Determination of Sulfur Amino Acids in Milk and Plant Proteins
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Determination of Sulfur Amino Acids in Milk and Plant Proteins Paul W. Johns 1 Received: 28 May 2020 / Accepted: 4 September 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract A method for the determination of the sulfur amino acids methionine and cysteine in milk- and plant-derived protein powders and in protein-based nutritional products is described. Samples and calibration standards are prepared by acid hydrolysis (6-M HCl, 110 °C, 24 h) with 3,3’dithiopropionic acid, evaporation, and resuspension. The released methionine and cysteine mixed disulfide are quantified by direct LC/UV, without pre- or post-column chromophore/fluorophore derivatization. Since tyrosine is also released by the acid hydrolysis and elutes in the same chromatogram, it was included in all determinations. Method performance has been defined by assessments of linearity (R2 averaged > 0.999, n = 10, for 5-point plots of each analyte), intermediate precision (within-day RSD < 2%, n = 3, and day-to-day RSD < 3%, n = 3 days, for each analyte), accuracy (cystine spike recovery = 101%; measured methionine, cystine, and tyrosine concentrations were 95–111% of published concentrations), selectivity (reagent blanks and peak purity found analyte bias ≤ 2%), and quantitation limit (25 mg of methionine and tyrosine, and 50 mg of cystine, per 100 g of protein powder). The method provides a relatively simple means for the accurate and precise determination of both sulfur amino acids in protein powders and in nutritional products, and may be performed by conventional LC/UV, without specialized amino acid analysis instrumentation. Keywords Methionine . Cystine . Cysteine . 3,3’-dithiodipropionic acid . Mixed disulfide . Disulfide exchange
Introduction The 2007 WHO/FAO/UNU minimum requirement for sulfur amino acid (methionine + cysteine) ranges from 22 mg (for adults) to 28 mg (for infants) per g of food protein (WHO/ FAO/UNU 2007). Food proteins that show promise as sustainable alternatives to milk-derived proteins, but that are also limiting or relatively low in sulfur amino acids (with or without digestibility correction), include fava bean, green and yellow pea, almond, soybean, and chickpea (Kalman 2014; Nosworthy et al. 2017; House et al. 2019; Ma et al. 2019; Rafii et al. 2020). Interest in the sulfur amino acids is not limited to compliance with indispensable amino acid concentration requirements; e.g., nutritional significance has also been associated with the ratio of methionine/cysteine (Sarwar et al. 1991; Shoveller et al. 2003; Kurpad et al. 2004; Pacheco et al. 2018), cysteine may be used as a marker of casein adulteration (Ballin 2006), and methionine may be used as a marker of peroxide treatment (Baxter et al. 2007). * Paul W. Johns [email protected] 1
Abbott Nutrition Division, Abbott Laboratories, 3300 Stelzer Road, Columbus, OH 43219, USA
Although the determination of methionine in proteins and nutritional products may be performed by the conventional acid hydrolysis/LC methods (including post-colum
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