SUMMARYBackground & aimsSignificant biological variation in macronutrient content of breast milk is an important barrier that needs to be overcome to meet nutritional needs of preterm infants. To analyze macronutrient content, commercial infrared milk analyzers have been proposed as efficient and practical tools in terms of efficiency and practicality. Since milk analyzers were originally developed for the dairy industry, they must be validated using a significant number of human milk samples that represent the broad range of variation in macronutrient content in preterm and term milk. Aim of this study was to validate two milk analyzers for breast milk analysis with reference methods and to determine an effective sample pretreatment. Current evidence for the influence of (i) aliquoting, (ii) storage time and (iii) temperature, and (iv) vessel wall adsorption on stability and availability of macronutrients in frozen breast milk is reviewed.MethodsBreast milk samples (n = 1188) were collected from 63 mothers of preterm and term infants. Milk analyzers: (A) Near-infrared milk analyzer (Unity SpectraStar, USA) and (B) Mid-infrared milk analyzer (Miris, Sweden) were compared to reference methods, e.g. ether extraction, elemental analysis, and UPLC-MS/MS for fat, protein, and lactose, respectively.ResultsFor fat analysis, (A) measured precisely but not accurately (y = 0.55x + 1.25, r2 = 0.85), whereas (B) measured precisely and accurately (y = 0.93x + 0.18, r2 = 0.86). For protein analysis, (A) was precise but not accurate (y = 0.55x + 0.54, r2 = 0.67) while (B) was both precise and accurate (y = 0.78x + 0.05, r2 = 0.73). For lactose analysis, both devices (A) and (B) showed two distinct concentration levels and measured therefore neither accurately nor precisely (y = 0.02x + 5.69, r2 = 0.01 and y = −0.09x + 6.62, r2 = 0.02 respectively). Macronutrient levels were unchanged in two independent samples of stored breast milk (−20 °C measured with IR; −80 °C measured with wet chemistry) over a period of 14 months.ConclusionsMilk analyzers in the current configuration have the potential to be introduced in clinical routine to measure fat and protein content, but will need major adjustments.
For preterm infants, it is common practice to add human milk fortifiers to native breast milk to enhance protein and calorie supply because the growth rates and nutritional requirements of preterm infants are considerably higher than those of term infants. However, macronutrient intake may still be inadequate because the composition of native breast milk has individual inter- and intra-sample variation. Target fortification (TFO) of breast milk is a new nutritional regime aiming to reduce such variations by individually measuring and adding deficient macronutrients. Added TFO components contribute to the final osmolality of milk feeds. It is important to predict the final osmolality of TFO breast milk to ensure current osmolality recommendations are followed to minimize feeding intolerance and necrotizing enterocolitis. This study aims to develop and validate equations to predict the osmolality of TFO milk batches. To establish prediction models, the osmolalities of either native or supplemented breast milk with known amounts of fat, protein, and carbohydrates were analyzed. To validate prediction models, the osmolalities of each macronutrient and combinations of macronutrients were measured in an independent sample set. Additionally, osmolality was measured in TFO milk samples obtained from a previous clinical study and compared with predicted osmolality using the prediction equations. Following the addition of 1 g of carbohydrates (glucose polymer), 1 g of hydrolyzed protein, or 1 g of whey protein per 100 mL breast milk, the average increase in osmolality was 20, 38, and 4 mOsm/kg respectively. Adding fat decreased osmolality only marginally due to dilution effect. Measured and predicted osmolality of combinations of macronutrients as well as single macronutrient (R2 = 0.93) were highly correlated. Using clinical data (n = 696), the average difference between the measured and predicted osmolality was 3 ± 11 mOsm/kg and was not statistically significant. In conclusion, the prediction model can be utilized to estimate osmolality values after fortification.
Commercially available milk analysers were originally developed for use in the dairy industry, but they are now used to analyse macronutrient content of breast milk in clinical studies and routine care of the premature or very low birthweight (VLBW) infants. Due to the different composition of cow and breast milk, these devices need to be validated against reference methods before they can be used in daily routine. However, current reference methods require a sample volume of 30-100 mL to analyse fat, protein and lactose. It is not feasible to obtain this volume of milk for research purposes, especially from VLBW infants as lactation may be delayed or impaired and the limited volume of breast milk must be provided to the infant. To support validation of milk analysers in both clinical and research settings, the aim of this study is to establish and validate micromethods for precise macronutrient analysis in small volume of breast milk and conduct a feasibility study of the micromethods as a post-validation. Methods include a modified Mojonnier ether extraction (fat), elemental analysis (protein) and ultra-performance liquid chromatography-tandem mass spectrometry (lactose). We were able to downsize volumes required for analysis of fat, protein and lactose to 1 mL, 260 μL and 100 μL; corresponding coefficients of variation are 1.7, 1.8 and 2.3%, respectively. The presented methods allow for reliable and precise analyses of macronutrients in ≤1.5 mL of breast milk and will be used to validate milk analysers.
Mackerel is known to be a rich source of omega‐3 family PUFAs. The acid value and conjugated dienoic acid value of mackerel, known as indices of oxidation, were determined. Fatty acids in both raw and broiled mackerels were analyzed by GC. PUFAs and saturated fatty acids were observed at a low level in broiled mackerel, possibly as a result of thermal degradation of the lipids. In addition, volatile components in mackerel extracted by solvent‐assisted flavor evaporation were analyzed by GC‐MS. In total, 38 volatile components were detected in raw mackerel, whereas 53 volatiles were found in broiled mackerel. Hydrocarbons and methyl‐ and/or ethyl‐substituted benzenes were quantitatively dominant. Levels of aldehydes and alcohols were significantly enhanced in broiled mackerel, as was the level of benzothiazole, which possibly forms as a result of the condensation of thermal degradation products from amino acids and/or proteins, and lipids.
Commercial infrared (IR) milk analyzers are being increasingly used in research settings for the macronutrient measurement of breast milk (BM) prior to its target fortification. These devices, however, may not provide reliable measurement if not properly calibrated. In the current study, we tested a correction algorithm for a Near-IR milk analyzer (Unity SpectraStar, Brookfield, CT, USA) for fat and protein measurements, and examined the effect of pasteurization on the IR matrix and the stability of fat, protein, and lactose. Measurement values generated through Near-IR analysis were compared against those obtained through chemical reference methods to test the correction algorithm for the Near-IR milk analyzer. Macronutrient levels were compared between unpasteurized and pasteurized milk samples to determine the effect of pasteurization on macronutrient stability. The correction algorithm generated for our device was found to be valid for unpasteurized and pasteurized BM. Pasteurization had no effect on the macronutrient levels and the IR matrix of BM. These results show that fat and protein content can be accurately measured and monitored for unpasteurized and pasteurized BM. Of additional importance is the implication that donated human milk, generally low in protein content, has the potential to be target fortified.
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