Combination of Machine Learning and Raman Spectroscopy for Determination of the Complex of Whey Protein Isolate with...

Macromolecules and their complexes remain interesting topics in various fields, such as targeted drug delivery and tissue regeneration. The complex chemical structure of such substances can be studied with a combination of Raman spectroscopy and machine learning. The complex of whey protein isolate (WPI) and hyaluronic acid (HA) is beneficial in terms of drug delivery. It provides HA properties with the stability obtained from WPI. However, differences between WPI-HA and WPI solutions can be difficult to detect by Raman spectroscopy. Especially when the low HA (0.1, 0.25, 0.5% w/v) and the constant WPI (5% w/v) concentrations are used. Before applying the machine learning techniques, all the collected data were divided into training and test sets in a ratio of 3:1. The performances of two ensemble methods, random forest (RF) and gradient boosting (GB), were evaluated on the Raman data, depending on the type of problem (regression or classification). The impact of noise reduction using principal component analysis (PCA) on the performance of the two machine learning methods was assessed. This procedure allowed us to reduce the number of features while retaining 95% of the explained variance in the data. Another application of these machine learning methods was to identify the WPI Raman bands that changed the most with the addition of HA. Both the RF and GB could provide feature importance data that could be plotted in conjunction with the actual Raman spectra of the samples. The results show that the addition of HA to WPI led to changes mainly around 1003 cm^-1 (correspond to ring breath of phenylalanine) and 1400 cm^-1, as demonstrated by the regression and classification models. For selected Raman bands, where the feature importance was greater than 1%, a direct evaluation of the effect of the amount of HA on the Raman intensities was performed but was found not to be informative. Thus, applying the RF or GB estimators to the Raman data with feature importance evaluation could detect and highlight small differences in the spectra of substances that arose from changes in the chemical structure; using PCA to filter out noise in the Raman data could improve the performance of both the RF and GB. The demonstrated results will make it possible to analyze changes in chemical bonds during various processes, for example, conjugation, to study complex mixtures of substances, even with small additions of the components of interest.