Phenolic and Fatty Acid Profile, and Protein Content of Different Walnut Cultivars and Genotypes (Juglans regia L.) Grown in the USA

ABSTRACT

Total phenol, phenolic compound, total oil, fatty acid, protein contents of walnut kernels from nine different cultivars and six genotypes grown in the USA were investigated. Total phenolic content was found to range from 540.08 to 1067.81 mg GAE/100 g dw, with three cultivars containing a value of or over 1000 mg GAE/100 g dw. A total of seven phenolic compounds were quantified, and their quantity were greatly varied among the cultivars/genotypes. Ellagic acid was the most abundant phenolic compound, varying from 20.03 to 97.23 mg/kg. Total oil component ranged from 53.17% to 65.92% with an average value of ca. 60%. As a saturated fatty acid type, myristic, palmitic, stearic, and arachidic acid; as an unsaturated acid type, linoleic, linolenic acid, oleic, and palmitoleic acid were quantified. Palmitic acid was the predominant saturated fatty acid compound and ranged from 5.74% to 9.49%; linoleic acid was the predominant unsaturated fatty acid compound and ranged from 58.96% to 66.07%. Protein content percentage was notably varied among cultivars and genotypes, ranging from 13.57% to 25.72% with an average value of ca. 21%. These results indicate that the walnut kernel is a good source of fat, phenolics, and proteins, and its abundance are greatly cultivar/genotype-dependent.

KEYWORDS:

• Walnut kernel
• walnut seed
• juglone
• ellagic acid
• palmitic acid

Introduction

Walnut, a member of Juglandaceae plant family, is one of the valued and widespread tree nut throughout the world owing to its rich nutritive content. The nut is widely planted especially in regions with a semitropical or temperate climate thanks to a high adaptation ability to different climatic conditions. Most of the walnut production takes place in China, the USA, Turkey, Iran, Ukraine, Romania, India, and Chile. The total area allocated for walnut production throughout the world is about 1.186.398 ha with an annual production of over 3.6 MT (FAO, 2018). China produces about half of this production with 1.7 MT. China is ensued by the USA, Iran, and Turkey with about 0.50, 0.45, and 0.21 MT, respectively (FAO, 2018). Located between the walnut gene canters and homeland, the USA is home to various walnut cultivars and varieties. Particularly, very rich walnut populations are found in California, and many walnut types have been selected from these populations.

In recent years, several studies have been carried out on identification and determination of biochemical, phytochemical, and antioxidant properties of walnut fruits and on their contribution to human nutrition and health. Of biochemical compounds, fat and derivates are the primary nutritional content of walnut kernels (Amaral et al., 2003). The walnut kernel has an oil high content up to 73.9% which consists of mainly unsaturated fatty acids (Martinez and Maestri, 2008). Linoleic acid is the major fatty acid type, followed by oleic acid and linoleic acid (Kosar et al., 2004; Zwarts et al., 1999). In connection with oil, fat-soluble vitamins A and E have been cited in walnut kernels as well (Amaral et al., 2005; Bujdosó et al., 2010). Other nutritive chemical components found in walnut kernels are proteins, carbohydrates, fibers, phenolics, flavonoids, and sterols (Beyhan et al., 2017; Colaric et al., 2005; Gharibzahedi et al., 2014; Ziarati and Aryapak, 2014). Walnut kernels are also rich in minerals such as iron, copper, zinc, and phosphorous (Cosmulescu et al., 2009; Ziarati and Aryapak, 2014).

Walnut’s oil has been lately studied for health-promoting effects by several scientists. Walnut carries significant amount of polyunsaturated fatty acids which are reported to have beneficiary effects on blood lipids, by lowering blood cholesterol, decreasing the rate of serum concentrations of low-density lipoprotein, and increasing high-density lipoprotein (Anderson et al., 2001; Mukuddem-Petersen et al., 2005). Furthermore, polyunsaturated fatty acids may play an important role in the prevention of coronary heart disease (Sabate et al., 1993).

Walnut kernel is a very good source phytochemicals including phenolic compounds such as juglone and catechin. Walnut’s exceptionally rich phytochemical contents make the nut one of the important antioxidative dietary sources. Antioxidants in walnut have been linked to protective and curative effects for various diseases such like cardiovascular disease, cancer, aging, and cataract (Anderson et al., 2001; Carvalho et al., 2010; Haddad et al., 2014; Slatnar et al., 2015; Trandafir et al., 2017). Antioxidative properties of walnut come from mainly phenolic compounds, fatty acids, and tocopherols (Beyhan et al., 2017; Colaric et al., 2005; Fukuda et al., 2003; Greve et al., 1992; Kornsteiner et al., 2006; Oliveira et al., 2008; Prasad, 1994). For example, it was reported that walnuts are good sources of ellagic acid and ellagitannins which are cancer chemo-preventive polyphenolic compounds found only in limited numbers of fruits and nut species (Cerdá et al., 2005).

Walnut kernels are a good source of protein as well. Walnut protein contains a total of 17 amino acids, including all essential amino acids (Liu et al., 2020). Walnut proteins are separated from the other vegetable proteins owing to the low lysine/arginine ratio which has been linked to a decline in progression of atherosclerosis (Sze-Tao and Sathe, 2000).

Our objective for the present work was to study and compare total phenol, phenolic compound, total oil, fatty acid, protein contents of walnut kernels from nine different cultivars and six promising genotypes grown in the USA. Display quotations of over 40 words, or as needed.

Materials and Methods

Plant Materials

Nine cultivars (‘Ivanhoe,’ ‘Franquette,’ ‘Howard,’ ‘Durham,’ Earliest,’ Solano,’ ‘Hartley,’ Robert Livermore,’ and ‘Chandler’) and five genotypes (95–014-3, PI59568, 03–001-2557, Robert Livermore NSB, Chenier, and Sinensis #5) were selected from the UC Davis Walnut Improvement Program in the USA. Walnut fruits were harvested at the commercial maturity stage depending on the cultivars or genotypes, with being picked up randomly from three different trees. Triplicate analysis was made with selected walnuts, and each replicate included 75 walnuts from each tree. Kernel samples were ground in a mortar and pestle into powder at the room temperature for the ensuing assays.

Quantification of Total Phenol and Phenolic Compounds

The analysis of total phenol content was carried out according to the Folin–Ciocalteu method (Spanos and Wrolstad, 1990). One ml aliquot extract (diluted 1:20 methanol) and 1 ml deionized water were mixed a 10-ml flask, followed by adding Folin–Ciocalteu reagent (500 µL). After 2 min, 4 ml 7.5% Na₂CO₃ solution was added to the mixture. After 2-h incubation at room temperature, the absorbance was measured at 745 nm (Perkin-Elmer, Lambda 5). Gallic acid was chosen as a standard phenolic compound. Total phenolic contents were reported in milligram equivalents of gallic acid (GAE) per 100 gr of dry weight. Phenolic extraction process was done according to the procedure employed by Kosar et al. (2004) and Trandafir et al. (2017) using HPLC UV detector (Shimadzu LC-20A). Powdered walnut kernels (0.5 g) mixed with acetone and water (1:4) were boiled under reflux for 30 min. After cooling, the mixture was filtered and volume to 10 ml by adding distilled water. Gallic acid, catechin, caffeic acid, syringic acid, p-coumaric acid, rutin trihydrate, ellagic aside, quercetin, naringenin, and juglone were selected as standards. A reverse phase column (5-μm Nucleosil C18; Supelco) was employed with a flow rate of 1 ml/min. The mobile phase composed of HCOOH-water (A; 2.5:97.5 v/v) and of acetonitrile-water (B; 2.5:97.5 v/v). The extracts were read at 280–360 nm to assess the phenolic compounds. The quantity of phenolic compounds was expressed in mg/kg.

Oil Extraction

Kernel was manually separated from the shell using a nutcracker. Oil extraction of kernel powder (25 g) was carried out with an automatic Soxhlet device for 4 h according to Bligh and Dyer (1959). Hexane was selected as an organic solvent, and methylation was executed with Boron trifluoride/methanol (AOAC, 2010). The oil content was calculated based on the weight difference of tubes before and after the experiment. The oil content was also used in ensuing fatty acid analysis.

Quantification of Fatty Acids

Fatty acid methyl esters acquired from boron trifluoride/methanol reagent were extracted through a GC (Clarus, 500) with an autosampler (Perkin Elmer) assembled with an FDI and a fused-silica capillary SGE column (100 m × 0.32 mm, ID 0.25 μm, BP20 0.25 UM; Perkin Elmer). The oven temperature was initially set to 140°C for 5 min, then increased to 200°C at a rate of 4°C/min, followed by another increase of to 220°C at a rate of 1°C/min. The injector and detector temperatures were set to 220°C and 280°C. The FAME mix standard consisting of 37 compounds was used for identification and quantification of fatty acids.

Protein Content

The protein content percentage was quantified indirectly by determining the total N content obtained by the Kjeldahl method (AOAC, 2010), utilizing a nitrogen-protein conversion factor (Kc = 6.25; % protein = Kc* % total N).

Statistical Analysis

Since the trees were neither on the same plot and nor treated equally, mean separation was not performed; each value is expressed as only mean± standard deviation.

Results and Discussion

Quantification of Phenolic Compounds

Total phenolic and phenolic compound contents of the walnut kernels are given Tables 1 and 2. Total phenol content was in the range of 540.08 (‘Chandler’) and 1067.81 mg GAE/100 g dw (‘Howard’), representing almost a twofold difference between two cultivars. ‘Howard,’ ‘Durham,’ and ‘Solana’ stood out from the other walnuts with rich phenolic content. Our results show both similarities (Tosun et al., 2011) and dissimilarities to those some works in which higher total phenolic content is cited (Kafkas et al., 2017; Kornsteiner et al., 2006; Labuckas et al., 2008). As expected ellagic acid was the most abundant phenolic compound followed by catechin, rutin tetrahydrate, naringin, juglone, caffeic acid, p-coumaric acid, and syringic acid, respectively. Ellagic acid content ranged from 20.03 (‘Hartley’) to 97.23 mg/kg (‘Chandler’), and the range was almost fivefold between the cultivars. ‘Howard,’ ‘Earliest,’ and ‘Chandler’ distinguished themselves from others with having abundant ellagic acid content of almost 90 or over 90 mg/kg while ‘Hartley’ and Chenier the lest abundant. Naringenin content varied from 4.19 (‘Chandler’) to 22.51 mg/kg (PI159568), depicting a more than fivefold difference between the lowest and highest. PI159568 genotype came out with carrying the maximal naringenin quantity when compared to the other walnuts while ‘Chandler’ cultivar the minimal. Catechin level was found between 6.29 (‘Howard’) and 45.27 mg/kg (‘Hartley’), accounting a very marked difference (more than sixfold) between two cultivars. ‘Hartley’ presented a very rich catechin level compared to the other walnuts while ‘Howard’ a poor catechin level. Rutin trihydrate amount greatly varied from 0.70 (‘Chenier’) and 45.99 mg/kg (‘Hartley’), representing a huge gap between the two cultivars. ‘Franquette’ and ‘Solana’ stood out from others with abundant amount of rutin trihydrate. All genotypes contained scarce amount of rutin trihydrate aside from Chenier and Sinensi #5.

Table 1. Total phenol (mg GAE/100 g dw), ellagic acid, naringenin, catechin, and quercetin contents of the walnut cultivars and genotypes (mg/kg)

Cultivar/genotype	Total phenol	Ellagic acid	Naringenin	Catechin	Rutin trihydrate
‘Ivanhoe’	670.07 ± 45.38	75.76 ± 3.39	13.29 ± 1.07	9.19 ± 0.94	13.49 ± 0.76
‘Franquette’	563.28 ± 14.59	35.70 ± 2.98	17.87 ± 3.81	35.04 ± 1.77	44.34 ± 1.39
‘Howard’	1067.81 ± 63.24	89.24 ± 5.57	13.98 ± 0.62	6.29 ± 0.68	30.05 ± 1.75
‘Durham’	1024.53 ± 70.72	66.72 ± 1.85	12.93 ± 2.59	16.55 ± 0.49	8.73 ± 0.81
‘Earliest’	597.32 ± 41.06	96.25 ± 2.60	16.08 ± 2.77	15.31 ± 1.25	15.25 ± 1.73
‘Solano’	1000.02 ± 58.71	60.48 ± 2.85	17.70 ± 0.96	27.51 ± 5.64	36.17 ± 1.04
‘Hartley’	688.07 ± 38.18	20.03 ± 2.46	12.27 ± 1.96	45.27 ± 5.53	45.99 ± 2.76
‘R. Livermore’	679.24 ± 55.44	84.55 ± 2.18	10.53 ± 0.30	10.67 ± 2.06	8.37 ± 0.26
‘Chandler’	540.08 ± 14.59	97.23 ± 3.70	4.19 ± 0.85	11.32 ± 1.38	31.91 ± 1.50
95–014-3	936.72 ± 22.84	75.70 ± 2.06	10.24 ± 3.93	31.34 ± 1.24	0.76 ± 0.07
PI159568	574.19 ± 21.5	76.45 ± 2.32	22.51 ± 3.28	19.36 ± 1.01	0.85 ± 0.05
03–001-2357	568.82 ± 14.22	80.21 ± 1.96	6.67 ± 0.60	19.97 ± 1.21	0.71 ± 0.10
R. Livermore NSB	549.89 ± 18.86	78.23 ± 2.52	13.67 ± 1.80	17.34 ± 0.82	5.04 ± 0.17
Chenier	701.29 ± 18.21	23.18 ± 2.28	13.90 ± 0.91	28.76 ± 0.77	0.70 ± 0.08
Sinensis #5	661.95 ± 18.62	52.87 ± 2.01	7.09 ± 0.27	28.30 ± 0.75	1.23 ± 0.15
Max value	1067.81	97.23	22.51	45.27	45.99
Min value	540.08	20.03	4.19	6.29	0.70
Avg Value	721.55	67.51	12.86	21.48	16.23

Each value is expressed as mean± standard deviation.

Table 2. Rutin trihydrate, juglone, p-coumaric acid, caffeic acid, and syringic acid contents of the walnut cultivars and genotypes (mg/kg)

Cultivar/genotype	Juglone	p-coumaric acid	Caffeic acid	Syringic acid
‘Ivanhoe’	13.00 ± 0.94	1.49 ± 0.44	0.68 ± 0.18	0.28 ± 0.04
‘Franquette’	9.84 ± 0.55	4.82 ± 1.07	6.43 ± 0.85	3.18 ± 0.65
‘Howard’	17.81 ± 0.49	2.38 ± 0.10	9.72 ± 0.25	1.11 ± 0.06
‘Durham’	9.53 ± 0.23	1.70 ± 0.21	1.34 ± 0.30	1.19 ± 0.08
‘Earliest’	9.34 ± 0.86	2.09 ± 0.12	6.62 ± 1.08	5.31 ± 0.78
‘Solano’	12.16 ± 2.78	1.80 ± 0.56	1.12 ± 0.06	1.35 ± 0.34
‘Hartley’	14.50 ± 1.30	6.41 ± 1.52	2.87 ± 1.21	1.44 ± 0.06
‘R. Livermore’	8.21 ± 0.68	1.12 ± 0.09	3.19 ± 0.08	1.36 ± 0.04
‘Chandler’	23.31 ± 1.79	0.65 ± 0.03	1.14 ± 0.09	1.89 ± 0.18
95–014-3	6.34 ± 0.93	5.49 ± 0.46	3.59 ± 0.25	1.03 ± 0.05
PI159568	9.61 ± 2.57	2.05 ± 0.07	1.75 ± 0.30	2.67 ± 0.11
03–001-2357	12.65 ± 0.80	1.92 ± 0.18	1.85 ± 0.12	2.65 ± 0.12
R. Livermore NSB	8.67 ± 0.23	2.10 ± 0.14	1.22 ± 0.11	2.32 ± 0.13
Chenier	2.26 ± 0.25	1.66 ± 0.18	1.20 ± 0.12	1.46 ± 0.09
Sinensis #5	2.44 ± 0.43	0.72 ± 0.04	1.23 ± 0.03	1.60 ± 0.11
Max value	23.31	6.41	9.72	5.31
Min value	2.26	0.65	0.68	0.28
Avg Value	10.64	2.43	2.93	1.93

Each value is expressed as mean± standard deviation.

Juglone content ranged from 2.26 (Chenier) 23.31 mg/kg (‘Chandler’), representing a huge 10-fold gap between them. ‘Chandler’ and ‘Howard’ displayed plentiful juglone content over 17 mg/kg while Chenier and Sinensis #5 scanty under 3 mg/kg. p-Coumaric acid value also greatly varied from 0.65 (‘Chandler’) to 6.41 mg/kg (‘Hartley’) among cultivars and genotypes. ‘Hartley’ and 95–014-3 stood out with a rich p-coumaric acid quantity over 5 mg/kg whereas ‘Chandler’ and ‘Sinensis #5ʹ with a poor quantity under 1 mg/kg. Caffeic acid level was found between 0.68 (‘Ivanhoe’) and 9.72 mg/kg (‘Howard’), with a very huge difference between the lowest and highest. ‘Howard’ stood out with an abundant caffeic acid level above 9 mg/kg whereas ‘Ivanhoe’ a poor value under 1 mg/kg. Syringic acid content notably varied from 0.28 (‘Ivanhoe’) to 5.31 mg/kg (‘Earliest’). ‘Earliest’ distinguished itself from the others with regard to syringic acid level of over 5 mg/kg while the others had a value between 1.03 and 3.18 excluding ‘Ivanhoe’ which had the lowest value of 0.28 mg/kg.

There are few literatures available regarding total phenol content or phenolic compound content of a walnut kernel. The quantities of both total and phenolic compounds reported in literatures (Bujdosó et al., 2010; Colaric et al., 2005; Li et al., 2006; Persic et al., 2018; Pycia et al., 2019; Zhang et al., 2009) were parallel to or higher compared to ours. We identified and quantified eight phenolic compounds in the cultivars and genotypes, however, as high as 16 polyphenolic compounds detected by other authors (Persic et al., 2018; Pycia et al., 2019). Persic et al. (2018) classified walnut phenolics as hydroxybenzoic acid derivates, hydroxycinnamic acid derivates, dicarboxylic acid derivates/isomers, and flavonoids. The aforementioned authors further indicated that phenolic compounds found in walnut kernels were mainly ellagic acid and quinic acid derivates. Phenolic compound abundance in walnuts is affected primarily by genotypes (Solar et al., 2006), and secondarily by growing conditions, location, and climatic conditions (Amaral et al., 2008). Ellagic acid is the most researched phenolic compound in walnuts and its abundance were greatly varied by cultivars and locations.

Quantification of Total Oil and Fatty Acids

Data of total oil and fatty acid content are presented in Tables 3 and 4. Total oil content percentage varied from 53.17 (‘R. Livermore’) to 65.92 (Chenier). All genotypes plus ‘Ivanhoe’ cultivar showed maximal oil content percentage of 60 over 60. As anticipated, total unsaturated content was much higher than total saturated content in the walnut cultivars and genotypes. Total saturated fatty acid level varied vaguely among the cultivars and genotypes, with lowest level of 8.37% represented by ‘Howard’ and highest level of 10.11 by ‘Solana.’ Palmitic acid was the prominent fatty acid type followed by stearic acid, arachidic acid, and myristic acid, respectively. Myristic and arachidic acid levels were however so low that it was undetectable in some cultivars and genotypes.

Table 3. Total lipid and saturated fatty acids content of the walnut cultivars and genotypes (%)

Cultivar/genotype	Total lipid	Saturated fatty acids
		Myristic acid (C14:0)	Palmitic acid (C16:0)	Stearic acid (C18:0)	Arachidic acid (C20:0)	Total saturated fatty acids
‘Ivanhoe’	65.39	0.04 ± 00	7.86 ± 0.03	2.43 ± 0.05	ND	10.33
‘Franquette’	56.29	0.06 ± 0.01	7.83 ± 0.03	2.04 ± 0.04	ND	9.93
‘Howard’	58.14	0.04 ± 00	5.74 ± 00	2.59 ± 00	ND	8.37
‘Durham’	57.04	0.03 ± 0.01	6.87 ± 0.06	2.39 ± 0.03	ND	9.29
‘Earliest’	54.85	0.04 ± 0.01	6.95 ± 0.17	2.4 ± 0.02	ND	9.34
‘Solano’	58.03	0.02 ± 0.01	9.49 ± 0.04	2.27 ± 0.02	0.08 ± 0.03	11.86
‘Hartley’	57.68	0.06 ± 0.03	7.09 ± 0.06	2.5 ± 0.07	0.12 ± 0.05	9.77
‘R. Livermore’	53.17	0.03 ± 00	8.03 ± 0.55	2.27 ± 0.22	0.06 ± 0.08	10.39
‘Chandler’	58.68	0.02 ± 00	6.46 ± 0.14	2.92 ± 0.02	0.06 ± 0.08	9.46
95–014-3	60.46	0.02 ± 00	7.16 ± 00	2.4 ± 0.02	ND	9.58
PI159568	59.65	0.02 ± 00	7.36 ± 0.06	2.73 ± 00	ND	10.11
03–001-2357	62.61	0.05 ± 0.05	6.73 ± 0.23	2.45 ± 0.08	0.08 ± 0.1	9.31
R. Livermore NSB	62.44	0.01 ± 0.01	6.59 ± 0.02	2.15 ± 0.01	0.02 ± 0.03	8.77
Chenier	65.92	ND	6.95 ± 0.04	3 ± 00	ND	9.03
Sinensis #5	61.18	ND	7.16 ± 0.01	2.44 ± 0.04	0.08 ± 0	9.68
Max value	65.92	0.06	9.49	3.00	0.12	11.86
Min value	53.7	0.01	5.74	2.04	0.02	8.37
Avg Value	59.43	0.03	7.22	2.47	0.07	9.68

Each value is expressed as mean± standard deviation. ND: non-detected

Table 4. Unsaturated fatty acids content of the walnut cultivars and genotypes (%)

Cultivar/genotype	Unsaturated fatty acids
	Polyunsaturated fatty acids (PUFA)			Monounsaturated fatty acids (MUFA)
	Linoleic acid	Linolenic acid	Total PUFA	Oleic acid	Palmitoleic acid	Total MUFA
‘Ivanhoe’	59.23 ± 0.13	13.24 ± 0.01	72.47	16.95 ± 0.05	0.24 ± 0.16	17.19
‘Franquette’	64.26 ± 0.02	11.57 ± 0.03	75.83	14.17 ± 0.02	0.08 ± 0.02	14.25
‘Howard’	64.34 ± 0.02	15.87 ± 0.02	80.21	11.36 ± 0.04	0.06 ± 00	11.42
‘Durham’	60.23 ± 0.05	9.7 ± 0.07	69.93	20.65 ± 0.05	0.13 ± 0.05	20.78
‘Earliest’	63.11 ± 0.04	9.88 ± 0.04	72.99	17.49 ± 0.19	0.13 ± 0.01	17.62
‘Solano’	61.5 ± 0.07	15.65 ± 0.05	77.15	10.85 ± 0.01	0.14 ± 00	10.99
‘Hartley’	65.26 ± 0.1	11.1 ± 0.02	76.36	13.74 ± 0.23	0.13 ± 0.03	13.87
‘R. Livermore’	61.69 ± 0.47	9.46 ± 0.39	71.15	18.34 ± 0.6	0.13 ± 0.01	18.47
‘Chandler’	58.96 ± 0.09	14.03 ± 0.02	72.99	17.48 ± 0.15	0.07 ± 0.02	17.55
95–014-3	60.83 ± 0.24	13.13 ± 0.03	73.96	15.99 ± 0.14	0.46 ± 0.42	16.45
PI159568	61.67 ± 0.04	10.56 ± 0.03	72.23	17.57 ± 0.02	0.09 ± 0.02	17.66
03–001-2357	60.00 ± 0.4	16.14 ± 0.17	76.14	14.31 ± 0.02	0.24 ± 0.12	14.55
R. Liver. NSB	64.31 ± 0.07	12.01 ± 0.01	76.32	14.78 ± 0.03	0.12 ± 0.02	14.90
Chenier	61.67 ± 0.01	9.08 ± 0.02	70.75	19.21 ± 0.03	0.08 ± 0.01	19.29
Sinensis #5	66.07 ± 0.18	8.41 ± 0.07	74.48	15.71 ± 0.02	0.14 ± 0.05	15.85
Max value	66.07	16.14	80.21	20.65	0.46	20.78
Min value	58.96	8.41	69.93	10.85	0.06	10.99
Avg Value	62.21	11.98	74.20	15.91	0.15	16.06

Each value is expressed as mean± standard deviation.

Myristic acid content percentage ranged very slightly from an undetectable value (Sinensis #5 and Chenier) to 0.06% (‘Hartley’). Palmitic acid content varied among cultivars, with the lowest percentage of 5.74 was displayed by ‘Howard’ while the highest of 9.49 by ‘Solana.’ ‘Solana’ distinguished itself from the others with having a value above 9%. Stearic acid content showed almost no variation among cultivars and genotypes. Chenier had the highest value of 3.00 whereas ‘Franquette’ the lowest value of 2.04%. Arachidic acid content was so low that it was detected in only half of the cultivars and genotypes in which values were recorded under 0.1% with the exception of ‘Hartley.’

Polyunsaturated fatty acid portion of the cultivars and genotypes were average fourfold higher than that of monounsaturated fatty acid portion. Linoleic acid as a polyunsaturated fatty acid registered the highest portion followed by linolenic acid, and oleic acid as monounsaturated fatty acid did the highest portion followed by palmitoleic acid. Polyunsaturated fatty acid portion was in the range of 69.93 (‘Durham’) and 80.21% (‘Howard’), accounting a slight variation among the cultivars and genotypes. ‘Howard’ stepped forth distinguished itself from the others with having a percentage value of over 80. Linoleic acid quantity slightly varied among the cultivars and genotypes, and ‘Sinensis #5ʹ were a much source of linoleic acid (66.07%) while ‘Chandler’ were a source less of linoleic acid (58.96%). Linolenic acid quantity ranged from 8.41 (Sinensis #5) to 16.14% (03–001-2357), accounting a twofold gap between the lowest and the highest. ‘Hovard,’ ‘Solana,’ and ‘03–001-2357ʹ came out as superlative cultivars or genotypes regarding linolenic acid quantity of over 15%.

Monounsaturated fatty acid portion of the cultivars and genotypes were found between 10.99 (‘Solano’) and 20.78% (‘Durham’), presenting a twofold gap between the highest and lowest. Oleic acid quantity varied from 10.85 (‘Solana’) to 20.65% (‘Durham’), indicating a twofold difference between two cultivars. ‘Durhan’ and Chenier distinguished themselves from the other cultivars and genotypes with regard to high oleic acid quantity. Palmitoleic acid quantity ranged from 0.06 (‘Howard’) to 0.46% (’95–013-3ʹ), presenting a very high variation. On the other hand, the quantities were so low even for the richest had value of under 0.5%.

Oil (fat), besides carbohydrate and protein, is the primary component of dry matter of walnuts (Pycia et al., 2019), thus oil content percentage is one of the valued parameters for walnuts. Average total oil content of the present study ca. 60% which fell in the range of several works around the world (Gao et al., 2018; Kafkas et al., 2017; Mitrovic et al., 1995; Pycia et al., 2019; Savage et al., 1999). We recorded the highest total oil of 65.92% from ‘Ivanhoe,’ on the other hand, a value of as high as 73.90% was cited in ‘Sorrento’ cultivar in Argentina by Martinez and Maestri (2008). We recorded palmitic acid as the major saturated fatty acid compound followed by stearic acid, and a trace amount of myristic and arachidic acid in walnuts, which is supported by several studies (Beyazit and Sumbul, 2012; Dogan and Akgul, 2005; Kafkas et al., 2017; Popa et al., 2011). In the present study, linoleic acid was the prominent polyunsaturated fatty acid followed by linolenic acid. Our results are consistent with previously published studies (Dogan and Akgul, 2005; Kafkas et al., 2017; Pereira et al., 2008). We found oleic acid was the major fatty acid compound followed by a trace amount of palmitoleic acid, which agrees previous findings (Kafkas et al., 2017; Popa et al., 2011; Zwarts et al., 1999).

Protein Content

Protein content of the walnut cultivars and genotypes is seen in Table 5. Protein content percentage was in the range of 13.57 (03–001-2357) and 25.72% (‘Franquette’), representing almost a twofold gap between the lowest and the highest. Majority of the cultivars or genotypes had a protein content value closed to or above 20% while ‘Chandler’ and 03–001-2357 presented a value under 15%. ‘Franquette’ stood out from others having a value over 25%. The protein content of walnut presented here was similar to ranges reported from different countries including New Zealand (Savage, 2001), China (Liu et al., 2020), Turkey (Ozcan, 2009), and Portugal (Amaral et al., 2003).

Table 5. Protein contents of the walnut cultivars and genotypes (%)

Cultivar/genotype	Protein
‘Ivanhoe’	22.18 ± 0.48
‘Franquette’	25.72 ± 0.09
‘Howard’	19.07 ± 0.81
‘Durham’	17.71 ± 0.12
‘Earliest’	24.25 ± 0.16
‘Solano’	24.64 ± 0.24
‘Hartley’	21.31 ± 0.44
‘R. Livermore’	21.45 ± 0.08
‘Chandler’	14.03 ± 0.46
95–014-3	17.77 ± 0.05
PI159568	21.23 ± 0.16
03–001-2357	13.57 ± 0.29
R. Livermore NSB	24.13 ± 0.28
Chenier	18.67 ± 0.29
Sinensis #5	22.01 ± 0.53
Max value	25.72
Min value	13.57
Avg Value	20.52

Each value is expressed as mean± standard deviation.

Conclusion

The current study revealed that the walnut kernels of various cultivars and genotypes possessed distinctive fatty acids, phenolics, and protein content. The cultivar ‘Ivanhoe’ contained the highest total oil content while ’Howard’ the highest polyunsaturated fatty acid and ‘Durhan’ the highest monounsaturated fatty acid content. Linoleic as an unsaturated fatty acid and palmitic acid as a saturated fatty acid were found as prominent fatty acid types. ‘Howard’ cultivar had the maximal total phenol content accompanied by ‘Durham’ and ‘Solano.’ Ellagic acid was recorded the most abundant phenolic compound, and its quantity was found to be the most prominent in ‘Chandler.’ A protein content percentage as high as 25.72 was recorded in ‘Franquette’ cultivar. These results obtained here indicate walnut kernels are very good natural source unsaturated fatty acids, phenolics, and protein which of most have been reported to have health-beneficiary effects.

Acknowledgments

The authors thank the UC Davis Walnut Improvement Program for supplying plant materials.