = 22.8 and ratio of performance to deviation (RPD) = 4.6. Monounsaturated, polyunsaturated and trans FAs provided r2 = 0.95, 0.90 and 0.97, RER = 20.4, 12.5 and 32.1 and RPD = 4.2, 2.8 and 5.2, respectively. The ability to predict the concentration of some saturated single FAs, also grouped according to their chain length, was also investigated. Satisfactory screening performances were found for C14, C16 and C18 FAs, as well as for the amount of the groups including medium (12–17 carbon atoms) and long (18–22 carbon atoms) FAs.

Bovine colostrum: Changes in lipid constituents in the first 5 days after parturition

Despite the great interest paid to protein components in colostrum, fat also plays an important role in the supply of essential nutrients to provide energy, increase metabolism, and protect the newborn calf against microbial infections. This work aimed to elucidate levels of different fat components in colostrum, in particular fatty acid (FA), triglyceride (TG), cholesterol, and phospholipid contents. Colostrum samples from primiparous and multiparous (3-5 lactations) Holstein dams, fed the same ration indoors, were collected on the first 5d after parturition, analyzed, and compared with milk samples from the same cows collected at 5mo of lactation. Fat content during the first 5d of milking did not vary. However, the proportion of short-chain saturated FA increased and that of long-chain FA decreased. The concentration of n-3 FA was higher on the first day of calving than on the other days, with clear differences in the number and type of n-3 FA. Conjugated linoleic isomers and trans FA slowly increased from d 1 to 5, reaching a maximum at 5mo of lactation. Changes in the distribution profile of TG were observed as lactation progressed, with a shift from a prevalence of high-carbon-number TG (C48-50) on d 1 to a bimodal distribution (maxima at C38 and C50) on d 5, characteristic of mid-lactation milk. Cholesterol content was high in the first hours after calving and rapidly decreased within 48h. Colostrum sampled on d 1 also had a high content of phospholipids. Phosphatidylethanolamine and sphingomyelin were, respectively, lower and higher in the first 5d than in mid-lactation milk. The influence of lactation number on colostrum fat composition was also considered and significant results were obtained for all FA groups (except for polyunsaturated and n-6 FA) and TG content.

Quantification of casein fractions and of some of their genetic variants in phosphate buffer by near infrared spectroscopy

Milk casein and casein fraction contents have a great influence on milk rennet properties and cheese yield so that the selection of dairy cattle with genetic characteristics suitable for milk transformation is of great interest to dairy farms and firms. The possibility of a rapid and accurate determination of these parameters would be very useful to predict milk aptitude to cheese making. This work aimed to determine casein fractions and their genetic variants content using near infrared (NIR) spectroscopy in reconstituted casein samples by comparing the performance of different NIR equipment (a monochromator instrument and a Fourier transform instrument) and different modes of measurement (reflectance and transflectance) in order to evaluate the best operative conditions for this application. Fifty-eight raw milk samples, collected from different farms in the Asturias region, Spain, were analysed for protein (TP%) and non caseinic nitrogen (NCN%) content using the Kjeldahl method. Casein content was calculated as the difference between TP and NCN content. Casein fractions (αs0-, αs1-, αs2-, κ-casein) and genetic variants of β-casein (βB- βA1-, βA2-casein) were determined by a capillary electrophoresis system. Samples were ultra-centrifuged to obtain native casein and then reconstituted in phosphate buffer (pH = 6.8) at the same original milk concentration, previously determined by the Kjeldahl method. Spectra were collected at 37°C with a FT-NIR instrument in transflectance mode and a monochrometer in both transflectance and reflectance mode. Partial least square (PLS) analyses performed on transflectance spectra showed good prediction ability for all variables—(min R2 = 0.80 for κ-casein; max R2 = 0.94 for βA2–casein), with the exception of αs2-casein. NIR spectroscopy has the ability to determine and quantify casein genetic variants and could be used to select milk for its final purpose and to predict the aptitude of milk to cheese-making.

Testing the suitability of different high-performance liquid chromatographic methods to determine aflatoxin M1 in a soft fresh Italian cheese.

Aflatoxin M1 (AFM1) is a toxic undesirable compound in milk. AFM1 affinity for caseins causes a concentration effect during milk process for dairy transformation. In spite of this, no official method of analysis, nor maximum tolerance level for aflatoxin M1 in cheese have been established. Thus, the aim of this work was to test the suitability of different HPLC methods for the AFM1 quantification in soft cheese samples at three different contamination levels (low, medium and high, at respectively nearly 30, 100 and 250 ng/kg). Nine participants were selected among Italian laboratories accredited by the Italian accreditation body (ACCREDIA) for HPLC toxin analysis. They were asked to analyze samples applying the method routinely used. The different applied methods were compared, and precision and accuracy parameters were evaluated. The main differences among HPLC procedures were registered at the level of extraction step. The use of an enzymatic digestion for the extraction of the toxin from cheese seemed to be particularly advantageous and the use of immunoaffinity columns seemed to be determinant for the improvement of sensitivity at low contamination levels. In general, the applied methods well discriminated the 3 levels of contamination, even though they performed better at the medium and high concentration levels (100 and 250 ng/kg) than at the low one (30 ng/kg). In fact relative standard deviation for reproducibility at low level was higher (60.1%) than the same value at medium and high levels (22.8% and 28.9%, respectively).