In was to study the evolution of quality factors

In viticultural
production, phenols generally, anthocyanins and condensed tannins especially
(proanthocyanidins) in the grape are important contributors to wine quality
(Glories, 1988). Anthocyanins are pigmented
compounds located in the skins of grape berries and are responsible for the red
coloration of the fruit and subsequent wines (Ribereau – Gayon and Glories,
1986). Tannins are responsible for the astringent component of the mouth feel
of a wine and are derived from both the seeds and skins of the grape (Downey et
al., 2003a). Tannins are also involved in the long-term color stability of
wines by forming pigmented polymers with anthocyanins (Zimman and Waterhouse, 2004). Also, a number of factors can influence
flavonoid accumulation in grapes, including variety, crop level and climatic
variables such as temperature and light (Downey et al., 2006).

Grape quality analysis was first
developed in France (Rousseau and Delteil, 2000)
and has been proposed as a reliable tool to evaluate berries for ripeness,
harvest scheduling and wine quality prediction (Winter et al., 2004). Wine descriptive analysis comprises a
series of quality techniques which intend to generate objective descriptions of
wines in terms of the perceived aroma and taste attributes and can be both
qualitative and quantitative (Vilanova and Soto, 2005).
Descriptive quality analysis has been very rarely applied to study the effect of
leaf senescence on the aroma properties of wine (Arnold and Bledsoe, 1990).

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Also, some many study analyzed certain grape textual proprieties
modifications during ripening stage (Torchio et al 2010). The influences of the
terroir effect on mechanical proprieties were also considered. However, still
more information about the contribution of quality behavior of grapevine to the
phenols composition diffusion process is required.

The aim of this work was
to study the evolution of quality factors of Vitis vinifera (L)
belonging to five different varieties during ripening and to investigate how these change and
phenols composition of grape berries has an effect on triploid and
diploids vine varieties and its degree of correlation to the grape quality in order to compare their composition. For that, sampling involved three introduced triploid
varieties: Superior, Early Sweet, Flame and two diploid local varieties: Abbou
and Aadari, the main varieties grown in the Marrakech region. The choice of
these varieties is justified mainly by their highest frequencies observed on the local
market and thus for export.

Materials and methods

Grape samples

The Vitis Vinifera varieties studied in 2017 was
cultivated in irrigated conditions. The vines were 15 to 20 years old and
belonged to three different areas Oudaya Marrakesh
region of Morocco (31° 33’39.1 “N 8° 14’59.3” W). Five  ( kg) grape were collected at 15-day
intervals from veraison to harvest were carefully collected from all
vines  and placed in a cooler during transport from the field to the
laboratory.  Physico-chemical and quality analyses were made at harvest time
and throughout the maturation period. Measurements were
replicated three times. 


Morphological analyzes

morphological characteristics of fruits are expressed
by the mean weight of the
fruit (Wm), the density
(?) and the form coefficient
(Cs). The form coefficient allows you to classify the varieties in three
categories of form including: Cf < 0.8: flattened shape; Cf > 1:
elongated shape and 0.8< Cf < 1: round form.   Physicochemical analyzes Physico-chemical analyzes were carried out on fruits green, ripening  and wall. These analyzes make it possible to assess fruit quality through the following parameters (Table 1). Total phenols contents determination Total phenol content of fruit pulp extracts was determined using the Folin-Ciocalteu assay method with modifications. 100 ?l of the extract is mixed with 250 µl of the freshly prepared Folin-Ciocalteu reagent and 500 ?l of 20% sodium carbonate (Na 2 CO 3). The whole is incubated at room temperature for 30 minutes and the reading is carried out against a blank using the UV-200 spectrophotometer  at 765 nm. A standard curve was drawn using catechin of different concentrations (10 mg/l, 20 mg/l, 300 mg/l, 40 mg/l and 80 mg/l). The concentration of total phenolics in the fruit pulp extract was calculated based on the equation from the standard curve and expressed in terms of catechin equivalent (cat g/100 g dry weight). Total  flavonoids contents determination The flavonoids content of the extracts was determined using the colorimetric method at Aluminum trichloride. A quantity of 100 ml of the extract was mixed with 0.4 ml of distilled water and subsequently with 0.03 ml of a sodium nitrite solution 5% NaNO2. 5 min after, 0.02 ml of a solution of AlCl3at 10% was added. To the mixture was added 0.2 ml of Solution of 1M Na2CO3 and 0.25 ml of distilled water after 5 min of rest. The whole is agitated by means of a vortex and the absorbance was measured at 510 nm. The results are expressed in milligrams equivalent of catechin per g of dry plant.   Condensed tannins Determination The condensed tannins content of the extracts was determined using the colorimetric method at the vanillin in an acid medium (Price et al., 1978). This method is based on the ability of vanillin to react with units of condensed tannins in the presence of acid to produce a colored complex measured at 500 nm. The extraction is carried out as follows: 5 ml of methanol (Price et al., 1978) are added to each tube containing 0.5 g of vine fruit powder, the mixture is homogenized every 5 min for 20 min, Then centrifuged (1700 g, 10 min at 25°C) to collect the supernatant. The vanillin extracts and reagents (8% equivalents of 37% HCl in methanol and 4% vanillin (Sigma-Aldrich, Germany) in methanol are maintained at 30°C prior to assay. 200 ?l of extract is added to 1000 ?l of vanillin reagent for the determination of condensed tannins. Blanks are prepared by replacing the reagent with the methanol-acid mixture; the tubes are maintained at 30°C. for 20 minutes, the absorbance is read at 500 nm. Catechin is used as a standard and the results are expressed in mg of catechin.   Statistical data treatment Significant differences between the varieties and levels of each parameter have been evaluated by analysis of variance (ANOVA). A test of Duncan was used to separate the means (P < 0.05). A classification of fruit maturity stage according to variety and quality factors according to phonols composition by PCA analysis is provided. All the statistical procedures have been carried out using the SPSS software 10.0. Chemicals All the chemicals reagents used for biochemical and enzyme analysis were of the analytical grade (Sigma-Aldrich). Results and discussion: During maturity, grape show a multitude modification in color, dimension and texture signifying that compositional changes are going on. The Technological and physicochemical data and different parameters of the quality assay at the moment of harvest are shown in figures 1, 2, 3 and table 1, representing the values of the parameters of development for the five Vitis Vinifera varieties explored.         Total phenols compounds: The evolution of total phenolic content during the vine fruits maturation for the five varieties and the three maturity stages is reported in Fig.1.A. A gradual decrease for the introduce varieties "Superior, Flame and Early sweet", was found according to an increase for the locales varieties, "Aadari and Abbou". The five varieties of fruits analyzed deduced, that the Adari variety contained the highest phenolic compounds at green and ripening stages (15.58 ± 0.35 and 22.67 ± 1.73 mg/g FW, respectively), while Abbou variety had a highest amount of phenolic concentration at ripe stage (37.91.03 ± 2.97 mg/g FW). At the green and ripening stages, Superior and Flame varieties had the lowest phenolic content (3.95 ± 0.68 ; 3.03 ± 0.45 mg/g FW, respectively). Our data indicated, besides the fact that grape genotype influence of total phenol content (Anttonen & Karjalainen, 2005), that late harvest (ripe fruit stages) in local varieties: Adari, Abbou significantly increased their phenols content.  Similar results have been reported by other researchers in blackberries (1786–2310 mg 100g-1 dw) and raspberries (1137–2112 mg 100g-1 dw). Some authors have reported an increase in total phenols and especially flavonoids content during the ripening of grapes (Munoz et al., 2007). The total phenols accumulated rapidly in the grapes during ripening after véraison. Our results support previous studies examining the influence of grape ripening on total anthocyanins (Kennedy et al., 2002). The phenols concentration decreased in introduced varieties with further increasing sugar concentration in the berry pulp; this can be explained by environment effects and/or genetic. During this period of grape development, the grapes were exposed to solar radiation, high temperatures and low irrigation. Berry temperature, additionally fuelled by berry skin exposure to sunlight, can slowly inhibit the biosynthesis of anthocyanins (Tarara et al., 2008). Other works have also described dramatic effects of the environment and culture conditions on polyphenols accumulation (Downey et al., 2006).    To summarize, this steeply accumulation in total phenolic content during maturation for local varieties is in good agreement with those reported by Gonzalez et al. (1991). They found that total phenolics accumulated gradually along the maturity stages. Several studies have evaluated the potential use of various berry metabolites associated with varietal character. Increases in total phenolic content have been associated with maturity. In a principal component analysis of various ripening indicators, phenolic content emerged as a key defining factor of grape maturity (Gonzalez et al., 1991). Also, Mainland and Tucker (2000) reported that the total phenolic content and anthocyanin of five blueberry cultivars increased with progressing maturity. In comparison with the introduced varieties, these results are in contrast with numerous researches, which indicated that the phenolic compounds were generally more abundant in the beginning of maturation. Their concentration tends to decrease with maturity in apricot (Dragovic- Uzelac et al., 2007), in the medlar fruit (Ayaz et al., 2008) and in 15 peach cultivars (Lee et al., 1990). To explain the evolution of phenolic content in the fruit, the pathway of phenolics synthesis must be investigated. It is known that the key enzyme of the phenylpropanoid pathway is phenylalanine ammonia lyase (PAL) which catalyze the initial and obligated phase in the biosynthesis of phenols compounds (Lancaster et al., 2000) by domination of L-phenylalanine to form trans-cinnamic acid with the release of NH3 and  initiate the phenylpropanoid pathway. However, it is unlikely that any single index of maturity will be discovered that can be indiscriminately applied in all growing conditions and to all varietals. Historical experience with specific vineyards and growing regions will continue to be a critical factor in determining the optimal maturity of the fruit.                                                                                      Flavonoïds and tannins content The flavonoids and tannins content in the pulp of five varieties studied since the stadium green fruit at harvest are presented in Fig 1B.  A gradual decrease was found, for the introduces varieties: Superior, Flame and  Early sweet, and locale: Adari and Abbou. There were marked significant (p<0.05) difference in the flavonoïdes and tannins content at different stages of maturity in locale (diploid) and in green stage for introduced ( triploid). The five varieties of fruits analyzed deduced that the Abbou and Adari locale variety contained the highest flavonoïdes and tanins compounds at green, ripening and ripe stages while, Superior and Flame varieties had the lowest flavonoïdes and tannins. Summarize, this steeply decreases in flavonoïdes and tannins content during maturation for locale and introduces. The flavonoids are involved in the color ; chalcones, aurones and flavones participate, with other pigments, in yellow color, by contrast, red colors, purple and blue are due primarily to anthocyanins (A. Fleuriet et al,. 1996). These accumulate in the vacuoles and their levels are particularly high in the air bodies and in epidermiques tissues and under epidermiques (A. Fleuriet et al,. 1996). We know currently the structure of about 260 anthocyanins including most of 6 anthocyanidines (aglycones) (Harborne and grayer, 1988; Mazza and Miniati, 1993). They differ mostly by the number of hydroxyl groups, their degree of methylation, the nature and number of sugars and aromatic acids or related aliphatic to the molecule as well as their position. In the skin grapes" Cabemet Sauvignon", twenty different anthocyanins were identified (Wulf and Nagel, 1978). Some blue pigments (commelinine and ternatine) can be very complex and reach of molecular masses close to small proteins. As well, the Grape has in particular of IA (+)-catechine, (-)-epicatechine, procyanidins dimeres and proanthocyanidins polymerisees more or less soluble which will have a very important role in the course of the vinification and aging of the wine (A. Fleuriet et al,. 1996). The tannins can form complexes with multiple molecules (proteins, polysaccharides, alkaloids, anthocyanins) and the process can be reversible or irreversible (Haslam, 1989; Haslam et al., 1992). The association polyphenols-proteins are affected in two times: The polyphenols are first of all traps at the level of the hydrophobic sites of protein and then they form of the connections hydrogens with the polar groupings of protein (Haslam et al., 1992). The astringency is a characteristic of young fruit and it disappears completely in the fruits walls. In some cases, this decrease is linked to a capital asset of the tannins by the other constituents of the fruit such as acetaldehyd or polysaccharide forms during the maturation (Haslam et al., 1992).   Physico-chemical characteristics of vine fruits The morphological, quality and physicochemical characteristics of the five varieties of vine studied are summarized in the Table 2. The very high water content > 70% by fresh weight of
mature fruits is a factor which reflects the
great perishability of the vine and
limits its ability to the storage to the ambient temperature. The other
chemical parameters clearly differentiate the varieties as well as the stages of
maturity. The levels of dry matter soluble (°Brix) obtained are more or
less identical between the varieties introduced and local authorities
with values more bottom 15.80% for the varieties Abbou and higher
16.53% for the Flame varieties on the mature stage. As can be seen
that the soluble dry matter content of equal to “°Brix” depend on the pH
and acidity of the fruits of vine and the varieties. The rate of
citric acid is in gradual decrease during maturation with the lowest 2.44 g/L
for the Flame variety and the highest 6.04 g/L for the superior
variety. It is the same for the ash content of 0.12 to 1.65%. The
value of pH was between 3.5 and 4.11 in ripe stage for the varieties
studied. The pH relatively low fruit mature (pH < 4.2) is an advantage from the point of view of stability (Rozier et al., 1985). The ability of rehydration allows appreciating the amount of water that can be absorbed by the powder or the fruit dry. The values of the capacity of rehydration understood between 25.25 % and 39.95% in green maturity stage for the varieties studied of vine but increases with maturation of fruit up to the values between 38.24% and 89.98%. With regard to the color, the variety Abbou presents a red color more intense than those of the Flame variety which in a yellow color reddish. For the varieties Superior, Early sweet and Adari have a yellowish color. In sum, the differences in physicochemical characteristics observed between the introduced and local varieties and/or the stage of maturation of the vine, could be related to the conditions of production of non-identical, to the genetic differences, environment and the pedology. In particular, the non-same varieties of vine would explain the constance observed at the level of their physicochemical parameters.                                             Relationship between maturity and phenolic compound The maturity index according the phenolic, flavonoids and tannins compounds of the five varieties of vine studied are summarized in the Fig 3. The criteria traditionally used to determine grape maturity are based on sugar content, which is normally measured in °Brix. Maturity index was determined in the report of sugar/acidity (in citric acid). Some differences were found in maturity index at the moment of harvest. As maturation progressed, the sugar content continued to increase and, at the moment of harvest, a fact that may be closely related to the high temperature reached during ripening period  (R. Gil-Mun˜oz et al. 2017). Vitrac et al., 2000, suggests that soluble sugars stimulate phenolic accumulation in grapevine tissue, the concentration of reducing sugars in the cytoplasm having been postulated as one of the regulators of the phenolic biosynthetic pathway. The highest maturity index was observed in Flame variety, where the phenolics compounds was lowers than in other varieties. The °Brix/acid ratio 67.57 (fig 2,A, B ou C.), indicating the beginning of over ripening stage of berries, and a °Brix/acid ratio 26.24  in superior variety at ripe stage indicating that the maturation is late for this variety. Moreover, the phenolic compounds, flavonoids, tannins contents were also reduced during maturity and the last harvest. For the locale varieties, the phenolic compounds accumulation coordinated with maturation index increase.  Several important classes of compounds are reported to change during maturation and ripening of the berries on the vine. These characters, however, do not transform in a highly coordinated fashion, and instead suggest a series of independently regulated pathways of synthesis. Each pathway again is influenced by the seasonal, biotic and/or abiotic factors, varieties and vineyard practices, and the effect varies with varietal characteristics (Jackson & Lombard, 1993). The total phenolics and the individual groups total flavonoids, tannins varied significantly during all the stages of grapes maturation  at the green stage showed significantly high contents of total phenolics (11.46 mg CE/g WF), flavonoids (2.25 mg CE/g WF), flavonols (1.28 mg CE/ g WF) for the introduced varieties.  These results are conflicting with those of local varieties where the levels of phenolic compounds, increased with maturation with the highest value is (26.7 mg CE/g WF) and similarly for flavonoids and tannins compounds. This could be attributed to the accumulation of water in  grapes during maturation, which enhances the hydrolysis of higher molecular weight phenolic compounds (Cimato et al., 1990). Amiot et al., (1989), also observed the decline in the contents of phenolic compounds during the maturation of olive fruit, which was accompanied by the accumulation of the hydrolysed products out of which some were even reported to be the non-phenolic compounds.               PCA analysis of maturity stages, technological and physicochemical characteristics A classification of grapes berries according to variety and maturation stage by PCA analysis is provided in fig. 3 and fig.4. Principal component analysis (PCA) is a powerful visualization tool for data evaluation, which can graphically represent intersample, intervariable relationships and provides a way to reduce the dimensionality of the data. PCA is an unsupervised method of pattern recognition in the sense that no grouping of the data has to be known before the analysis. Using PCA, class membership is easy to indicate on a score plot. The percentage of variance captured is 52.20% and 23.71% for the PC1 and PC2, respectively. The distribution of the sample on the PC1 and PC2 reflects the influence of the morphological and physicochemical compounds on the stages of maturation based on the different varieties. (Fig 3) shows Pearson's correlation coefficients and Principal component analysis between morphological and physicochemical components in five varieties of vine fruit. The phenolic compounds, flavonoids and tanins was positively (p<0.05) correlated together with proteins content. In the other hand, a highly significant (p<0.001) correlation values among form coefficient and weight. The "°brix", ash content, maturity index and sugar content was also highly correlated (p<0.01). In the opposite, there is no correlation between the precedent parameters and acidity and density.