The Application of FTIR Spectroscopy and Chemometrics for the Authentication Analysis of Horse Milk
Milk is a good source of protein needed for human development. Milk also contains numerous bioactive molecules, which protect against microbial infection and inflammation and contribute to immune maturation and healthy microbial colonization [1, 2]. Due to price discrepancy, expensive milk was adulterated with cheaper price milk to get economic profits. In the milk industry, horse’s milk (HM) is extravagant milk to produce in comparison to cow’s milk (GM) and goat’s milk (GM). HM is far more nutritious than any other milk, along with CM and GM. HM contains only 44 calories per 100 grams, compared to 64 for cows and 70 for human milk . HM had a similar composition compared to human milk for whey protein and casein, but metabolic profiles examined different HM to human milk [4, 5]. Therefore, HM may be an adulteration target with CM and GM. The adulteration practice of dairy products, involving milk, was typically done by substituting or diluting high price milk with cheaper price milk .
Milk authenticity is an important issue nowadays, not only for producers and consumers but also for the regulatory bodies, as consequently, some analytical methods capable of detecting the adulteration practice and quantifying the adulterants are needed . These methods included ultraperformance liquid chromatography-tandem triple quadrupole mass spectrometry (UPLC-TOF MS) by determining the peptide markers  and metabolomics approach , LC-MS based on ion-trap for proteomics  and peptide analyses , GC-MS and GC-FID by determining fatty acid composition , differential scanning calorimetry (DSC) coupled with machine learning detecting the thermal profile of authentic and adulterated milk , and ICP-MS discriminating milk by geographical origin clustering . Chromatographic-based techniques coupled with MS detectors are widely used detection methods, despite these methods being expensive, involving sophisticated instruments, and needing competent analysts. To this difficulty, an easy and reliable technique based on vibrational spectroscopy authenticated milk from adulterated milk.
Vibrational spectroscopy (Raman and infrared spectroscopy), based on the interaction of samples with electromagnetic radiation in the infrared region, is one of the fingerprinting techniques widely reported for the authentication analysis of dairy products, including milk, especially in combination with chemometrics . Chemometrics is the appliance of mathematical and statistical techniques to extract the chemical responses into more understandable information such as pattern recognition patterns and discrimination . Raman spectroscopy and chemometrics of pattern recognition applied for milk authenticity offer a reliable and easy method. Near-infrared was also successful for the authentication analysis of organic milk , while raw milk from reconstituted milk was determined using midinfrared  and determination of different milk species . Now, reports are available related to the authenticity of horse milk; for this reason, this study is aimed at developing FTIR spectroscopy and chemometrics for authentication analysis of HM from cow milk (CM) and goat milk (GM).
2. Materials and Methods
The horse’s milk samples were collected from a farm in West Nusa Tenggara. Cow milk (CW) and goat milk (GM) were available from farms in Yogyakarta, Indonesia. All samples were stored in a refrigerator at -4°C before being used for analysis.
2.2. Preparation of Calibration Samples
Calibration samples prepared a set of 75 calibration samples. HM was mixed with CM and GM in the concentration binary mixture range of 0-100%. Validation samples comprising of HM, CM, and GM evaluated the calibration models. FTIR spectral measurement subjected all samples. The composition of HM in a binary mixture with GM as well as HM in a binary mixture with CM.
2.3. Linear Discriminant Analysis (LDA)
LDA was used for discrimination between HM and HM adulterated with CM and GM. The samples consisted of pure HM and HM mixed with CM and GM at different concentrations covering 1-100%. Discrimination between authentic and adulterated HM constructed Cooman’s plot.
2.4. Scanning FTIR Spectra
Spectrophotometer FTIR (FTIR Nicolet iS20) using detector DTGS (deuterated triglycine sulfate) was connected to software OMNIC® and Windows®. The samples were directed placed into multibounce attenuated total reflectance (ATR) crystal, scanned using a resolution of 8 cm-1 and number scanning of 64. All spectra were measured at the midinfrared region (4000–650 cm-1) using air as background. All spectra were recorded to the absorbance mode to facilitate quantitative analysis according to the Lambert-Beer law. The data obtained was managed using the software of TQ Analyst®.
2.5. Chemometrics Analysis
TQ Analyst is used for chemometrics analysis, including LDA and multivariate calibrations (PLSR and PCR). LDA assessed discrimination between authentic and adulterated HM by accuracy levels. In addition, multivariate calibrations were evaluated by the root mean square error of calibration (RMSEC), root mean square error of prediction (RMSEP), and coefficient of determination ().
3. Results and Discussion
In this study, FTIR spectroscopy in the midinfrared region (4000-650 cm-1) combined with chemometrics of multivariate calibration and supervised pattern recognition of linear discriminant analysis (LDA) determined authentication analysis of HM from CM and GM. FTIR spectra are considered fingerprint tools for analytical purposes, including to assess milk authenticity, due to specific peaks and shoulders indicating functional groups presented in the valuated samples. Figure 2 reveals FTIR spectra of milk, namely, horse milk (HM), cow milk (CM), goat milk (GM), and ternary mixture milk which had similar features. The identification of functional groups of these milk spectra is shown in Table 2. However, three spectra of HM, CM, and GM were distinguished from peak intensities as fingerprint property. These differences could be exploited as regions to optimize for chemometrics analysis. The peak at wavenumbers () of 3320 cm-1 was due to -OH stretching vibration coming from water contents of milk in Figures 3 and 4. The wavenumbers of 1700-1500 cm-1 corresponded to amide groups (amide I and amide II) as specific in proteins and nucleic acids. Specifically, absorption peaks presented characterized the amide bands at 1635 cm-1 and 1455 cm-1 . These peaks also were optimized during LDA and multivariate calibrations .
FTIR spectra in combination with chemometrics of linear discriminant analysis (LDA) were successfully applied for the classification between authentic horse milk (HM) and HM adulterated (GM and CM) without any misclassification observed. In addition, PLSR could provide the quantitative analysis of adulterants (GM and CM) reliably. The developed method is a fast and green analytical technique because it avoids the use of chemicals and solvents.
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