Confocal Raman Microspectroscopy of Dairy Emulsions: Ratiometric Features of Dehydration and Matrix Thermal History

Introduction: Identification of reconstituted milk is complicated because conventional heat-treatment markers reflect cumulative time-temperature load and do not always distinguish spray drying from intensive heating of liquid milk. Raman spectroscopy can register changes in protein, carbohydrate, and lipid regions without chemical sample preparation. However, physically and chemically interpretable ratiometric features that allow the spectral response of pasteurized milk to be compared with changes formed during drying and retained after reconstitution remain insufficiently developed.

Objective: To identify ratiometric features in Raman spectra that differ among raw, pasteurized, and reconstituted milk and to assess differences between samples produced from whole milk powder obtained under two drying regimes.

Materials and Methods: A single batch of milk was studied in raw form, after pasteurization at 76, 88, and 98 °C, after drying under regimes characterized by outlet-air temperatures of ≤70 and approximately 95 °C, and after reconstitution to a calculated total-solids content of 12.1%. Spectra were recorded using an M532 Raman microscope at 532 nm and analyzed from the integrated areas of bands at 1004, 1070, 1265, 1440, and 1660 cm^-1. The S₁₆₆₀/S₁₂₆₅, S₁₀₇₀/S₁₂₆₅, S₁₀₇₀/S₁₄₄₀, and S₁₀₇₀/S₁₀₀₄ ratios were calculated. Three technical measurements were performed for each sample.

Results: In the reconstituted samples, the integral area of the phenylalanine band, S₁₀₀₄, was 2.7 and 4.6 times higher than that in the raw milk. Conversely, the empirical index S₁₀₇₀/S₁₀₀₄ in the reconstituted milk decreased significantly to values of 2.17 and 2.56 (compared to 7.58 in the raw milk). The area of the S₁₄₄₀ band decreased by approximately 45 and 31%. The S₁₀₇₀/S₁₄₄₀ index increased from 0.21 in the raw milk to 0.30 and 0.48 in the reconstituted samples.

Conclusions: The model series revealed ratiometric features sensitive to the combined effects of drying, reconstitution, and heat-treatment regime. The observed values should be regarded as preliminary ranges rather than diagnostic thresholds. Validation using independent batches is required, with control of raw-milk composition, homogenization, storage, and between-day measurement variability.

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