Determination of Ca, P, K, Na, and Mg in Australian Retail Pasteurised Milk Using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP OES).

A rapid and simple inductively coupled plasma atomic emission spectrometry (ICP OES) method was developed and validated for the determination of macroelements including calcium (Ca), phosphorus (P), potassium (K), sodium (Na), and magnesium (Mg) in Australian retail pasteurised milk. The milk samples were digested using the mixture of 70% HNO3 and 30% H2O2 (2 : 1, v/v) in an open-tube digester block at 120°C for 4 h. The validated ICP OES method showed good linearity for all elements (R 2 > 0.9993). The method limits of quantification (LOQ) for Ca, P, K, Na, and Mg were 19.85, 8.97, 100.8, 41.92, and 11.56 µg·g-1, respectively. Recoveries were in the range of 91.54-116.0%. Repeatability and interday reproducibility expressed as the relative standard deviation (% RSD) was below 5.0%. The contents of macroelements in 6 retail pasteurised milk samples were between 1099.32 and 1348.65 µg·g-1 (Ca), 914.01 and 1091.21 µg·g-1 (P), 1362.76 and 1549.74 µg·g-1 (K), 288.89 and 323.22 µg·g-1 (Na), and 97.62 and 110.57 µg·g-1 (Mg). Principal component analysis (PCA) revealed that retail pasteurised milk samples were distinctly separated into four groups on the first two principal components (PCs). The difference in the macroelement content between milk brands might be affected by milk regions.

Fatty acid profiling of Western Australian pasteurised milk using gas chromatography-mass spectrometry.

The fatty acid composition of Western Australian commercial pasteurised milk was profiled using gas chromatography-mass spectrometry (GC-MS). A total of 31 fatty acids (FA) were identified in the milk samples. The majority of FA were medium-chain fatty acids (MCFA) with 6-13 carbon atoms and long-chain fatty acids (LCFA) with 14-20 carbon atoms. The results of principal component analysis (PCA) showed significant differences in the levels of MCFA and LCFA in the different milk samples. The levels of MCFA and LCFA ranged from 10.09 % to 12.12% and 87.88% to 89.91% of total FA, respectively. C10:0 and C12:0 were the major components of MCFA comprising 3.46% and 4.22% of total FA, while C16:0 and C18:1 (cis 9-octadecenoic acid) represented the majority of LCFA with the levels of 26.18% and 23.34% of total FA, respectively. This study provides new insight into the FA composition of Western Australian pasteurised milk and differences in FA profiles which influence human health.

Application of ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (Orbitrap™) for the determination of beta-casein phenotypes in cow milk.

The objective of this study was to determine the ß-CN phenotypes in cow milk collected from HF and cross-bred HF dairy cattle in Phu Dong, Vietnam. In total, 85 samples of raw milk were collected from 85 individual cows. Beta-casein (ß-CN) phenotypes in cow milk were determined using ultra-high performance liquid chromatography coupled to high resolution mass spectrometry (UHPLC-HRMS). The results showed that three ß-CN variants A1, A2 and I were detected and identified in the milk samples. Five ß-CN phenotypes A1A1, A1A2, A1I, A2A2 and A2I were found with percentages of 0.035, 0.400, 0.059, 0.482 and 0.024, respectively. The higher proportion of ß-CN phenotype A2A2 compared to other phenotypes was expected because of changes in dairy cow breeding in Phu Dong, Vietnam.

Degradation of ß-casomorphins and identification of degradation products during yoghurt processing using liquid chromatography coupled with high resolution mass spectrometry.

Liquid chromatography-high resolution mass spectrometry (LC-HRMS) was used to investigate the degradation of ß-casomorphin 5 (ß-CM5) and ß-casomorphin 7 (ß-CM7) by Streptococcus thermophilus and/or Lactobacillus delbrueckii ssp. bulgaricus, and to identify the degradation products forming during yoghurt processing. Bovine UHT milk was fermented with: (i) a single strain of L. delbrueckii ssp. bulgaricus, (ii) a single strain of S. thermophilus and (iii) the mixture of S. thermophilus and L. delbrueckii ssp. bulgaricus to pH4.5 and then stored at 4°C for 1 and 7days. Results showed that L. delbrueckii ssp. bulgaricus and/or S. thermophilus completely degraded ß-CM5 and ß-CM7 upon fermentation to pH4.5 and degradation products were significantly influenced by bacteria strains and storage time. Four peptides, ß-CNf60-61 (YP), ß-CNf62-63 (FP), ß-CNf64-66 (GPI) and ß-CNf62-66 (FPGPI) were tentatively identified through high resolution MS/MS experiments; however, it was not possible to confirm if either milk protein or ß-casomorphins was a source releasing these peptides. Nonetheless, in this study peptides YP and GPI were released by L. delbrueckii ssp. bulgaricus. This is the first time GPI has been identified and thus future investigation of its bioactivity is warranted.

Evaluation of a Commercial Sandwich Enzyme-Linked Immunosorbent Assay for the Quantification of Beta-Casomorphin 7 in Yogurt Using Solid-Phase Extraction Coupled to Liquid Chromatography-Tandem Mass Spectrometry as the "Gold Standard" Method.

This study investigated beta-casomorphin 7 (BCM7) in yogurt by means of LC-tandem MS (MS/MS) and enzyme-linked immunosorbent assay (ELISA) and use LC-MS/MS as the "gold standard" method to evaluate the applicability of a commercial ELISA. The level of BCM7 in milk obtained from ELISA analysis was much lower than that obtained by LC-MS/MS analysis and trended to increase during fermentation and storage of yogurt. Meanwhile, the results obtained from LC-MS/MS showed that BCM7 degraded during stages of yogurt processing, and its degradation may have been caused by X-prolyl dipeptidyl aminopeptidase activity. As a result, the commercial sandwich ELISA kit was not suitable for the quantification of BCM7 in fermented dairy milk.

Formation and Degradation of Beta-casomorphins in Dairy Processing.

Milk proteins including casein are sources of peptides with bioactivity. One of these peptides is beta-casomorphin (BCM) which belongs to a group of opioid peptides formed from ß-casein variants. Beta-casomorphin 7 (BCM7) has been demonstrated to be enzymatically released from the A1 or B ß-casein variant. Epidemiological evidence suggests the peptide BCM 7 is a risk factor for development of human diseases, including increased risk of type 1 diabetes and cardiovascular diseases but this has not been thoroughly substantiated by research studies. High performance liquid chromatography coupled to UV-Vis and mass spectrometry detection as well as enzyme-linked immunosorbent assay (ELISA) has been used to analyze BCMs in dairy products. BCMs have been detected in raw cow's milk and human milk and a variety of commercial cheeses, but their presence has yet to be confirmed in commercial yoghurts. The finding that BCMs are present in cheese suggests they could also form in yoghurt, but be degraded during yoghurt processing. Whether BCMs do form in yoghurt and the amount of BCM forming or degrading at different processing steps needs further investigation and possibly will depend on the heat treatment and fermentation process used, but it remains an intriguing unknown.