New portable optical sensors for the assessment of winegrape phenolic maturity based on berry fluorescence
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1 New portable optical sensors for the assessment of winegrape phenolic maturity based on berry fluorescence Z.G. Cerovic 1,2, N. Moise 3, G. Agati 4, G. Latouche 2, N. Ben Ghozlen 1,2 and S. Meyer 1,2 1 CNRS, Laboratoire d'ecologie Systématique et Evolution, UMR 8079, Orsay, F Equipe de Biospectroscopie Végétale, Univ. Paris-Sud, Orsay, F-91405, France 3 FORCE-A, Univ. Paris-Sud, Orsay, F Istituto di Fisica Applicata 'Nello Carrara', CNR, I Sesto Fiorentino, Italy zoran.cerovic@u-psud.fr Abstract Grape phenolic maturity is usually analysed by destructive wet chemistry in the laboratory. Yet, for precision agriculture more rapid and nondestructive methods are needed. Therefore, in addition to measurements of fruit colour, a new optical method was recently proposed. It is based on the screening of fruit chlorophyll fluorescence that allows both flavonol and anthocyanin contents of intact berry skin to be assessed. Here we present preliminary results obtained with two commercial devices, Dualex FLAV and Dualex ANTH, and a prototype, Multiplex, all based on this new method. We found that Multiplex has strong potential for an application in the vineyard for precision viticulture or for crop evaluation at the weighbridge. Keywords: grape quality sensors, chlorophyll fluorescence, epidermal absorbance Introduction Quality wine can be made only from good quality grapes; therefore, good viticultural practice is constantly gaining in importance. Several destructive and non-destructive methods are used in viticulture for crop evaluation and as decision support both at the plant and fruit level (Krstic et al., 2003). Optical methods are particularly appropriate for precision viticulture that tries to answer the problem of spatial and temporal crop heterogeneity, especially in fruit maturity. The optimal berry sugar content and acidity, known as technological maturity, are attained at different times in vineyard blocks. The grape phenolic maturity is even more heterogeneous (Bramley, 2005) and is attained later than technological maturity (Keller et al., 1998). Selective harvesting has been proposed as a solution to this problem based on zonal vineyard management and on-thego quality sensing (Bramley, 2005). The assessment of grape quality inside the zones has relied mostly on destructive laboratory analysis, although new optical techniques based on near infrared spectroscopy linked to chemometrics is emerging (Gishen et al., 2005). Estimation of skin anthocyanins (ANTH) through direct berry colour measurement without extraction have also been used (Carreño et al., 1995). More recently, a method was proposed to assess skin content of phenolics. It is based on their screening of excitation of chlorophyll fluorescence (ChlF). The method is applicable to winegrape leaves (Kolb & Pfündel, 2005) and fruits (Kolb et al., 2003; Agati et al., 2007) using either UV light for flavonols (FLAV), or visible light for ANTH. A visible beam for which the epidermis is transparent is used as a reference (cf. Agati et al., 2007). Assessment of FLAV and ANTH has also been attempted in apple skin by devices originally developed for leaves (Hagen et al., 2006). In summer 2005 we had 1
2 Figure 1. Spectra of the main berry skin absorbers compared to the light source used in Dualex FLAV, Dualex ANTH and Multiplex. Pictures on the right: (approximately at the same scale): Multiplex illuminating a bunch of Chardonnay (top) and the Dualex leaf-clip (bottom). the opportunity to test the new Dualex ANTH in addition to the leaf-clip Dualex FLAV, and a new prototype hand-held sensor Multiplex. The latter stands for multiple excitation fluorescence sensor and was derived from our work on fluorescence lidars (Ounis et al., 2001). Dualex measurements were performed on berry caps in order to calibrate the method on grapes. Multiplex measurements were performed on whole bunches to evaluate the potential use of fluorescence as signature of phenolic maturity. The comparison of red grape to white grape varieties during the whole season revealed two fluorescence ratios as potential signatures of grape phenolic maturity. Materials and methods Each week from mid July to the end of September two bunches of grapes (Vitis vinifera L.) of three varieties, Pinot Noir, Pinot Meunier and Chardonnay, were sampled from the experimental vineyard Fort Chabrol in Epernay, France (Long ' E, Lat ' N). Bunches were shipped to the laboratory in Orsay where all spectroscopic measurements were performed on the next day after harvesting. Dualex measurements and extractions of flavonoids The Dualex (FORCE-A, Orsay, France) technology, involving the use of ChlF excitation screening by UV-absorbing compounds of the epidermis, was described by Goulas et al. (2004). In the new Dualex ANTH the original UV-A LED is replaced by a green sampling LED emitting at 528 nm, and is therefore dedicated to the measurement of ANTH (Figure 1). At véraison, which occurred on August 16 (DOY 228), 30 berries were chosen to cover six different grades of colour from green to dark violet. There was a single blue-coloured berry (cf. uppermost point in Figure 2). Berry caps of 7-mm diameter were cut with a cork borer and razorblade and placed on a cover slip for microscopy, the flesh side on the glass. This preparation was then inserted in Dualex ANTH and Dualex FLAV. The juice of the remaining berry part was used 2
3 for total soluble solids (TSS) measurements expressed in Brix (Pocket PAL-1, Atago, Japan). The caps were frozen in liquid nitrogen and stored at -80 C until freeze-dried and ground. After a triple extraction in acidified 50% methanol, extract absorption spectra were recorded and FLAV and ANTH contents calculated as described in Agati et al. (2007). FLAV and ANTH were expressed in equivalent quercitrin and oenin, with molar absorptivities of 9.7 µmol -1 cm 2 and 28.5 µmol -1 cm 2, at 375 nm and 530 nm, respectively. Multiplex fluorescence measurements The details of the Multiplex prototype will be presented elsewhere (patent pending). It is a hand-held optical sensor controlled by a computer. It has three LED-matrice light sources: UV-A (UV), green (G) and red (R), pulsed at 3.3 khz, and three synchronised photodiode detectors protected by interference filters for fluorescence recording: bluegreen (BGF), red (RF) and far-red (FRF). Here we concentrated on the signal ratios most important for phenolic maturity, based on FRF UV, FRF G, FRF R and UV-excited BGF signals. We present the ratios and not individual signals because only the formers are able to avoid changes in orientation and distance when measuring in the vineyard. Multiplex was used indoors although it can be used in the field under moderate daylight. Each bunch was measured twice on the two sides of the two bunches, so each point in Figure 3 is a mean of 8 measurements. After Multiplex measurements six berries were sampled individually for their sugar content (TSS in Brix) and then the ph of the pooled juice was measured. Curve fitting and statistical analysis were performed using Igor Pro 4.0 software (WaveMetrics, USA). Results and Discussion Dualex Dualex ANTH gave a very good estimation (r 2 = 0.98, fit standard error = 0.083) of the skin ANTH content, albeit for an exponential relationship as expected from our recent spectroscopic studies (Agati et al, 2007) (Figure 2). This non-linearity above 100 Figure 2. Validation of Dualex optical sensors. Absorbance of extracts of both Pinot Noir and Pinot Meunier at characteristic wavelengths is compared to anthocyanins measured by Dualex ANTH (A) and flavonols measured by Dualex FLAV (B). Dotted lines indicate the 95% confidence interval. 3
4 nmol cm -2 is due to incomplete ANTH and chlorophyll spatial separation in the skin, and to inherent limits of absorption spectroscopy at high concentrations. FLAV were also measured reliably by Dualex FLAV (r 2 = 0.91, fit standard error = 0.112) showing tendencies towards saturation at higher skin contents (above 200 nmol cm -2 ). This is due to the three-fold smaller molar absorptivity for FLAV at their measuring wavelength compared to ANTH (cf. Figure 1). One of the solutions to extend the working span of Dualex ANTH would be to use a less absorbed sampling wavelength. Thanks to their low absorptivity in UV-A (Figure 1) ANTH will not interfere much with the measurements of FLAV. The 375/530 nm absorptivity ratio for the former is only (Figure 1). The FLAV do not contribute to absorbance at 530 nm at all (Figure 1). So, Dualex devices can be used to follow optically the two flavonoid families to about halfway up the maturation curve (DOY 240) (Figure 3). Thereafter individual berries can show saturation especially for ANTH. This is to be expected since extracted ANTH then give absorbances of over 20 per cm 2 of skin. Multiplex Both the ph (not shown) and sugar concentration increased along a typical maturation curve despite the fact that only two bunches per cultivar were sampled each week (Figure 3). At véraison the intra-bunch variability was very large, with coefficients of variations for Brix of the order of 25% (not shown) even among the berries of the same colour, green or red. Individual red berries had characteristics that will be attained by the whole bunch only two weeks later. The Multiplex fluorescence index for FLAV was defined here as log (FRF R /FRF UV ) like in Dualex FLAV. As can be seen from Figure 3B, it changed little with time, with a small tendency to decrease. It was weakly and negatively correlated to the sugar content, with a r 2 of only 0.35, 0.30 and 0.12 for Chardonnay, Pinot Figure 3. Changes in technological and phenolic maturity during the summer Fluorescence ratios measured on whole bunches with Multiplex are compared to the sugar content of the berry juice (inserts). 4
5 Meunier and Pinot Noir, respectively (not shown). On the other hand, the FORCE-A Multiplex index for ANTH, defined as an inverted normalised log (FRF R /FRF G ), and the BGF/FRF UV ratio were far more responsive to ANTH accumulation with grape maturation. This is due to the screening of ChlF excitation by the accumulation of ANTH, as seen with Dualex ANTH. But soon after véraison, FRF R and FRF UV were also decreased by ANTH accumulation in addition to FRF G that was already very low. So, changes in FRF R and FRF UV substantially influenced the Multiplex indices, the FRF G and BGF signals being one order of magnitude smaller (not shown). As FLAV seems to change little during ripening (Figure 3B) the increase in BGF/FRF UV (Figure 3D) can either be due to an accumulation of ANTH or to a decrease in chlorophyll. The behaviour of Multiplex indices was very similar for Pinot Noir and Pinot Meunier showing good correlation to the grape sugar content (insert Figure 3). As expected, ANTH related Multiplex indices changed little in white grapes (Chardonnay) and were therefore totally disconnected from technological maturity (inserts Figure 3). Anthocyanins, flavonols and phenolic maturity In a skin-disc study on Pinot Noir grapes, Price et al. (1995) have shown that sunexposed bunches have ten times more quercetin glycosides in their berry skins compared to their shaded counterparts, and that sun-exposure did not affect ANTH. So, since FLAV and ANTH skin contents are differently controlled by environmental constraints they may not be correlated, as seen here. Still, FLAV are important for wine as co-pigmentation factors that stabilise ANTH colour. It is therefore important to have a means to assess them in each berry. Dualex can fulfil that need for research on light intensity, temperature, nitrogen and other nutrient effects on accumulation of phenolics. Preliminary tests have shown that, taking certain precautions, Dualex measurements can be performed even on whole berries. In the vineyard the largest problem for maturity assessment in general is the representativity of the sample. A large variability exists among vines, grape bunches and even among berries inside a single bunch. So, the faster and the friendlier the method, the larger and the more representative will be the analysis. For the multiplex type sensor the number of samples to be measured is not limited, and in theory even total harvest can be sensed through on-line measurement. In addition, for maturation surveys, thanks to the non-destructive nature of the measurements, marked bunches representative of the block can be followed during the whole season. Technological and phenolic maturity are not attained at the same time (Keller et al, 1998). Phenolic maturity is the last to be attained, therefore optical signals dependent on ANTH accumulation could be used alone as a real-time decision support for selective harvesting. Conclusions Both types of optical sensors tested can be useful for viticulture. The leaf-clip type Dualex is inherently more precise and can yield quantitative data on ANTH and FLAV in individual berries. These would be valuable for studies on environmental effects on grape maturation. The non-contact sensor Multiplex, on the other hand, has real potential for precision viticulture. Still, it needs further comparison to other optical sensors and calibrations against grape skin phenolics quantified by wet chemistry. 5
6 Acknowledgements This project received financial support from the CNR/CNRS bilateral cooperation project n We would like to thank Alexandre Tonnellier & Laurent Panigai of CIVC (Comité Interprofessionnel des Vins de Champagne) for the supply of grapes and useful advice. References Agati G., Meyer S., Matteini P. and Cerovic Z. G Assessment of anthocyanins in grape (Vitis vinifera L.) berries using a non-invasive chlorophyll fluorescence method. Journal of Agricultural and Food Chemistry Bramley R Understanding variability in winegrape production systems. 2. Within vineyard variation in quality over several vintages. Australian Journal of Grape and Wine Research Carreño J., Martínez A., Almela L. and Fernández-López J. A Proposal of an index for the objective evaluation of the color of red table grapes. Food Research International Gishen M., Dambergs R. G. and Cozzolino D Grape and wine analysis - enhancing the power of spectroscopy with chemometrics. A review of some applications in the Australian wine industry. Australian Journal of Grape and Wine Research Goulas Y., Cerovic Z. G., Cartelat A. and Moya I Dualex: A new instrument for field measurements of epidermal UV-absorbance by chlorophyll fluorescence. Applied Optics Hagen S. F., Solhaug K. A., Bengtsson G. B., Borge G. I. A. and Bilger W Chlorophyll fluorescence as a tool for non-destructive estimation of anthocyanins and total flavonoids in apples. Postharvest Biology and Technology Keller M., Arnink K. J. and Hrazdina G Interaction of nitrogen availability during bloom and light intensity during veraison. II. Effects on anthocyanin and phenolic development during grape ripening. American Journal of Enology and Viticulture Kolb C. A., Kopecky J., Riederer M. and Pfündel E. E UV screening by phenolics in berries of grapevine (Vitis vinifera). Functional Plant Biology Kolb C. A. and Pfündel E. E Origins of non-linear and dissimilar relationships between epidermal UV absorbance and UV absorbance of extracted phenolics in leaves of grapevine and barley. Plant, Cell and Environment Krstic M., Moulds G., Panagiotopoulos B. and West S Growing quality grapes to winery specifications: quality measurement and management options for grapegrowers, Winetitles: Adelaide, 101 pp. Ounis A., Cerovic Z. G., Briantais J.-M. and Moya I Dual excitation FLIDAR for the estimation of epidermal UV absorption in leaves and canopies. Remote Sensing of Environment Price S. F., Breen P. J., Valladao M. and Watson B. T Cluster sun exposure and quercetin in Pinot noir grapes and wine. American Journal of Enology and Viticulture
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