Month: December 2018

Application of portable micro near infrared spectroscopy to the screening of extractable polyphenols in grape skins: A complex challenge.

By Berta Baca-Bocanegra, José Miguel Hernández-Hierro, Francisco José Heredia, Julio Nogales-Bueno

Phenolic compounds are secondary metabolites that not only have well-known health benefits but also add several sensory characteristics to wine. Red grapes (Vitis vinifera L.) contain about four grams of phenolic material per kilo. Most of them are found in berry solid parts and are transferred to the wine during the fermentation stage. Consequently, two important topics in oenology research have typically been the study of phenolic compounds present in grape solid parts and how well these compounds are transferred to wine (Ribéreau-Gayon, et al., 2006; Waterhouse, 2002). Among other techniques, near infrared (NIR) spectroscopy has been applied to study these topics.
A brief bibliographic review can show the strong evolution suffered by NIR spectroscopy during the last decades. A number of important oenological parameters, such as total soluble solids, pH, phenolic compounds (phenolic acids, flavanols, flavonols or anthocyanins), variety, geographical origin, etc., have been determined using traditional NIR spectroscopy (Cozzolino, et al., 2004; Ferrer-Gallego, Hernández-Hierro, Rivas-Gonzalo, & Escribano-Bailón, 2011; Herrera, Guesalaga, & Agosin, 2003). In a further step, NIR hyperspectral imaging added spatial information of the samples to the NIR spectrum and new and more suitable methods could be developed (Baca-Bocanegra, et al., 2016; Nogales-Bueno, et al., 2016). Among these studies, NIR hyperspectral imaging has been used to develop screening methods to measure extractable phenols in grapes (Nogales-Bueno, Baca-Bocanegra, Rodríguez-Pulido, Heredia, & Hernández-Hierro, 2015). However, despite the fairly good results obtained by NIR spectroscopy in the prediction of different parameters in the wine sector, most of these studies are carried out in the laboratory and involve the transport of samples. To solve this problem, interest has shifted toward the development of portable vis/NIR systems using Linear Variable Filter (LVF). This filter is an important innovation in optical system design and miniaturization due to the fact that it does not need any external components because all the needed parts are incorporated into its design. This kind of device might allow acquiring NIR spectra in vineyards, directly on-the-vine. Although limited information is still available with regards to this technology on the enology sector, their use could be significantly hindered by the varying conditions of field measurements.
In this work, a feasibility study on the use of a portable micro NIR spectroscopy device for the “in vineyard” screening of extractable total phenolic content, extractable flavanol content and extractable anthocyanin content (EPC, EFC, EAC) in red grape skins has been developed. For that purpose, quantitative (PLS) and qualitative (LDA, DPLS and Pearson’s similarity index) approaches were applied to two different sets of grape samples.
Grape spectra collection was carried out in two different seasons and following two different methodologies for the spectra acquisition (Figure 1). These methodologies were designed in order to optimize the procedure for “in vineyard” grape spectra acquisition (Baca-Bocanegra, Hernández-Hierro, Nogales-Bueno, & Heredia, 2019). Briefly, in 2016 season, grape spectra were collected directly on the bunch while in 2017 season, the engaging grapes were picked from the bunch and, just after that, grape skins were manually separated from the whole grapes and placed at the bottom of quartz cuvettes to collect the spectra.

Figure 1. Schematic representation of the experimental design. Taken from Baca-Bocanegra, et al. (2019).

For both seasons, quantitative calibrations produced higher errors than those obtained in our previous study developed using a bench top hyperspectral system (Nogales-Bueno, et al., 2015) but in accordance with the high errors previously obtained by Guidetti, Beghi, and Bodria (2010) for the estimation of extractable anthocyanins and polyphenols in grapes using a portable device. With regard to the aforesaid hyperspectral imaging study, similar InGaAs sensor was used for predicting the same reference parameters in similar samples and applying similar chemometrics. Therefore, it is proven that the measurement of whole grapes or grape skins in field with the MicroNIR system is not as efficient as the in-lab hyperspectral methodology applied in our previous study.
Moreover, qualitative analyses (LDA, DPLS and Pearson's similarity index) produced high errors in the classification of samples with low or high extractable polyphenols contents. A comparative between the generated models is shown in Figure 2.


Figure 2. Receiver operating characteristic (ROC) curves of different chemometric tools applied (LDA, DPLS and Pearson's similarity index) for each parameter (EPC, EFC and EAC) and each season (2016 and 2017). Internal and external validation results are shown. Taken from Baca-Bocanegra, et al. (2019).

Receiver operating characteristic (ROC) curves shows that the measurement of grape skin in quartz cuvettes, carried out in 2017 season, resulted in a slight improvement in the percentages of samples correctly classified according their EPC, EFC and EAC levels, although they were not good enough for considering them useful models.
Therefore, the procedure reported here does not present enough accuracy for the “in vineyard” screening of extractable polyphenols in red grape skins. Although the aforesaid device has been developed for its use out of lab, vineyard environmental conditions might play a critical role on its use. Furthermore, heterogeneity of analyzed grapes and the own features of the berries (size, geometry or skin grape thickness) may also have influence on the obtained data and especial attention should be paid in further studies.



This work has been carried out by the “Food Colour and Quality” research group (Universidad de Sevilla, Spain). This group investigates on different areas related to the quality of food and the control of the production. Among these research areas, we can highlight the development and application of rapid quality control methodologies, especially Tristimulus Colorimetry, and the relationships between colour, chemical composition and visual assessment of several food products such as wines and grapes, citric juices, red fruits, olive oil, honey, etc. Within this research group, the spectroscopy section develops different studies for the control of food quality by means of several spectral tools. From left to right: Berta Baca Bocanegra, Julio Nogales Bueno, María Lourdes González-Miret Martín, José Miguel Hernández Hierro, Francisco José Rodríguez Pulido and Francisco José Heredia Mira.


Baca-Bocanegra, B., Hernández-Hierro, J. M., Nogales-Bueno, J., & Heredia, F. J. (2019). Feasibility study on the use of a portable micro near infrared spectroscopy device for the “in vineyard” screening of extractable polyphenols in red grape skins. Talanta, 192, 353-359.
Baca-Bocanegra, B., Nogales-Bueno, J., Rodríguez-Pulido, F., González-Miret, M., Hernández-Hierro, J., & Heredia, F. (2016). Near infrared hyperspectral imaging: recent applications in the oenological and viticultural sectors. NIR news, 27(6), 14-18.
Cozzolino, D., Kwiatkowski, M. J., Parker, M., Cynkar, W. U., Dambergs, R. G., Gishen, M., & Herderich, M. J. (2004). Prediction of phenolic compounds in red wine fermentations by visible and near infrared spectroscopy. Analytica Chimica Acta, 513(1), 73-80.
Ferrer-Gallego, R., Hernández-Hierro, J. M., Rivas-Gonzalo, J. C., & Escribano-Bailón, M. T. (2011). Determination of phenolic compounds of grape skins during ripening by NIR spectroscopy. Lwt-Food Science and Technology, 44(4), 847-853.
Guidetti, R., Beghi, R., & Bodria, L. (2010). Evaluation of grape quality parameters by a simple VIS/NIR system. Transactions of the ASABE, 53(2), 477-484.
Herrera, J., Guesalaga, A., & Agosin, E. (2003). Shortwave-near infrared spectroscopy for non-destructive determination of maturity of wine grapes. Measurement Science and Technology, 14(5), 689-697.
Nogales-Bueno, J., Baca-Bocanegra, B., Rodríguez-Pulido, F. J., Heredia, F. J., & Hernández-Hierro, J. M. (2015). Use of near infrared hyperspectral tools for the screening of extractable polyphenols in red grape skins. Food Chemistry, 172, 559-564.
Nogales-Bueno, J., Rodríguez-Pulido, F. J., Baca-Bocanegra, B., González-Miret, M. L., Heredia, F. J., & Hernández-Hierro, J. M. (2016). Hyperspectral Imaging - A Novel Green Chemistry Technology for the Oenological and Viticultural Sectors. In P. Gorawala & S. Mandhatri (Eds.), Agricultural Research Updates, vol. 12 (pp. 45-56 ). New York: Nova Science Publishers, Inc.
Ribéreau-Gayon, P., Dubourdieu, D., Doneche, B., Lonvaud, A., Glories, Y., Maujean, A., & Branco, J. M. (2006). Handbook of Enology, The Microbiology of Wine and Vinifications West Sussex, England: J. Wiley & Sons.
Waterhouse, A. L. (2002). Wine phenolics New York, New york: The New York Academy of Sciences.

Posted by in Chemistry, Viticulture

Chronobiology in the vineyard

By Suzy Rogiers and Francesca Moroni

What is chronobiology?
Chronobiology refers to the periodic rhythms of organisms in response to solar or lunar cycles. Chronobiology is a phenomenon that occurs in many behavioural, physiological and metabolic processes of living organisms. Cycling rhythms in biological organisms can occur with change in seasons (such as flower development or hibernation), to lunar-monthly (marine animal breeding), to changes that cycle approximately every 24 hours (the sleep-wake cycle in many animals), to cycles that last less than 24 hours (REM periods during sleep). Some of these cycles are driven by changes in the external environment while others are endogenous and not dependent on external stimuli.

Biological clocks in the vineyard
Evidence for chronobiology can be found in many aspects of viticulture. Buds swell and burst every spring with warmer temperatures and longer days. The onset of flowering is also driven by changes in day length and its timing is important to reproductive success. Grapevines can sense longer days in spring through the light receptors located in green tissues. Alternatively, leaf senescence is activated as day length shortens in autumn and the vine prepares for dormancy. Co-ordination between cells and tissues of the shoot tips, leaves, woody components and roots is critical to successful regulation of growth and development (Figure 1). This may explain the presence of multiple clocks in different tissue types within any one organism.


The vineyard is a complex ecosystem with its resident and visiting organisms inherently undertaking seasonal and daily adjustments to the changing surroundings. For instance seasonal bird migration into and out of the vineyard is one aspect of an annual cycle. These same birds will likely have diurnal rhythms in eating behaviour and digestion, body temperature, immunity response and hormone release among others. Nocturnal grazing of insects and caterpillars on succulent leaf tissues revolve around a 24 h cycle. Butterflies are active during the day while moths are active at night. Mushrooms and other fungi release spores either diurnally or nocturnally. Flowers of weeds growing on the vineyard floor may only release scent molecules during a specific time of to conserve energy. Honeybees visiting these flowers are trained to feed on them at that specific time of day. The same weeds undergo photosynthesis and stomatal movements in a diurnal cycle. Even the microbes living on the berry surface likely undergo rhythmic changes in metabolic processes.

Circadian rhythms
The approximate 24 hour endogenous cycle is referred to as a circadian rhythm and is common in plants. The daily tracking of the sun by leaves of the mimosa plant (first described in 1729 by French astronomer Jean Jacques d’Ortous de Mairan), the opening and closing of tulip petals, the tracking of the sun by the sunflower, and carbohydrate metabolism are good examples of this type of rhythm. Circadian rhythms are driven by a self-sustained 24 h internal clock. These rhythms persist for some time despite the absence of environmental cues such as changes in light or temperature, but they are able to adjust (entrain) to these stimuli. For instance, the opening and closing of a flower is circadian, not just diurnal, if it continues after the plant is placed in continuous darkness. However, after some time the rhythm is lost, illustrating the importance of environmental cues in maintaining the rhythm. Circadian rhythms gives an organism the competitive advantage of ‘anticipating‘ change in its environment so that it can respond appropriately. For examples, Charles Darwin and his son Francis (1880) hypothesised that circadian leaf movements might be important for heat transfer. They found that a vertical leaf position reduces heat loss at night so that the plants are less likely to freeze.


Circadian root growth
Growth of plant organs can also follow circadian rhythms. The elongation of cells in leaves and stems can follow such oscillations. This and much other circadian behaviour are under transcriptional and translational regulation with underlying multiple interlocked feedback loops.
In plant science, root growth studies are not as common as those of the above ground components due to the difficulty of monitoring roots in the soil medium; this is especially challenging for large perennial plants with widespread root systems that grow far and deep. These difficulties have been partially overcome by rhizotrons (transparent windows placed in the soil) with scanners and cameras that allow frequent image acquisition. Using such methods we have shown that root growth in grapevines follows a diurnal rhythm with a maximum at sunset and a minimum at sunrise, despite constant soil temperature and moisture (Figure 2). Slow root growth by the end of the night can be explained by the depletion of starch reserves from the roots. Stressful soil temperatures that are too warm will dampen the maximum rate of root growth but will not shift its diurnal pattern. 


Exposing vines to shorter daylight in controlled environment chambers also did not disrupt the diurnal pattern demonstrating that root growth is under circadian control. Interestingly, diurnal shoot growth patterns did shift with changes in day length indicating that, in grapevines, shoot elongation is not under endogenous regulation.
There is no doubt that temperature and light are critical drivers for grape berry growth and development and leaf photosynthesis provides the carbon source to drive these processes. However the hidden half of the vine is integral to water and nutrient uptake and a better understanding of the environmental and endogenous factors that impinge on root functioning will result in better wines.


Figure 1. Grape berry development is a complex process integrating endogenous and environmental stimuli. How many of these individual physiological, metabolic and genetic processes are driven by endogenous or exogenous rhythms? Source: Rogiers et al., Potassium in the grape berry: transport and function. Frontiers in Plant Science 8: 1629. doi: 10.3389/fpls.2017.01629

Biological clocks and wine perception
We have demonstrated that grapevine biological clocks will impinge on berry development and thus berry composition and the resultant wine style, but do human biological clocks have an impact on how a wine is perceived? The subjective experience of a wine is influenced by appetite, time of day and the physical and social environment of the taster. How our innate circadian rhythms interact with our ability to perceive, decode and process the sensory stimuli offered by wine may well influence our physiological and behavioural response. Little is known about how circadian clocks control taste and other chemosensory systems but hopefully some light will be shed on this multifaceted subject so that we may enjoy our wine to the fullest.


Figure 2. Root growth in grapevines follows a circadian rhythm with rapid growth at sunset and slow growth at sunrise.

Suzy Rogiers is Principal Research Scientist with NSW Department of Primary Industries. She is also adjunct Professor at Western Sydney University and a member of the National Wine & Grape Industry Centre in Australia. Her research is focussed on the impacts of heat, drought and unpredictable weather on crop productivity. She has researched water-use efficiency, nutrition and carbon acquisition in horticultural crops with a focus on wine grapes. She has a particular interest in fruit colour, flavour, aroma and nutritional profile in response to environmental conditions (light, temperature, water) and cultural practises (canopy management, orchard floor management, irrigation, nutrition, clonal selection and rootstocks). She is also interested in the endogenous regulation of fruit development and chronobiology.
Francecsa Moroni is a Sydney based artist and student of Design in Architecture. She composed the images for this article.


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Posted by in Viticulture