Month: December 2018

Immobilization of yeasts in oak chips or cellulose powder for use in bottle-fermented sparkling wine

By Carmen Berbegal and Isabel Pardo

High quality sparkling wines using the traditional or “champenoise” method include two fermentations carried out by Saccharomyces cerevisiae (Garofalo et al. 2018). The starter cultures that are used to develop the second fermentation inside the capped bottles need to possess several additional technological properties to those yeasts used in the first fermentation. They have to be selected in order to survive in stress conditions, in particular this yeast must be chosen for its ability to ferment high-acidity and low-pH wines, and it must be ethanol-tolerant (Garofalo et al. 2016). A way to protect yeast cells from stress in the second fermentation is through the cell immobilization (Nedović et al. 2015). Moreover, this mechanism can reduce the use of riddling agents. Thus, the objectives of our project were to initially develop yeast immobilization processes on two wine-compatible supports: oak chips and cellulose powder; secondly, to study the effects of type of yeast and type of immobilization support on the fermentation kinetics, the deposition rate of lees and the volatile composition of finished sparkling wine; finally, to compare the fermentation parameters of the wines inoculated with immobilized or non-immobilized cells.
We immobilized two different Saccharomyces cerevisiae yeast strains in oak chips and cellulose powder by lyophilization: the Saccharomyces cerevisiae strain Enolab55A (55A) isolated from an organic Spanish wine from Utiel-Requena D.O.P. in Spain and the commercial yeast IOC18-2007. As shown in Figure 1, the lyophilized Saccharomyces cerevisiae cells were adhered to the surfaces of oak chips/cellulose powder.

Figure 1. Electron microscopy photograph showing Saccharomyces cerevisiae cells immobilized on oak chips (A and B) and on cellulose powder (C and D) by lyophilization.

All the vinification trials were run in a base wine that consisted in a coupage of 80% Macabeo and 20% Chardonnay. We distributed the base wine in transparent glass bottles, the “tirage liqueur” and we added the immobilized oak chips/cellulose powder yeasts (1 g/L), or a free cell yeast culture (2×106 CFU/mL) (Fig. 2). No riddling agents were added. Each week during the first month and at the end of the second month of ageing, we opened three bottles per experiment to determine the residual sugars and ethanol concentrations (Frayne et al. 1986). After 9 months, we measured yeast sediment deposition efficiency by considering the total time that the gyropalette required to make wines transparent and we determined the volatile composition of the resulting sparkling wines at the end of the ageing period (Ortega et al. 2011).

Figure 2. Outline of the different vinification trials carried out in Bodega Dominio de la Vega winery S.L. (D.O.P. Utiel-Requena, Spain).

Our results showed that the final sugar and ethanol concentrations were similar in the sparkling wines fermented with the free cells or those immobilized on oak chips/cellulose powder cells, regardless of the yeast strain used; glucose and fructose were consumed in under 60 days and ethanol increased by 1.5% with the second fermentation. During the riddling performed automatically with a gyropalette, the immobilized yeasts on oak chips/cellulose powder settled in the neck of the bottles 3-fold faster than in the bottles that contained free cells. Completely transparent wines were obtained without having to resort to riddling agents, such as bentonite, which results in less manipulated and more natural products (Figure 3).

Figure 3. Sparkling wine bottles during the riddling and disgorging procedures.

When the volatile aroma analysis was done, thirty-two volatile compounds were identified in sparkling wines. We found significant differences in the formation of esters, acids, alcohols, aldehydes and lactones depending on the yeast used and the immobilization support. From both supports, oak chips were the more appropriate support for yeast immobilization. Despite the differences observed in the profile of volatile wines, at the sensory level no significant differences appeared between the fermented wines with both yeast types and those with the free or immobilized yeast with the different immobilization substrates.
It is important to highlight that during the sparkling wine-making process, the second fermentation occurs under very particular conditions for yeasts; the base wine has high alcohol content, and not all strains can grow and ferment under these conditions. Our results suggest that this new technology can be used with the organic yeast 55A as an alternative to commercial starter IOC18-2007, and that the use of immobilization supports reports the advantages of rapid yeast elimination and not adding bentonite, and does not have a negative impact on the wine sensory profile.

Those interested in a longer length report can download the working paper at:

This work was supported by the “Programa Valoritxa i Transfereix” 2013 (Ref: UV-CPI13274-159983) of the Universitat de València and carried out by Carmen Berbegal, Lucía Polo, Mª José García-Esparza, Victoria Lizama, Sergi Ferrer, Isabel Pardo and people from the winery ‘Dominio de la Vega’.

Carmen Berbegal is a PostDoc researcher from Enolab group at the University of Valencia (Spain). In addition to the University of Valencia education, she has been formed as a microbiologist in the National Collection of Industrial and Marine Bacteria (Scotland), in the University of Patras (Greece) and in the University of Foggia (Italy). All her investigations are focused on wine microbiology, mainly on lactic acid bacteria and on applied aspects related to the use of microorganisms to solve technological problems in the wine industry. She has been involved in the characterization and selection of lactic bacteria such as Oenococcus oeni and Lactobacillus plantarum to design different starter cultures to improve ‘wine qualities’, to avoid the growth of undesirable microorganisms, and to study the formation and the control of undesirable toxic compounds produced by lactic acid bacteria, such as biogenic amines. Reserchgate profile: , Scholar profile:


Isabel Pardo holds a degree in Biology and a PhD in Biology from the University of Valencia. She obtained the position of Full Professor at the Univ. Valencia in 2012. Co-author of more than 140 scientific papers and 240 congress communications. Regular editor/reviewer of many scientific and technical journals related to Microbiology and Oenology. Supervisor of 12 doctoral theses, one of them with Extraordinary Doctorate Prize, and supervisor of graduate/postgraduate students. Participation in 57 R+D+i Projects financed in competitive calls of Regional Administrations or public and private entities (in 9 of them as Principal Researcher), and in not competitive 37 Contracts, research agreements or R&D+i projects of Regional Administrations or public and private entities (in 7 of them as Principal Researcher). Co-author of a Spanish patent and of another International. She is member of different networks and associations related to microbiology, food and wine: CYTED, Gienol, Microcluster Vitivinicultura, Technological Platform of Wine, RedBal, Red Sicura. She is an evaluator of the National Agency for Evaluation and Prospective (ANEP) that depends on the Secretariat of State for Research, Ministry of Science and Innovation of Spain. She has extensive in Wine Microbiology.



Frayne, R.F., 1986. Direct analysis of the major organic components in grape must and wine using high performance liquid chromatography. Am. J. Enol. Vitic. 37, 281-287.
Garofalo, C., Arena, M., Laddomada, B., Cappello, M., Bleve, G., Grieco, F., Beneduce, L., Berbegal, C., Spano, G., Capozzi, V., 2016. Starter cultures for sparkling wine. Fermentation 2, 21.
Garofalo, C., Berbegal, C., Grieco, F., Tufariello, M., Spano, G., Capozzi, V., 2018. Selection of indigenous yeast strains for the production of sparkling wines from native Apulian grape varieties. Int J Food Microbiol 285, 7-17.
Nedović, V., Gibson, B., Mantzouridou, T.F., Bugarski, B., Djordjević, V., Kalušević, A., Paraskevopoulou, A., Sandell, M., Šmogrovičová, D., Yilmaztekin, M., 2015. Aroma formation by immobilized yeast cells in fermentation processes. Yeast 32, 173-216.
Ortega, C., Lopez, R., Cacho, J., Ferreira, V., 2001. Fast analysis of important wine volatile
compounds development and validation of a new method based on gas chromatographic- flame ionisation detection analysis of dichloromethane microextracts. J. Chromatogr. A 923, 205–214.

Posted by in Enology, Food Science and Technology

Polyphenols in grapevine leaves: unravelling vein and blade specific traits.

By A Ferrandino, O. Kedrina-Okutan, V. Novello

The genus Vitis comprises thousands of genotypes. Vitis vinifera (L.) represents one of the most widely cultivated plant species worldwide, table and wine grape cultivation playing pivotal roles in the economy of many countries, including old-tradition areas for wine grape cultivation (mainly countries around the Mediterranean basin), countries where cultivation has rapidly increased and new emerging regions. The environmental impact of viticulture is high due to a number of reasons: i) repetitive monoculture in traditional cultivation areas; ii) hilly cultivation conditions that imply soil erosion; iii) accumulation of copper in soils due to largely used copper-based fungicides.
However, the most striking impact of viticulture in the environment is the vast use of fungicides, to limit downy and powdery mildews, rots, all very dangerous and economically detrimental grapevine pathogens diffused in many world and European cultivation areas. According to the last published European Statistics on Plant Protection Products (PPP; European Commission Eurostat Data 1992-2003) 83% of total PPP was represented by fungicides and grapevine cultivation alone used 75% of fungicides sold in Europe (at that time represented by 25 Countries). The necessity to investigate and propose solutions to limit a further widespread in the use of these products is evident. Possible solutions include different alternatives, spreading from mechanical approaches, through the optimization of PPP distribution in the vineyard, to genetics, by searching and implanting low-susceptibility or resistant varieties, where allowed, and to agronomy. This last point requires a deep knowledge of the plant behavior, involving disciplines such as plant physiology and biochemistry. Considering that many grapevine pathogens, both those with leaf localization and those that localize in other organs, induce symptoms in leaves, we have recently investigated the polyphenolic accumulation and profile of constitutive polyphenols of leaf veins and blades in healthy plants of Vitis species and of Vitis vinifera varieties (Kedrina-Okutan et al., 2018). The importance of grapevine polyphenols is well known and detailed in berries; however, plant polyphenols play many different and important biological roles, taking part into the plant-defense mechanisms through molecular communication with pathogens, signals for the establishment of the infection, the activation of plant disease-resistance genes, the formation of elicitors, the activation of elicitor receptors and, finally gene regulation. Moreover, in the case of grapevine, vegetative organs, including leaves, are used in traditional plant-based medicine and as fresh food. Recent studies demonstrated that grapevine leaves have beneficial effect on human health due to their antioxidant properties, with anti-inflammatory, antibacterial, anticancerogenic and antiviral roles.

Studying the polyphenolic composition of some Vitis vinifera genotypes in veins and blades separately, we found different compositional traits and changes during the season. In healthy grapevine blades, regardless the cultivar, anthocyanins, dihydromyricetin-rhamnoside, hexosides of dihydroquercetin and dihydrokaempferol were absent as they exclusively accumulated in veins. Vein anthocyanin profile was generally characterized by the prevalence of acyl-derivatives, particularly in Sangiovese (Caramanico et al., 2017), and in Cabernet Sauvignon whereas, in line with what happens in berries, Pinot noir did not accumulate acylated-anthocyanins, at all. Barbera leaves displayed the highest complexity, accumulating seven different types of anthocyanins, Pinot noir and Grenache, the lowest (one or two types of molecules). Flavonol concentrations and profiles were cultivar-related, as well. They accumulated both in blades and in veins with Barbera and Grenache blades displaying the highest concentration, Nebbiolo the lowest.
Astilbin (dihydroquercetin-rhamnoside) was the exclusive flavanonol detected in blades and the prevalent one in veins. Specific traits were found in Nebbiolo leaves that showed the highest concentrations of flavanonols and the widest profile differentiation respect to other biotypes. Nebbiolo, Pinot noir and Cabernet Sauvignon displayed high concentration of the flavan-3-ol, epigallocatechin gallate in veins, particularly early in the season. Epigallocatechin gallate is the only flavan-3-ols listed by the Italian Health Ministry among “Other nutrients and molecules with nutritional and physiological effects”, thanks to its high antioxidant capacity, conferring anticarcinogenic, cardio and neuroprotective properties. Among non-flavonoid polyphenols, grapevine leaf extracts presented important concentration of hydroxycinnamic acids both in blades and in veins. Their profile did not differ respect to that of berries, except in Cabernet Sauvignon veins and blades where a caffeoyl hexoside was identified. Among the studied varieties, Barbera leaves showed the highest concentrations, particularly in blades.

Knowledge derived from the experimentation we have undertaken opens new and interesting fields of inquiry on several aspects. One is represented by the possible relation occurring between leaf flavonol accumulation and Vitis vinifera varieties response to water stress conditions, being known that flavonols are dampers of the reactive-oxygen species accumulation, mediated by abscisic acid (the hormone that regulates stomata opening) (Watkins et al., 2017). This, together with the higher concentration of flavonols in specific genotypes, could contribute to explain the cultivar-related stomata opening mechanism. Another field of investigation relies on the fact that innate lower or higher levels of susceptibility to pathogens could be related to the accumulation of higher or lower amounts of polyphenols, being demonstrated that constitutive higher amounts of flavonols can hinder the development of Plasmopara viticola (Latouche et al., 2013). The individuation of specific molecules that, being constitutively higher in specific genotypes could be an element of protection against reactive oxygen species induced by stressors, including those of biotic origin, would be of great help to contribute to the understanding of Vitis genotypes specific tolerance to or avoidance of damages induced by pathogens.


One of the most important and applicative consequence that can derive from this kind of knowledge relies on the future possibility of driving the accumulation of specific molecules, once demonstrated that also in ‘in field’ conditions they can contribute to limit the pathogen development. Or, to understand, through an in-depth study of grapevine leaf polyphenols if alternative cultivation management, such as the organic management of the vineyard, could provide specific polyphenolic accumulation and profiles, able to help the plant innate response to pathogens. Moreover, it represents a starting point for future deepening about grapevine and vineyard by-products as a source of bioactive phenolic compounds to be destined to numerous uses.
Those interested in a longer length report can download the working paper at:


Alessandra FERRANDINO, PhD (orcid/org0000-0002-1567-5874) is Researcher at the Department of Agriculture, Forest and Food Sciences (DISAFA) of Turin University.
She teaches ‘Secondary metabolites in grapevine’ and ‘Viticulture’ at Master Degrees of Turin University (MD in Viticulture and Enology Sciences and MD in Agricultural Sciences of DISAFA). Her research is focused on the impacts of abiotic (water stress) and biotic (Flavescence dorée, Plasmopara viticola) stress on grapevine secondary metabolite accumulation in fruits and in vegetative organs, contributing to understand how to drive berry quality and how to exploit innate phenomena of resilience to biotic stress. She has experience in grapevine polyphenol and volatile assessment through traditional and innovative techniques of analysis. She is reviewer of many International Journals. She has developed national and International scientific collaborations.


PhD candidate in Agricultural, Forestry and Food Sciences at University of Turin (Italy).
- graduated in Food Engineering and Product Development, Master from Tallinn University of Technology (Estonia).
- during 2017 took part in collaboration research activity in department of Biotechnology of Natural Products and Fruit Science at Technical University of Munich (Germany).
- experienced in polyphenol profiles composition analysis in plant based materials, using advanced chromatography techniques such as HPLC-DAD-UV and HPLS-ESI-MS/MS.
- Doctorate research project focused on investigation of constitutive polyphenolic compounds in various Vitis species to give a new insight about natural bioactive compounds and accumulation trends in grapevine leaves.


Novello Vittorino UNITO-DISAFA
- Full professor at Department of Agriculture, Forest and Food Sciences (DISAFA) of University of Turin.
- President of the Master of Viticulture and Enology Sciences, network among the Universities of Turin, Milan, Foggia, Palermo and Sassari, partner of the European Consortium EMaVE (European Master of Viticulture and Enology)
- Component of the Italian Delegation at OIV (International Organization of Vine and Wine). President of OIV Viticulture Commission and component of the Scientific and Technical Committee of OIV.
- Associated Editor of the open-access journal OENO-one, and referee of many international scientific journals.



Caramanico, L.; Rustioni, L.; De Lorenzis, G. Iron deficiency stimulates anthocyanin accumulation in grapevine apical leaves. Plant Physiol. Biochem. 2017, 119, 286–293.

Kedrina-Okutan, O.; Novello, V.; Hoffmann,T.; Hadersdorfer, J.; Occhipinti, A.; Schwab, W.; Ferrandino, A. Constitutive polyphenols in blades and veins of grapevine (Vitis vinifera L.) healthy leaves. J. Agric. Food Chem. 2018, 66, 10977−10990.

Latouche, G.; Bellow, S.; Poutaraud, A.; Meyer, S.; Cerovic, Z.G. Influence of constitutive phenolic compounds on the response of grapevine (Vitis vinifera L.) leaves to infection by Plasmopara viticola. Planta 2013, 237 (1), 351−361.

Watkins, J.; Chapman, J. M.; Muday, G. K. Abscisic acid induced reactive oxygen species are modulated by flavonols to control stomata aperture. Plant Physiol. 2017, 175 (4), 1807−1825.

European Commission Eurostat Data 1992-2003. The use of plant protection products in the European Union. Ed. Pierre Nadin.

Posted by in Chemistry, Viticulture

Effects of grape pomace on insulin sensitivity: towards a whole use of natural materials in wine production

By Jara Pérez-Jiménez

Grape pomace is the main byproduct originated during wine production. It is constituted by a mixture of peels, seeds and remaining pulps and it represents about a 30% of grapes used for wine production. The disposal of this material is mostly a problem for wineries, which commonly pay for their removal. Some uses previously suggested for wine pomace are low benefit, e.g., animal nutrition, while other ones require sophisticated systems, such as the preparation of grape seed extracts.
However, grape pomace itself is a relevant source of bioactive compounds. Indeed, this material may be classified as antioxidant dietary fibre [1], i.e., natural materials which present a very high content in dietary fibre and phenolic compounds, being them either free in the food matrix [2] or associated with dietary fibre [3]. The latter fraction corresponds to the so-called non-extractable polyphenols, with increasing interest in nutrition and health [4], being the major fraction of grape pomace polyphenols [3].
Nevertheless, the potential uses of grape pomace as whole matter -only subjected to drying and milling- in nutrition and health remain mostly to be explored. In this way, we previously reported that supplementation with grape antioxidant dietary fibre improved lipid profile in hypercholesterolemic subjects [5]. Similarly, other researchers showed that grape pomace improved blood pressure and glycaemia in male subjects with at least one cardiometabolic alteration [6].
In our last study, we decided to focus on Metabolic Syndrome, a cluster of risk factors predisposing to type 2 diabetes and cardiovascular diseases, and with increasing prevalence in developed countries [7]. Among the underlying mechanisms of Metabolic Syndrome, insulin resistance is a key one, so strategies aimed to ameliorate this parameter may have a potential health impact. Indeed, several animal studies with grape seed extracts have shown beneficial effects in the modulation of insulin resistance [8, 9], but we wanted to perform a clinical trial on the effect of whole grape pomace.
Grape pomace from Tempranillo cultivar was kindly provided by Roquesan Wineries, in Spain. It was freshly collected, freeze-dried, milled and packed in 8g envelopes, as shown in Figure 1. The clinical trial was conducted with subjects who exhibited overweight/obesity plus at least one additional factor of Metabolic Syndrome (hyperglycemia, hypertension, hypertriglyceridemia or low HDL cholesterol); the mean number of Metabolic Syndrome risk factors among the subjects was 2.8. A total of 49 subjects participated in the study and they consumed a daily dose of the product for 6 weeks, without altering their usual dietary habits. Additionally, there was a 6 weeks control period, where subjects had the same follow-up that during supplementation, in order to test possible modifications due to the participation in the study (it was not possible to find an appropriate placebo). Both periods were separated by 4 weeks and, since it was a cross-over study, subjects were randomly assigned into two arms, one starting with grape pomace supplementation and the other one starting with the control period. Blood samples were collected at the beginning and at the end of each period. And half of the subjects were subjected to an Oral Glucose Tolerance Test (oral dose or 75 g of glucose) before and after the supplementation period.

Figure 1. Preparation of grape pomace for the clinical trial.

Several cardiometabolic markers were evaluated in the biological samples; also, blood pressure and anthropometry were collected during each visit. There was no modification in most of the parameters measured (as expected, intestinal movements improved after supplementation, as consequence of the high dietary fiber content of grape pomace), but relevant results were obtained regarding insulin metabolism. In particular, there was a significant decrease in fasting insulinaemia after grape pomace supplementation (from 8.5 + 4 to 5.5 + 4.5 µU/mL), without modification in fasting glucose levels. Thus, grape pomace supplementation caused an increase in insulin sensitivity (as measured by QUICKI index, quantitative insulin sensitivity check index) and a decrease in insulin resistance (as measured by HOMA index, homeostatic model assessment); these results are shown in Figure 2. Moreover, a non-significant tendency towards an improvement in postprandial insulin was observed. Overall, these effects seemed to be stronger in subjects with higher basal insulin values.

Figure 1. Effect of grape pomace supplementation in: a) HOMA index and b) QUICKI index. Light bars, control period; dark bars, supplementation period.

Therefore, our clinical trial showed that daily supplementation with a dose (8 g suspended in water) easily included in a common diet may have beneficial effects in insulin resistance. The study was recently published in a recognized scientific journal [10]. Currently, we are evaluating potential underlying mechanisms in the effects observed, as well as the potential reasons explaining why not all subjects responded to the treatment, i.e., differences between responders and non-responders. Further studies should be performed in order to confirm the results we obtained. But we consider that this clinical trial, together with previous research on the use of grape pomace, highlights how this by-product currently neglected should be considered as a rich source of bioactive compounds with several applications in food industry (preservative, ingredient, dietary supplement) and other sectors (cosmetics, pharmacy). Therefore, and particularly in a context of increasing interest on circular economy, exploring strategies towards the exploitation of grape pomace may allow to increase the sustainability of wine making.

Those interested in a longer length report can download the working paper at:!divAbstract

Jara Pérez-Jiménez completed holds a PhD in Food Science and Technology. She has worked in several research centres and universities in Spain and France focused on the study of food bioactive compounds, in particular polyphenols, using a multidisciplinary approach (studies on food composition, development of an online database, preclinical and clinical trials, observational studies). Currently, she is a Tenured Scientist at the Institute of Food Science, Technology and Nutrition (ICTAN-CSIC) in Madrid. She is co-author of more than 60 papers in international scientific journals (> 3,800 citations, h-index: 30). She is also co-inventor a patent transferred to a national company, has been coeditor of a book published by the Royal Society of Chemistry (United Kingdom) has been invited speaker in universities form United Kingdom, France, Spain, Mexico and Chile. Additionally, she was a member of the Experts Committee on Human Nutrition of the French Agency of Food Safety in 2015-18. Finally, she regularly develops dissemination activities, for which she has received several awards.
Several other researchers from ICTAN-CSIC participated in this study: Daniel Martínez-Maqueda, Angélica Gallego-Narbón, Belén Zapatera, Pilar Vaquero and Fulgencio Saura- Calixto.


1. Saura-Calixto F (1998) Antioxidant dietary fiber product: A new concept and a potential food ingredient. J. Agric. Food Chem. 46: 4303-4306
2. Kammerer D, Claus, A, Carle, R, Schieber, A (2004) Polyphenol screening of pomace from red and white grape varieties (Vitis vinifera L.) by HPLC-DAD-MS/MS. J. Agric. Food Chem. 52: 4360-4367
3. Pérez-Ramírez IF, Reynoso-Camacho, R, Saura-Calixto, F, Pérez-Jiménez, J (2018) Comprehensive Characterization of Extractable and Nonextractable Phenolic Compounds by High-Performance Liquid Chromatography-Electrospray Ionization-Quadrupole Time-of-Flight of a Grape/Pomegranate Pomace Dietary Supplement. J. Agric. Food Chem. 66: 661-673
4. Pérez-Jiménez J, Díaz-Rubio, ME, Saura-Calixto, F (2013) Non-extractable polyphenols, a major dietary antioxidant: Occurrence, metabolic fate and health effects. Nutr Res Rev 26: 118-129
5. Pérez-Jiménez J, Serrano, J, Tabernero, M, Arranz, S, Díaz-Rubio, ME, García-Diz, L et al (2008) Effects of grape antioxidant dietary fiber in cardiovascular disease risk factors. Nutr 24: 646-653
6. Urquiaga I, D’Acuña, S, Pérez, D, Dicenta, S, Echeverría, G, Rigotti, A et al (2015) Wine grape pomace flour improves blood pressure, fasting glucose and protein damage in humans: A randomized controlled trial. Biological Res. 48:
7. Scuteri A, Laurent, S, Cucca, F, Cockcroft, J, Cunha, PG, Mañas, LR et al (2015) Metabolic syndrome across Europe: Different clusters of risk factors. Eur. J. Prev. Cardiol. 22: 486-491
8. Castell-Auví A, Cedó, L, Pallarès, V, Blay, MT, Pinent, M, Motilva, MJ et al (2012) Procyanidins modify insulinemia by affecting insulin production and degradation. J. Nutr. Biochem. 23: 1565-1572
9. Meeprom A, Sompong, W, Suwannaphet, W, Yibchok-Anun, S, Adisakwattana, S (2011) Grape seed extract supplementation prevents high-fructose diet-induced insulin resistance in rats by improving insulin and adiponectin signalling pathways. Br. J. Nutr. 106: 1173-1181
10. Martínez-Maqueda D, Zapatera, B, Gallego-Narbón, A, Vaquero, MP, Saura-Calixto, F, Pérez-Jiménez, J (2018) A 6-week supplementation with grape pomace to subjects at cardiometabolic risk ameliorates insulin sensitivity, without affecting other metabolic syndrome markers. Food Function 9: 6010-6019

Posted by in Health

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.

Those interested in a longer length report can download the working paper at:


Bordage, S., Sullivan, S., Laird, J., Millar, A.J., Nimmo, H.G., 2016. Organ specificity in the plant circadian system is explained by different light inputs to the shoot and root clocks. New Phytol. 212, 136–149.Clarke, S.J., Lamont, K.J., Pan, H.Y., Barry, L.A., Hall, A., Rogiers, S.Y., 2015. Spring rootzone temperature regulates root growth, nutrient uptake and shoot growth dynamics in grapevines. Austr. J. Grape Wine Res. 21, 479–489.
James, A.B., Monreal, J.A., Nimmo, G.A., Kelly, C.L., Herzyk, P., Jenkins, G.I., Nimmo, H.G., 2008. The circadian clock in Arabidopsis roots is a simplified slave version of the clock in shoots. Science 322, 1832–1835.
Mahmud ,K.P., Holzapfel, B.P., Smith, J.P., Guisard, Y., Nielsen S., Rogiers, S.Y. 2018. Circadian regulation of grapevine root and shoot growth and their modulation by photoperiod and temperature. J.Plant Physiol. 222: 86-93.
Mahmud, K.P., Smith, J.P., Rogiers, S.Y., Guisard, Y., Nielsen, S., Holzapfel, B.P. 2018. Diurnal Root Growth Dynamics in Mature Grapevines. Acta Hort. 1205: 555-562.
Mora-García, S., de Leone, M.J., Yanovsky, M., 2017. Time to grow: circadian regulation of growth and metabolism in photosynthetic organisms. Curr. Opin. Plant Biol. 35,84–90.
Nozue, K. and Maloof, J.N., 2006. Diurnal regulation of plant growth. Plant, Cell Environ. 29, 396-408.
Simon, N.M.L., Dodd, A.N., 2017. A new link between plant metabolism and circadian rhythms? Plant Cell Environ. 40, 995–996.
Yazdanbakhsh, N., Sulpice, R., Graf, A., Stitt, M., Fisahn, J., 2011. Circadian control of root elongation and C partitioning in Arabidopsis thaliana. Plant Cell Environ. 34,877–894.

Posted by in Viticulture