Effects of drought-tolerant grapevine rootstocks on the mineral contents and fatty acid compositions of grape seeds

Abstract

BACKGROUND:

As the interest in foods with positive effects on human health has increased in recent decades, the importance of the mineral contents and oil compositions of grape seeds has been better understood. However, research on grape seeds to date has largely focused on effects on health and the usability of grape seeds in the food industry and animal feeds. In agricultural research, grape seeds have generally been evaluated as a source of genes in breeding new varieties and as propagation material.

OBJECTIVE:

Unlike previous studies, this study not only investigates the effect of variety on the changes of pomological properties, fatty acid compositions, and oil, protein, and mineral contents of grape seeds; it also aims to determine the effects of grape rootstocks and growing seasons on those changes.

METHODS:

The seeds of five grape varieties (Red Globe, Trakya Ilkeren, Ata Sarisi, Hatun Parmagi, and Horoz Karasi) grafted onto drought-tolerant grape rootstocks (1103P and 110R) were analyzed during the growing seasons of 2017 and 2018.

RESULTS:

Seed size, moisture and oil contents, major fatty acids (palmitic, oleic, and linoleic), and levels of saturated fatty acids, monounsaturated fatty acids, unsaturated fatty acids, and boron varied by variety, rootstock, and season. However, the effects of growing seasons and varieties on the seeds were not limited to these findings; effects were also seen for seed vitality, protein content, and N, K, Ca, and Fe levels of the seeds, among others.

CONCLUSIONS:

Genotype was found to be the main factor causing changes in the fatty acid compositions and mineral contents of grape seeds. When the factors that we investigated were ordered based on the changes they caused for seeds in general, the sequence that appeared was genotype > growing season > grape rootstock.

Keywords

Grape seed seed oil seed protein fatty acids seed minerals grape rootstock seasonal variation

1 Introduction

Although grapes of the species Vitis vinifera L., which are cultivated for their fruit, are botanically seed-propagated plants, they are propagated vegetatively today [1]. Many researchers have reported that grape seeds can be used in a variety of areas besides plant cultivation and that new areas of usage will continue appearing in the future [2 –6]. Various early studies were undertaken, such as that conducted by St. Leger et al. [7], but since the discovery of the so-called French paradox by Renaud and Lorgeril [8], the positive effects on human health of the phytochemical compounds contained by grape seeds have become better understood, such as their contributions to reducing the effects of oxidative stress, strengthening the central nervous system, repairing DNA damage, reducing high blood pressure triggered by cholesterol, and healing dermal wounds [9 –17]. With these effects being revealed more clearly, the interest in both grapes and grape seeds has increased [18, 19]. Previous studies revealed that grape seeds, largely considered to be winery waste, can be utilized in the feed industry as an important source of protein and oil in animal feeds [20 –22]. The fact that grape seeds containing significant amounts of oil are also rich in mineral substances and phenolic compounds suggests that there may be an increase in their use as raw materials in the cosmetics industry in the near future [23].

It has been reported that the seeds of varieties of Vitis vinifera L. contain oil at ratios ranging from 3.9% to 22.4%, while ratios range from 10.8% to 21.4% for seeds of varieties of Vitis labrusca [24 –27]. The amount of oil in grape seeds may vary depending on the variety, the ecological conditions of the place where cultivation is performed, and the ripening state of the grapes [28 –30]. In oils extracted from seeds of varieties of Vitis vinifera L., the amounts of linoleic acid and oleic acid were found to be higher compared to the contents of many oils of vegetable origin [31 –34]. However, the levels of unsaturated fatty acid (UFA) are notably higher than those of saturated fatty acid (SFA) in grape seed oil [35 –37].

The seeds of Vitis vinifera L. were found have protein contents ranging from 5.9% to 15.3%, but these ratios again vary depending on the grape genotype and the ecological conditions of the place where cultivation is performed [24 , 38–41]. Feeds that contain grape seed additives were found to increase live weight in chickens [42] and rabbits [43], but the same effect was not observed in fish [44]. Despite studies reporting that adding grape seeds to feeds increases egg weight in chickens [45] and milk yield in cows [46] and sheep [47], Nudda et al. [48] reported that such a dietary regimen had no effect on milk yield in sheep. These different results in animal studies may be due to seeds being obtained from different grape varieties, grapes being cultivated in regions with different ecological conditions, or differences in the methods used in cultivation. These variable results also indicate the need for more studies on grape seeds.

Considering that 46,516,791 tons of grapes are processed yearly for food products derived from vines and grape must, and that the seed-panicle ratio is about 3% (w/w), it can be said that approximately 1,395,503 tons of grape seeds emerge as waste from the food industry every year [49]. Studies have revealed that the seeds of different grape varieties differ from each other in terms of some other seed properties (weight, ash, moisture, size) in addition to their oil and protein contents [24 , 50]. Likewise, the compositions of the mineral substances in seeds are known to vary depending on the variety [32, 40]. Although differences were found to occur mostly under the control of the genotype, the effects of the grape rootstocks used in vineyards on seed properties of the grafted grape varieties have not yet been sufficiently studied. In this study conducted to eliminate this deficiency in the literature, the changes that occurred in seeds as a result of grafting different table grape varieties onto two drought-tolerant rootstocks were explored.

2 Materials and methods

2.1 Experimental site and plant material

This study was conducted between 2017 and 2018 in Sanliurfa, Turkey. The seeds of five table grape varieties (Red Globe, Trakya Ilkeren, Ata Sarisi, Hatun Parmagi, and Horoz Karasi) grafted onto drought-tolerant grape rootstocks (1103P and 110R) were analyzed. The grape varieties studied were grafted onto the rootstocks in 2014 and the vinestocks were grown in a double-arm cordons system (distances of 1.5×3 m). The study designed 3 replicates and 27 grapevines (9 grapevines used in each replication) were used for each rootstock/variety combination. No irrigation was carried out in the seasons in which the experiment was conducted. Data on precipitation and temperature in the region where the vineyard was located are shown in Fig. 1.

Fig. 1

Climatic data of the region where the study was conducted.

The soil of the vineyard where the study was conducted has a slightly alkaline nature and low organic matter content (Table 1). However, the lime content is significantly high. The highest P₂O₅, K₂O, Cu, Zn, and Mn contents were found at a depth of 0–30 cm, but their levels decreased as the depth increased. The Fe and Mg levels of the soil, on the other hand, increased as the depth increased.

Table 1

Soil properties (physical and chemical) of the vineyard where the experiment was conducted

	0–30 cm	30–60 cm	60–90 cm	90–120 cm
N (%)	0.139	0.133	0.102	0.095
P₂O₅ (kg da^–1)	4.36	0.47	0.22	0.16
K₂O (kg da^–1)	95.1	63.6	61.1	56.9
Ca (ppm)	272.3	325.7	304.7	293.3
Mg (ppm)	10.71	15.80	16.83	18.41
Fe (mg L^–1)	0.405	0.738	0.770	0.833
Cu (mg L^–1)	1.174	1.103	0.909	0.848
Zn (mg L^–1)	0.175	0.070	0.060	0.018
Mn (mg L^–1)	2.726	1.675	0.982	0.958
pH	7.61	7.82	7.88	7.94
EC (ds m^–1)	1.39	1.12	0.90	0.81
Lime (%)	25.8	22.7	21.4	28.6
Organic matter (%)	0.93	0.73	0.71	0.64
Saturation	65.7	68.3	69.3	70.0

The seeds were extracted from the grapes of each variety after the grapes reached harvest maturity (16–18.5°Brix). After this process, the seeds were washed for 2 minutes in pure water to make sure that there was no pulp or must residue on them, water droplets were removed using a piece of paper towel, and the seeds were then dried for 72 hours at 24°C. After the completion of the drying process, the seeds were stored in a glass desiccator until analysis.

2.2 Determination of pomological properties

One hundred randomly selected seeds of the examined varieties were weighed on a precision scale with accuracy of 0.001 g to determine the seed weights [51]. Twenty grams of randomly selected seeds were counted with four replications and the number of seeds was multiplied by 50 to calculate the number of seeds per 1 kg.

Measurements of seed lengths and widths were made using a digital caliper with accuracy of 0.001 mm with four repetitions; each repetition involved 30 seeds. Moisture contents in the seeds were determined using the high constant temperature oven method [52]. Seed vitality was determined with Agaoglu’s method [53].

2.3 Determination of fixed oil and fatty acid composition

Seeds (10 g) were dried for 72 hours at 65°C in the laboratory and then ground and subjected to oil extraction (6 hours) with hexane in a Soxhlet device [25 , 55]. After the extraction process, balloon flasks were kept in a drying oven at 60°C for 24 hours in the laboratory to separate the hexane from the oil-hexane mixture. The weight percentage of oil was determined as % w/w [27].

The derivatization process was performed using Slover and Lanza’s method [56]. Determination of fatty acids was performed with a Nexis GC-2030 Shimadzu (Kyoto, Japan) gas chromatograph. A FID detector and TR-CN100 column (100 m×0.25 mm×0.20μm) (Barcelona, Spain) were used. Readings were taken at a detector temperature of 240°C with an injection volume of 1μL and flow rate of 1 mL min^-1. Hydrogen was used as the carrier gas. In the obtained chromatograms, the peaks were defined based on retention times and the ratios of fatty acids were determined (Fig. 2). Fixed oil content and fatty acid composition analysis were performed in three repetitions.

Fig. 2

Chromatogram sample used to determine ratios of fatty acids (Ata Sarisi/1103 P). ^*Fatty acid peaks 1- Butyric acid (C4:0) 2-Caproic acid (C6:0) 3-Myristic acid (C14:0) 4-Palmitic acid (C16:0) 5-Palmitoleic acid (C16:1) 6-Heptadecanoic acid (C17:0) 7-Stearic acid (C18:0) 8-Cis-oleic acid (C18:1n-9c) 9-Trans-elaidic acid (C18:1n-9t) 10-Cis-linoleic acid (C18:2n-6c) 11-Arachidic acid (C20:0) 12-Cis-11-eicosenoic acid (C20:1) 13-Linolenic acid (C18:3n6) 14-Cis-11,14-eicosadienoic acid (C20:2) 15-Behenic acid (C22:0) 16-Cis-11,14,17-eicosatrienoic acid (C20:3n3) 17-Cis-4,7,10,13,16,19-docosahexaenoic acid (C22:6n3).

2.4 Determination of ash, protein, and mineral contents

Seeds (0.5 g) were randomly placed into a porcelain crucible and burned in a muffle furnace for 2 hours at 200°C and then for 4 hours at 500°C. The samples were then weighed on a precision scale and the amount of ash (% w/w) was determined [57]. After determining the amount of ash, the ashes of the seeds were dissolved in a 1 N HCl acid solution and the minerals were determined using an ICP-MS device [58].

Seeds (0.5 g) that were previously dried in the drying oven for 72 hours at 65°C were ground and burned with the wet decomposition method. The amount of % N was determined with the Kjeldahl distillation method [59]. The obtained % N value was multiplied by a coefficient of 6.25 to determine the total amount of protein [30].

2.5 Statistical analysis

Analysis of variance (one-way ANOVA) was conducted using Minitab (ver. 18) statistical software in order to determine the effects of grape varieties, grape rootstocks, and growing seasons on the changes in all properties examined (seed pomological properties, fixed oil, ash, protein, mineral contents, and fatty acid composition) in this study. Significant differences between averages were indicated by grouping them on the basis of the Tukey test.

3 Results and discussion

3.1 Pomological features

The 100-seed weights of the examined grape varieties were found to change depending on rootstock-variety interactions, and changes were also observed in the averages according to the seasons in which the study was carried out. The heaviest seeds were found for the Ata Sarisi/1103P grafting combination, while the lightest seeds were found for Trakya Ilkeren/110R (Table 2). Grape rootstocks and growing seasons were found to have no statistically significant effects on seed weight. When grape varieties were examined independently of the rootstocks onto which they were grafted, it was found that the variety with the heaviest seeds in both seasons was Ata Sarisi, while the variety with the lightest seeds was Trakya Ilkeren. Although these results were in parallel with those of Uslu and Dardeniz [51], who previously studied wine grape varieties, the obtained values were lower than the lower limit values reported by Fernandes et al. [26] and Kamiloğu and Üstün [60]. Kısakürek [61], on the other hand, reported higher seed weights for the Horoz Karasi and Hatun Parmagi varieties. The conclusion of those researchers that seed weight may vary depending on genotype supports our findings in the present study. However, different researchers have reported many environmental factors affecting vineyards, which are likely to cause some changes in the structure of grape berries (berry size, phytochemical composition, enzyme activities, etc.) [62 –64].

Table 2
Changes of pomological properties of seeds by rootstocks, grape varieties, and seasons

Rs. Variety 100 seed weight (g) Num. of seeds (seeds kg^-1) Seed width (mm) Seed length (mm) Humidity (% w/w)

1103 P R.G. 3.14±0.10 c^ 31267±465 e^ 4.10±0.08 c^ 7.48±0.12 b^ 5.16±0.72 ns

T. I. 2.56±0.30 e 40202±1444 a 3.64±0.05 e 5.79±0.15 g 2.71±0.59

A. S. 4.00±0.14 a 25397±1214 e 4.31±0.17 a 6.86±0.14 c 3.64±0.17

H. P. 3.17±0.17 c 31422±1674 d 3.72±0.46 e 6.02±0.17 f 4.17±0.43

H. K. 2.79±0.13 d 36867±1140 bc 4.30±0.23 ab 6.83±0.36 c 5.30±0.46

110 R R.G. 3.23±0.16 c 34952±5394 c 4.22±0.15 ab 7.87±0.28 a 4.02±0.13

T. I. 2.48±0.12 e 38982±1511 ab 3.63±0.07 e 5.80±0.07 g 2.74±0.42

A. S. 3.74±0.10 b 27122±1588 e 4.19±0.24 bc 6.60±0.18 d 2.77±0.28

H. P. 3.16±0.09 c 31522±1473 d 3.88±0.52 d 6.33±0.28 e 3.85±0.29

H. K. 2.80±0.12 d 35742±487 c 4.24±0.07 ab 6.77±0.14 c 4.54±1.31

Rs. Av. 1103 P 3.13±0.53 ns 33031±5292 ns 4.01±0.36 ns 6.59±0.64 b^ 4.20±1.09 a^

110 R 3.08±0.45 33664±4830 4.03±0.35 6.67±0.72 a 3.59±0.94 b

Var. Av. R.G. 3.19±0.14 b^ 33109±4178 c^ 4.16±0.13 b^ 7.67±0.29 a^ 4.59±0.77 ab^

T. I. 2.52±0.23 d 39592±1569 a 3.64±0.06 d 5.80±0.11 d 2.72±0.49 c

A. S. 3.87±0.18 a 26259±1636 d 4.25±0.21 a 6.73±0.20 b 3.21±0.51 c

H. P. 3.17±0.13 b 31472±1536 c 3.80±0.48 c 6.17±0.26 c 4.01±0.39 b

H. K. 2.79±0.12 c 36304±1030 b 4.27±0.17 a 6.80±0.27 b 4.92±1.01 a

Year Av. 2017 3.10±0.53 ns 33656±6121 ns 4.10±0.28a^ 6.64±0.62 ns 3.64±0.77 b^**

2018 3.11±0.39 33038±4229 3.94±0.40b 6.63±0.74 4.14±1.25 a

Rs.	Variety	100 seed weight (g)	Num. of seeds (seeds kg^-1)	Seed width (mm)	Seed length (mm)	Humidity (% w/w)
1103 P	R.G.	3.14±0.10 c^**	31267±465 e^**	4.10±0.08 c^**	7.48±0.12 b^**	5.16±0.72 ns
	T. I.	2.56±0.30 e	40202±1444 a	3.64±0.05 e	5.79±0.15 g	2.71±0.59
	A. S.	4.00±0.14 a	25397±1214 e	4.31±0.17 a	6.86±0.14 c	3.64±0.17
	H. P.	3.17±0.17 c	31422±1674 d	3.72±0.46 e	6.02±0.17 f	4.17±0.43
	H. K.	2.79±0.13 d	36867±1140 bc	4.30±0.23 ab	6.83±0.36 c	5.30±0.46
110 R	R.G.	3.23±0.16 c	34952±5394 c	4.22±0.15 ab	7.87±0.28 a	4.02±0.13
	T. I.	2.48±0.12 e	38982±1511 ab	3.63±0.07 e	5.80±0.07 g	2.74±0.42
	A. S.	3.74±0.10 b	27122±1588 e	4.19±0.24 bc	6.60±0.18 d	2.77±0.28
	H. P.	3.16±0.09 c	31522±1473 d	3.88±0.52 d	6.33±0.28 e	3.85±0.29
	H. K.	2.80±0.12 d	35742±487 c	4.24±0.07 ab	6.77±0.14 c	4.54±1.31
Rs. Av.	1103 P	3.13±0.53 ns	33031±5292 ns	4.01±0.36 ns	6.59±0.64 b^**	4.20±1.09 a^**
	110 R	3.08±0.45	33664±4830	4.03±0.35	6.67±0.72 a	3.59±0.94 b
Var. Av.	R.G.	3.19±0.14 b^**	33109±4178 c^**	4.16±0.13 b^**	7.67±0.29 a^**	4.59±0.77 ab^**
	T. I.	2.52±0.23 d	39592±1569 a	3.64±0.06 d	5.80±0.11 d	2.72±0.49 c
	A. S.	3.87±0.18 a	26259±1636 d	4.25±0.21 a	6.73±0.20 b	3.21±0.51 c
	H. P.	3.17±0.13 b	31472±1536 c	3.80±0.48 c	6.17±0.26 c	4.01±0.39 b
	H. K.	2.79±0.12 c	36304±1030 b	4.27±0.17 a	6.80±0.27 b	4.92±1.01 a
Year Av.	2017	3.10±0.53 ns	33656±6121 ns	4.10±0.28a^**	6.64±0.62 ns	3.64±0.77 b^**
	2018	3.11±0.39	33038±4229	3.94±0.40b	6.63±0.74	4.14±1.25 a

Statistical differences (^**: p < 0.01) between the mean values of rootstock-variety groups, rootstocks, varieties, and seasons are shown with different letters. ns: Not significant.

A statistically significant (p < 0.01) difference was found between the grafting combinations studied for the evaluation of the number of seeds per 1 kg, as in the 100-seed weight assessment. The strong negative correlation (r = –0.845, p < 0.001) found between 100-seed weight and number of seeds per 1 kg showed that these findings supported each other. On the other hand, the effects of rootstocks and seasons on the number of seeds were found to be statistically insignificant.

Seed dimensions (both length and width) were found to statistically differ in both seasons. The longest seeds were found for the Red Globe/110R grafting combination and the widest seeds for the Ata Sarisi/1103P grafting combination. The seeds of Trakya Ilkeren were found to have similar dimensions and be considerably smaller than the others on both rootstocks. The reason why the Red Globe variety was found to have the longest seeds in both seasons is the fact that the seeds of this variety have a long beak. The effects of the grape rootstocks used in the vineyard and the growing season on the seed sizes of the grape varieties were found to be limited. In addition, a positive correlation between seed width and length (r = 0.713, p < 0.001) was revealed. Despite possible variations of seed dimensions depending on environmental factors, the shape and dimensions of seeds are primarily under the control of the genotype [50]. Our finding that the seed sizes varied depending on genotype is in parallel with the findings of Uslu and Dardeniz [51] and Mironeasa et al. [30].

The moisture contents of the seeds of the examined rootstock-variety interaction groups were observed to statistically differ from each other across growing seasons, but such differences were not observed in yearly averages. When varieties were compared independently of rootstocks, the moisture contents of the seeds were found to vary depending on the grape varieties in both growing seasons. The highest moisture content was found for the Horoz Karasi variety (4.92% v/w), while the lowest moisture content was found for Trakya Ilkeren (2.72% v/w). The finding that Horoz Karasi had much higher moisture contents than other varieties was in parallel with the findings reported by Gök Tangolar et al. [40]. Our findings were also consistent with the findings of previous studies that reported that the moisture content of seeds varies depending on grape varieties [24 , 66].

Grape rootstocks were found to affect the moisture content of the seeds of the varieties grafted onto them. Serra et al. [67] stated that the 110R rootstock is more drought-tolerant than 1103P. Our findings, on the other hand, showed that this may be different in non-irrigated vineyards that have reached yield age. McCarthy et al. [68] reported that these two rootstocks provided similar efficiency of the varieties grafted onto them in wet conditions, whereas the 1103P rootstock provided higher productivity compared to 110R under drought stress.

It was determined that the moisture content of the seeds differed by years as the moisture contents were higher in the 2018 season compared to the 2017 season. This change was observed for most of the studied varieties, excluding Trakya Ilkeren. This was the consequence of the fact that climatic features differ depending on growing seasons.

3.2 Seed vitality and ash, protein, and oil contents of the seeds

In the 2018 season, there was more precipitation in the vineyard where the experiment was conducted, and the seasonal distribution of precipitation had an effect on the seed properties examined in this study. The rainfall after fruit set in May of the 2018 season led to a longer preservation of moisture levels in the soil. Because of this, not only the seed moisture but also the vitality rate of the seeds and the ash, protein, and oil contents were positively affected. In the second season of this study, statistically better results were achieved for all mentioned properties in comparison to the first season (Table 3). In studies conducted with different plant species, the seed weight and the oil and protein contents of plants whose water requirements were met were found to be higher compared to those of plants whose water requirements could not be met at a sufficient level [69 –71]. Our findings are in parallel with the literature in this regard.

Table 3
Changes in the vitality of seeds and their ash, protein, and oil contents by rootstocks, varieties, and seasons

Rootstock Variety Vitality (%) Ash (% w/w) Protein (% w/w) Oil (% w/w)

1103 P Red Globe 35.00±22.80 ns 2.95±0.17 ns 6.78±0.16 ns 12.00±2.95 ns

Trakya Ilkeren 57.50±23.40 2.98±0.31 7.91±0.22 12.53±2.46

Ata Sarisi 81.67±19.15 2.80±0.31 8.35±0.71 13.75±2.93

Hatun Parmagi 75.83±20.10 2.82±0.21 9.00±0.70 13.52±1.85

Horoz Karasi 27.50±18.37 2.85±0.18 6.89±0.70 8.10±2.20

110 R Red Globe 41.67±17.51 2.71±0.15 6.52±0.21 13.82±1.02

Trakya Ilkeren 65.83±8.01 2.88±0.13 8.37±0.37 13.80±1.46

Ata Sarisi 75.00±26.10 3.03±1.27 8.10±0.80 14.30±1.59

Hatun Parmagi 75.83±14.29 3.07±0.09 9.33±0.69 13.21±0.71

Horoz Karasi 51.67±19.41 2.83±0.28 6.69±0.30 10.49±0.97

Rootstock Av. 1103 P 55.50±29.19 ns 2.88±0.24 ns 7.79±1.01 ns 11.98±3.13b^

110 R 62.00±21.60 2.90±0.57 7.80±1.18 13.12±1.78a

Variety Av. Red Globe 38.33±19.69 c^ 2.83±0.20 ns 6.65±0.23 c^ 12.91±2.31 a^

Trakya Ilkeren 61.67±17.23 b 2.93±0.23 8.14±0.38 b 13.16±2.04 a

Ata Sarisi 78.33±22.09 a 2.92±0.89 8.23±0.73 b 14.02±2.27 a

Hatun Parmagi 75.83±16.63 ab 2.94±0.20 9.16±0.68 a 13.36±1.35 a

Horoz Karasi 39.58±22.00 c 2.84±0.23 6.79±0.52 c 9.29±2.05 b

Year Av. 2017 45.17±23.29 b^** 2.77±0.25b^* 7.44±0.90 b^ 11.33±2.46 b^

2018 72.33±20.42 a 3.01±0.53a 8.15±1.16 a 13.77±2.11 a

Rootstock	Variety	Vitality (%)	Ash (% w/w)	Protein (% w/w)	Oil (% w/w)
1103 P	Red Globe	35.00±22.80 ns	2.95±0.17 ns	6.78±0.16 ns	12.00±2.95 ns
	Trakya Ilkeren	57.50±23.40	2.98±0.31	7.91±0.22	12.53±2.46
	Ata Sarisi	81.67±19.15	2.80±0.31	8.35±0.71	13.75±2.93
	Hatun Parmagi	75.83±20.10	2.82±0.21	9.00±0.70	13.52±1.85
	Horoz Karasi	27.50±18.37	2.85±0.18	6.89±0.70	8.10±2.20
110 R	Red Globe	41.67±17.51	2.71±0.15	6.52±0.21	13.82±1.02
	Trakya Ilkeren	65.83±8.01	2.88±0.13	8.37±0.37	13.80±1.46
	Ata Sarisi	75.00±26.10	3.03±1.27	8.10±0.80	14.30±1.59
	Hatun Parmagi	75.83±14.29	3.07±0.09	9.33±0.69	13.21±0.71
	Horoz Karasi	51.67±19.41	2.83±0.28	6.69±0.30	10.49±0.97
Rootstock Av.	1103 P	55.50±29.19 ns	2.88±0.24 ns	7.79±1.01 ns	11.98±3.13b^**
	110 R	62.00±21.60	2.90±0.57	7.80±1.18	13.12±1.78a
Variety Av.	Red Globe	38.33±19.69 c^**	2.83±0.20 ns	6.65±0.23 c^**	12.91±2.31 a^**
	Trakya Ilkeren	61.67±17.23 b	2.93±0.23	8.14±0.38 b	13.16±2.04 a
	Ata Sarisi	78.33±22.09 a	2.92±0.89	8.23±0.73 b	14.02±2.27 a
	Hatun Parmagi	75.83±16.63 ab	2.94±0.20	9.16±0.68 a	13.36±1.35 a
	Horoz Karasi	39.58±22.00 c	2.84±0.23	6.79±0.52 c	9.29±2.05 b
Year Av.	2017	45.17±23.29 b^**	2.77±0.25b^*	7.44±0.90 b^**	11.33±2.46 b^**
	2018	72.33±20.42 a	3.01±0.53a	8.15±1.16 a	13.77±2.11 a

In each column, statistical differences (^*: p < 0.05, ^**: p < 0.01) between the mean values of rootstock-variety groups, rootstocks, varieties, and seasons are marked with different letters. ns: Not significant.

The studied rootstock-variety combinations were found to not statistically differ from each other in terms of seed vitality or ash, protein, and fixed oil contents. However, when the varieties were compared to each other, statistically significant differences were found in terms of properties other than ash content. The effect of rootstocks was limited to the oil contents of the seeds (p < 0.01), and they did not cause statistically significant changes in other properties.

It was found that the Ata Sarisi/1103P combination had higher seed vitality compared to other grafting combinations in this study. Although it was not found to be statistically significant, the 110R rootstock had higher values by approximately 4–9% compared to the 1103P rootstock in terms of both seasons and the average of the seasons. This may be because 110R rootstock enables the berries of the grafted varieties to complete their development earlier. According to Ristic and Iland [72], the development of grape seeds follows a process in parallel with the grapes reaching harvest maturity. However, Kennedy et al. [73] reported that the seeds of the Cabernet Sauvignon variety fully developed 40 days before harvest. Fredes et al. [74] reported that the effects of total polyphenol index and total anthocyanin change on the color of the seed coat of the Carménère grape variety continued after the water-soluble dry matter reached 16% Brix in grapes. Based on our findings, it can be said that the use of 110R rootstock both shortens the ripening time of grape berries and increases the vitality of seeds in studied grape varieties.

In the second season of the experiment and in the average of the seasons, there was no statistically significant difference between varieties in terms of the ash content of their seeds (p > 0.05), but the varieties differed from each other in terms of this feature in the 2017 season (p < 0.01). The rootstocks, on the other hand, were found to not affect the ash content of the seeds. The variety with the highest ash contents in its seeds was Hatun Parmagi (2.91% w/w) in the 2017 season and Ata Sarisi (3.36% w/w) in the 2018 season. A positive correlation was found between the fixed oil contents and ash contents of the seeds (r = 0.261, p < 0.05). It was also determined that the amount of ash in the seeds varied depending on the growing season. The seeds from the 2018 season contained higher amounts of ash compared to those from the 2017 season. The amounts of ash that we determined were similar to the findings obtained by previous researchers [24 , 65].

In terms of protein amount, the seeds belonging to the studied varieties were found to differ from each other in both seasons and seasonal average. The highest amount of protein (9.16%) was found in the seeds of the Hatun Parmagi variety. In both seasons, the protein contents of the seeds belonging to the Red Globe and Horoz Karasi varieties were found to be lower than those of other varieties. Our finding of differences between the varieties in terms of protein contents of the seeds was also reported by other researchers in previous studies [24 , 75]. However, increased amounts of protein in the grape seeds were found in parallel with the increase in the oil content of the grape seeds (r = 0.521, p < 0.001). There was no statistically significant difference between rootstocks in terms of the protein contents of the seeds in the average of the seasons in which the study was conducted, but significant differences (p < 0.05) were found for individual seasons. It was observed that the protein contents of the seeds increased with the 110R rootstock in the 2017 season and with the 1103P rootstock in the 2018 season. These differences observed between seasons confirmed the findings of Serra et al. [67] but were inconsistent with the finding of McCarthy et al. [68], as the latter showed that these rootstocks have similar properties in conditions where the moisture level of the soil is sufficient.

The oil contents of the seeds varied from 6.28% to 14.45% w/w (p < 0.01) in the 2017 season and from 9.92% to 16.30% w/w (p > 0.05) in the 2018 season. However, there was no difference between the groups of rootstock-variety interactions in terms of the average of the seasons (p > 0.05). When grape varieties were evaluated independently of rootstocks, the highest oil content was found in the Hatun Parmagi variety (12.79% w/w) in the 2017 season and in the Ata Sarisi variety (15.93% w/w) in the 2018 season. The lowest oil content was found in Horoz Karasi (7.99–10.60% w/w) in both seasons. According to the average of the seasons, Ata Sarisi was the variety with the most oil content in its seeds, followed by Hatun Parmagi, Trakya Ilkeren, Red Globe, and Horoz Karasi, respectively.

The extraction method used for obtaining oil from grape seeds can affect the amount and the composition of that oil [39 , 77]. The seed oil ratios that we found were within the lower and upper limit values determined by researchers who previously analyzed the oil contents of grape seeds using the same method [25, 27]. In addition, it was reported previously by other researchers [25, 75] that the seeds of the Red Globe and Hatun Parmagi varieties contain more oil than those of Horoz Karasi.

3.3 Fatty acid compositions

In the oil contents of the varieties analyzed in this study, we found different numbers (15–24) of fatty acids, which differed between varieties, although most shared some common features. Previous studies showed that grape seed oil may contain from 5 to 34 different fatty acids [54 , 78]. Since some fatty acids that we found in this study were present in trace amounts, the findings for 15 common fatty acids are presented here.

It was found that linoleic acid was the fatty acid present in the highest amount for each rootstock-variety interaction (Table 4). Linoleic acid was followed by oleic acid, palmitic acid, and stearic acid, respectively. The order of fatty acids determined in this study was in accord with the findings of previous studies [25–27 , 79–81]. The highest amount of linoleic acid was found in the Trakya Ilkeren/1103P combination and the highest amount of oleic acid was found in the Hatun Parmagi/110R combination. The palmitic and linoleic acid contents of Horoz Karasi were higher while the stearic and oleic acid contents were lower on both rootstocks compared to the Hatun Parmagi variety. Palmitic, palmitoleic, heptadecanoic, linoleic, arachidic, and docosahexaenoic acids were found to show statistically significant changes depending on rootstock-variety interactions, but it was observed that other fatty acids did not show changes. SFAs and polyunsaturated fatty acids (PUFAs) showed changes according to rootstock-variety interactions while monounsaturated fatty acids (MUFAs) did not show changes.

Table 4
Fatty acid compositions of seed oil in groups of grape rootstock-variety interactions

Fatty Acid 1103 P 110 R

R.G. T.I. A.S. H.P. H.K. R.G. T.I. A.S. H.P. H.K.

C4:0 0.26±0.04 ns 0.30±0.06 0.24±0.03 0.23±0.07 0.28±0.04 0.28±0.06 0.47±0.29 0.30±0.06 0.44±0.24 0.30±0.05

C6:0 0.35±0.05 ns 0.40±0.07 0.32±0.03 0.30±0.10 0.36±0.05 0.38±0.08 0.56±0.31 0.40±0.07 0.30±0.17 0.41±0.08

C14:0 0.08±0.01 ns 0.14±0.13 0.08±0.01 0.06±0.00 0.11±0.02 0.08±0.05 0.19±0.15 0.10±0.02 0.08±0.02 0.10±0.00

C16:0 10.09±1.15 b^ 08.94±0.33b 9.63±1.15 b 8.20±0.34 b 11.86±1.16 ab 10.31±1.54 b 14.28±7.04 a 10.35±1.04 b 9.30±0.92 b 11.69±0.83 ab

C16:1 0.17±0.03 b^ 0.11±0.01d 0.21±0.05 a 0.13±0.01 cd 0.25±0.05 a 0.16±0.02 bc 0.16±0.06 bc 0.23±0.05 a 0.16±0.03 bc 0.24±0.03 a

C17:0 0.08±0.02 b^** 0.08±0.01b 0.06±0.00 b 0.07±0.00b 0.07±0.00 b 0.06±0.01 b 0.12±0.04 a 0.10±0.03 ab 0.12±0.06 a 0.09±0.02 ab

C18:0 4.15±0.17 ns 5.25±0.31 4.00±0.36 4.40±0.08 4.06±0.16 4.22±0.08 6.93±2.55 4.12±0.42 4.24±0.28 4.07±0.18

C18:1 c(n-9) 18.37±0.66 ns 15.01±0.91 22.72±0.15 31.05±1.80 18.67±0.85 18.87±0.72 19.54±5.62. 23.91±0.58 31.64±2.78 19.47±0.90

C18:1 t(n-9) 0.99±0.08 ns 0.78±0.03 1.02±0.12 0.87±0.10 1.04±0.08 0.93±0.11 0.93±0.38 1.15±0.17 0.79±0.44 1.05±0.04

C18:2 c(n-6) 64.11±0.87 ab^* 67.90±0.63 a 60.57±1.12 abc 60.57±2.44 abc 61.93±2.25 abc 63.33±2.88 ab 53.34±19.20 bc 58.03±1.13 abc 51.57±3.44 c 61.31±1.57 abc

C18:3(n-6) 0.25±0.01 ns 0.31±0.00 0.28±0.01 0.23±0.00 0.29±0.16 0.24±0.03 0.21±0.14 0.27±0.02 0.25±0.01 0.35±0.02

C20:0 0.16±0.01 b^* 0.18±0.00b .0.12±0.00 b 0.17±0.01 b .0.18±0.00 b .0.16±0.01 b 0.25±0.11 a .0.13±0.01 b 0.17±0.01 b 0.17±0.00 b

C20:1 0.15±0.02 ns 0.14±0.00 0.15±0.02 0.17±0.00 0.13±0.00 0.10±0.05 0.13±0.09 0.14±0.02 0.16±0.02 0.13±0.01

C20:3 (n-3) 0.31±0.04 ns 0.35±0.09 0.27±0.03 0.26±0.10 0.29±0.06 0.37±0.12 0.58±0.37 0.39±0.07 0.42±0.08 0.37±0.04

C22:6 (n-3) 0.33±0.12 b^** 0.32±0.13b .0.24±0.15 b 0.16±0.04 b .0.28±0.05 b .0.29±0.20 b 0.77±0.68 a .0.28±0.10 b .0.18±0.05 b 0.22±0.09 b

∑ SFA 15.16±1.02 b^* 14.98±0.25b 14.46±0.77 b 13.42±0.44 b 16.91±1.11 b 15.48±1.77 b 22.80±10.36 a 15.48±0.87 b 14.66±0.87 b 16.83±0.55 b

∑ MUFA 19.68±0.58 ns 16.04±0.89 24.09±0.17 32.22±1.88 20.08±0.98 20.06±0.82 20.77±6.100 25.43±0.53 32.75±2.51 20.89±0.96

∑ PUFA 65.00±0.76 ab^* 68.88±0.74a 061.36±0.96 a-d 54.27±2.32 cd 062.80±2.05 a-d 0.64.24±2.64 abc .54.90±18.35 bcd .58.67±0.98 a-d 52.43±3.34 d .62.25±1.54 a-d

Fatty Acid	1103 P	110 R
C4:0	0.26±0.04 ns	0.30±0.06	0.24±0.03	0.23±0.07	0.28±0.04	0.28±0.06	0.47±0.29	0.30±0.06	0.44±0.24	0.30±0.05
C6:0	0.35±0.05 ns	0.40±0.07	0.32±0.03	0.30±0.10	0.36±0.05	0.38±0.08	0.56±0.31	0.40±0.07	0.30±0.17	0.41±0.08
C14:0	0.08±0.01 ns	0.14±0.13	0.08±0.01	0.06±0.00	0.11±0.02	0.08±0.05	0.19±0.15	0.10±0.02	0.08±0.02	0.10±0.00
C16:0	10.09±1.15 b^**	08.94±0.33b	9.63±1.15 b	8.20±0.34 b	11.86±1.16 ab	10.31±1.54 b	14.28±7.04 a	10.35±1.04 b	9.30±0.92 b	11.69±0.83 ab
C16:1	0.17±0.03 b^**	0.11±0.01d	0.21±0.05 a	0.13±0.01 cd	0.25±0.05 a	0.16±0.02 bc	0.16±0.06 bc	0.23±0.05 a	0.16±0.03 bc	0.24±0.03 a
C17:0	0.08±0.02 b^**	0.08±0.01b	0.06±0.00 b	0.07±0.00b	0.07±0.00 b	0.06±0.01 b	0.12±0.04 a	0.10±0.03 ab	0.12±0.06 a	0.09±0.02 ab
C18:0	4.15±0.17 ns	5.25±0.31	4.00±0.36	4.40±0.08	4.06±0.16	4.22±0.08	6.93±2.55	4.12±0.42	4.24±0.28	4.07±0.18
C18:1 c(n-9)	18.37±0.66 ns	15.01±0.91	22.72±0.15	31.05±1.80	18.67±0.85	18.87±0.72	19.54±5.62.	23.91±0.58	31.64±2.78	19.47±0.90
C18:1 t(n-9)	0.99±0.08 ns	0.78±0.03	1.02±0.12	0.87±0.10	1.04±0.08	0.93±0.11	0.93±0.38	1.15±0.17	0.79±0.44	1.05±0.04
C18:2 c(n-6)	64.11±0.87 ab^*	67.90±0.63 a	60.57±1.12 abc	60.57±2.44 abc	61.93±2.25 abc	63.33±2.88 ab	53.34±19.20 bc	58.03±1.13 abc	51.57±3.44 c	61.31±1.57 abc
C18:3(n-6)	0.25±0.01 ns	0.31±0.00	0.28±0.01	0.23±0.00	0.29±0.16	0.24±0.03	0.21±0.14	0.27±0.02	0.25±0.01	0.35±0.02
C20:0	0.16±0.01 b^*	0.18±0.00b	.0.12±0.00 b	0.17±0.01 b	.0.18±0.00 b	.0.16±0.01 b	0.25±0.11 a	.0.13±0.01 b	0.17±0.01 b	0.17±0.00 b
C20:1	0.15±0.02 ns	0.14±0.00	0.15±0.02	0.17±0.00	0.13±0.00	0.10±0.05	0.13±0.09	0.14±0.02	0.16±0.02	0.13±0.01
C20:3 (n-3)	0.31±0.04 ns	0.35±0.09	0.27±0.03	0.26±0.10	0.29±0.06	0.37±0.12	0.58±0.37	0.39±0.07	0.42±0.08	0.37±0.04
C22:6 (n-3)	0.33±0.12 b^**	0.32±0.13b	.0.24±0.15 b	0.16±0.04 b	.0.28±0.05 b	.0.29±0.20 b	0.77±0.68 a	.0.28±0.10 b	.0.18±0.05 b	0.22±0.09 b
∑ SFA	15.16±1.02 b^*	14.98±0.25b	14.46±0.77 b	13.42±0.44 b	16.91±1.11 b	15.48±1.77 b	22.80±10.36 a	15.48±0.87 b	14.66±0.87 b	16.83±0.55 b
∑ MUFA	19.68±0.58 ns	16.04±0.89	24.09±0.17	32.22±1.88	20.08±0.98	20.06±0.82	20.77±6.100	25.43±0.53	32.75±2.51	20.89±0.96
∑ PUFA	65.00±0.76 ab^*	68.88±0.74a	061.36±0.96 a-d	54.27±2.32 cd	062.80±2.05 a-d	0.64.24±2.64 abc	.54.90±18.35 bcd	.58.67±0.98 a-d	52.43±3.34 d	.62.25±1.54 a-d

Fatty acid codes: Butyric acid (C4:0), Caproic acid (C6:0), Myristic acid (C14:0), Palmitic acid (C16:0), Palmitoleic acid (C16:1), Heptadecanoic acid (C17:0), Stearic acid (C18:0), Cis-oleic acid (C18:1n-9c), Trans-elaidic acid (C18:1n-9t), Cis-linoleic acid (C18:2n-6c), Linolenic acid (C18:3n6), Arachidic acid (C20:0), Cis-11-eicosenoic acid (C20:1), Cis-11,14,17-eicosatrienoic acid (C20:3n3), Cis-4,7,10,13,16,19-docosahexaenoic acid (C22:6n3). In each line, statistical differences (^*: p < 0.05, ^**: p < 0.01) between the mean values of the rootstock-variety groups are shown with different letters. ns: Not significant.

A relationship was determined between the oil content of the seeds of the grape varieties and some fatty acids in these seed oils. Although most of the fatty acids analyzed did not change depending on the oil content of the seeds, palmitic, trans-elaidic, linolenic, and palmitoleic acids changed. As the oil contents of the seeds increased, contents of palmitic acid (r = –0.276, p < 0.05), trans-elaidic acid (r = –0.282, p < 0.05), linolenic acid (r = –0.292, p < 0.05), and palmitoleic acid (r = –0.544, p < 0.001) were found to decrease. We found no significant relationship between the amount of oil in the seeds and SFA, MUFA, or PUFA levels.

It was determined that the protein contents of the seeds and the fatty acid compositions were significantly related to each other. The amounts of oleic acid (r = 0.578, p < 0.001) and eicosenoic acid (r = 0.483, p < 0.001) increased in parallel with increases in the amount of protein, while linoleic acid (r = –0.355, p < 0.01), linolenic acid (r = –0.294, p < 0.05), palmitic acid (r = –0.263, p < 0.05), palmitoleic acid (r = –0.451, p < 0.001), and trans-elaidic acid (r = –0.271, p < 0.05) decreased. In relation to these changes, the MUFA contents of seeds with high amounts of protein also increased (r = 0.564, p < 0.001), while PUFA contents decreased dramatically (r = –0.370, p < 0.01).

When the examined varieties were evaluated independently of the rootstocks, they were found to differ from each other in terms of fatty acids other than butyric, caproic, and linolenic acids (Table 5). The highest linoleic acid content was found in the Red Globe grape variety. On the contrary, the seeds with the highest oleic acid contents were obtained from the Hatun Parmagi grape variety, while the lowest contents were obtained from Trakya Ilkeren. The Trakya Ilkeren grape variety was found to have the highest SFA values. The highest MUFA values were found for Hatun Parmagi, while the highest PUFA values were found for Red Globe. When the fatty acids in grape seed oil were classified according to their saturation states, they were ranked in the order of PUFA > MUFA>SFA, similar to the findings of previous studies [26, 77].

Table 5

Changes of the compositions of fatty acids in seed oil by grape varieties

Fatty Acid	Red Globe	Trakya Ilkeren	Ata Sarisi	Hatun Parmagi	Horoz Karasi
C4:0	0.27±0.05 ns	0.39±0.22	0.27±0.05	0.34±0.20	0.29±0.05
C6:0	0.36±0.07 ns	0.48±0.23	0.36±0.07	0.30±0.13	0.38±0.07
C14:0	0.08±0.04 b^**	0.17±0.14 a	0.09±0.02 b	.0.07±0.02 b	00.10±0.01 ab
C16:0	10.20±1.30 ab^**	11.46±5.59 a	..9.99±1.11 ab	.8.75±0.88 b	11.78±0.97 a
C16:1	0.16±0.03 c^**	0.14±0.05 d	0.22±0.05 b	0.0.15±0.03 cd	0.24±0.04 a
C17:0	0.07±0.02 b^**	0.10±0.03 a	.0.08±0.03 ab	.0.10±0.05 a	00.08±0.02 ab
C18:0	4.19±0.13 b^**	6.09±1.94 a	4.06±0.38 b	04.32±0.21 b	4.06±0.16 b
C18:1 c (n-9)	18.62±0.71 c^**	17.28±4.51 c	23.31±0.74 b	31.35±2.25 a	19.07±0.93 c
C18:1 t (n-9)	0.96±0.10 ab^**	0.86±0.27 b	1.08±0.16 a	00.83±0.30 b	01.05±0.06 ab
C18:2 c (n-6)	63.72±2.07 a^**	60.62±15.02 a	59.30±1.71 a	52.59±3.04 b	61.62±1.88 a
C18:3 (n-6)	0.25±0.02 ns	0.26±0.10	0.27±0.02	0.24±0.01	0.32±0.11
C20:0	0.16±0.01 bc^**	0.21±0.08 a	0.13±0.01 c	.0.17±0.01 b	0.17±0.01 b
C20:1	0.12±0.05 b^*	0.14±0.06 ab	00.15±0.02 ab	.0.17±0.01 a	00.13±0.01 ab
C20:3 (n-3)	0.34±0.09 ab^*	0.47±0.28 a	0.33±0.08 b	.00.34±0.12 ab	0.33±0.07 b
C22:6 (n-3)	0.31±0.16 b^**	0.54±0.52 a	0.26±0.12 b	.0.17±0.04 b	0.25±0.08 b
∑ SFA	15.32±1.39 b^**	18.89±8.09 a	14.97±0.95 b	14.04±0.92 b	16.87±0.84 ab
∑ MUFA	19.87±0.70 c^**	18.41±4.83 c	24.76±0.79 b	32.49±2.13 a	20.48±1.01 c
∑ PUFA	64.62±1.89a^**	61.89±14.38 a	60.16±1.55 a	53.35±2.90 b	62.53±1.75 a

Fatty acid codes: Butyric acid (C4:0), Caproic acid (C6:0), Myristic acid (C14:0), Palmitic acid (C16:0), Palmitoleic acid (C16:1), Heptadecanoic acid (C17:0), Stearic acid (C18:0), Cis-oleic acid (C18:1n-9c), Trans-elaidic acid (C18:1n-9t), Cis-linoleic acid (C18:2n-6c), Linolenic acid (C18:3n6), Arachidic acid (C20:0), Cis-11-eicosenoic acid (C20:1), Cis-11,14,17-eicosatrienoic acid (C20:3n3), Cis-4,7,10,13,16,19-docosahexaenoic acid (C22:6n3). In each line, statistical differences (^*: p < 0.05, ^**: p < 0.01) between the mean values of the varieties are shown with different letters. ns: Not significant.

In the studied varieties, the rootstocks were found to have a limited effect on the fatty acid compositions of the seeds. It was observed that linoleic acid contents increased with the use of the 1103P rootstock, while oleic and palmitic acid contents increased with the use of 110R (Table 6). Compared to the 110R rootstock, 1103P was found to cause higher PUFA contents of seeds. On the other hand, MUFAs and SFAs were found at higher rates in the seeds of grape varieties grafted onto 110R rootstock.

Table 6

Effects of grape rootstocks and growing seasons on fatty acid compositions of grape seeds

Fatty Acid	1103 P	110 R	2017	2018
C4:0	0.26±0.05 b^**	0.36±0.18 a	0.37±0.18 a^**	0.26±0.05 b
C6:0	0.34±0.07 ns	0.41±0.18	0.41±0.17 a^*	0.34±0.08 b
C14:0	0.09±0.06 ns	0.11±0.08	0.13±0.09 a^**	0.07±0.01 b
C16:0	9.68±1.56 b^**	11.19±3.53 a	11.72±3.42 a^**	9.15±0.99 b
C16:1	0.17±0.06 b^**	0.19±0.05 a	0.21±0.06 a^**	0.15±0.03 b
C17:0	0.07±0.01 b^**	0.10±0.04 a	0.10±0.04 a^**	0.07±0.01 b
C18:0	4.37±0.52 ns	4.72±1.56	4.56±1.61 ns	4.53±0.43
C18:1 c (n-9)	21.17±5.69 b^**	22.69±5.58 a	22.76±6.26 a^**	21.09±4.91 b
C18:1 t (n-9)	0.94±0.13 ns	0.97±0.28	1.02±0.27 a^**	0.89±0.13 b
C18:2 c (n-6)	61.62±5.03 a^**	57.52±9.42 b	56.81±9.30 b^**	62.33±4.52 a
C18:3 (n-6)	0.27±0.07 ns	0.27±0.08	0.27±0.08 ns	0.27±0.07
C20:0	0.16±0.02 ns	0.17±0.06	0.18±0.06 a^*	0.16±0.02 b
C20:1	0.15±0.02 ns	0.13±0.05	0.13±0.04 ns	0.14±0.04
C20:3 (n-3)	0.43±0.07 a^**	0.30±0.18 b	0.43±0.18 a^**	0.29±0.08 b
C22:6 (n-3)	0.27±0.12 ns	0.35±0.37	0.45±0.33 a^**	0.17±0.05 b
∑ SFA	14.99±1.37 b^*	17.05±5.33 a	17.46±5.22 a^**	14.58±0.96 b
∑ MUFA	22.42±5.70 b^**	23.98±5.61 a	24.13±6.22 a^**	22.27±4.98 b
∑ PUFA	62.46±5.10 a^**	58.56±9.05 b	57.95±9.04 b^**	63.07±4.54 a

Fatty acid codes: Butyric acid (C4:0), Caproic acid (C6:0), Myristic acid (C14:0), Palmitic acid (C16:0), Palmitoleic acid (C16:1), Heptadecanoic acid (C17:0), Stearic acid (C18:0), Cis-oleic acid (C18:1n-9c), Trans-elaidic acid (C18:1n-9t), Cis-linoleic acid (C18:2n-6c), Linolenic acid (C18:3n6), Arachidic acid (C20:0), Cis-11-eicosenoic acid (C20:1), Cis-11,14,17-eicosatrienoic acid (C20:3n3), Cis-4,7,10,13,16,19-docosahexaenoic acid (C22:6n3). In each line, statistical differences (^*: p < 0.05, ^**: p < 0.01) between the mean values of the rootstocks and growing seasons are shown with different letters. ns: Not significant.

Only three of the fatty acids in the seed oils (stearic acid, eicosenoic acid, and linolenic acid) showed similar rates in both seasons, while others showed changes depending on the seasons. Linoleic acid was obtained at higher rates in the 2018 season and other fatty acids in the 2017 season. In addition, the SFA and MUFA contents of the seeds were found to be higher in the 2017 season, while PUFA contents were higher in the 2018 season. Based on these findings, it can be said that the fatty acid composition of grape seeds is affected by seasonal climate changes in the cultivation area. As a matter of fact, the vineyard area where the samples were taken was the same in both years, and the soil characteristics and applied cultural practices were not changed. The difference between the two years especially depended on the available water capacity variation of soil, which was changed according to the precipitation regime.

3.4 Mineral contents

It was found that the seeds belonging to different groups of rootstock-variety interactions did not differ statistically from each other in terms of their mineral contents, except Zn (Table 7). The highest Zn content was found in Ata Sarisi/1103P. However, the seeds taken from this variety that were grafted onto 110R rootstock were also rich in Zn content, in comparison to the findings of Kamel et al. [32]. When the varieties were compared among themselves, their seeds were observed to differ from each other according to mineral contents. When the minerals in grape seeds were ordered according to their quantities, the following ranking appeared: N > Ca >K >P >Mg >Mn >B >Fe >Cu >Zn. Gök Tangolar et al. [40] reported a similar order in their study on different grape varieties (4 colored and 2 white V. vinifera L. varieties) and grape rootstocks (Salt Creek and Cosmo 2) at Çukurova conditions.

Table 7
Changes of mineral compositions of seeds according to rootstocks, grape varieties, and seasons

Rs. Var. N (%) P (%) K (%) Ca (%) Mg (%) Fe (ppm) Mn (ppm) Cu (ppm) Zn (ppm) B (ppm)

1103 P R.G. 1.09±0.03ns 0.30±0.01ns 0.52±0.03ns 0.81±0.07ns 0.16±0.01ns 20.18±2.97ns 43.45±8.63 ns 12.25±0.89ns 8.45±0.65c^* 29.20±0.89ns

T.I. 1.27±0.04 0.32±0.01 0.47±0.07 0.91±0.08 0.18±0.02 27.93±6.66 48.45±7.82 13.23±0.47 12.03±1.70ab 31.30±2.02

A.S. 1.34±0.11 0.30±0.03 0.54±0.11 0.68±0.08 0.12±0.01 21.20±2.51 31.95±3.98 12.45±0.99 13.53±1.25a 34.88±5.82

H.P. 1.44±0.11 0.29±0.07 0.37±0.09 0.76±0.19 0.16±0.04 20.93±5.55 35.30±10.27 11.10±2.33 10.85±3.12abc 25.05±2.86

H.K. 1.07±0.11 0.27±0.02 0.50±0.06 0.82±0.09 0.14±0.00 20.75±0.40 26.23±1.45 12.33±0.53 8.43±0.97c 28.65±2.09

110 R R.G. 1.04±0.03 0.29±0.00 0.55±0.03 0.77±0.07 0.16±0.01 21.80±3.01 34.13±6.90 12.33±1.03 10.95±2.51abc 34.40±2.33

T.I. 1.34±0.06 0.29±0.02 0.44±0.08 0.81±0.07 0.15±0.01 25.88±8.47 42.48±5.04 13.33±0.75 10.60±0.23abc 33.13±4.88

A.S. 1.30±0.13 0.29±0.01 0.55±0.09 0.70±0.06 0.12±0.01 17.65±1.94 31.65±3.86 10.88±0.55 12.90±0.99a 40.23±6.31

H.P. 1.49±0.11 0.29±0.05 0.38±0.05 0.70±0.12 0.16±0.03 18.68±4.63 35.70±9.46 10.35±1.23 9.65±2.22bc 27.30±2.11

H.K. 1.10±0.05 0.26±0.01 0.45±0.02 0.80±0.07 0.13±0.00 16.65±0.15 27.58±1.44 11.53±0.65 8.98±0.57bc 29.40±3.48

Rs Av. 1103 P 1.25±0.16ns 0.30±0.04ns 0.48±0.10ns 0.80±0.13ns 0.15±0.03ns 22.20±4.92ns 37.08±10.50ns 12.27±1.34a^* 10.66±2.61ns 29.82±4.43b^

110 R 1.25±0.19 0.28±0.03 0.47±0.09 0.75±0.09 0.14±0.02 20.13±5.47 34.31±7.48 11.68±1.35b 10.62±2.01 32.89±5.95a

Var. Av. R.G. 1.06±0.04c^ 0.29±0.01ab^* 0.53±0.03ab^ 0.79±0.07ab^ 0.16±0.01a^ 20.99±2.98b^ 38.79±8.90ab^ 12.29±0.92ab^ 9.70±2.18bc^ 31.8±3.19bc^

T.I. 1.30±0.06b 0.31±0.02a 0.45±0.07c 0.86±0.09a 0.16±0.02a 26.90±7.34a 45.46±7.01a 13.28±0.60a 11.31±1.38ab 32.2±3.69b

A.S. 1.32±0.12b 0.30±0.02a 0.55±0.09a 0.69±0.07c 0.12±0.01b 19.43±2.83b 31.80±3.74bc 11.66±1.12bc 13.21±1.12a 37.6±6.43a

H.P. 1.47±0.11a 0.29±0.06ab 0.37±0.08d 0.73±0.16bc 0.16±0.03a 19.80±5.01b 35.50±9.42b 10.73±1.82c 10.25±2.65bc 26.2±2.67d

H.K. 1.09±0.08c 0.26±0.01b 0.48±0.05bc 0.81±0.08ab 0.13±0.01b 18.70±2.16b 26.90±1.55c 11.93±0.70bc 8.70±0.81c 29.0±2.77cd

Year Av. 2017 1.19±0.14b^ 0.28±0.04ns 0.51±0.10a^ 0.72±0.11b^ 0.15±0.03ns 22.71±6.74a^ 35.21±9.69ns 11.81±1.69ns 10.31±2.69ns 33.67±6.34a^**

2018 1.30±0.19a 0.30±0.02 0.44±0.06b 0.83±0.09a 0.15±0.02 19.62±2.44b 36.18±8.71 12.15±0.94 10.97±1.83 29.04±2.94b

Rs.	Var.	N (%)	P (%)	K (%)	Ca (%)	Mg (%)	Fe (ppm)	Mn (ppm)	Cu (ppm)	Zn (ppm)	B (ppm)
1103 P	R.G.	1.09±0.03ns	0.30±0.01ns	0.52±0.03ns	0.81±0.07ns	0.16±0.01ns	20.18±2.97ns	43.45±8.63 ns	12.25±0.89ns	8.45±0.65c^*	29.20±0.89ns
	T.I.	1.27±0.04	0.32±0.01	0.47±0.07	0.91±0.08	0.18±0.02	27.93±6.66	48.45±7.82	13.23±0.47	12.03±1.70ab	31.30±2.02
	A.S.	1.34±0.11	0.30±0.03	0.54±0.11	0.68±0.08	0.12±0.01	21.20±2.51	31.95±3.98	12.45±0.99	13.53±1.25a	34.88±5.82
	H.P.	1.44±0.11	0.29±0.07	0.37±0.09	0.76±0.19	0.16±0.04	20.93±5.55	35.30±10.27	11.10±2.33	10.85±3.12abc	25.05±2.86
	H.K.	1.07±0.11	0.27±0.02	0.50±0.06	0.82±0.09	0.14±0.00	20.75±0.40	26.23±1.45	12.33±0.53	8.43±0.97c	28.65±2.09
110 R	R.G.	1.04±0.03	0.29±0.00	0.55±0.03	0.77±0.07	0.16±0.01	21.80±3.01	34.13±6.90	12.33±1.03	10.95±2.51abc	34.40±2.33
	T.I.	1.34±0.06	0.29±0.02	0.44±0.08	0.81±0.07	0.15±0.01	25.88±8.47	42.48±5.04	13.33±0.75	10.60±0.23abc	33.13±4.88
	A.S.	1.30±0.13	0.29±0.01	0.55±0.09	0.70±0.06	0.12±0.01	17.65±1.94	31.65±3.86	10.88±0.55	12.90±0.99a	40.23±6.31
	H.P.	1.49±0.11	0.29±0.05	0.38±0.05	0.70±0.12	0.16±0.03	18.68±4.63	35.70±9.46	10.35±1.23	9.65±2.22bc	27.30±2.11
	H.K.	1.10±0.05	0.26±0.01	0.45±0.02	0.80±0.07	0.13±0.00	16.65±0.15	27.58±1.44	11.53±0.65	8.98±0.57bc	29.40±3.48
Rs Av.	1103 P	1.25±0.16ns	0.30±0.04ns	0.48±0.10ns	0.80±0.13ns	0.15±0.03ns	22.20±4.92ns	37.08±10.50ns	12.27±1.34a^*	10.66±2.61ns	29.82±4.43b^**
	110 R	1.25±0.19	0.28±0.03	0.47±0.09	0.75±0.09	0.14±0.02	20.13±5.47	34.31±7.48	11.68±1.35b	10.62±2.01	32.89±5.95a
Var. Av.	R.G.	1.06±0.04c^**	0.29±0.01ab^*	0.53±0.03ab^**	0.79±0.07ab^**	0.16±0.01a^**	20.99±2.98b^**	38.79±8.90ab^**	12.29±0.92ab^**	9.70±2.18bc^**	31.8±3.19bc^**
	T.I.	1.30±0.06b	0.31±0.02a	0.45±0.07c	0.86±0.09a	0.16±0.02a	26.90±7.34a	45.46±7.01a	13.28±0.60a	11.31±1.38ab	32.2±3.69b
	A.S.	1.32±0.12b	0.30±0.02a	0.55±0.09a	0.69±0.07c	0.12±0.01b	19.43±2.83b	31.80±3.74bc	11.66±1.12bc	13.21±1.12a	37.6±6.43a
	H.P.	1.47±0.11a	0.29±0.06ab	0.37±0.08d	0.73±0.16bc	0.16±0.03a	19.80±5.01b	35.50±9.42b	10.73±1.82c	10.25±2.65bc	26.2±2.67d
	H.K.	1.09±0.08c	0.26±0.01b	0.48±0.05bc	0.81±0.08ab	0.13±0.01b	18.70±2.16b	26.90±1.55c	11.93±0.70bc	8.70±0.81c	29.0±2.77cd
Year Av.	2017	1.19±0.14b^**	0.28±0.04ns	0.51±0.10a^**	0.72±0.11b^**	0.15±0.03ns	22.71±6.74a^**	35.21±9.69ns	11.81±1.69ns	10.31±2.69ns	33.67±6.34a^**
	2018	1.30±0.19a	0.30±0.02	0.44±0.06b	0.83±0.09a	0.15±0.02	19.62±2.44b	36.18±8.71	12.15±0.94	10.97±1.83	29.04±2.94b

In each column, statistical differences (^*: p < 0.05, ^**: p < 0.01) between mean values of the rootstock-variety groups, rootstocks, varieties, and seasons are shown with different letters. ns: Not significant.

In general, our results, except for Ca and Mn, were within or close to the limit values specified by previous researchers [32 , 82] and are in line with the literature in this respect. On the other hand, the Ca contents of the seeds were found to be higher according to Özcan et al. [41], while the contents of Mn were found to be higher according to Gök Tangolar et al. [40]. The cause of these differences between studies may be the varieties included or the different mineral levels in the soils of the cultivation regions where the vineyards were located.

Compared to the other varieties evaluated in this study, Trakya Ilkeren was found to have richer mineral contents with seeds containing higher amounts of P, Ca, Mg, Fe, Mn, and Cu. The Ata Sarisi variety, on the other hand, differed from the other varieties because its seeds contained high amounts of K, Zn, and B. The seeds of Hatun Parmagi were found to be similar to the seeds of Red Globe in terms of Mg content, and a high amount of N was also found in the seeds of this variety. The mineral contents of the seeds of Horoz Karasi were found to be lower than those of the other varieties considered in this study. Similarly, the mineral contents of the seeds of this variety were reported to be quite low by Gök Tangolar et al. [40]. The K, Ca, Mg, and Fe levels that we found in the seeds of Horoz Karasi were also very similar to the values found by Gök Tangolar et al. [40]. Based on this similarity, it can be stated that it was the genotype that directly controlled the accumulation of certain mineral substances in the seeds of these grape varieties. In addition, the higher N (p < 0.001) and Zn (p < 0.01) contents of the seeds of colored varieties and the higher Ca (p < 0.001) and Cu (p < 0.001) contents of the seeds of white varieties suggested that the mineral contents of seeds may be associated with the color of the grape skin.

It was found that the rootstocks had no effect on the mineral contents of the seeds, except for Cu and B. Although it was observed that the 1103P rootstock had a positive effect on the uptake of some minerals in the study seasons, this finding was not reflected in the seasonal average. The positive effect of the 110R rootstocks on the B contents of seeds was found for both study seasons and the average of the seasons. On the other hand, the use of the 1103P rootstock led to increased Cu concentrations in the seeds.

The effects of the growing seasons on the mineral contents of grape seeds were found to be limited. In samples taken in the first season of the study, the P, Fe, and B contents were found to be higher, while in samples taken in the second season, the N and Ca contents were found to be higher. The amount of precipitation in the 2018 season, which was greater than that in the previous growing season, may have caused an inhibition of K and B uptake while increasing Ca uptake by activating the lime in the soil. Kacar and Katkat [83] reported decreased B uptake in plants caused by increases in soil pH or the amount of lime in the soil due to a variety factors. Sultana et al. [84] found that an increased amount of lime in the soil caused increased Ca uptake in wheat. Although Elzam and Hodges [85] reported that Ca may have an effect on K uptake, Gluhic et al. [86] claimed that the active lime content of the soil is not the only factor that affects the K level in grape leaves. In addition, the difference in precipitation between seasons may negatively affect Fe uptake by causing HCO₃ formation [87]. This also explains the higher Fe content in the seeds in the 2017 season in the present study.

When the mineral contents of the seeds were analyzed, the levels of some minerals were found to be associated with some others. Increased P levels in the seeds led to an increase in other minerals (Table 8). However, the relation of N and B levels with other minerals is generally quite limited. The only statistically significant negative correlation that we found in this study was between N and K (r = –0.516, p < 0.01).

Table 8

Correlations of minerals in grape seeds with each other (^*: p < 0.05, ^**: p < 0.01)

	N	P	K	Ca	Mg	Fe	Mn	Cu	Zn
P	0.309^*
K	–0.516^**	0.371^**
Ca	–0.021	0.592^**	0.015
Mg	0.175	0.706^**	0.051	0.609^**
Fe	0.093	0.603^**	0.401^**	0.348^**	0.632^**
Mn	0.175	0.704^**	0.419	0.581^**	0.791^**	0.577^**
Cu	–0.093	0.629^**	0.379^**	0.571^**	0.439^**	0.668^**	0.416^**
Zn	0.343^**	0.674^**	0.390^**	0.216	0.219	0.431^**	0.359^**	0.366^**
B	–0.225	0.304^*	0.808^**	–0.175	–0.111	0.325^*	0.111	0.226	0.490^**

4 Conclusion

The fact that grape seeds with significant protein and oil contents are also an important source of minerals and PUFAs indicates that their use in both human diets and animal feed will have positive health effects. As a matter of fact, our findings showed that the grape seeds contain more than 8% oil, and 6.5% protein and seed oil contain a high amount of linoleic (51.5–67.9%) and oleic (15.0–31.6%) acids. In addition to this, it also found that grape seeds contain a high amount of Ca, Fe, and Mg which are necessary for human and animal diets. However, when planning the production of grapes with the intent to provide raw materials for the pharmaceutical, cosmetics, or oil industries in the future, rootstocks and varieties should be selected while taking into consideration that the fatty acid compositions of grape varieties may change depending on both the variety and the rootstock on which it is grafted. In addition, it is worth emphasizing that the amount of oil in the seeds, the number of seeds in grapes of different varieties, and the yield of grapes per hectare are other important factors in determining the amount of seed oil to be obtained from vineyard areas.

Although the effect of production season on the pomological properties of seeds was found to be limited in this study, it is necessary to more comprehensively explore the changes detected in the oil, protein, ash, fatty acid, and mineral contents of grape seeds. Therefore, the effects of different irrigation regimes and extreme temperatures on grape seeds should be investigated in future studies.

Footnotes

Acknowledgments

This work was supported by the Harran University Scientific Research Projects Coordinatorship (Project no: 19022).

Conflict of interest

The authors have no conflict of interest to report.

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