In viticulturalproduction, phenols generally, anthocyanins and condensed tannins especially(proanthocyanidins) in the grape are important contributors to wine quality(Glories, 1988). Anthocyanins are pigmentedcompounds located in the skins of grape berries and are responsible for the redcoloration of the fruit and subsequent wines (Ribereau – Gayon and Glories,1986). Tannins are responsible for the astringent component of the mouth feelof a wine and are derived from both the seeds and skins of the grape (Downey etal., 2003a). Tannins are also involved in the long-term color stability ofwines by forming pigmented polymers with anthocyanins (Zimman and Waterhouse, 2004).
Also, a number of factors can influenceflavonoid accumulation in grapes, including variety, crop level and climaticvariables such as temperature and light (Downey et al., 2006). Grape quality analysis was firstdeveloped 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 aseries of quality techniques which intend to generate objective descriptions ofwines in terms of the perceived aroma and taste attributes and can be bothqualitative and quantitative (Vilanova and Soto, 2005).Descriptive quality analysis has been very rarely applied to study the effect ofleaf senescence on the aroma properties of wine (Arnold and Bledsoe, 1990).Also, some many study analyzed certain grape textual proprietiesmodifications during ripening stage (Torchio et al 2010). The influences of theterroir effect on mechanical proprieties were also considered. However, stillmore information about the contribution of quality behavior of grapevine to thephenols composition diffusion process is required.The aim of this work wasto study the evolution of quality factors of Vitis vinifera (L)belonging to five different varieties during ripening and to investigate how these change andphenols composition of grape berries has an effect on triploid anddiploids vine varieties and its degree of correlation to the grape quality in order to compare their composition. For that, sampling involved three introduced triploidvarieties: Superior, Early Sweet, Flame and two diploid local varieties: Abbouand Aadari, the main varieties grown in the Marrakech region.
The choice ofthese varieties is justified mainly by their highest frequencies observed on the localmarket and thus for export.Materials and methodsGrape samplesThe Vitis Vinifera varieties studied in 2017 wascultivated in irrigated conditions. The vines were 15 to 20 years old andbelonged to three different areas Oudaya Marrakeshregion of Morocco (31° 33’39.1 “N 8° 14’59.
3″ W). Five ( kg) grape were collected at 15-dayintervals from veraison to harvest were carefully collected from allvines and placed in a cooler during transport from the field to thelaboratory. Physico-chemical and quality analyses were made at harvest timeand throughout the maturation period. Measurements werereplicated three times. Morphological analyzesThemorphological characteristics of fruits are expressedby the mean weight of thefruit (Wm), the density(?) and the form coefficient(Cs). The form coefficient allows you to classify the varieties in threecategories of form including: Cf < 0.8: flattened shape; Cf > 1:elongated shape and 0.8< Cf < 1: round form.
Physicochemical analyzesPhysico-chemical analyzes were carriedout on fruits green, ripening and wall. Theseanalyzes make it possible to assess fruit quality through the followingparameters (Table 1). Total phenols contents determinationTotal phenol content of fruit pulpextracts was determined using the Folin-Ciocalteu assay method withmodifications. 100 ?l of the extract is mixed with 250 µl of the freshlyprepared Folin-Ciocalteu reagent and 500 ?l of 20% sodium carbonate (Na 2 CO3). The whole is incubated at room temperature for 30 minutes and the readingis carried out against a blank using the UV-200 spectrophotometer at 765 nm. A standard curve was drawn usingcatechin of different concentrations (10 mg/l, 20 mg/l, 300 mg/l, 40 mg/l and80 mg/l). The concentration of total phenolics in the fruit pulp extract wascalculated based on the equation from the standard curve and expressed in termsof catechin equivalent (cat g/100 g dry weight).Total flavonoids contents determinationThe flavonoids content of the extracts was determined using thecolorimetric method atAluminum trichloride.
A quantity of 100 ml of the extract was mixed with 0.4 mlof distilled water and subsequently with 0.03 ml of a sodium nitrite solution5% NaNO2.
5 min after, 0.02 ml of a solution of AlCl3at 10% was added. To themixture was added 0.2 ml of Solution of 1M Na2CO3 and 0.25 ml of distilledwater after 5 min of rest. The whole is agitated by means of a vortex and theabsorbance was measured at 510 nm. The results are expressed in milligramsequivalent of catechin per g of dry plant.
Condensed tannins Determination The condensed tannins content of theextracts was determined using the colorimetric method at the vanillin in anacid medium (Price et al., 1978).This method is based on the ability of vanillin to react with units ofcondensed tannins in the presence of acid to produce a colored complex measuredat 500 nm.
The extraction is carried out as follows: 5 ml of methanol (Price etal., 1978) are added to each tubecontaining 0.5 g of vine fruit powder, the mixture is homogenized every 5 minfor 20 min, Then centrifuged (1700 g, 10 min at 25°C) to collect thesupernatant. The vanillin extracts and reagents (8% equivalents of 37% HCl inmethanol and 4% vanillin (Sigma-Aldrich, Germany) in methanol are maintained at30°C prior to assay. 200 ?l of extract is added to 1000 ?l of vanillin reagentfor the determination of condensed tannins. Blanks are prepared by replacingthe 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 astandard and the results are expressed in mg of catechin. Statistical data treatmentSignificant differences between the varieties and levels of eachparameter have been evaluated by analysis of variance (ANOVA). A test of Duncanwas used to separate the means (P < 0.05). A classification of fruitmaturity stage according to variety and quality factors according to phonolscomposition by PCA analysis is provided. All the statistical procedures havebeen carried out using the SPSS software 10.
0. ChemicalsAll the chemicals reagents used for biochemical and enzyme analysis wereof 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. TheTechnological and physicochemical data and different parameters of the qualityassay at the moment of harvest are shown in figures 1, 2, 3 and table 1, representingthe 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 maturationfor the five varieties and the three maturity stages is reported in Fig.1.A. Agradual 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 containedthe highest phenolic compounds at green and ripening stages (15.58 ± 0.35 and22.
67 ± 1.73 mg/g FW, respectively), while Abbou variety had a highest amountof phenolic concentration at ripe stage (37.91.03 ± 2.97 mg/g FW).
At the greenand ripening stages, Superior and Flame varieties had the lowest phenoliccontent (3.95 ± 0.68 ; 3.03 ± 0.45 mg/g FW, respectively). Our data indicated,besides the fact that grape genotype influenceof total phenol content (Anttonen & Karjalainen, 2005),that late harvest (ripe fruit stages) in local varieties: Adari, Abbousignificantly 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-1dw). Some authors have reported an increase in total phenols and especiallyflavonoids content during the ripening of grapes (Munoz et al.
, 2007). The total phenols accumulated rapidly in thegrapes during ripening after véraison. Our results support previous studiesexamining the influence of grape ripening on total anthocyanins (Kennedy etal., 2002).
The phenols concentrationdecreased in introduced varieties with further increasing sugar concentrationin the berry pulp; this can be explained by environment effects and/or genetic.During this period of grape development, the grapes were exposed to solarradiation, high temperatures and low irrigation. Berry temperature,additionally fuelled by berry skin exposure to sunlight, can slowly inhibit thebiosynthesis of anthocyanins (Tarara et al., 2008).Other works have also described dramatic effects of the environment and cultureconditions on polyphenols accumulation (Downey et al., 2006). To summarize, this steeplyaccumulation in total phenolic content during maturation for local varieties isin good agreement with those reported by Gonzalez et al.
(1991). They found that total phenolics accumulatedgradually along the maturity stages. Several studies have evaluated thepotential use of various berry metabolites associated with varietal character.Increases in total phenolic content have been associated with maturity. In aprincipal component analysis of various ripening indicators, phenolic content emerged as a key defining factorof grape maturity (Gonzalez et al., 1991).Also, Mainland and Tucker (2000) reported that the total phenolic content andanthocyanin of five blueberry cultivars increased with progressing maturity.
Incomparison with the introduced varieties, these results are in contrast withnumerous researches, which indicated that the phenolic compounds were generallymore abundant in the beginning of maturation. Their concentration tends todecrease 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 phenoliccontent in the fruit, the pathway of phenolics synthesis must be investigated.It is known that the key enzyme of the phenylpropanoid pathway is phenylalanineammonia lyase (PAL) which catalyze the initial and obligated phase in thebiosynthesis of phenols compounds (Lancaster et al.
, 2000)by domination of L-phenylalanine to form trans-cinnamic acid with the releaseof NH3 and initiate the phenylpropanoidpathway. However, it is unlikely that any single index of maturity will bediscovered that can be indiscriminately applied in all growing conditions andto all varietals. Historical experience with specific vineyards and growingregions will continue to be a critical factor in determining the optimalmaturity of the fruit. Flavonoïds and tannins contentThe flavonoids and tannins content in the pulp of five varietiesstudied since the stadium green fruit at harvest are presented in Fig 1B. A gradualdecrease was found, for the introduces varieties: Superior, Flame and Early sweet, and locale: Adari and Abbou. Therewere marked significant (p<0.05) difference in the flavonoïdes and tanninscontent at different stages of maturity in locale (diploid) and in green stagefor introduced ( triploid). The five varieties of fruits analyzed deduced that the Abbou and Adari locale variety contained thehighest flavonoïdes and tanins compounds at green, ripening and ripe stages while, Superior and Flame varieties had the lowestflavonoïdes and tannins.
Summarize, this steeply decreases in flavonoïdes andtannins content during maturation for locale and introduces. The flavonoids areinvolved in the color ; chalcones, aurones and flavones participate, with otherpigments, in yellow color, by contrast, red colors, purple and blue are dueprimarily to anthocyanins (A. Fleuriet et al,. 1996). These accumulate in the vacuoles and their levels are particularlyhigh in the air bodies and in epidermiques tissues and under epidermiques (A.Fleuriet et al,. 1996).
We knowcurrently the structure of about 260 anthocyanins including most of 6 anthocyanidines (aglycones) (Harborne andgrayer, 1988; Mazza and Miniati, 1993). They differ mostly by the number ofhydroxyl groups, their degree of methylation, the nature and number of sugarsand aromatic acids or related aliphatic to the molecule as well as theirposition. In the skin grapes” Cabemet Sauvignon”, twenty differentanthocyanins were identified (Wulf and Nagel, 1978).Some blue pigments (commelinine and ternatine) can be very complex and reach ofmolecular masses close to small proteins. As well, the Grape has in particularof IA (+)-catechine, (-)-epicatechine, procyanidins dimeres andproanthocyanidins polymerisees more or less soluble which will have a veryimportant role in the course of the vinification and aging of the wine (A.Fleuriet et al,. 1996).
The tannins canform 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 areaffected in two times: The polyphenols are first of all traps at the level ofthe hydrophobic sites of protein and then they form of the connectionshydrogens with the polar groupings of protein (Haslam et al., 1992). The astringency is a characteristic ofyoung 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 otherconstituents of the fruit such as acetaldehyd or polysaccharide forms duringthe maturation (Haslam et al., 1992).
Physico-chemical characteristics of vine fruitsThe morphological, quality and physicochemical characteristicsof the five varieties of vine studied are summarized in the Table 2.The very high water content > 70% by fresh weight ofmature fruits is a factor which reflects thegreat perishability of the vine andlimits its ability to the storage to the ambient temperature. The otherchemical parameters clearly differentiate the varieties as well as the stages ofmaturity. The levels of dry matter soluble (°Brix) obtained are more orless identical between the varieties introduced and local authoritieswith values more bottom 15.80% for the varieties Abbou and higher16.53% for the Flame varieties on the mature stage. As can be seenthat the soluble dry matter content of equal to “°Brix” depend on the pHand acidity of the fruits of vine and the varieties. The rate ofcitric acid is in gradual decrease during maturation with the lowest 2.
44 g/Lfor the Flame variety and the highest 6.04 g/L for the superiorvariety. It is the same for the ash content of 0.12 to 1.65%. Thevalue of pH was between 3.5 and 4.
11 in ripe stage for the varietiesstudied. The pH relatively low fruit mature (pH < 4.2) is anadvantage from the point of view of stability (Rozier et al.
, 1985). The abilityof rehydration allows appreciating the amount of water that canbe absorbed by the powder or the fruit dry. The values of thecapacity of rehydration understood between 25.25 % and 39.95% in greenmaturity stage for the varieties studied of vine but increaseswith 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 moreintense than those of the Flame variety which in a yellow color reddish. Forthe varieties Superior, Early sweet and Adari have a yellowishcolor. In sum, the differencesin physicochemical characteristics observed between the introducedand local varieties and/or the stage of maturation of the vine, could berelated to the conditions of production of non-identical, to the geneticdifferences, environment and the pedology. In particular, the non-samevarieties of vine would explain the constance observed at the level oftheir physicochemical parameters. Relationship between maturity and phenolic compoundThe maturity index according thephenolic, flavonoids and tannins compounds of the five varieties of vinestudied are summarized in the Fig 3.
The criteria traditionally usedto determine grape maturity are based on sugar content, which is normallymeasured in °Brix. Maturity index was determined in the report of sugar/acidity(in citric acid). Some differences were found in maturity index at the momentof 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 hightemperature reached during ripening period (R. Gil-Mun˜oz et al. 2017).
Vitrac et al., 2000, suggests that solublesugars stimulate phenolic accumulation in grapevine tissue, the concentrationof reducing sugars in the cytoplasm having been postulated as one of theregulators of the phenolic biosynthetic pathway.The highest maturity index was observed in Flame variety, where the phenolicscompounds was lowers than in other varieties. The °Brix/acid ratio 67.57 (fig2,A, B ou C.), indicating the beginning of over ripening stage of berries, anda °Brix/acid ratio 26.
24 in superiorvariety at ripe stage indicating that the maturation is late for this variety.Moreover, the phenolic compounds, flavonoids, tannins contents were alsoreduced during maturity and the last harvest. For the locale varieties, thephenolic compounds accumulation coordinated with maturation indexincrease. Several important classes ofcompounds are reported to change during maturation and ripening of the berrieson the vine. These characters, however, do not transform in a highlycoordinated fashion, and instead suggest a series of independently regulated pathways of synthesis. Each pathwayagain is influenced by the seasonal, biotic and/or abiotic factors, varietiesand vineyard practices, and the effect varies with varietal characteristics(Jackson & Lombard, 1993).
The totalphenolics and the individual groups total flavonoids, tannins variedsignificantly during all the stages of grapes maturation at the green stage showed significantly highcontents 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 oflocal varieties where the levels of phenolic compounds, increased withmaturation with the highest value is (26.7 mg CE/g WF) and similarly for flavonoidsand tannins compounds. This could be attributed to the accumulation of water in grapes during maturation, which enhancesthe hydrolysis of higher molecular weight phenolic compounds (Cimato et al., 1990).
Amiot et al., (1989), also observed thedecline in the contents of phenolic compounds during the maturation of olivefruit, which was accompanied by the accumulation of the hydrolysed products outof which some were even reported to be the non-phenolic compounds. PCA analysis of maturity stages, technological andphysicochemical characteristics A classification of grapes berries according to variety andmaturation stage by PCA analysis is provided in fig.
3 and fig.4. Principalcomponent analysis (PCA) is a powerful visualization tool for data evaluation,which can graphically represent intersample, intervariable relationships and providesa way to reduce the dimensionality of the data. PCA is an unsupervised methodof pattern recognition in the sense that no grouping of the data has to beknown before the analysis. Using PCA, class membership is easy to indicate on ascore plot. The percentage of variance captured is 52.
20% and 23.71% for thePC1 and PC2, respectively. The distribution of the sample on the PC1 and PC2reflects the influence of the morphological and physicochemical compounds onthe stages of maturation based on the different varieties. (Fig 3) showsPearson’s correlation coefficients and Principal component analysis betweenmorphological 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 highlysignificant (p<0.001) correlation values among form coefficient and weight.The "°brix", ash content, maturity index and sugar content was also highlycorrelated (p<0.
01). In the opposite, there is no correlation between theprecedent parameters and acidity and density.