Evaluation of sample preparation methods for NMR-based metabolomics of cow milk

Abstract

The quality of milk metabolome analyzed by nuclear magnetic resonance (NMR) is greatly influenced by the way samples are prepared. Although this analytical method is increasingly used to study milk metabolites, a thorough examination of available sample preparation protocols for milk has not been reported yet. We evaluated the performance of eight milk preparation methods namely (1) raw milk without any processing; (2) skimmed milk; (3) ultrafiltered milk; (4) skimming followed by ultrafiltration; (5) ultracentrifuged milk; (6) methanol; (7) dichloromethane; and (8) methanol/dichloromethane, in terms of spectra quality, repeatability, signal-to-noise ratio, extraction efficiency and yield criteria. A pooled sample of milk was used for all protocols. Skimming, ultracentrifugation and unprocessed milk protocols showed poor NMR spectra quality. Protocols involving multiple steps, namely methanol/dichloromethane extraction, and skimming followed by ultrafiltration produced inadequate results for signal-to-noise ratio parameter. Methanol and skimming associated to ultrafiltration provided good repeatability results compared to the other protocols. Chemical-based sample preparation protocols, particularly methanol, showed more efficient metabolite extraction compared to physical preparation methods. When considering all evaluation parameters, the methanol extraction protocol proved to be the best method. As a proof of utility, methanol protocol was then applied to milk samples from dairy cows fed a diet with or without a feed additive, showing a clear separation between the two groups of cows.

Development of a device for cleansing cow udder teats and testing it under industrial conditions

“…Nerve receptors of the udder skin become excited by tactile influences, that is, during the extraction of first milk squirts at the beginning of milking, the udder washing, massage, attachment of the milking machine cups as well as pulsation of the cup rubber during milking. To achieve optimal stimulation, it is necessary that the duration of a certain combination of previous operations is at least 60 s [13][14][15]. Since most operations are manual, they need to be reduced in time to achieve higher productivity of the milking operator labor in automated milking processes.…”

Selected Instrumental Techniques Applied in Food and Feed: Quality, Safety and Adulteration Analysis

“…In this regard, we suggest Sundekilde and coworkers [ 63 ] and Maher and Rochfort [ 64 ] as primers. Besides, Yanibada and coworkers [ 65 ] have prepared a thorough paper regarding the sample preparation methods recommended before metabolomics analysis for bovine milk. Additionally, Bertram [ 66 ] highlighted the use of metabolomics in milk and meat to assess authenticity and quality.…”

Detection of bovine milk adulteration in caprine milk with N-acetyl carbohydrate biomarkers by using 1H nuclear magnetic resonance spectroscopy

“…All other compounds were confirmed by using the Chenomx built-in library ver. 10.0, the Human Metabolome Database (https: / / hmdb .ca/ ; Wishart et al, 2018), and previous references (Klein et al, 2010;Sundekilde et al, 2013;Yanibada et al, 2018;Garwolińska et al, 2020).…”

“…Higher sensitivity than specificity means that the model better recognized adulterated milk (FP > FN). Further ROC curve analysis was performed to evaluate the AUC values for N-acetyl carbohydrates between the Literature (Klein et al, 2010;Sundekilde et al, 2013;Wishart et al, 2018;Yanibada et al, 2018;Garwolińska et al, 2020).…”