Synchronous Front-Face Fluorescence Spectra: A Review of Milk Fluorophores

Abstract

Milk is subjected to different industrial processes, provoking significant physicochemical modifications that impact milk's functional properties. As a rapid and in-line method, front-face fluorescence can be used to characterize milk instead of conventional analytical tests. However, when applying fluorescence spectroscopy for any application, it is not always necessary to determine which compound is responsible for each fluorescent response. In complex matrixes such as milk where several variables are interdependent, the unique identification of compounds can be challenging. Thus, few efforts have been made on the chemical characterization of milk' fluorescent spectrum and the current information is dispersed. This review aims to organize research findings by dividing the milk spectra into areas and concatenating each area with at least one fluorophore. Designations are discussed by providing specific information on the fluorescent properties of each compound. In addition, a summary table of all fluorophores and references cited in this work by area is provided. This review provides a solid foundation for further research and could serve as a central reference.

Keywords: excitation emission matrix; fluorophores; front-face fluorescence; skim milk; synchronous fluorescence spectroscopy.

Figure 1

A simplified Jablonski diagram to illustrate fluorescence process.

Figure 2

Excitation and emission spectra.

Figure 3

Principles of right-angle and front-face fluorescence configurations.

Figure 4

Areas of interest established on heatmap fluorescence of reconstituted skimmed milk powder (“low heated”, spray-dried, pH = 6.5, solubility = 99%, WPNI ≥ 7 mg·g⁻¹ and 800 cfu·g⁻¹ milk; Chr. Hansen SL, Jernholmen, Denmark). Milk spectra were acquired at 20 °C on a fluorescence spectrophotometer (Cary Eclipse Fluorescence Spectrophotometer, Agilent Technologies, Santa Clara, CA, USA) equipped with 15 W lamp “press Xenon lamp” and a “front-face” geometry accessory at 35° (Solid Sample Holder Accessory and Cuvette Kit, Agilent Technologies). The excitation was scanned from 250 to 550 nm at 600 volts with 1 nm intervals and an initial Delta of 20 nm, a Delta increment of 10 nm from the corresponding excitation wavelength and a Delta stop of 220 nm (excitation and emission slit widths of 5 nm); A1–A10: areas 1 to 10.

Figure 5

Schematic figure showing the decomposition of the decrease in total fluorescence intensity of tryptophan (ΔI) into two fractions, one associated with the change of the peak height (ΔI_F) and another with the displacement of the emission maximum (ΔI_λ). Change in the peak wavelength (Δλ) can be seen as a shift from λ₀ to λ_T with intensities shown as Iλ₀ and Iλ_T. Reprinted with permission from Ref. [6]. Copyright 2020, Elsevier.

Figure 6

Emission spectra excited at 370 nm of reconstituted skim milk powder heat treated at 80 °C at six holding times.Reprinted with permission from Ref. [13]. Copyright 2016, Universitat Autònoma de Barcelona.