Comparison between Hyperspectral Imaging and Chemical Analysis of Polyphenol Oxidase Activity on Fresh-Cut Apple Slices

The assessment of the quality of fresh-cut apple slices is important for processing, storage, market value, and consumption. Determination of polyphenol oxidase activity (PPO) in apples is critical for controlling the quality of the ﬁnal product (i.e., dried apples and juices). Hyperspectral imaging (HSI) is a nondestructive, noncontact, and rapid food quality assessment technique. It has the potential to detect physical and chemical quality attributes of foods such as PPO of apple. The aim of this study was to investigate the suitability of HSI in the visible and near-infrared (VIS-NIR) range for indirect assessment of PPO activity of fresh-cut apple slices. Apple slices of two cultivars (cv. Golden Delicious and Elstar) were used to build a robust detection algorithm, which is independent of cultivars and applied treatments. Partial least squares (PLS) regression using the 7-fold cross-validation method and method comparison analysis (Bland–Altman plot, Passing–Bablok regression, and Deming regression) were performed. The 95% conﬁdence interval (CI) bands for the Bland–Altman analysis between the methods were − 4.19 and 13.11, and the mean diﬀerence was 3.7 e − 12 . The Passing–Bablok regression had a slope of 0.8 and an intercept of 7.6. The slope of the Deming regression was 0.8 within the CI bands of 0.56 and 1.10. These results show acceptable performance and no signiﬁcant deviation from linearity. Hence, the results demonstrated the feasibility of HSI as an indirect alternative to the standard chemical analysis of PPO enzyme activity.


Introduction
Processed foods such as fresh-cut and dried apples are becoming more popular due to their nutritional, functional, and convenience properties. However, processed apple products encounter a challenge in meeting the quality demands of consumers [1]. During apple processing, the occurrence of browning reactions can lead to aesthetically unappealing final products. e development of brown pigments during processing is an important criterion in the determination of the quality of fresh-cut apples and dried apple slices [2]. e colour, occurring on the product surface, can be attributed to the activities of polyphenol oxidase visually appealing products with a close-to-fresh produce appearance [10].
Several techniques have been extensively investigated to estimate the browning reaction and to control the discolouration of apple slices [11][12][13][14][15][16]. However, there are limited treatments that could be convenient for small-and medium-scale farmers to inhibit the PPO activity. Additionally, consumers are becoming more health-conscious with a preference for products containing no or a low amount of industrial preservatives. Ascorbic acid (AA), citric acid (CA), hot-water blanching (HWB), steam blanching (SB), and their combinations could be suitable treatments for maintaining a good visual appearance of apple slices [17]. Moreover, these treatments could be affordably utilized by all farmers without extensive technical background.
Previous studies have focused on destructive methods for measuring enzymatic reactions in apples. However, these methods are time-and resource-consuming and make use of chemicals during the analysis with a potential to adversely affect both human health and the environment [5,18]. In recent years, various techniques for analysing food quality attributes have been developed to replace standard methods. Noninvasive imaging methods, including HSI, have a reputation of being cost-effective in the long term and could eliminate the laboriousness associated with the traditional standard methods [19,20]. However, the main drawback of HSI is the data dimensionality and size. e resulting images are called data cubes with two spatial dimensions and one spectral dimension at different wavebands, in addition to the signal noise and highly correlated features along the spectral range. e noise and inadequate information can be discarded by handling HSI data which consequently reduces the computational load [21]. HSI has been successfully applied for reliable and accurate measurement of sensory, chemical, and physical properties of foods, such as PPO in mushroom [22], PPO in lychee pericarp [23], moisture content in white button mushroom [24], and moisture content and colour in apple slices [25]. Since PPO exists in multiple forms, its expression may differ among species and cultivars [26]. Algorithms already developed from HSI for mushroom products cannot be directly transferred to other products. Consequently, extensive experiments are required for the development of appropriate algorithms by conducting calibration, validation, and testing.
A robust and accurate statistical analysis is a prerequisite for the proper validation and interpretation of new methods [27]. In many algorithms, the correlation coefficient (r) is used to investigate the effectiveness of the model. However, the correlation coefficient (r) is a criterion to evaluate the linear association between variables but does not signify a good agreement between the two methods. erefore, the correlation coefficient (r) does not provide confirmation if newly developed techniques perform as well as or even better than standard methods [28]. Ideally, the magnitude of measurement error is known from measurement systems analysis and the guide to the expression of uncertainty in measurement (GUM) [29]. is indicates that measurement systems are subject to certain measurement errors that require the utilization of special experimental designs. Ordinary least squares (OLS) regression assumes only an error in the X-direction as it requires known fixed values for X (independent variable).
is approach is not suitable for method comparison, in which an error in the Y-(dependent variable) direction as well as in the X-direction must be assumed, since both methods to be compared have a specific known or unknown measurement uncertainty. e minimization of the sum of the squares can, therefore, not be achieved with an ordinary linear regression in the vertical direction. erefore, statistical approaches such as method comparison are applied to calibrate and validate new methods by assessing repeatability, relative precision, robustness, and reproducibility of the new method [30,31]. e agreement between methods is deemed good when the paired measures show a high correlation and low random scattering. In the Deming and Passing-Bablok regressions, this is indicated by an intercept which is not confidently different from 0 and a slope which is not confidently different from 1. Furthermore, the error should not be dependent on the measured value and should not have any curvature or any other systematic pattern. e Deming regression considers errors in both X-and Y-directions and the optimization is precisely orthogonal to the fitted linear function. e angle of the line depends on the measurement uncertainty in both Xand Y-directions [32]. e Passing-Bablok regression is more robust against extreme values or differences (possible outliers) and nonnormal distributed measurement errors [33]. Since each method covers a specific context in which the method comparison is evaluated, both methods are applied. e Bland-Altman plot analysis of differences and agreement offers a visual evaluation between the two methods. Each of the paired measures is represented on the graph by assigning the mean of the two methods the X-value and the difference between the two methods the Y-value within the limits of agreement (LOA). e LOA are determined by using the mean and the standard deviation (SD) of the differences between two methods; 95 percent of the data points should lie within ±2 SD of the mean difference. However, this does not evaluate whether the agreement is sufficient or suitable to use one method or the other. It simply quantifies the bias (the mean of the differences between the two methods) and the range of agreement. Like the regression approach, LOA shows unsystematic scattering of errors when the methods match well [34].
To the best of our knowledge, no research comparing the HSI method and the standard chemical analysis method in measuring PPO activity in fresh-cut apples has been documented. Moreover, previous studies have only developed algorithms specifically for individual cultivars and treatments. In this study, a robust model that is independent of treatments and cultivars was developed.
is study assessed the agreement between HSI and the standard chemical analysis method of determining PPO activity in apple slices of Golden Delicious and Elstar cultivars subjected to several antibrowning treatments. Experimental activities included (1) selecting the most useful VIS-NIR features by using the Variable Importance in the Projection (VIP) plot; (2) developing a model to predict PPO enzyme activity of apple slice regardless of the cultivar and treatment; and (3) validating the model using method comparison.

Materials, Methods, and Statistics
2.1. Samples Preparation. Two apple cultivars "Golden Delicious" and "Elstar" were purchased from a local supermarket (Göttingen, Germany) and the university farm of the University of Kassel, Hessische Staatsdomäne Frankenhausen (Grebenstein, Germany), respectively. ey were visually assessed for uniformity in ripeness and size. e apples were then stored at 4°C in a laboratory refrigerator; afterwards they were conditioned at room temperature before experimental preparations. During the experiments, the apples were washed and the centre cores were removed using 2.5 cm diameter stainless-steel corer (Lurch, Hildesheim, Germany) and sliced to 5 mm thickness using an electrical slicer (Graef, Allesschneider Vivo V 20, Arnsberg, Germany).
A total of 192 apples were used for the experiments, and 576 apple slices were treated with various antibrowning treatments. e experimental design was based upon a splitplot design with 64 runs created using the Design-Expert software version 10 [35]. Before the experiments, three slices were extracted from selected apples. After applying treatments and holding for 60 min at room temperature, hyperspectral images were taken and afterwards PPO activity was analysed. e average value of the three slices was calculated for each run.

Antibrowning Treatments.
Both HWB and SB treatments were performed in a temperature-controlled water bath with a maximum capacity of 22 litres (L) and interior dimensions of 350 × 220 × 290 mm (Memmert GmbH Co., WNB22, Schwabach, Germany). SB involved injecting steam through a stainless-steel perforated wire tray with stands where the apple slices were placed. e temperatures of water and steam generated in the water bath were measured with a K292 data logger thermometer (Voltcraft, Switzerland). Slices were dipped in hot water of 50°C, 60°C, and 70°C ensuring that the slices were uniformly under the water. e slices were exposed to steam of 65°C, 75°C, and 85°C. After the blanching treatment, slices were either immediately cooled for 2 min at room temperature or directly pretreated by dipping in an acid solution for 3 min at room temperature; then the residual PPO enzymatic activity was evaluated. Dipping treatments were performed using either 1% (w/v) AA or 1% (w/v) CA or a combination of both solutions. After treatment, the apple slices prepared for use as control samples and treated samples were exposed to ambient temperature for 60 min after the slicing operation (e.g., control slice (a), slice immediately after treating with 70°C HWB + 1% AA + 1% CA (b), and 70°C HWB + 1% AA + 1% CA treated slice exposed for 60 min at room temperature (c)), as presented in Figure 1.
e HSI system included a camera mod.
V10 E PFD (Specim Spectral Imaging Ltd., Oulu, Finland) coupled with a Schneider 35 mm lens (Xenoplan 1.9/35, Schneider Optische Werke GmbH, Bad Kreuznach, Germany) and a linear translation stage (Specim Spectral Imaging Ltd., Oulu, Finland). e camera captures slice images in the visible (VIS) and the near-infrared (NIR) range between 400 and 1000 nm at 1.5 nm increments. ree 60 W halogen GU10 bulbs illuminated the platform on which the slices were placed. During scanning, the translation stage moved at a speed of 8 mm s −1 and the exposure time was 20 msec. e distance between camera lens and the surface of apple slices was set at 270 mm. ree slices per experiment were scanned. Each complete image consisted of those three slices and the white reference tile. e apple samples were saved in the ENVI 3.6 format (Research Systems, Inc., USA) for further processing. e hyperspectral images were calibrated with both white and dark references. ese references were acquired to correct the raw images by removing random noise, unevenness in the light source intensity and to normalize the output of the camera. e white reference consisted of a ceramic tile of 200 × 24 mm with an overall image spatial resolution of 1632 × 1302 pixels. e dark reference was obtained by closing the shutter of the camera. e relative reflectance spectrum for each pixel in the image (R(λ xy )) was calculated using the following equation with a spatially averaged white reference illumination spectrum W(λ xy ), dark reference image (Dλ), and a sample irradiance spectrum S(λ xy ): e average relative reflectance spectrum for each sample was then calculated automatically.
Hyperspectral preprocessing methods, such as normalization, multiple scattering correction (MSC), and the first derivative, were executed to improve original spectra by eliminating noise and extracting informative spectral variables before model development [36]. In this study, normalization was used to scale the data to ensure each input parameter (pixel) has a similar data distribution and average values.
Image segmentation, background and dead pixel removal, noise removal, and average reflectance calculation were carried out using MATLAB 2015a ( e MathWorks Inc., Natick, MR, USA), following the methods used in a previous study [25].

Colour Assessment.
e surface colour of the apple slices was measured with a CR-400 Chroma Meter (Minolta, Osaka, Japan). e measurements were based on the CIELab colour space with the CIE Standard Illuminant C and 2degree observer angle. e colorimeter was calibrated using a standard white reflector plate with values of Y (92.4), x (0.316), and y (0.3322). Four replications were carried out for each sample by performing two colour measurements on each side of the slices. e results were expressed in terms of luminance (L * ), redness (a * ), and yellowness (b * ) [37].

PPO Enzyme Activity (Wet-Chemistry Method)
2.5.1. Enzyme Extraction. Enzyme extraction was carried out in accordance with Massantini et al. [38]. Ten grams of apple samples were homogenized with 20 mL of chilled sodium phosphate buffer (PBS, 0.1 M, pH 6.5) containing 1.5% (w/v) polyvinylpolypyrrolidone and 1% (v/v) Triton X 100 . e homogenization was performed under ice-cooling conditions using an Ultra-Turrax T25 (IKA Instruments Ltd., Staufen, Germany). e mixture was centrifuged at 12,400 × g for 20 min at 4°C using a centrifuge mod. Eppendorf 5416 (Sigma-Aldrich, Darmstadt, Germany). e supernatant was then filtered through a Macherey-Nagel (MN) 640 w filter paper. Finally, the crude enzymatic extract was stored at −80°C until use.

Enzyme Activity
Measurement. PPO activity was measured by following the method developed by Moscetti et al. [39] with a slight modification. e assay was performed using a spectrophotometer mod. HP 8453 UV-Vis system (Agilent Technologies GmbH, Waldbronn, Germany). Catechol (Sigma-Aldrich, St. Louis, Missouri, USA) was used as a substrate. e final reaction mixture contained 1.5 mL of catechol (40 mM), 2.3 mL of PBS (0.1 M, pH 6.5), and 0.2 mL of crude enzyme. Changes in the absorbance at 420 nm were monitored for 2 min upon oxidation of the substrates catalysed by the enzyme. One unit of enzyme activity (U) was defined as an increase in absorbance of 0.001 min −1 . Enzyme activity was measured in duplicate.

Statistical Analysis, Modelling Technique, and Method
Comparison. Partial least squares (PLS) regression and principal component analysis (PCA) are the common methods for multivariate analysis in the field of chemometrics. ese methods help to analyse a set of dependent variables from a large set of independent variables or predictors [40]. Models are built by extracting successive orthogonal latent factors that maximize the covariance structure between the response and predictor variables in a robust way. PCA is then performed on the preprocessed spectra collected from all samples. PCA uses orthogonal transformation to convert highly dimensional and highly correlated data into linearly uncorrelated variables known as principal components (PCs). A particular original wavelength variable has a specific loading on each PC. High loading indicates high importance of a specific wavelength on a PC. PCs explain the descending importance of explainable variation in datasets. Loadings of the first few PCs with the greatest contribution are used to identify important wavelengths [41,42].
Based on the waveform and multicollinearity of the hyperspectral data, the following steps were performed for regression model development: (1) PCA was carried out before developing the calibration model to visualize any relevant and interpretable structure in the data and to detect the outliers. PCA was also performed to correlate specific quality attributes such as water content, colour, and PPO in the wavebands in accordance with the literature [21,43]; (2) regression model development was conducted using the full spectrum with PLS. Fully cross-validated PLS models were developed to predict PPO activity. e optimum number of latent factors was used to build a robust PLS model to obtain the best prediction performance. e optimum number of PLS-latent variables for each feature and the number of factors that produced the least predicted residual errors sum of squares (PRESS) were selected as the optimum values [44]. ese optimum factors were selected to guard against over-and underfitting problems and to model the variability in the data. Due to the limited number of observations, the data was not split into a training and validation subsets. However, the regression model was cross-validated using the 7-fold cross-validation (CV) method [45,46].
Additionally, the Variable Importance in the Projection (VIP) was used to select the important wavelengths from the PLS models [31,47]. e VIP scores obtained by PLS regression can be used to select the most influential variables or predictors, x. e jth x-variable can be estimated by the following equation: Slice immediately a er treating with 70°C HWB + 1% AA + 1% CA (0 min) Slice treated under 70°C HWB + 1 % AA + 1% CA exposed for 60 min at room temperature (c) Figure 1: Illustration of the appearance of fresh-cut apple slices at different conditions: (a) control slice; (b) slice immediately after treating with 70°C HWB + 1% AA + 1% CA; and (c) 70°C HWB + 1% AA + 1% CA treated slice exposed for 60 min at room temperature.

Journal of Spectroscopy
where P is the total number of variables, A is the total number of components, SS a is the sum of squares explained by the a th component, SST is the total variance explained by all the components, and (W aj /W a ) 2 is calculated using loading weight vectors W aj for each component and represents the importance of the variable j for component a. e predictor variable whose VIP score is greater than 0.8 is considered as an important variable in this study considering the average of squared VIP score [48].
In this study, PLS was applied to find linear relationships between hyperspectral features and PPO activity through a nonlinear iterative partial least squares (NIPALS) algorithm.
Due to the low signal-to-noise ratio at the lower end of the spectral range, only the wavebands in the spectral range from 500 to 1009 nm were used to evaluate the data. Both apple cultivars and all treatments were used to develop the PLS regression model and for method validation. e basic assumption was that the measurement methods were  Journal of Spectroscopy independent of the sample treatment and apple cultivar for the purposes of model robustness [52]. Data handling and statistical analysis were both performed using JMP Pro 14.2.0 software (SAS Institute Inc., Cary, NC, USA). Figures 2(a)-2(c), the PPO, a * , and b * parameters have VIP scores greater than 0.8. e wavelength around 677 nm was found to be important for the determination of PPO activity. e development of brown colour at approximately 677 nm is also noticeable in the a * and b * parameters as shown in Figures 2(b) and 2(c).

Characteristics of Spectral Profiles of Apple Slices.
e average reflectance spectra extracted from regions of interest (ROIs) of apple slices treated with different treatments are shown in Figure 3 in the wavelength range of 500-1000 nm. A similar overall trend throughout the whole wavelength range was observed for all samples. e wavelength at around 677 nm was found to be an interesting absorption region, which was as well as that observed from the VIP plot. e changes in the spectral profiles for slices treated with different treatments showed similar spectral curves and slight differences in reflectance values. For instance, Elstar slices treated with HWB 60 + 1% AA + 1% CA showed nearly no spectral patterns, which might be due to the inactivation of PPO activity. is might be due to the loss of main secondary structure elements of PPO mainly during the thermal treatment [53].

Regression Analysis to Check Bias between Methods
Using the Intercept and the Slope as Indicators. PLS regression analysis was performed using 4 latent variables provided by the minimum root mean PRESS (Figure 4(a)). e comparison between measured (reference laboratory method) and predicted values of PPO activity by PLS is shown in Figure 4(b). A strictly paired dataset was built by the laboratory-measurement values as X-variable and the predicted values by HSI as Y-variable for the method comparison analysis. Both the Deming regression (red line) and the Passing-Bablok regression (blue line) were performed.
It can be observed that the values obtained from the reference chemical method and the predicted values from the PLS model are randomly scattered around the line of equality (Figure 4(b)). e slope of the Deming regression was 0.80 and that of the Passing-Bablok regression was 0.81, both of which are not confidently different from 1. e intercept of the Passing-Bablok regression was 7.62, which is also not confidently different from 0 (Table 1). e orthogonal fit ratio was found to be 0.64 (Table 2).

e Bland-Altman Plot to Check General Agreement Instead of Correlation between Methods.
e Bland-Altman plot analysis of differences and agreement is provided in Figure 5. e plot was constructed by plotting the difference between the predicted data from PLS and the measured PPO activity against the mean of both methods. e plot displays an acceptable agreement between noninvasive HSI technique and the chemical method (95% CI: −22.4 to 22.4) ( Table 3). As shown in Table 4, the mean difference (mean bias) was found to be 3.7e −12 with a standard deviation (SD) of 11.43.

Discussion
e suitability of noninvasive measurement using HSI for detecting PPO activity on fresh-cut apple slices was investigated by comparison with the standard chemical analysis method. According to Mollazade [54], the most suitable wavelength range for the determination of PPO activity in Button mushroom was 470-640 nm. In this study, X-variables (spectral wavelengths) with a VIP score greater than 0.8 were found to be important for modelling the metric (Yvariables). Around 677 nm, a VIP score plot greater than 0.8 indicated that the pigment changes such as chlorophyll a were perhaps related to PPO and brown colour development [55].
is could also be due to functional pigments (i.e., chlorophyll a and the accessory pigments) which absorb at wavelengths shorter than 700 nm [56,57]. Jian et al. [58] used the visible and shortwave near-infrared (VIS/SWNIR) technique in the range 400-1100 nm to evaluate the bruising susceptibility in Golden Delicious apple. eir findings showed a change in colour as a result of chlorophyll content at around 680 nm during prediction of the bruising of apples at maturity stage. Other studies have also shown that PPO activity leads to browning reactions [5,14,23,59]. erefore, the results from this study are in agreement with previous studies indicating relative reflectance spectra at 677 nm. e mean and the standard deviation of differences between the predicted and measured PPO obtained in this study were in agreement with the statistical limits defined by Bland and Altman [51]. However, a full-scale measurement system analysis could not be applied as this study only assessed whether the HSI method can be used as an alternative to the standard chemical analysis method. Consequently, a real measurement system analysis is necessary to confirm the repeatability and reproducibility of the HSI     method in a robust setup [29]. Two independent methods measuring the same variable might have their own inherent error. e dispersion observed in Figure 3 could be due to physical and chemical heterogeneity of the sample which is indicated by outliers outside the LOA [60]. Additionally, critical patterns in the plots indicated constant bias as well as slopes and curves (nonconstant bias) or variance heterogeneities [51,60]. e orthogonal fit ratio of 0.64 proved that both measurement methods had different measurement uncertainties. Nevertheless, the two approaches were found to be comparable since the mean difference is close to zero and the standard deviation of the differences between measured values reveals no systematic variation from the mean of the measurement pairs. Furthermore, the standard deviation of difference between the noninvasive and invasive methods was significantly small showing an acceptable precision, and thus the two methods can be used interchangeably to predict the enzyme activity.

Conclusions
e investigation on apple PPO activity is a fundamental challenge for the food industry and to the researchers. Proper control of browning reactions prevents adverse effects on the visual appearance of foods as perceived by the final consumer. is study investigated the suitability of HSI as a noninvasive method for the measurement of PPO on fresh-cut apples. Results showed that the changes in PPO activity of slices were mainly at wavelengths around 677 nm as is indicated by the VIP plot and spectral profile. A robust algorithm that is independent of both cultivars and applied treatments was developed. For this purpose, method comparison (Bland-Altman, Passing-Bablok, and Deming regressions) was conducted to assess HSI as an alternative method to the standard chemical method. us, the study clearly indicates that PPO measurement using HSI is applicable for automated real-time and in-line measurement.
is study provides an important first step towards the adoption of HSI as a nondestructive method of establishing enzyme activity in apples irrespective of cultivar. In our case, 7-fold cross-validation technique was performed, which makes the chance of overfitting much lower by striking a balance between modelling the intrinsic structure of the data and modelling the noise. Moreover, several hundred samples for both calibration (training) and validation sets are sufficient at the proof-of-concept stage. However, the technique should be validated more extensively before implementation in industry. us, the data is essential for evaluating model performance using independent data to assess the suitability of the developed models.
Further research and in-depth analysis of the spectral changes and chemometrics of enzyme activity in apple slices from different cultivars will be needed to develop a more precise and robust model.

Data Availability
e data used to support the findings of the study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.

Authors' Contributions
Luna Shrestha, Boris Kulig, and Barbara Sturm conceived and designed the experiments. Luna Shrestha performed the whole experiments and was the lead author of the manuscript. Luna Shrestha and Boris Kulig analysed the data and the interpretation of the results. Boris Kulig and Roberto Moscetti helped in structuring the manuscript and the interpretation of the results. Barbara Sturm also supported in terms of improvement of the manuscript style and content as well as English language. Elke Pawelzik provided all types of facility and supervision to conduct the laboratory work. Riccardo Massantini, Elke Pawelzik, and Oliver Hensel contributed their critical comments to improve the manuscript.