Near Infrared Laboratory on shoulder Portable NIR Solutions.
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1 Near Infrared Laboratory on shoulder Portable NIR Solutions. Igor Nazarov 1 ; Dr. Robert L. Wample 2 ; Oren Kaye 2 ; Dr. Antonio Odair Santos 2 ; Krystal Goulart 2 1 Brimrose Corporation of America, Baltimore, MD U.S.A. 2 California State University. Department of Viticulture and Enology, Fresno,CA U.S.A. Abstract The goal of this research was to determine if near infrared spectroscopy (NIR) could be integrated into vineyard and winery applications. Le Vigneron, a portable NIR spectrometer developed by Brimrose Corporation USA, was used to collect spectra (in the range from 1100 to 2300nm) from wine grapes and wine products. These spectra were then compared with values determined by standard reference methodologies. Subsequent calibration modeling using partial regression analysis (PLS1) with cross validation produced the following results, using a sampling set of more than 6,000 grape berries for fruit chemistry and fifty wine samples for wine chemistry. For grapes at or near harvest maturity, the follow Standard Errors of Predictions (SEP) were determined: Brix (.91), ph (.75), TA (.76) and anthocyanins (.82). In wines, correlation coefficients were greater then 0.9 for ethanol. These results demonstrate NIR s capabilities as an analytical tool for the grape and wine industry. Further work, using more robust data sets will be necessary, to improve the NIR s commercial applications in the future. INTRODUCTION The wine industry uses a variety of chemical parameters to measure and asses grape quality. These parameters include Brix (Baume), ph, titratable acidity, phenolic content and color. The ability to quickly and accurately measure these parameters either in the field or at a sampling station can vastly improve wine quality, by giving the winemaker an opportunity to designate grapes into different quality programs based on their chemistries. Once processed into wine, winemakers measure a variety of other chemical parameters such as ethanol, ph, malic acid, SO 2 concentration, phenolic content and residual sugar to maximize wine quality and mitigate microbial problems in finished wine products. Many of the common analytical procedures for these assays are impractical to perform regularly because of time constraints or because of storage and disposal costs for the required reagents. Indirect chemical analysis using NIR spectroscopy, therefore offers a variety of advantages over standard bench top analytical procedures. For starters, NIR spectroscopy can perform multiple analyses simultaneously. Once calibrated, these analyses can be performed instantaneously without the need for chemical reagents or fragile laboratory glassware. Equally as important, an NIR spectrometer is easy to operate, significantly reducing variability from operator error. The accuracy, speed and repeatability of NIR spectroscopy allows for its consideration as an integral tool for research and commercial wine production. Continued development of this technology will lead to further precision in a wider variety of important assays and determinations. 355
2 Figure 1. Schematic of the Acousto Optics Tunable Filter (AOTF) The principle of the Acousto- Optic Tunable Filter (AOTF) is based upon the acoustic diffraction of light in an an-isotropic medium. The solid-state device consists of a piezo-electric transducer bonded to a birefringent crystal. When the transducer is excited by an applied RF signal, acoustic waves are generated in the crystal. The propagating acoustic wave produces a periodic modulation of the refractive index. This provides a moving phase grating that under proper conditions will diffract portions of an incident light beam. For a fixed acoustic frequency, a narrow band of optical frequencies satisfies the phase matching conditions and is cumulatively diffracted. As the RF frequency is changed, the center of the optical band-pass is changed accordingly so that the phase matching condition is maintained. Near Infrared Spectroscopy is currently being used as a quantitative tool, which relies on chemometrics to develop calibrations relating a reference analysis of the constituent to that of the NIR optical spectrum. The mathematical treatment of NIR data includes Multi- Linear Regression (MLR), Principle Component Analysis (PCA), and Principle Component Regression (PCR), Partial Least Squares (PLS) and discriminate analysis. All of these algorithms can be used singularly or in combination to yield the resultant goal of quantitative prediction and qualitative description of the constituents of interest. Brimrose Corporation of America has developed a portable, handheld Near-Infrared (NIR) spectrometer with no moving parts for use in vineyards and wineries. The unit, dubbed Le Vigneron, utilizes AOTF-NIR (acousto-optic tunable filter) technology and is backed up by powerful chemometric-statistical software. Rather than performing chemical analyses of grapes, fruits, or wine; Le Vigneron measures chemical parameters indirectly, by comparing the spectral characteristics of a sample with a predetermined, calibrated model. Quantitative Analyses such as Brix, ph, acidity, and color, can be determined in the vineyard using a battery pack, and a wireless laptop computer. Figure 2. NIR Hand-held Analyzer is taking readings for Brix, ph, TA in vineyard without removing it from vine 356
3 MATERIAL AND METHODS Thousands of spectra from grapes were used to build the calibrations over two seasons at California State University Fresno and at wineries in Australia. Calibrations were built using individual berry or collective composites from many berry samples, depending on the specific assay. Robust calibrations based on many grape varieties have allowed for the construction of general predication models for Brix, ph, TA, color organic acids, potassium and amino nitrogen. In all of these models the predicted results were close to the reference values. Additionally, individual models were created for Cabernet Sauvignon and Merlot. By keeping one grape variety in the model, an element of variability was removed and the model s results were improved. An attachable probe allows the same NIR unit to be used for analyses in the winery cellar. This stainless steel transfelctance probe can be used to measure different parameters in aging wine on a barrel by barrel basis. Analyses such as % alcohol, ph, TA, or Figure 3. NIR Analyzer with Probe for Wine testing malic acid can be performed instantaneously, without the need for laboratory glassware or hazardous chemical reagents. Methodology (grapes): Researchers at California State University Fresno collected cluster samples (5-10) from more than 50 vineyards and treatment block across California s San Joaquin Valley. From these samples, berries were randomly selected for spectral analysis using NIR spectroscopy with wavelength range: nm and wavelength increment 2 nm. Immediately after scanning, these grapes were analyzed by established methodologies. Additional analyses (not published here) were performed by Fourier Transformed Infrared Analysis (FTIR) using a Foss 120 Grapescan unit. Parameter Reference Method Spectrometric Method Brix Refractometer FTIR ph ph meter FTIR Titratable Acidity Titration FTIR Color Malvidin-3-Glucoside Assay Malic Acid FTIR Tartartic Acid FTIR NH 3 FTIR Potassium FTIR Methodology (wines): Fifty to 150 red wines were sampled for various analyses. Wines were scanned using a submersible transflectance probe attachment with 1 mm pathlength. The following analyses were performed. Additional FTIR analysis was performed using a Foss 120 Winescan unit. 357
4 Calibrations: NIR spectra were processed into units of absorbance 1 st derivative or 2 nd derivatives. PLS multivariate regression analysis was then performed using the maximum number of reliable components (factor scores) in Unscrambler chemo-statistical software. Regression models were subsequently compared and manipulated to produce the highest possible calibration coefficients with correspondingly high validation coefficients. RESULTS Some results from this calibration work are included below. The following abbreviations will be used for all models: Calibration Correlation Coefficient = CC Validation Correlation Coefficient = VC Number of Principle Components = PC Standard Error of Prediction = SEP Result (grapes): Figure 4 : Calibration for Brix using a standard digital (temperature correcting) refractometer for reference values CC = , VC = , PC=11, SEP =
5 Figure 5: Calibration for ph using a calibrated ph meter for reference values CC = , VC = , PC = 13, SEP= 0.13 Figure 6: Calibration for color determined by assay of malvidin-3-glucoside (mg/g) of fresh fruit for reference values CC =.8192, VC =.6972, PC = 10, SEP =
6 Results (wine): Figure 7: Calibration for Ethanol content in wine CONCLUSION Based on the correlation coefficients, NIR has proven its potential for vineyard applications. Continued research being conducted at CSU Fresno will improve upon these calibrations coefficients and provide for newer more robust assays. Other work at CSU Fresno will incorporate GPS technology into the NIR unit such that on the vine analyses can be geo-located and referenced using GIS software. This unprecedented, real-time mapping will allow for more efficient (precision) farming applications. For wineries, NIR technology can be used for analysis and quality control. Currently, we have determined that NIR can be used to accurately measure ethanol content (CC =.9). Continued research is being conducted to determine if NIR can be used to measure malic acid concentration, as well as SO 2 content in wine products. The same technology can successfully be used for on-line measurements of fruits or vegetables. Viticulturists familiar with Le Vigneron have suggested that the device can be used to measure plant-water status by analyzing the moisture content of leaves and fruit. Other attributes such as nutrient utilization and photosynthetic activity may also be measured this same way. In vineyards, leaf study looks to be a promising area for research. A Le Vigneron unit with a rotating sample cup has also been used to determine important qualitative characteristics of crushed olives (aceituna) and the oils they produce. These qualitative characteristics include moisture and fat content. A set of crushed olive 360
7 samples were scanned using the Le Vigneron, and the corresponding spectra were processed by chemometric, algorithmic modeling to produce a regression model for both moisture and fat content. The regression models have correlation coefficients of 0.9 and 0.8 respectively. Moisture values ranged from 35 % to 65 % and the fat content ranged from 15 % to 35%. The standard error for each model indicate that spectra analyzed with Le Vigneron, can be used to accurately and precisely determine these parameters for the olive oil industry. The next step to improve quality control is to develop regression models for moisture and fat content on whole olives as well phenol content and fatty acids profiles. After creating such regression models, the Le Vigneron can be taken to the field to predict olive quality prior to harvesting. As with winery applications, a transflectance probe can be attached to the Le Vigneron enabling it to be used at various points in the oil production stream. Analyses for acidity, degree of saturation, contaminants and free fatty acids can be read by submerging the liquid probe into the oil vats at multiple points of processing and clarification. Overall, the results of these applications prove the feasibility of measuring different quantitative parameters in various fruits and their products. Future work involving continuous calibration improvement for existing models will improve the precision and reliability of the instrument. Pairing the Le Vigneron with GPS devices will provide a quality profile any olive orchard or vineyard. The high degree of portability as well as the durable, solid state structure of AOTF-NIR technology make the Le Vigneron a versatile tool for all aspects of wine and olive oil production Un laboratoire proche_infrarouge sur l épaule : solutions PIR portables Résumé Le but de cette recherche était de déterminer si la spectroscopie proche infrarouge (PIR) peut être intégrée dans les applications viti-vinicoles. Le Vigneron, un spectromètre portable développé par Brimrose Corporation (USA), a été mis en œuvre pour collecter des spectres (dans le domaine nm) de baies et de produits vinicoles. Ces spectres ont ensuite été comparés avec les valeurs obtenues par des méthodes de référence standard. Les étalonnages de modèles associés, basés sur la régression linéaire partielle (PLSR : Partial Least Square Regression) avec validation croisée ont produit les résultats suivants, sur un jeu de plus de 6000 baies pour la chimie du fruit et de 50 échantillons pour la chimie du vin. Pour les fruits mûrs ou proches de la maturité, les erreurs standard de prédiction (SEP) suivantes ont été déterminées : Brix (0,91), ph (0,75), TA (0,76) et anthocyanes (0,82).Pour les vins, les coefficients de corrélation étaient supérieurs à 0,9 pour l éthanol. Ces résultats démontrent le potentiel du PIR en tant qu outil d analyse pour l industrie viti vinicole. Des travaux complémentaires basés sur des jeux de données plus robustes seront nécessaires pour améliorer les applications commerciales du PIR dans le futur. 361
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