Lactoferrin content in bovine milk: Validation of radial immunodiffusion method and predictive ability of mid-infrared spectroscopy

Lactoferrin (LF) is an iron-binding globular glycoprotein, responsible for the iron transfer through the bowel and for the regulation of the quantity being absorbed by it. It is produced by mucosal epithelial cells of the mammals, and it is mostly expressed in the colostrum and in the milk. Lactoferrin is considered a multifunctional protein, since its positively charged net and widespread delivery, it has a lot of physiological functions, such as antibacterial, antiviral, antifungal, anti-inflammatory, anti-tumor, osteogenic and antioxidant ability. The concentration of LF in the human milk and colostrum is higher than the one in the bovine milk, therefore the LF supplementation in the commercial milk and mainly the infant one is of great importance. The concentration of the protein in the milk depends on multiple factors such as species, breed, milk yield, diet, stage of lactation, lactation number, lactation period and health conditions of the animal. Several methods have been reported for the quantification of its content in milk and in dairy products, but none of them can be used in the industry and in the laboratories as a simple, cheap, fast, precise, and routine-based approach. In this project, the radial immunodiffusion technique was first validated and then used to quantify LF of raw milk samples, aiming to create a dataset of LF content combined with the correspondent mid-infrared spectra, in order to develop a model to predict LF during routine infrared analyses. Descriptive statistics for LF content in processed commercial cow milk samples were calculated from data obtained from radial immunodiffusion analysis. The validation of the radial immunodiffusion reference method was achieved by time-related and operators-related repeatability and reproducibility in treatment-related (no treated, frozen, with preservatives) cow raw milk samples and by the linearity of the reference standard method. Repeatability of milk lactoferrin content calculated as relative standard deviation of 5 measurements within day, regarding raw cow milk samples, frozen and with preservatives samples ranged from 3.72% to 4.52%, 3.56% to 5.34%, and 2.43% to 5.66%, respectively, while within operators, it ranged from 3.04% to 4.03%, 0.79 to 4.94%, and 2.43% to 5.50%, respectively. The overall reproducibility of milk lactoferrin content, calculated as relative standard deviation of 45 measurements across 3 days of analysis and 3 operators, was 5.99% for the raw untreated, 6.96% for the frozen and 7.41% for the milk samples with preservatives. iii The mean lactoferrin content among 1,048 individual Holstein cows’ raw milk samples was 12.14 mg/dl, with a high coefficient of variation (68.69%), while among the commercial milk samples, the whole fat pasteurized milk had the highest content (16.51 mg/dl) with a coefficient of variation of 6.36%. Mid-infrared spectroscopy prediction models developed through partial least squares approach had a coefficient of determination in cross validation of 0.58 and root mean squared error of 5.22 mg/dl.