1 CIR-Center for Integrated Research – University Campus Bio-Medico di Roma ... 100 virtual sensor responses was obtained from a single physical sensor.
MULTIDIMENSIONAL APPROACH TO SOLANACEAE’S NUTRITIONAL AND GUSTATIVE ASPECTS S. GRASSO 1, F. GENOVA 2, M . SANTONICO 1, G. PENNAZZA 1, V. LOCATO 2, L. DE GARA 2, D. ACCOTO 3, A. SUDANO 3, A. D‟AM ICO 4, W. M ARM O 5. 1
CIR-Center for Integrated Research – University Campus Bio-Medico di Roma Lab of Electronics for Sensor Systems 2 CIR-Center for Integrated Research – University Campus Bio-Medico di Roma Food Science e Human Nutrition Unit 3 CIR-Center for Integrated Research – University Campus Bio-Medico di Roma Laboratory of Biomedical Robotics and Biomicrosystems 4 Department of Electronic Engineering – University of Rome Tor Vergata 5 Antica Azienda Agricola Brocchieri - Roma
An innovative approach based on a potentiometric sensor has been employed for peppers characterization. Despite a great number of hot pepper varieties are cultivated worldwide and are consumed, a simple method to discriminate them does not exist yet. A multidimensional system centered on a liquid sensor was used to successfully characterize five different pepper species by means of an easy and rapid procedure. T he information deriving from cyclic voltammetry analyses was acquired by the electronic system and further evaluated through a multivariate data analysis approach. T he calculated models showed an efficiency of 100% in the classification of the hot peppers and were able to predict with good accuracy the antioxidant content of the pepper exemplars.
1. Introduction Pepper is the fruit of plants from the genus Capsicum L., which belongs to th e large Solanaceae family. Peppers are commonly called “hot” or “sweet” depending on their spiciness when consumed. The difference between the two kinds of peppers resides in the amount of a specific alkaloid, the capsaicin, which the plant accumulates in the fruits as a deterrent against premature consumption. Nevertheless, due to the many other beneficial properties, peppers have become a common cooking ingredient worldwide. Taxonomy of Capsicum includes 39 existing species and nowadays 5 of these are domesticated and cultivated since ancient times (5.200 – 3.400 B.C.) [1]. From domesticated species a great number of varieties derives, different in colors, shapes and spiciness. Despite a so wide choice of peppers, easy identification criteria are currently not available and hot peppers are commonly identified only on the basis of their spiciness. This classification method is reductive from a nutritional standpoint, because peppers do not contain only capsaicin, but also several vitamins and micro-nutrients [2]. Here we present a novel electronic system (BIONOTE) based on a liquid sensor array that is able to discriminate from different pepper species by means of a simple analytical procedure.
2. Materials & Methods 2.1. Sample selection Pepper fruits of the five domesticated species (Capsicum annuum, Capsicum baccatum, Capsicum chinense, Capsicum frutescens, Capsicum pubescens) were harvested on the basis of the correct maturation state and the absence of abnormalities. 2.2. Liquid analysis One gram of fresh tissue was isolated from each pepper exemplar paying attention to leave out both seeds and placenta. Samples were fine homogenized in 30 mL of double distilled water (pH 7.2) using an UltraTurrax instrument and the extracts were clarified through a centrifugation at 8.000 rcf and 4 °C. Finally, supernatants were collected and stored in ice up to the analysis. Electronic interface and sensors employed in the liquid analyses were the same as described in Santonico et al. 2013 [3]. Cyclic voltammetry in the range from −1 to 1 Volt was performed using a triangular function at 10 mHz and a sampling interval of 1 second. 2.3. Trolox Equivalent Antioxidant Capacity (TEAC) For lipophilic extraction, one gram of fresh tissue was isolated from each pepper exemplar as described above. Samples were homogenized in a volume of 5 mL of acetone (Sigma-Aldrich, Milan, Italy) using mortar and pestle with the addition of a small amount of quartz sand. Homogenates were clarified through a centrifugation at 8.000 rcf and 4 °C and the lipophilic fractions were collected. Remaining pellets were let to dry under flow hood to eliminate any acetone trace, thus were resuspended with 2 mL of 50 mM sodium phosphate buffer (pH 7.5). Mixtures were agitated vigorously for 1 minute and the hydrophilic extracts were recovered from the upper phase after a centrifugation. The antioxidant potential of either the hydrophilic and lipophilic pepper extracts was evaluated through the TEAC assay [4]. Results were derived from a standard curve of Trolox (Sigma-Aldrich, Milan, Italy) ranging from 3 to 15 µM. 3. Results and Discussion Pepper exemplars from the five domesticated species underwent extraction procedures before being characterized by means of either electrochemical and biochemical analyses. A screen-printed gold electrode (Aux.: Pt; Ref.: Ag) fabricated by DropSens S.L. (Llanera (Asturias), Spain) was employed as liquid sensor, while the electronic interface controlling it granted on the reference electrode a voltage input in the form of a triangular function and converted the out current from the working electrode in a an output voltage value. An array of 100 virtual sensor responses was obtained from a single physical sensor applying a 10 mHz triangular function as voltage input signal and recording one
output value per second. By means of this setup, each pepper extract was evaluated through five consecutive independent measures and reproducibility of the system was confirmed by voltammetric curves overlapping (Fig. 1).
Figure 1. Cyclic voltammogram of five C. frutescens consecutive independent measures.
A data set array comprising all the acquired values was constructed and it was further analyzed using multivariate data analysis techniques. The score plot of the first two Principal Components (PCs) shows the ability of the system to sharply discriminate between the five pepper species (Fig. 2). Additionally, a Partial Least Square Discriminant Analysis (PLS-DA) model was calculated showing an efficiency of 100% in the classification of the hot peppers in four independent repetitions each.
Figure 2. Scores plot of the first two PCs of the PCA model built on the collected data.
From a biochemical point of view, the antioxidant potential of either the hydrophilic and lipophilic pepper extracts was evaluated through the TEAC assay. Results showed quite differences between the two kinds of fraction in terms of antioxidant molecules concentration. In fact, while C. frutescens showed the highest value among the hydrophilic ext racts, its lipophilic antioxidant molecules resulted only the 25% of C. chinense„s ones (Fig. 3). In general, the estimated total antioxidant capacity of the lipophilic pepper extracts was about one order of magnitude higher than hydrophilic counterparts.
Figure 3. Hydrophilic and lipophilic extracts evaluation by T EAC assay.
Finally, a single data set comprising all the electrochemical and biochemical features was evaluated through a multivariate data analysis approach. PLS-DA model highlighted the excellent ability of BIONOTE to predict antioxidant content within hydrophilic extracts with a Root Mean Square Error in Cross Validation, using the Leave One Out criterion, of 0.29 μmol/g (Fig. 4).
Figure 4. PLS-DA model performed on antioxidant molecules concentrations versus predicted ones.
On the contrary, the PLS-DA model did not seem to be able to calculate the lipophilic antioxidant molecules concentration with the same good accuracy. This discrepancy could be fundamentally attributed to the water based nature of the extract analyzed by the liquid sensor. In conclusion, BIONOTE system has demonstrated to recognize peppers of different species and to predict some biochemical features, thus opening the way to an easy-to-use device for farmers, manufacturers and consumers that actually lack of systems to analyze peppers and peppers derived products easily. References [1] Perry, L., et al. Starch fossils and the domestication and dispersal of chili peppers (Capsicum spp. L.) in the Americas. Science, 315 (5814), 986-988 (2007) [2] M. Materska and I. Perucka. Antioxidant Activity of the Main Phenolic Compounds Isolated from Hot Pepper Fruit (Capsicum annuum L.). J. Agric. Food Chem. 53 (5), 1750–1756 (2005) [3] M. Santonico, G. Pennazza, S. Grasso, A. D‟Amico, M. Bizzarri. Design and T est of a Biosensor-Based Multisensorial System: A Proof of Concept Study. Sensors 13, 16625-16640 (2013) [4] Re R. et al. Antioxidant Activity applying an improved ABTS Radical Cation Decolorization Assay. Free Radical Biology & Medicine. 26, 1231-1237 (1999)
