Elsevier

Food Chemistry

Volume 124, Issue 4, 15 February 2011, Pages 1760-1769
Food Chemistry

Analytical Methods
Monitoring biochemical changes during grape berry development in Portuguese cultivars by NMR spectroscopy

https://doi.org/10.1016/j.foodchem.2010.08.015Get rights and content

Abstract

1H nuclear magnetic resonance (NMR) was applied for the metabolic profiling of grapes from three Portuguese cultivars including ‘Trincadeira’, ‘Aragonês’, and ‘Touriga Nacional’, at four developmental stages. Two kinds of extraction methods including deuterated NMR solvent extraction and solid phase extraction (SPE) were used for the metabolomic analysis and all the metabolites detected in 1H NMR were elucidated by two-dimensional NMR techniques as well as the in-house NMR chemical shift database. Multivariate data analyses were also performed to identify overall metabolic differences. Trincadeira was found different from the other two cultivars, having low phenolic contents as compared to other cultivars. The initial stages showed comparatively high phenolics and organic acid contents like caftaric and malic acid while the later stages showed higher glucose and fructose levels. Veraison was found to be a metabolically critical stage of berry development. On the basis of these findings distribution of metabolites among different cultivars at different developmental stages is discussed.

Introduction

Grapevine (Vitis spp.) is one of the most economically important and widely cultivated fruit crops across the world. The total arable land for grape production is about 8 million hectares (Vivier & Pretorius, 2002) with a production of 68.9 million metric tons in 2006. These figures place grapes second among other famous fruits like bananas, oranges, and apples, produced throughout the year. The grape berry is used as a fresh or dried fruit, for the extraction of its juice, and most importantly in wine production. Around 71% of the total grape production is made into wine, a commodity which has a far greater economic value than the grapes. In addition to their economic importance, an increasing number of health benefits have been attributed to grapes and wine. For instance, grapes are known to have antioxidant, cardioprotective, anti-inflammatory, and anti-cancer activities (Ali, Maltese, Choi, & Verpoorte, 2009a).

Due to the above mentioned importance, knowledge regarding the development and maturation of grape berries is of great economical interest. Climacteric fruit such as tomatoes and apples have been well studied but comparatively less is known about the development and ripening of non-climacteric fruits e.g. grapes and strawberry (Giovannoni, 2004, Given et al., 1988). Considerable scientific efforts have been made to understand the complex series of physical and biochemical changes of grape berries during their development cycle (Coombe, 1992). But today, the major concerns of the viticulturists are size, colouration, control of ripening, acidity, and volatile and non-volatile contents of the grape berry.

The Portuguese wines are traditionally made by blending generally three or four varieties. However, due to the exceptional characteristics of Touriga Nacional, Trincadeira and Aragonês, monovarietal red wines are produced with these varieties. Their specific aromas have recently attracted the interest of vine growers and viticulturists abroad with the aim of introducing new competitive products into the market. Touriga Nacional is considered to be one of the most important native varieties in Portugal. This variety produces dark, full-bodied, powerful and aromatic wines with floral notes similar to the scent of purple violet blossoms. It frequently shows blackberry, blueberry, rock rose, and rosemary notes. Aragonês also called Tinta Roriz produces full-bodied, inky and highly aromatic wines. The grape has fine and delicate aromas of pepper and berries. Aragonês has high yields and is indispensable in the blend of a good Porto wine. Trincadeira is most suited to grow in hot, dry and very bright areas. It is not easy to grow, however, having irregular yields and prone to infection with pathogenic moulds. But in good years, Trincadeira will produce great wines. Trincadeira variety is also known to have excellent acidity, soft tannins and abundant, intense aromas of plum and blackberry, resulting in elegant and balanced wines. Blending of different Portuguese wines like Trincadeira with Aragonês or Touriga Nacional result in top quality cocktail Portuguese wines.

Chemical characterisation of the phenotype of an organism has become the focal point of many researchers in recent years. The analysis of these low molecular weight compounds seems to reflect the physiological activities of an organism or tissue under different conditions. The observable chemical profile or fingerprint shown by the plant tissue, ‘metabolome’, is highly complex and consists of a vast variety of chemicals which differ in their structure and function. Due to this, it is unlikely that a single analytical method could provide information about all the metabolites, considering their chemical diversity, and at the same time be unbiased, rapid, reproducible, and stable over time, while requiring only simple sample preparation. Many platforms are being used for the high throughput analysis of plant metabolites, but vary according to their sensitivity (Kopka, Fernie, Weckwerth, Gibon, & Stitt, 2004).

Apart from its routine use in the identification, characterisation, and structure elucidation of molecules, nuclear magnetic resonance (NMR) spectroscopy is now increasingly popular in the area of metabolome analysis (Son et al., 2009). NMR spectroscopy has been criticised for its low sensitivity but on the other hand, presents great advantages being nondestructive, and requiring simple sample preparation in relatively short time. This feature has the additional benefit of helping to preserve the integrity of the sample and its components. Its non-selectiveness made it an ideal choice for the profiling of the broad range metabolites (Dixon et al., 2006).

In combination with different multivariate data analyses tools, such as principal components analysis (PCA), NMR has been used for the fingerprinting or metabolic profiling of various sample types (Brescia et al., 2002, Charlton et al., 2002). This combination has been very useful for the characterisation of different plant species (Kim, Choi, Erkelens, Lefeber, & Verpoorte, 2005), and cultivars as well (Ali et al., 2009b). In the study of grapevine, recently, the coupling of metabolic analysis with transcriptional analysis has also been applied to the profiling of two grape cultivars with different resistance capabilities to pathogenic fungi (Figueiredo et al., 2008). In the case of berries, NMR coupled to multivariate analyses has been used to study the effects of growing areas, vintage, and soil (Pereira et al., 2005), and the influence of microclimate on metabolic profiles of grape berries (Pereira et al., 2006). Many of grape berry metabolic profiling studies have focused mainly on the amino acid or sugar contents while ignoring the phenolic composition of the sample. This may be due to the low signal quality or signal overlapping of phenolics in the NMR spectra.

In this study, we sought to obtain a metabolic characterisation of the different developmental stages of grape berries from three different important Portuguese cultivars. To highlight these differences, one-dimensional 1H NMR with additional two-dimensional NMR techniques, coupled with principal component analysis (PCA), partial least-squares-discriminant analysis (PLS-DA), and orthogonal partial least squares (OPLS) analysis were applied.

Section snippets

Grape cultivars and sampling

Three elite Portuguese cultivars i.e. Trincadeira, Touriga Nacional, and Aragonês, were used in this study. Five biological replicates of each cultivar of 80–100 berries from 8 to 10 plants were collected in 2008 corresponding to the developmental stages of EL 32 (green), 35 (veraison), 36 (ripe), 38 (harvest) (EL refers to the modified Eichhorn and Lorenz developmental scale as described by Coombe (1995)). Each biological replicate contained berries from a single row of plants. Four rows

Visual analysis of 1H NMR spectra

The corresponding berry weight for each stage at the time of sample collection is shown in Fig. 1A. In this study four different developmental stages (EL 32, 35, 36, and 38) of the grape berries from three different Portuguese cultivars were analysed. For metabolic profiling of the berries two kinds of extraction methods were used including extraction directly with deuterated NMR solvents and solid phase extraction (SPE) with C18 resins. In the 1H NMR spectrum, the area between δ 0.8 and 4.0

Biochemistry of grape berry development

The time required for the growth of berries in Vitis vinifera has been defined differently by different authors. As a grape berry grows, it goes through a complex series of physical and biochemical changes including modifications in size, composition, colour, texture, flavour, and pathogen resistance. The growth pattern of a developing grape berry exhibits a double sigmoid growth pattern (Fig. 1B) which can be divided into three phases (Coombe, 1992). At the beginning (phase I), the berry

Conclusions

Grape berries are undoubtedly among the most important fruit species because of their use in wine making. Grape biochemistry shows a great diversity in terms of structure and function ranging from simple amino acids and sugars to highly complex polymers of condensed tannins. The understanding of grape berry development and the metabolic fate of different classes of compounds is imperative in order to control and improve different quality traits of grapes and ultimately wine flavour. Since grape

Acknowledgements

This work was supported by ERA-PG in Portugal under the project Genomic Research-Assisted breeding for Sustainable Production of Quality GRAPEs and WINE (http://urgi.versailles.inra.fr/projects/GRASP/). The authors thank Higher Education Commission (HEC) of Pakistan for the financial support of Kashif Ali.

References (28)

  • B.G. Coombe

    Research on development and ripening of the grape berry

    American Journal of Enology and Viticulture

    (1992)
  • B.G. Coombe

    Growth stages of the grapevine: Adoption for a system for identifying grapevine growth stages

    Australian Journal of Grape and Wine Research

    (1995)
  • S. de Bolt et al.

    l-Tartaric acid synthesis from vitamin-C in higher plants

    Proceedings of the National Academy of Sciences USA

    (2006)
  • R.A. Dixon et al.

    Applications of metabolomics in agriculture

    Journal of Agricultural and Food Chemistry

    (2006)
  • Cited by (102)

    • <sup>1</sup>H NMR and UPLC-HRMS-based metabolomic approach for evaluation of the grape maturity and maceration time of Touriga Nacional wines and their correlation with the chemical stability

      2022, Food Chemistry
      Citation Excerpt :

      Indeed, this result is expected, since the maturation causes a decrease of organic acid, which are either converted to sugars or used as carbon and energy sources for respiration (Conde et al., 2007). Previously, Ali et al. (2011) used 1H NMR and chemometrics to monitor the biochemical changes during the ripening of Touriga Nacional berries. They observed that the unripe grapes (green and veraison stages) were characterized by higher levels of phenolics and organic acids, whereas the ripe and harvest stages were found with more amounts of amino acids and sugars.

    • Metabolomics: An Emerging Tool for Wine Characterization and the Investigation of Health Benefits

      2019, Engineering Tools in the Beverage Industry: Volume 3: The Science of Beverages
    View all citing articles on Scopus
    View full text